i ©^roTenoidS CAROTENOIDS Sole distributors for the U.S.A. and Canada: Elsevier Book Company, Inc., 250 Fifth Avenue, New York i For the British Commonwealth except Canada: Cleaver-Hume Press, Ltd, 42a South Audley Street, London. W. i Printed in The Netherlands by Meijer's Boek- en Handelsdrukkerij, Wormerveer i' •.V CAROTENOIDS PAUL KARRER Director of the Chemical Institute of the University of Ziirich ERNST JUCKER Research Assistant at the Chemical Institute of the University of Zurich TRANSLATED AND REVISED BY ERNEST A. BRAUDE Lecturer in Organic Chemistry, Imperial College of Science and Technology, London ELSEVIER PUBLISHING COMPANY, INC NEW YORK AMSTERDAM LONDON BRUSSELS 1950 Original title: Carotinoide Published 1948 by Verlag Birkhauser A.G., Basel AUTHOR'S FOREWORD The first monograph on carotenoids was written in 1922 by L. S. Palmer {Carotenoids and Related Pigments, New York). In view of the state of the subject at the time, this author could say rela- tively little about the chemistry of these natural pigments and his work therefore consisted mainly of a summary of the knowledge then available concerning the distribution and the biological signif- icance of the carotenoids. In 1934, L. Zechmeister's excellent book Carotinoide (Berlin) appeared, in which the great advances which had been made in the chemistry of these polyene pigments during the period 1927-1934 were described. Since then, the unravelling of the chemical nature of the carotenoids has further advanced and progress has also been made in the elucidation of their biological significance. A great deal of material has thus accumulated during a relatively short period. It was the desire to sift and collate the extensive literature on carotenoids which led to the writing of the present monograph on this class of natural pigments. Special attention has been paid not only to the chemistry but also to the distribution and biological significance of the carotenoids. It is hoped that the numerous tables will help to clarify the relationships between the different pigments. P. Karrer, E. Jucker Zurich, August 1948 TRANSLATOR'S FOREWORD In the present English edition of Professor P. Karrer's and Dr E. Jucker's book, a number of corrections have been made, and a certain amount of new material, covering some of the more impor- tant investigations published since the appearance of the Swiss edition, has been added. I am indebted to my wife for assistance in preparing the trans- lation, and to Dr B. C. L. Weedon for help in checking the proofs. E. A. Braude London, January 1950 \<<>V *hf<^y>^/ CONTENTS ^ -f^ #^ General Part Page Introduction 3 L The mode of occurrence of carotenoids in plants and animals. The detection and estimation of carotenoid pigments 5 1 . Mode of occurrence in plants 5 2. Mode of occurrence in animals 5 3. Detection and estimation 6 4. Colour reactions 7 5. Spectroscopy 8 6. Colorimetry 8 7. Fluorescence spectra 8 References 8 II. The formation of carotenoids in plants and their physiological significance 10 1. Mode of formation 10 2. Function of carotenoids in plants 10 3. Function of carotenoids in the animal organism 11 a) Carotenoids as provitamins A 11 b) Function of carotenoids in the visual process 16 References 17 III. The isolation of carotenoids 20 1. Extraction of carotenoids 20 2. Separation into hypophasic and epiphasic carotenoids 21 3. Separation of the carotenoid mixtures of the two phases 23 4. Crystallisation 28 References 28 IV. The chemical constitution of carotenoids 29 References 37 V. The cis-trans isomerism of carotenoids 38 References 42 VI. Methods of elucidating the constitution of carotenoids 43 1. Determination of the number of double bonds 43 2. Determination of side-chain methyl groups 44 3. Determination of isopropylidene groups 45 4. Determination of hydroxyl groups 45 5. Determination of methoxyl groups 45 6. Detection and estimation of carbonyl groups 46 7. Determination of carboxyl groups 46 8. Oxidation with permanganate and ozone 47 9. Partial degradation of carotenoids with permanganate and chromic acid 47 10. Thermal degradation 48 C^(>P6 viii CONTENTS Page 11. Determination of Optical rotation 49 1 2 . Relationships between chemical constitution and biological properties 49 13. Relationships between chemical constitution and colour 50 14. Comparison with partially synthetic polyene pigments 50 15. Determination of molecular weight 51 References 5i VII. Relationships between the chemical constitution and colour of carotenoids 53 References 59 VIII. The synthesis of carotenoids 60 References 64 IX. The distribution of carotenoids in nature 66 A. Carotenoids in plants 67 1. Phanerogams 67 a) Carotenoids in unexposed parts of plants 67 b) Carotenoids in exposed parts of plants 67 c) Carotenoids in blossoms 69 d) Carotenoids in fruit and seeds 73 2. Cryptogams 7^ B. Carotenoids in animals 82 1. Invertebrates 82 a) Arthropods 83 b) Molluscs 84 c) Echinoderms 86 d) Worms 87 e) Coelenterates and sponges 88 /; Chordata 89 2. Vertebrates 9° a) Mammals 90 b) Birds 91 c) Fish 95 d) Amphibia 98 e) Reptiles 98 f) Miscellaneous 98 References 99, 108 Special Part ^. Carotenoid hydrocarbons of known constitution 113 1. Lycopene 113 2. Prolycopene 125 3. /5-Carotene 126 4. a-Carotene 15° 5. y-Carotene 161 6. Pro-y-carotene 164 References 165 XI. Carotenoids of known structure containing hydroxyl groups 171 1. Lycoxanthin 17^ 2. Rubixanthin 172 CONTENTS ix Page 3. Cryptoxanthin 175 4. Zeaxanthin 180 5. Antheraxanthin 191 6. Violaxanthin I93 7. Auroxanthin 196 8. Xanthophyll I97 9. Xanthophyll epoxide and its transformation products 206 10. Flavoxanthin 207 11. Chrysanthemaxanthin 211 12. Lycophyll 213 References 214 XII. Carotenoids of known or largely known structure containing one ormore carbonyl groups 218 I . /?-Citraurin 218 2. Rhodoxanthin 221 3. Myxoxanthin 225 4. Myxoxanthophyll 227 5. Astacene and Astaxanthin 229 6. Capsanthin 240 7. Capsorubin 251 References 253 XIII. Carotenoid carboxylic acids 256 1. Bixin 256 2. Crocetin 272 3. Azafrin 281 References 290 XIY . Carotenoids of partly or completely unknown structure 295 1 . Rhodoviolascin 295 2. Rhodopin 297 3. Rhodovibrin 298 4. Rhodopurpurin 299 • 5. Flavorhodin 299 6. Aphanin 3°° 7. Aphanicin 3^4 8. Flavacin 3^7 9. Aphanizophyll 3^8 10. Fucoxanthin 3^9 11. Gazaniaxanthin 3^3 12. Celaxanthin S^S 13. Petaloxanthin 3^7 14. Sarcinin and Sarcinaxanthin 3^9 15. Taraxanthin 320 16. Eschscholtzxanthin 323 17. Echinenone 324 18. Pectenoxanthin 326 19. Pentaxanthin 326 20. Sulcatoxanthin 3^7 21. Glycymerin 328 22. Cynthiaxanthin 3^8 23. Torulin 329 CONTENTS Page 24. Torularhodin 330 25. Actinioerythrin 333 26. Violerythrin 333 27. <5-Carotene 334 28. Fenicotterin 334 29. Oscillaxanthin 335 30. Trollixanthin and Trollichrome 335 31. Haematoxanthin 337 32. Canaryxanthophyll and Picofulvin 337 33. Leprotin 338 34. Salmon acid 339 35. Asterin acid 339 36. Mytiloxanthin 339 37. Carotenoid from the blossoms of satin oak 340 38. Carotenoids from diatoms, brown algae and dinofiagellates .... 340 39. Carotenoid from Neurospora 340 References 341 Crystalline form of some carotenoids (Plates) 345 Light absorption curves of some carotenoids 349 Index of vegetable and animal sources of carotenoids 363 Subject index 377 GENERAL PART Introduction The term carotenoids refers to a group of pigments, yellow to red in colour, which are widely distributed in the vegetable and animal kingdoms, and are distinguished by the following features : they are generally composed of isoprene residues, usually eight, arranged in such a way that in the middle of the molecule two methyl groups are present in i : 6 positions, while all other side-chain methyl groups occupy i : 5 positions. The general structure of the carotenoids is of the aliphatic or aliphatic-alicyclic type and their chromophoric systems contain numerous conjugated carbon-carbon double bonds. All carotenoids are soluble in fats and lipoids; the term lipochronies is derived from this property. The only water-soluble carotenoids are those which, owing to the presence of acidic groups (e.g. carboxyl or enol groups), are able to form water-soluble alkali salts, or have acquired lyophilic properties by esterification with sugar residues (e.g. in crocin). In view of their general chemical structure, carotenoids may be regarded as a sub-group of the polyene pigments. However, the latter also include pigments not composed of isoprene residues, but containing an unbranched aliphatic chain of conjugated double bonds (e.g. the diphenylpolyenes). A revised nomenclature for carotenoids has recently been proposed*, but in the present monograph the individual pigments are mostly referred to by the names given to them by their discoverers. The great interest which the carotenoids have aroused during the last twenty years is conditioned not only by their interesting chemical structure but also by their biological and physiological importance. Several of these pigments are pro-vitamins of vitamin A and thus play an essential part in the animal and human organism. Their significance in the vegetable kingdom has so far been less thoroughly investigated but there can be little doubt that here also they fulfil important functions. The Nomenclature of the Carotenoid Pigments (Report of the Committee on Bio- chemical Nomenclature of the National Research Council, accepted by the Nomenclature, Spelling and Pronunciation Committee of the American Chemical Society — Chem. Eng. News 24 (1946) 1235. — Report of the 'Commissions de Reforme de la Nomenclature de Chimie organique at de Chimie biologique'. London, July 1947). 4 INTRODUCTION As the following chapters will show, between 70 and 80 carotenoids have been found in nature up to the present time. They can all be related to one parent substance, lycopene. By means of simple chemical changes such as cyclisation, double bond migration, partial hydrogenation, introduction of hydroxyl-, keto-, or methoxyl-groups, or introduction of an oxygen bridge, etc., the whole range of pigments can be derived from this parent substance. Their visible Ught absorption comprises a range of about 300 m/u, (ca. 400-700 mju). The carotenoids thus represent one of the most striking examples of the mani- fold variation of a parent substance by the vegetable and animal cell. CHAPTER I Mode of occurrence of carotenoids in plants and animals Detection and estimation of carotenoid pigments I. MODE OF OCCURRENCE IN PLANTS Relatively little information is available regarding the mode of occurrence of carotenoids in plants. In view of their non-polar nature, the majority of natural polyene pigments are insoluble in water and do not normally occur dissolved in the celLfluid. An, exception is provided by crocetin which occurs in the cell fluid in the form of its water-soluble gentiobiose ester, crocin. Water solubility can be conferred not only by esterification with sugars, as in the case of crocin, but also by combination with proteins. Esterification can occur with the carotenoid carboxylic acids (e.g. crocetin, bixin and azafrin), while combination with proteins has so far been observed mainly with polyene pigments (e.g. astacene) occurring in animals. (Compare Menke^). The majority of vegetable carotenoids occur in the chromatophores. They seldom occur crystalline, but are usually present in colloidal suspension in the cell hpoids or in admixture with solid or semi-solid fats. Menke^ has recently found that certain carotenoids in the plastids are combined with proteins; this is in accord with the observations of Junge^ regarding the mode of occur- rence of carotenoids in animals. According to Goldowski and Podolskaja^ the carotenoids of the sunflower seeds occur in the aqueous and not in the oily phase. This is in contrast to the findings of Savellis^, according to whom the carotenoids are present in separate lipoid droplets in the chloroplasts. It will be clear from these partly contradictory results that this question has not yet been fully clarified. For further data and examples, reference should be made to the original literature^. 2. MODE OF OCCURRENCE IN ANIMALS Many attempts have been made in recent years to determine the fate of carotenoids taken up with the vegetable food by the animal organism. It has References p. 8-g. 6 OCCURRENCE, DETECTION, ESTIMATION I been found that part of the pigments is excreted unchanged, while the re- mainder is absorbed. The absorbed carotenoids are either deposited in the fat tissues, nerve tissues, inner organs, etc., or converted into other substances which often fulfil important physiological functions in the animal organism (e.g. vitamin A). In the animal organism, the carotenoids either occur dissolved in fats or combined with protein in the aqueous phase. Colloidal solutions are also observed^. A typical example of a carotenoid-protein complex is the astaxanthin proteid, ovoverdin. This water-soluble chromoproteid occurs, for example, in the green eggs of the lobster and in many other Crustacea (cf. p. 230). Asta- xanthin occurs as the fat-soluble carboxylic acid ester in the red hypodermis of the lobster, and the retina of the chicken also contains at least two different esters of this pigmenf^. JuNGE^ has recently carried out investigations on the pigments of insects and has observed that many of these appear to be carotenoid-protein complexes. Thus, phytoxanthins (e.g. xanthophyll) as well as epiphasic carotenoids (e.g. jS-carotene) have been found to be constituents of such chromoproteids. The mode of occurrence of carotenoids in blood serum is also of importance. The present view is that the carotenoids here occur in the aqueous phase com- bined with lipoids and proteins^. Von Euler and Adler^'' have estabhshed the occurrence of carotene in the retina. According to experiments by Brunner and collaborators^\ the pigment here occurs in the colloidal state. 3. detection and estimation In order to establish the presence of carotenoids in natural sources, the dried materials (e.g. leaves or blossoms) are treated with certain reagents (e.g. concentrated sulphuric acid), which produce characteristic colourations. Ac- cording to MoLiscH^^^ -j-i^g polyene pigments are best detected by first destroying the surrounding substances, e.g. fats, and only then applying the colour tests. In practice, the material is first treated with concentrated aqueous alcoholic alkali, which dissolves the fats and sets free the carotenoids. At the same time, the phytoxanthin esters* are hydrolysed and the phytoxanthins are liberated. In this way, crystalline carotenoids are often obtained and can be recognised under the microscope. Their presence can also be shown by colour reactions. Recently, however, it has become increasingly usual to isolate the carotenoids first and to characterise them subsequently. For this purpose the micro- method of KuHN and Brockmann^^ ^g often employed. It must be emphasised. In nature, phytoxanthins often occur esterified as colour waxes, e.g. physalien, helenien. References p. 8-g. 4 COLOUR REACTIONS 7 however, that for the complete identification of a carotenoid it is necessary to carry out a chromatographic purification and to isolate the pigment in the crystalline state. 4. COLOUR REACTIONS The polyene pigments are well known to give blue or violet solutions with a variety of strong acids such as concentrated sulphuric acid, hydrochloric acid, perchloric acid, trichloracetic acid, and with acid chlorides, such as antimony trichloride or arsenic trichloride. These colourations, although not specific, can be used as qualitative tests^*. (a). Reaction with concentrated sulphuric acid: This reaction is carried out by carefully forming a layer of concentrated sulphuric acid under an ethereal solution of the pigment. The sulphuric acid layer acquires an intense dark blue to blue-violet or, occasionally, greenish-blue colour which disappears on the addition of water^^. (b). Other strong acids: Fuming nitric acid produces a transient blue colouration. A number of observations have also been published recently regarding the blue colouration produced by concentrated aqueous hydro- chloric acid. It appears that the following carotenoids colour concentrated aqueous hydrochloric acid blue: (i). Aldehydes, e.g. j3-citraurin, ^-apo-2-carotenal. (ii). Some carotenoids containing several hydroxyl groups, e.g. fucoxanthin, azafrin. , (iii). Carotenoid epoxides and their furanoid transformation products, e.g. violaxanthin, auroxanthin, xanthophyll epoxide, flavoxanthin, j8-carotene di- epoxide, aurochrome. In practice, the hydrochloric acid reaction is carried out in the following way : The pigment is dissolved in a little ether and concentrated aqueous hydro- chloric acid is added to the solution. After shaking, the acid layer is coloured blue. With a number of hydrocarbon epoxides, such as a-carotene mono- epoxide, the blue colouration is very weak and only persists for a short time. For further details, reference should be made to the description of this reaction in the sections on individual carotenoids. (c). Antimony trichloride in chloroform solution (Carr-Price reagent) : Similarly to vitamin A, carotenoids give dark blue colourations with the Carr-Price reagent^^. These blue colourations often have characteristic absorption maxima, and can be used for the quantitative estimation of carotenoids^'. References p. 8-g. 8 OCCURRENCE. DETECTION, ESTIMATION I 5. SPECTROSCOPY An important part of the characterisation of a carotenoid is the determi- nation of its absorption maxima. (With regard to the relationships between constitution and colour, and extinction curves, cf. page 53). On examining the solution of a carotenoid, usually in carbon disulphide or petroleum ether, in a spectrometer, two or three sharp absorption bands can usually be observed. Their positions can be accurately determined (approximately to within 0.5 m/z) and represent the wavelengths of the absorption maxima. These data are characteristic for each carotenoid and together with other physical constants are used for its identification. (Detailed light absorption data will be quoted in the description of the individual pigments in later sections). By means of the photographic method of determining solution spectra, as developed, for in- stance, by VON Halban, Kortum and Szigeti^^, or by other suitable means, the complete absorption curves can also be determined^'. 6. COLORIMETRY There are numerous methods for the colorimetric determination of a caro- tenoid, all of which have in common the comparison of an unknown quantity of the pigment with a standard solution. Potassium dichromate^", azobenzene^^, bixin22 and jS-carotene^^ have been used as standard substances. Instruments not requiring standard solutions have also been employed^*. It is necessary to separate the carotenoids before their colorimetric determination, otherwise t. misleading results are obtained. 7. fluorescence spectra Following the determination of the fluorescence spectra of different diphenyl- polyenes by Hausser and collaborators^^, Dhere^^ examined vitamin A, j8-carotene and lycopene at — 180° in this respect. The determination of fluo- rescence spectra has not, however, found general application. REFERENCES 1. W. Menke, Naturwissenschaflen 28 (1940) 31. 2. H. JuNGE, Z. physiol. Chem. 268 (1941) 179. 3. A. M. GoLDOwsKi and M. S. Podolskaja, Chem. Centr. igjg II 2437. 4. R. Savelli, Protoplasma 2g (1938) 601; C. igjS II 2126. 5. H. MoLiscH, Ber. deut. Botan. Ges. 36 (1918) 281. — K. Noack, Biochem. Z. 183 (1927) 135. — A. GuiLLiERMOND, Compt. rend. 76^(1917) 232. — Courchet, tImm. Scj.wai. (7) (1888) 263. — R. KuHN and H. J. Bielig, Ber. 73 (1940) 1080. 6. Cf. W. Straus, Dissertation, Zurich, (1939) p. 50. 4 REFERENCES g 7. R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. 8. H. JuNGE, Z. physiol. Chem. 268 (1941) 179. 9. L. Palmer, /. Biol. Chem. 23 (1915) 261. — H. van den Bergh, P. Muller and J. Broek-Meyer, Biochem. Z. 108 (1920) 279. — Bendien and Snapper, Biochem. Z. 261 (1933) I. — E. Lehnartz, Einfiihrung in die chemische Physiologie, 1931, p. 353. — E. V. DAniel and T. Beres, Z. physiol. Chem. 238 (1936) 160. — S. P. L. Sorensen, Kolloid-Z. 53 (1930) 306. — W. Kraus, Dissertation, Zurich 1939. 10. H. V. Euler and E. Adler, Arkiv. Kemi Mineral. Geol. B 11 (1933) No. 20. 11. O. Brunner and co-workers, Z. physiol. Chem. 236 (1935) 257. 12. H. MoLiscH, Mikrochemie der Pflamen, Jena, 1923, p. 253. 13. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 41. 14. Cf. also: H. Molisch, Mikrochemie der Pflanzen, 1923. — C. van Wisselingh, Flora 7 (1915) 371- — L. Zechmeister, Carotinoide, Berlin (1934), pp. 82, 126, 235. 15. R. Kuhn and A. Winterstein, Helv. Chim. Acta 11 (1928) 87, 116, 123, 144. 16. F. H. Carr and E. A. Price, Biochem. J. 20 (1926) 497. 17. P. Karrer and H. Wehrli, 23 Jahre Vitamin-A Forschung, Nova Acta Leopoldina, New series, i (1933) 175. — A. Winterstein and C. Funk, G. Kleins Handbuch der Pflanzenanalyse 4 (1933) 1041. — L. Zechmeister and L. v. Cholnoky, Ann. 465 (1928) 288. • — B. V. Euler and P. Karrer, Helv. Chim. Acta 15 (1932) 496. 18. H. V. Halban, G. Kortum and B. Szigeti, Z. Elektrochem. 42 (1936) 628. 19. Cf. F. Zscheile, Botan. Rev. 7 (1941) 587. 20. R. Willstatter and A. Stoll, Untersuchnngen iiber Chlorophyll, 19 13. — G. Fraps and co-workers, /. Agr. Research 53 (1936) 713. — K. Sjoberg, Biochem. Z. 240 (1931) 156. — S. W. Clausen and A. B. McCoord, /. Biol. Chem. 113 (1936) 89. 21. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 41. 22. H. N. Holmes and W. H. Bromund, /. Biol. Chem. 112 (1936) 437. 23. H. R. Bruins, J. Overhoff and L. K. Wolff, Biochem. J. 25 (1931) 430. 24. Ferguson and Bishop, Analyst 61 (1936) 515. — Munsey, /. Assoc. Offic. Agr. Chemists 21 (1938) 331. — H. v. Euler, H. Hellstrom and M. Rybdom, Mikrochemie, Pregl- Festschrift 1929, p. 69. 25. K. W. Hausser and co-workers, Z. physik. Chem. (B) 2g (1935) 391; 2g (1935) 363. 26. C. Dh6re, Fortschr. Chemie Org. Nat. II (1939) 301. CHAPTER II The formation of carotenoids in plants and their physiological significance I. MODE OF FORMATION No experimental evidence, only hypotheses, exist at present with regard to the mode of formation of carotenoids in plants. It therefore appears prema- ture to come to any definite conclusions on this subject. Karrer, Helfenstein, Wehrli and Wettstein^ consider the possibility that lycopene may be formed from phytyl aldehyde by a benzoin condensation or by a pinacol reduction, followed by dehydrogenation. Carotenoids containing fewer than 40 carbon atoms in the molecule may be formed by oxidation of C^^ carotenoids^. Several investigations have been made concerning the morphological changes which take place in fruit during the ripening process. Some of these are referred to on p. iig^. 2. THE FUNCTION OF CAROTENOIDS IN PLANTS Although numerous investigations have been carried out within recent years concerning the significance of carotenoids in the vegetable organism, our present knowledge of this subject is still very scanty and no final opinion can be formed. The first studies in this field by Willstatter and his school* attempted to demonstrate a possible influence of carotenoids on the processes of respiration and assimilation, but with negative results. More recently a number of workers have sought to determine the influence of carotenoids on sexual reproduction. The following is a brief summary of the results so far obtained. For details the original literature should be consulted. Willstatter and Stoll^ examined the function of carotene and xantho- phyll in green leaves, in which the two carotenoids occur in fairly constant proportion to chlorophyll. They were unable to detect any definite influence of the two pigments on respiration. According to Noack^, carotene and xantho- phyll fulfil the role of light filters for chlorophyll, whereas Went^ has expressed References p. ly-ig. 3 FUNCTION IN TNE ANIMAL ORGANISM n the opinion that they are more Hkely to function as protectors for sensitive enzymes in the cell. Warburg and Negelein^ considered that carotene and xanthophyll are photochemically active in assimilation. Fodor and Schoen- FELD^ have recently reported that colloidal carotene solutions can act as hydro- gen acceptors, and consider the possibility that they fulfil a similar function in the respiratory process. It has also been suggested^ that certain carotenoids (e.g. y-carotene) play a part in the reproduction of algae. There appears to be no certain foundation for this hypothethis. Some investigators^^ assume that carotenoids influence the growth of plants, and Buexxixg^^ has recently attempted to identify the pigment concerned in the phototropy of plants with ^-carotene. According to Kuhx, Moewus and Jerchel^-, crocin (crocetin digentiobiose ester), as well as cis- and /raws-crocetin dimethyl ester play a part in the re- production of the unicellular algae chlamydomonas eugamctos f. simplex. It appears that crocin can induce the formation of flagella in the gametes, while mixtures of trans- and c/s-crocetin methyl ester convert the motile, but still infertile gametes into male and female gametes. The ratio of cis- to trans- crocetin methyl ester determines whether male or female gametes are formed. These experiments require further confirmation. As the result of recent investigations (cf. p. 66) which show that various carotenoid epoxides are widely distributed in plants, it has been suggested that these compounds play a part in the transport of oxygen or in oxidation re- actions^^. All these investigations are still at a preliminary stage and further researches will be required in order to elucidate the importance of carotenoids in plants. 3. THE FUNCTION OF CAROTENOIDS IN THE ANIMAL ORGANISM We are somewhat better informed regarding the significance of certain carotenoids in the animal organism, but again many important problems still require to be elucidated. A number of carotenoids are converted into vitamin A in the animal organism and therefore play the part of pro-vitamins. Further- more, carotenoids play a part in the visual process, but their function is as yet incompletely understood. a. Carotenoids as Pro-vitamins A • Numerous papers have been published dealing with the function of carote- noids as pro-vitamins A^*. Only a brief summary of the definitely established facts will be given here. For further details, the original literature should be consulted. References p. ly—ig. 12 FORMATION IN PLANTS II Steenbock and his collaborators were the first to suggest, about 30 years ago, that a connection exists between the yellow plant pigments (carotene) and vitamin A.^^ During the following years, the question of the growth- promoting properties of carotene was investigated by different workers. The results of these investigations were very contradictory and a definite solution of this problem was only achieved in 1929 by B. v. Euler, H. v. Euler and Karrer^^. The results of their investigations definitely proved that carotene possesses qualitatively the same biological effect (resumption of arrested growth) as vitamin A, and was therefore probably related to the latter. This result at first appeared difficult to understand as carotene is a deeply coloured crystalline substance, quite different from the pale yellow vitamin A*. Later experiments by Karrer and collaborators elucidated the constitution of vitamin A^'^ and of jS-carotene^^ and the relationship between the two compounds. The chemical structure of j3-carotene is such that by the uptake of water it can be converted into two molecules of vitamin A, and the growth-promoting properties of ^-carotene thus find their explanation. CH3 CHs CH, CH, C CH3 CH3 CH3 CH3 c /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH,j CHj C-CHg I HgC-C CHj \/ 2 H.0 \/ CHo CH, C CHo CHo /\ I I CHa C-CH=CH-C=CHCH=CH-C=CH-CHoOH •I II CHo C" CHo Vitamin A CHa In agreement with this view, experiments reported by T. Moore showed that rats kept on a vitamin A-free diet, and whose livers contained practically no vitamin A, again accumulated vitamin A in the liver after being given /S-carotene^^. y-Carotene, which contains only cme j8-ionone ring in the molecule, and can therefore only form one molecule of vitamin A by the addition of water, exhibits much weaker growth-promoting properties than jS-carotene^". Very little is yet known about the mechanism by which ^-carotene and other pro- vitamins A are converted into the vitamin. It has been assumed that this process depends on a ferment, carotenase. It is very probable that the reaction occurs in the liver^i or in the intestine^^. In animals deficient of Cristalline vitamin A was not yet known at the time. References p. ly-ig. 3 FUNCTION IN THE ANIMAL ORGANISM 13 vitamin A, the conversion of pro-vitamins into vitamin A takes place rapidly and fairly completely (up to 70 or 80 %). If the organism is saturated with vitamin A, however, or if high doses of pro-vitamins are given, then only a small proportion is converted into the vitamin-^. For this reason, carotene and other carotenoids are always found in the faeces. The form in which the pro-vitamins A are supplied to the organism is of decisive importance for their absorption and conversion into vitamin A. If the carotenoids are dissolved in animal or vegetable fats, they are easily taken up ; if, on the other hand, solutions in paraffin oil or ethyl oleate are used, hardly any absorption takes place^*. Ignorance of these facts is partly responsible for the contradictory results recorded in the early literature concerning the activity of carotene^*. Very recently a number of attemps have been made to convert /3-carotene into vitamin A in vitro. Although some of these attempts are claimed to have been successful, this problem cannot yet be regarded as finally solved, as it has not been possible to isolate the vitamin A formed in a pure state and to establish its identity with certainty. Willstaedt^^ reports a transformation of this type, using liver preparations, while Hunter and Williams^^ obtained traces of vitamin A by the action of hydrogen peroxide on ^-carotene and sub- sequent reduction of the aldehyde produced. It is an interesting fact that not all mammals have the same capacity for converting pro-vitamins A into vitamin A. The most suitable experimental animal appears to be the rat^^. Guinea pigs^^, rabbits-^, pigs^° and cattle^^ possess the capacity to a reduced extent, dogs only to a very small extent^^, whereas in cats it is completely absent^^. Chicken also appear capable of trans- forming /3-carotene into vitamin A^*. The facts regarding fresh water and salt water fish are not yet completely known, but it appears that fish are capable of converting pro-vitamins A into vitamin Aj^^ (and vitamin Ag)^®. After recognition of the fact that carotene consists of several isomers (cf. p. 125), and that a-carotene also possesses vitamin A activity, though to a reduced extent, a number of different investigations were begun with the view to elucidating the relationships between the structure of a compound and its vitamin A activity. In the course of these investigations several naturally occurring carotenoids were recognised as pro-vitamins A and a number of partly synthetic carotenoids were also shown to possess growth-promoting properties. Before dealing with the theoretical aspects of these results, a summary is given here of the compounds which possess vitamin A activity (see table i, p. 14). As all these compounds (with the exception of vitamin A methyl ether and vitamin A acid) will be dealt with in detail in later sections, the reader is merely referred to the alphabetical index at this stage. The relationships which exist between the vitamin A activity of a compound References p. ly-ig. 14 FORMATION IN PLANTS II TABLE 1 PRO-VITAMINS A Naturally occurring Partially synthetic Totally synthetic a-Carotene /3-Carotene mono-epoxide Vitamin-A methyl ^-Carotene /S-Carotene di-epoxide ether* y-Carotene Dihydroxy-|8-carotene Vitamin-A acid * * a-Carotene epoxide Semi-/5-carotenone Citroxanthin = Mutato- Semi-j3-carotenone monoxime chrome Anhydrosemi-jS-carotenone Cryptoxanthin Luteochrome Myxoxanthin /3-Apo-2-carotenal Aphanin )S-Apo-2-carotenal oxime Echinenone )5-Apo-4-carotenal oxime Torularhodin a-Carotene diiodide ^-Carotene diiodide Product from zeaxanthin -j- PBrg Product from xanthophyll + PBr3 /3-Apo-2-carotenol W. Oroshnik, /. Am. Chem. Soc. 67 (1945) 1627. — O. Isler, W. Huber, A. Ronco and M. Kofler, Experientia 2 (1946) 31. - — See also Festschrift 'Emil Barell', Basle 1946, P- 31- J. F. Arens and D. A. van Dorp, Nature 157 (1946) 190. — P. Karrer, E. Jucker and E. Schick, Helv. Chim. Acta 2g (1946) 704. — I. M. Heilbron, E. R. H. Jones, and D. G. O'SuLLiVAN, Nature 157 (1946) 485; /. Chem. Soc, 1946, 866. and its chemical constitution have now been clarified. In order to exhibit vitamin A activity a compound must contain an unsubstituted j8-ionone ring and the unsaturated side-chain present in axerophtol (vitamin A). a-Semi- carotenone^' possesses the unsaturated side chain but not the ^-ionone ring and is biologically inactive. ^-Euionone^^ contains an unsubstituted ^-ionone ring but not the complete side chain and is incapable of replacing vitamin A in experiments with animals*. Steric relationships also play a part in the biological activity of a pro- vitamin A. Most of the investigations in this field are due to Zechmeister and I. M. Heilbron, E. R. H. Jones and their collaborators (of. I. M. Heilbron, Pedlar Lecture, /. Chem. Soc, 1948, 3S6; I. M. Heilbron, E. R. H. Jones and R. W. Richardson, ibid., 1949, 287; I. M. Heilbron, E. R. H. Jones, D. G. Lewis and B. C. L. Weedon, ibid., 1949, 2023) have recently synthesized a number of partly demethylated and acetylenic analogues of vitamin A acid and have shown that they exhibit some growth-promoting properties. This work is providing important new evidence concerning the relationships between chemical constitution and vitamin A activity. References p. ly-ig. 3 FUNCTION IN THE ANIMAL ORGANISM 15 his collaborators^^. These workers have shown that in general the greatest biological activity is exhibited by those pro-vitamins which possess a ^raws- configuration throughout*. The following table** demonstrates these rela- tionships. TABLE 2 RELATIONSHIP BETWEEN VITAMIN A ACTIVITY AND STERIC CONFIGURATION OF SOME CAROTENOIDS* )?-Carotene, /ya>2S-configuration throughout 10 0% Neo-/3-carotene U (probably 1 cis-linkage) 3 8 % Neo-^-carotene B (probably 2 c/s-linkages) 53% a-Carotene, /yaws-configuration throughout 53% Neo-a-carotene U (probably 1 cis-linkage) 1 3 % Neo-a-carotene B (probably 2 cfs-linkages) 1 ^ % y-Carotene, ira^s-configuration throughout * * 2 8 % Pro-y-carotene (probably 5 ji^-linkages) 4 4 % The potency of pure ^-carotene is used as a standard (100 %). ** According to R. Kuhn and H. Brockmann, Klin. Wochschr. 12 (1933) 972, y-carotene has the same vitamin A potency as a-carotene, instead of half the potency as shown here. The fact that a-carotene mono-epoxide, ^-carotene mono-epoxide, /3-carotene di-epoxide and luteochrome all exhibit vitamin A activity although (with the exception of jS-carotene mono-epoxide) they do not possess an unsubstituted j3-ionone ring, deserves special mention"*". It may be deduced that these com- pounds are partly de-oxygenated in the organism of the rat. TABLE 3 COMPARISON OF THE BIOLOGICAL ACTIVITY OF SOME CAROTENOID EPOXIDES*^ Carotenoid Active daily dose, y )S-Carotene a-Carotene epoxide /?-Carotene di-epoxide Luteochronie * 2.5 10 17 18 P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 429, 430. y-Carotene isolated from mimulus blossoms (p. 162) is an exception to this rule. ** Taken without alteration from L. Zechmeister and co-workers, Arch. Biochem, 7 (^945) 247. References p. ly-ig. i6 FORMATION IN PLANTS II b. The Function of Carotcnoids in the Visual Process Some carotenoids appear to play a part in the process of visional. Although there has been a good deal of theoretical speculation as well as experimental work in this connection, this field of research is still in an early stage of develop- ment and it is not yet possible to draw any definite conclusions. The following observations are merely meant to provide a brief summary. The suggestion that visual purple is a carotenoid was first made by Boll^^ about 70 years ago. For some time afterwards this question was not further investigated, presumably because science as a whole had not yet sufficiently progressed. In 1923, Blegvad^^ ^nd in 1924, Bloch^* showed that vitamin A deficiency results in xerophthalmia, a sclerotic inflammation of the eyes. In 1925, Fridericia and Holm'^^ found that night blindness (hemeralopia) is a direct consequence of vitamin A deficiency which must be related to the incapacity of forming visual purple in the retinal rods. After the relationships between certain processes in the eye and vitamin A or the carotenoids had thus been demonstrated, several investigations were begun with the view to isolating the pigments concerned from the eye and to identifying them. However, this task proved to be a very difficult one. The eyes of animals only contain very small quantities of pigments and, furthermore, these substances are unstable and to some extent sensitive to light. The first success was achieved by von Euler and Adler*'^, who isolated compounds of a carotenoid nature from the pigmented epithehal layer of bull and fish eyes. Shortly afterwards, Wald*'' proved the presence of vitamin A in the retina of bulls and frogs. Later, Wald*^ sfiowed that the retina of frogs contained xanthophyll ester and recently Wald and Zussman'*^ found strongly coloured oily discs in the pupils of many birds and reptiles. In the chicken these discs are red, golden and yellow-green and from them three carotenoids can be isolated, one of which appears to be identical with esterified astacene, while the other two are of as yet unknown constitution. Honigmann^" has reported to have found a photolabile pigment in the retina of young chicken. This pigment appears to be of a carotenoid nature and to be similar to, but not identical with, rhodopsin and porphyropsin (cf. below). The investigations just described show that carotenoids or very similar pigments occur in the eyes of numerous animals. Their function has not yet been clarified, but it seems possible that they act as light filters which ensure that only rays of certain wavelengths enter the inner part of the eye and reach the photolabile substance. The question then arises as to the nature of the photolabile substance. Wald^^ showed that after a brief illumination of the eyes of frogs or mammals, a new pigment with different spectral properties is formed from the rhodopsin (visual purple). He suggested the name of References p. ly-ig. 3 FUNCTION IN THE VISUAL PROCESS 17 retinene for this yellow pigment. Retinene is subsequently converted into vitamin A and the latter is converted back into rhodopsin in the dark. To some extent retinene can also be directly transformed into rhodopsin. Both retinene and vitamin A always occur in the eye combined with protein^^^ j^g transformations described can be summarised schematically as follows: Rhodopsin .71 Vitamin A^ + Protein < Retinene + Protein According to Wald^^, retinene can also be obtained directly without illumination from eyes adapted to darkness by the extraction of visual purple with chloroform. This fact, as well as certain other considerations, led Wald to believe that rhodopsin is a carotenoid (retinene) -protein combination which can be destroyed by illumination, heat, or suiviable solvents such as chloroform, thus liberating the protein-bound carotenoid (retinene). It should be mentioned that neither retinene nor rhodopsin have ever been obtained in the crystalline state or analysed. Nevertheless, it cannot be doubted that the chromophoric system of the carotenoids is utilised in the act of vision. Rhodopsin, ret^ene and vitamin A have also been found in the eyes of mammals and salt water fish°^ e.g. Prionotus carolinus, Centropristes stiratus, Stenotomus chrysops, and the cycle between these substances again appears to be the same. Many fresh water fish contain a different light-sensitive pigment, por- phyropsin^^, in place of rhodopsin. The reactions involved in the visual process of such fresh water fish, e.g. Morone americana, Perca flavescens, and Esox rcticulatus were examined by Wald^^ who found that they were similar in character to those occurring with rhodopsin. During the illumination of por- phyropsin, retinenCg is formed, which is in turn converted into vitamin Ag. Morton and his collaborators have recently shown^^ that retinene^ is identical with vitamin A^ aldehyde and retinenCg with vitamin A^ aldehyde. REFERENCES 1. P. Karrer, a. Helfenstein, H. Wehrli and A. Wettstein, Helv. Chim. Acta 13 (1930) 1084. 2. R. KuHN and Ch. Grundmann, Ber. 65 (1932) 898, 1880. — R. Kuhn and A. Winter- stein, Naturwissenschaften 21 (1933) 527; Ber. 6j (1934) 344; (>5 (1932) 646. — R. Kuhn, Forsch. u. Fortschr. 9 (1933) 426. — R. Kuhn and A. Deutsch, Ber. 66 (i933) 883. P. Karrer and T. Takahashi, Helv. Chim. Acta 16 (1933) 287. 3. A very detailed description of these processes will be found in L. Zechmeister's Monograph Die Carotinoide, BerUn, 1934, Springer- Verlag. Carotenoids 2 i8 FORMATION IN PLANTS II 4. Cf. R. WiLLSTATTER and A. Stoll, Untersuchungen uber die Assimilation der Kohlen- saure, Berlin, 1918. 5. K. NoACK, Z. Botan. ly (1925) 481. 6. F. A. F. C. Went, Rec. irav. botan. neerland. i (1904) 106. 7. O. Warburg and E. Negelein, Z. physik. Chem. 106 (1923) 191; Naturwissenschaften 10 (1923) 647. 8. A. FoDOR and R. Schoenfeld, Biochem. Z. 233 (1931) 243. 9. R. Emerson and D. Fox, Proc. Roy. Sac. B 128 (1940) 275. — Cf. P. Karrer and coworkers, Helv. Chim. Acta 26 (1943) 2121. 10. A. H. Blaauw, Z. Botan. 6 (1914) 641; 7 (1915) 465. — H. Borris, Planta 22 (1934) 644. — W. V. BuDDENBROCK, GrundHss der vergleichenden Physiologic i (1937) i°- — S. Hecht, Naturwissenschaften 13 (1925) 66. 11. E. BuNNiNG, Planta 26 (1937) T^9'> ^7 (^937) 148, 583. 12. R. KuHN, F. MoEwus and D. Jerchel, Ber. yi (1938) 1541. For further references s'ee Jahrbiicher fiir wissenschaftliche Botanik. 13. P. Karrer, E. Jucker, J. Rutschmann and K. Steinlin, Helv. Chim. Acta 28 (1945) 1 149. 14. P. Karrer and H. Wehrli, 25 Jahre Vitamin-A Forschung, Nova Acta Leopoldina, New series i (1933) 175. — U. Solmssen, Dissertation, Ziirich, 1936. — G. R. Rosen- berg, Chemistry and Physiology of the Vitamins, New York 1945, p. 38. — Cf. Otto Walker, Dissertation, Ziirich, 1935. 15. H. Steenbock, M. T. Sell, E. M. Nelson and M. V. Buell, /. Biol. Chem. 46 (1921) Proceed. XXXII. — Cf. H. Steenbock et al.. Science 50 (1919 )352; /. Biol. Chem. 41 (1920) 163; 40 (1919) 501; 51 (1922) 63. For later, partly contradictory investiga- tions see: J. C. Drummond and co-workers, Biochem. J . ig (1925) 1047. x6. B. V. Euler, H. v. Euler and P. Karrer, Helv. Chim. Acta 12 (1929) 278. 17. ^Karrer, R. Morf and K. Schopp, Helv. Chim. Acta 14 (1931) 1036, 1431; Helv. Chim. Acta 16 (1933) 557. — P. Karrer and R. Morf, Helv. Chim. Acta 16 (1933) 625. 18. Cf. p. 133. 19. Th. Moore, Biochem. J. 24 (1930) 692. — Cf. H. BROCKMANNandM.-L. Tecklenburg, Z. physiol. Chem. 221 (i933) ii7- — R- Kuhn and H. Brockmann, Z. physiol. Chem. 200 (1932) 246. — R. Kuhn and H. Brockmann, A. Scheunert and M. Schieblich, Z. physiol. Chem. 221 (1933) 129. — H. v. Euler, P. Karrer and A. Zubrys, Helv. Chim. Acta ly (1934) 24. 20. P. Karrer, H. v. Euler, H. Hellstrom and M. Rydbom, Svensk. Kem. Tid. 43 (1931) 105. 21. H. S. Olcott and D. C. McCann, /. Biol. Chem. g4 (1931) 185. — B. Ahmad, Biochem. J . 25 (1931) 1 195. — J. L. Rea and J. C. Drummond, Z. Vitaminforsch. i (1932) 177. — • J. G. Brazer, a. C. Curtis, Arch. Internal. Med. 65 (1940) 90. 22. J. Glover, T. W. Goodwin and R. A. Morton, Biochem. J. 41 (1947) Proceed. XLV; C. E. Wiese, J. W. Mehl and H. J. Devel, Arch. Biochem. 15 (1947) 75; J. Glover, T. W. Goodwin and R. A. Morton, Biochem. J. 43 (1948) 512; F. H. Mattson, /. Biol. Chem. iy6 (1948) 467. 23. Cf. L. Zechmeister, Erg. physiol. Biol. Chem. exp. Parmakol. 39 (1937) 148- — C. A. Baumann, B. M. Riising and H. Steenbock, /. Biol. Chem. loy (1934) 705- 24. J. C. Drummond, B. Ahmad and R. Morton, /. Soc. Chem. Ind. (London) ^9 (1929) Transact. 291. — Cf. W. Duliere, R. A. Morton and J. C. Drummond, Chem. Centr. 1930, I, 1639. 25. H. WiLLSTAEDT, Enzymologia 3 (1937) 228. — Cf. also L. E. Baker, Proc. Soc. Exp. Biol. Med. 33 (1935) 124. — H. S. Olcott and D. C. McCann, /. Biol. Chem. 94 (1931) 185. — J. L. Rea and J. C. Drummond, Z. Vitaminforsch. i {1932) 177. — H. v. Euler and E. KLussmann, Arkiv. Kemi Mineral. Geol. B 11 (1932) 6. — B. Wolff and T. Moore, Lancet 223 (1932) 13. — J. C. Drummond and R. J. McWalter, Biochem. J. 27 (1933) 1342;/. Physiol. (London) 83 (1934) 236. 26. R. F. Hunter and N. E. Williams, /. Chem. Soc. 1945, 554. REFERENCES 19 27. B. Ahmad and K. Malik, Indian J. Med. Research 20 (1933) 1033. 28. H. Brockmann and M.-L. Tecklenburg, Z. physiol. Chem. 221 (1933) 117. 29. Idem, ibid., see also H. Rosenberg, Chemistry and Physiology of the Vitamins, New York 1945, p. 55. 30. Th. Moore, Biochem. J. 25 (1931) 2131. 31. Th. Moore, Biochem. J. 26 (1932) i. 32. A. J. CooMBES, G. L. Ott and W. Wisnicky, North. Am. Veterinarian 21 (1940) 601. 33. H. Rosenberg, cf. ref. 30. 34. N. S. Capper, JM. W. McKibbin, J. H. Prentice, Biochem. J. 25 (1931) 265. 35. H. Rosenberg, Chemistry and Physiology of the Vitamins, p. 50, 73, New York 1945. 36. R. A. Morton and R. H. Creed, Biochem. J. 33 (1939) 318. 37. P. Karrer, H. v. Euler and U. Solmssen, Helv. Chim. Acta ly (1934) 1169. 38. P. Karrer and co-workers, Helv. Chim. Acta 15 (1932) 878; 17 (1934) 3. 49. L. Zechmeister and co-workers, Arch. Biochem. 5 (1944) 107; 7 (1945) 247. 40. H. V. Euler, Helv. Chim. Acta 28 (1945) 1150. 41. Cf. the review by P. Karrer, Dociimenta Ophthalmologica, 1938, p. 259. 42. F. Boll, Arch. Anat. Physiol. Anat. Abt. 1877, 4. 43. Blegvad, Dissertation, Kopenhagen, 1923. 44. Block, 7. Dairy Sci. U.S.A. 7 (1924) i. 45. Fridericia and Holm, Am. J. Physiol. 73 {1925) 63. 46. H. V. Euler and E. Adler, Arkiv. Kemi Mineral. Geol. B 11 (1933), Nr. 20, 21. 47. G. Wald, Nature 132 (1933) 316; 134 (1934) 65; /. Gen. Physiol. 18 (1934) 905- 48. G. Wald, /. Gen. Physiol, jg {1935) 351. 49. G. Wald and H. Zussman, Nature 140 (1937) i97- 50. HoNiGMANN, Arch. Ges. Physiol. 189 (1921) i. Cf. G. Wald, Nature 140 (1937) 545- 51. G. Wald, /. Gen. Physiol, ig (1935) 351. 52. G. Wald, /. Gen. Physiol. 21 (1937) 93. •* 53. G. Wald, Nature 136 (1935) 9i3- 54. E. Kottgen and C. Abelsdorff, Z. Psychol. Physiol. Sinnesorgane 12 (1896) 161. 55. G. Wald, Nature 139 (1937) 1017. 56. S. Ball, T. W. Goodwin and R. A. Morton, Biochem. J. 40 (1946) Proceed. LIX. — R. A. Morton, Nature, London 153 (1944) 69. — R. A. Morton and T. W. Goodwin, Nature, London, 153 (1944) 405. — S. Ball, T. W. Goodwin and R. A. Morton, Biochem. J. 42 (1948) 516. CHAPTER III The isolation of carotenoids The isolation of carotenoids from vegetable or animal sources often presents difficulties, especially if extensive experience in this field is lacking. An attempt will therefore be made in the present section to describe some general methods of isolation. These have to be adapted in particular cases to take account of the mode of occurrence of the pigments and of the materials which accompany them. The usual course of isolation consists of the following parts: 1. Preparation of the materials to be examined and extraction of the carotenoids. 2. Division of the carotenoids into hypophasic and epiphasic constituents. 3. Separation of the pigments in the two phases and preparation in a crystalline condition. The individual steps are dealt with in more detail in the following section. No attempt has been made to summarise all the usual methods employed; instead, a few of the well-tried and common methods are given which are successful in most cases. T. EXTRACTION OF CAROTENOIDS Before the extraction of the carotenoids, the vegetable or animal material must be dried (dehydrated). With blossoms or fruit or other parts of plants, this is most easily accomplished by drying in the sun (preferably in a current of air), or in a well-aired room at 40-50° C. It is important that the material should be spread in thin layers and should be turned from time to time in order to achieve as uniform a drying as possible and in order to prevent fermentation which would destroy the carotenoids. If it is not possible to dry the material in this way, as is sometimes the case with algae, marine plants, or animals, the dehydration may be carried out by submersion in solvents such as acetone, methanol, ethanol, etc. The extraction can be carried out by means of a wide variety of solvents. The following are most commonly used: benzene, petroleum ether, ether (free References p. 28. 2 HYPOPHASIC AND EPIPHASIC CAROTENOIDS 21 from peroxide), carbon disulphide, chloroform (free from hydrochloric acid), ethanol, methanol and acetone. Extraction at room temperature is carried out, by allowing the material to stand with the solvents in wide-necked bottles or percolators in a carbon dioxide or nitrogen atmosphere. If large amounts of material have to be extracted, large metal extractors can be used to advantage and also make it possible to work at higher temperatures. The extracts must be concentrated in vacuum as quickly as possible, and the concentrates thus obtained must be kept sealed up under vacuum in large ampoules until they are worked up. 2. SEPARATION INTO HYPOPHASIC AND EPIPHASIC CAROTENOIDS As a result of the pioneering work of Willstatter and Stoll^, it is usual to divide the extracted carotenoids into two groups by partition between two immiscible solvents. Carotenoids with two or more hydroxyl groups are thus obtained as hypophasic pigments and those without hydroxyl groups as epi- phasic pigments. Mono-hydroxy compounds such as cryptoxanthin and rubi- xanthin occupy an intermediate position and are found in the epiphase as well as in the hypophase. The following table provides a summary of the epiphasic and hypophasic carotenoids (table 4, p. 22). As most of the hydroxylated carotenoids occur in nature in the form of esters as the so-called "pigment-waxes", it is necessary to saponify before partition between methanol and petroleum ether. This can be done by means of about 12 % methanolic potassium hydroxide at room temperature. The carotenoid mixture to be saponified is dissolved in petroleum ether and a requisite quantity of alkali is added. If the resulting mixture is homogenous it is simply allowed to stand for about 20 hours. If two phases are formed, the mixture must be shaken mechanically. In either case, the space above the solution should be filled with an inert gas (e.g. hydrogen or nitrogen) in order to prevent aerial oxidation. In some cases a solution of sodium methoxide in methanol is preferred to methanolic potassium hydroxide. Saponification can also be carried out at more elevated temperatures (about 60-70° C.) ; in this case an alkali concentra- tion of about 5% is usually sufficient. After saponification is complete, petroleum ether is added, followed by sufficient water to result in a separation into two phases. The upper phase contains mainly the epiphasic carotenoids and the lower phase mainly the hypophasic carotenoids. The petroleum ether phase is then repeatedly ex- tracted with methanol, and the methanol layer is repeatedly extracted with petroleum ether, and the appropriate extracts are combined. The solution of epiphasic pigments is washed with water, dried over sodium sulphate, concen- References p. 28. ISOLATION III TABLE 4 DIVISION OF THE NATURAL CAROTENOIDS INTO HYPOPHASIC AND EPIPHASIC PIGMENTS ON THE BASIS OF PARTITION BETWEEN PETROLEUM ETHER AND 90 % METHANOL Almost equally Epiphasic distributed between epiphase and hypophase Hypophasic Actinioerythrin Celaxanthin Antheraxanthin Aphanin Gazaniaxanthin Auroxanthin Aphanicin' Cryptoxanthin Aphanizophyll a-Carotene Lycoxanthin Astacene a-Carotene epoxide Rhodoxanthin Astaxanthin )S-Carotene Rubichrome Azafrin y-Carotene Rubixanthin Bixin 5-Carotene ( ?) Sarcinaxanthin Capsanthin Citroxanthin = Mutato- Capsorubin chrome j8-Citraurin Echinenone Chrysanthemaxanthin Flavorhodin Crocetin Haematoxanthin Cynthiaxanthin Leprotin Flavoxanthin Lycopene Fucoxanthin Mycoxanthin Glycymerin Pro-y-carotene Canary-xanthophyll Pro-lycopene Lycophyll Rhodopsin Mytiloxanthin Rhodopurpurin Myxoxanthophyll Rhodovibrin Oscillaxanthin Rhodoviolascin Pectenoxanthin Sarcinin Pentaxanthin Torulin Petaloxanthin Picofulvin Salmon acid Satinwood carotenoid Sulcatoxanthin Taraxanthin Torularhodin Trollixanthin Violaxanthin Violerythrin Xanthophyll Xanthophyll epoxide Zeaxanthin References p. 28. 3 SEPARATION OF THE CAROTENOID MIXTURES 23 trated in vacuum, and is then subjected to chromatography on suitable ad- sorbents. The aqueous methanoHc solution is diluted with water and extracted with ether. After washing and drying the ethereal solution, the solvent is re- moved by distillation in vacuum and the residue is dissolved in a suitable solvent and also subjected to chromatography. 3. SEPARATION OF THE CAROTENOID MIXTURES OF THE TWO PHASES The method almost invariably employed at the present time for the separation of natural carotenoid mixtures is Tswett's adsorption analysis. It is probably no exaggeration to say that the extraordinary development of carotenoid research during the last 20 years is mainly due to the application of Tswett's chromatographic method and it therefore appears appropriate to describe it in some detail^. However, since a number of excellent monographs dealing with chromatography are already available, only the practical appli- cations of the method to the separation of carotenoid pigments will be dealt with here. Although TswETT^, a Russian botanist, published the first description of chromatographic adsorption analysis as early as 1906, and although he drew attention to the scope and manifold applications of his method, chromatography was but seldom made use of during the following 25 years^. It was not until 1931, when the older methods of separation had finally proved inadequate, that adsorption analysis was re-introduced by Kuhn, by Karrer, and by Zechmeister, although it had occasionally been used by Dhere^ during the intervening period. The following short summary wiU give an indication of the progress subsequently achieved. TABLE 5 NUMBER OF NATURAL CAROTENOIDS ISOLATED DURING THE PERIOD 1922-1946 Period Number of carotenoids isolated Up to 1922 „ ,, 1933 ,. ,, 1937 „ „ 1948 7 about 15 ,, 30 ,, 80 A decisive factor in this rapid development was the fact that the adsorptive capacity of the polyene pigments is strongly influenced by relatively small differences in molecular structure. It is therefore possible to separate even References p. 28. 24 ISOLATION III closely related pigments on the chromatographic column. A classical example of the efficiency of the method is the separation of y-carotene from the a- and jS-components of crude carotene, although the proportion of y-carotene only corresponds to about one part in a thousand. Even differences in steric con- figuration of the pigments are sufficient for a quantitative separation, as was first shown by the investigations of Winterstein and Stein^ on cis- and trans- crocetin methyl esters. The strengths of adsorption of different carotenoids on a given adsorbent bear a definite relationship to their chemical structures. Hydroxyl substituents exert the largest influence in this respect. (Carotenoids containing carboxyl groups are not considered here). Of two carotenoids otherwise possessing the same structure, that containing a larger number of a) hydroxyl groups, b) carbonyl groups, c) esterified hydroxyl groups, or d) double bonds, is adsorbed more strongly. The effectiveness of functional groups on the strength of adsorption de- creases in the sequence : hydroxyl group, carbonyl group, esterified hydroxyl group, double bond. The following table provides a summary of the positions of some caro- tenoids (those whose functional groups are known) on the chromatogram. At the top of the table are the pigments which are most strongly adsorbed, at the bottom those which are least strongly adsorbed. In practice, adsorption analysis is carried out by percolating the solution of the carotenoids through a long column of suitable adsorbent. The amount of adsor- bent used depends on the amount of pigments to be separated. The individual zones are then "developed" by further washing with the same (or a different) solvent. The solution of carotenoids is poured into a tube which is filled to the extent of about ^/g with the adsorbent and connected to an evacuated bottle. As soon as the solution has been almost completely adsorbed by the column, the latter is washed through with an organic solvent (usually the same as that employed to prepare the solutions) until an optimum separation of the zones has been achieved. It is of great importance that the chromatogram should never be allowed to dry, as this results in the destruction of the polyene pigments by aerial oxidation, and in a shrinking of the upper part of the column and a distortion of the zones. As soon as the individual pigments have been separated as shown by the formation of colourless zones between the in- dividual coloured layers, the "development" is stopped. The adsorption column is extruded from the tube and the individual zones are mechanically divided. They are immediately placed in prepared vessels already filled with References p. 28. SEPARATION OF THE CAROTENOID MIXTURES TABLE 6 THE POSITION OF CAROTENOIDS IN THE CHROMATOGRAM Carotenoid Hydroxyl groups Carbonyl groups Ether groups Conjugated ethylenic bonds Isolated ethylenic bonds Myxoxanthophyll Fucoxanthin 6 4-6 1 0 ? 10 10? 0 ? Astaxanthin 2 2 0 11 0 Capsorubin Capsanthin Auroxanthin 2 2 2 2 1 0 0 0 2 9 10 7 0 0 2 Violaxanthin 2 0 2 9 0 Antheraxanthin 2 0 1 10 0 Lycophyll * Eschscholtzxanthin 2 2 0 0 0 0 13 12** 0 0 Flavoxanthin 2 0 1 9 1 Chrysanthemaxanthin Xanthophyll epoxide Zeaxanthin 2 2 2 0 0 0 1 1 0 9 10 11 1 0 0 Xanthophyll Rubichrome 2 1 0 0 0 1 10 10 1 1 Lycoxanthin 1 0 0 13 0 Rubixanthin 1 0 0 11 1 Cryptoxanthin Rhodoxanthin 1 0 0 2 0 0 11 12 0 0 Myxoxanthin Aphanin Citroxanthin 0 0 0 1 1 0 0 0 1 11 11 9 1 0 1 Flavochrome 0 0 1 8 2 a-Carotene epoxide Physalien Helenien 0 0 Di-ester Di-ester 1 9 11 10 1 0 1 Lycopene Pro-lycopene y-Carotene 0 0 0 0 0 0 0 0 0 11 11 11 2 2 1 Pro-y-carotene 0 0 0 11 1 ^-Carotene 0 0 0 11 0 a-Carotene 0 0 0 10 1 The relative positions of antheraxanthin and lycophyll in the chromatogram are still uncertain. Possibly only ii of the double bonds are conjugated. an eluting solvent. When the whole chromatogram has thus been separated, the solutions of pigments from the different zones are filtered, the solvent is removed by distillation and the residues are crystallised. If the separation thus achieved References p. 28. 26 ISOLATION III is incomplete, the eluting solvent, usually methanol, can be removed by shaking with water and the remaining solution dried, concentrated and again subjected to chromatography. Adsorbents. The following materials, in finely divided form, have been used for the chromatographic separation of carotenoids: alumina. Fuller's earth, calcium carbonate, calcium hydroxide, kaolin, kieselguhr, magnesium oxide, talcum, zinc carbonate, norite A, and others. The following 4 adsorbents are, however, adequate for most purposes: aluminium oxide*, calcium hydroxide, calcium carbonate, and zinc carbonate. Using these adsorbents it should be possible to carry out successfully all chromatographic separations of carotenoids. Alumina AlgOg: Suitable for the separation of carotenoid hydrocarbons, but its use has recently decreased owing to its high cost. Calcium hydroxide Ca(0H)2: Calcium hydroxide was introduced for the separation of carotenoid hydrocarbons by Karrer and Walker' in 1933 and has since become the adsorbent most widely used for this purpose. It is cheap and allows a complete separation of the epiphasic carotenoids. Calcium carbonate CaCOg : Calcium carbonate was iirst employed by Tswett for the separation of carotenoids. Since 1 931, it has frequently been used for the separation of phytoxanthins. Zinc carbonate ZnCOa: Zinc carbonate has frequently been employed, especially within recent years. It was introduced by Karrer and is very suitable for the separation of phytoxanthins. These are adsorbed somewhat more strongly on zinc carbonate than on calcium carbonate. The following is the sequence of adsorbents of decreasing activity : Alumina, aluminium hydroxide, magnesium oxide, calcium oxide, calcium hydroxide, zinc carbonate, calcium carbonate, calcium sulphate, calcium phosphate, talcum, sucrose, inulin'. Solvents. The purity of the solvents used is of the greatest importance for the success of a chromatogram. The solvents must be dry and free from impurities, such as alcohol, pyridine, sulphur-containing compounds, etc. For the chromatography of epiphasic carotenoids, petroleum ether (b.p. 70-80°) or a mixture of petroleum ether with benzene or ether are commonly used. Recently petroleum ether-acetone mixtures have frequently been employed** For hypophasic carotenoids it is usual to employ benzene or a mixture of benzene and ether or petroleum ether. Other solvents such as carbon di- sulphide, ethyl acetate, etc. are also used, although the first-named solvents are adequate in most cases. The following sequence of solvents, which is taken Different standardised grades of alumina are commercially available. For alumina according to H. Brockmann, cf. Neuere Methoden der praparativen organischen Chemie, 1943. P- 553- ** See p. 42, reference 10. References p. 28. SEPARATION OF THE CAROTENOID MIXTURES 27 from the excellent work of Hesse', is one of decreasing adsorptive capacity of the solutes : petroleum ether, carbon tetrachloride, trichloroethylene, benzene, methylene dichloride, chloroform, ether, ethyl acetate, acetone, w-propyl alcohol, ethanol, methanol, water, pyridine. -Adsorbent 'Glass tube Cottonwool Rubber stoppers Experimental. A suitable adsorbent and solvent, and the size of the chromatogram column, are first selected by means of preliminary small-scale experiments. The chro- matogram tube is then filled with the adsorbent. A variety of more or less complicated arrangements for large-scale chromatography have been de- scribed. Only a very simple apparatus which is readily as- sembled, inexpensive, and adequate for all purposes will be described here. It consists of a suction flask, a short glass tube ca. 10 mm in diameter and two good rubber stoppers. The stoppers are arranged back-to-back on the glass tube; one stopper is placed in the suction flask and the other in the chromatogram tube, as shown in the accompanying figure. The filling of the tube with the adsorbent can be carried out in various ways. The usual procedure is to add a small amount of the adsorbent at a time and to press it down with a half-bored cork attached to a glass rod. For larger tubes, Zechmeister recommends a wooden stopper, the end of which has a diameter corresponding to two-thirds of that of the tube. After one layer has been well pressed down, more adsorbent is added and the operation is repeated until the tube is suffi- ciently full. It is important that the tube should be well and evenly filled, otherwise distorted colour-zones are obtained which are difficult to separate. When the tube has been filled, the vacuum is connected and the pressing and tapping (from the outside) is continued until the adsorbent no longer moves. If this is done, no shrinking should occur when solvent is added. WiNTERSTEiN and SxEiN^ recommend that for filling large Arrangement for chro- tubes the adsorbent should be moistened with the solvent matographic analysis and then poured into the tube. When the column is ready, the solution of carotenoids is poured on and allowed to penetrate completely. More solvent is then added to "develop" the chromato- gram. The development is best carried out by connecting the suction flask to the vacuum and then isolating the water pump by means of a screw tap. When the rate of flow of the solvent becomes too small, the flask is re-evacuated. It is important that the rate of flow should be neither too great nor too small. If the rate is too great, the zones tend to be blurred, while if it is too small, no sharp layers are formed because the rate of diffusion of the pigments then exceeds the rate of flow. After development is complete, the column is sucked dry until it has the appearance of a compact mass which can be extruded without falling to pieces. After mechanical separation of the coloured zones, the pigments of the individual layers are eluted with the solvent previously employed, but containing a little methanol(ca. 2-5 %). The eluates are evaporated to dryness in vacuum and the residue from each zone is either crystallised or, if the pigment is still non-homogeneous, again subjected to chromatography. References p. 28. 28 ISOLATION III 4. CRYSTALLISATION The crystallisation of carotenoids requires considerable practice, especially if small amounts of material are involved. It is not always possible to recrystal- lise a carotenoid from a single solvent. Solvent mixtures consisting of one component in which the pigment is easily soluble, and a second component in which the pigment is sparingly soluble, sometimes have to be employed. No attempt will be made here to give a complete list of all the solvents which have been used, since more information on this point will be found in later sections dealing with individual carotenoids. Epiphasic carotenoids can often be recrystallised from light petroleum or from ether-methanol, or benzene- methanol mixtures. For hypophasic pigments, benzene-methanol or ether- methanol mixtures are often used. Methanol alone is also employed. Certain carotenoids (e.g. violaxanthin, fucoxanthin, zeaxanthin) can be precipitated by adding water to a methanolic solution of the pigment covered with a layer of light petroleum. The water is added in very small portions and crystallisation can often be induced by scratching the sides of the vessel with a glass rod. REFERENCES 1. R. WiLLSTATTER and A. Stoll, Untersuchungen iiber aas Chlorophyll, Berlin 191 3; Untersuchungen iiber die Assimilation der Kohlensaure, Berlin 1918. — Cf. also R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 41. 2. An extensive literature dealing with chromatography is available. The following mono- graphs should be consulted: L. Zechmeister and L. v. Cholnoky, Die chromato- graphische Adsorptionsanalyse, Berlin 1938. — H. Brockmann, Die chromatographische Adsorption, in: Neuere Methoden der praparativen organischen Chemie, Berlin 1943. — Gerhard Hesse, Adsorptionsmethoden im chemischen Laboratorium, Berlin 1943. — H. G. Cassidy et al., "Chromatography" in: Annals of the New York Academy of Sciences, 49 (1948) 141-326. 3. M. TswETT, Ber. deut. botan. Ges. 24 (1906) 316, 384. — M. Tswett, The chromophylls in the vegetable and animal kingdoms (in Russian), Warsaw, 1910. 4. Cf. L. S. Palmer, Carotinoids and related Pigments, New York, 1922. 5. C. Dhere, Compt. rend. 158 (1914) 64-66. — Cf. also C. Dhere, Candollea 10 (1943) 60. 6. A. WiNTERSTEiN and G. Stein, Z. physiol. Chem. 220 (1934) 251. 7. Cf. G. Hesse, Adsorptionsmethoden im chemischen Laboratorium, Berlin, 1943, p. 31. 8. Cf. H. Brockmann, Die chromatographische Adsorption, in: Neuere Methoden der praparativen organischen Chemie, Berlin, 1943. 9. A. Winterstein and G. Stein, Z. physiol. Chem. 220 (1933) 273. — Cf. D. C. Castle, A. E. GiLLAM, I. M. Heilbron and H. W. Thompson, Biochem. J. 28 (1934) 1702. CHAPTER IV The chemical constitution of the carotenoids Approximately 80 natural carotenoids are at present known. The constitu- tions of about 35 have been completely or largely elucidated and they are all closely related chemically. They all belong to the class of polyenes, their most characteristic structural feature being the large number of conjugated double bonds. Another characteristic feature is that of the 50 carotenoids the empirical formulae of which are known, 45 contain 40 carbon atoms, and only 5 have a different number of carbon atoms in the molecule. The fact that there is some connection between the carotenoids and isoprene was first recognised by Willstatter and Mieg^. To-day we know that isoprene is a constituent unit of the carotenoid pigments, which may be regarded as consisting of 8 isoprene molecules. It is characteristic of all carotenoids that the arrangement of the isoprene residues becomes reversed in the centre of the carotenoid molecule so that the central methyl groups occupy i : 6 instead of 1:5 positions^. The formula of lycopene is an example of this structural principle : CHa CHq CHi CHo C CH3 CHj CH3 CH3 C / ■ \ I I I \ CH CH-CH4:CH-C=CHCH:^iCH-C=CHCHiiCHCH = C-CHit:CHCH=C-CH:|:CH-CH CH CHg C*CH3 . H3C*C CH2 X. / Lycopene \ y, CH2 CHj The important principle of the "reversal" of the central isoprene units has given rise to the hypothesis that a carotenoid molecule may be formeid in the plant by the combination of two identical residues, e.g. two partially de- hydrogenated phytyl groups, by a linking of the two terminal carbon atoms. The following table, which contains the structural formulae of all naturally occurring carotenoids of known structure, shows the close structural relationship between these compounds. Formally, they can all be related to lycopene. y-Carotene, /3-carotene and a-carotene can be formed from lycopene by ring closure at one or both ends of the molecule, while bixin and crocetin can be References p. jy. 30 CHEMICAL CONSTITUTION IV formed by oxydative degradation. From lycopene itself and from the three carotenes, numerous other pigments can then be derived by the introduction of oxygen, hydroxyl, methoxyl or carbonyl groups. Finally, the oxidation of certain of these compounds can give rise to the aldehydes and carboxylic acids of the carotene series such as j3-citraurin and azafrin. TABLE 7 FORMULAE OF THE NATURALLY OCCURRING CAROTENOIDS OF KNOWN STRUCTURE CH. CH, CH3 CH3 \V \V C CH3 CH3 CH3 CH3 c y I I I I \ CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH I II 1 I CH2 C-CHs HsC-C CHa Lycopene \ / CH„ CH, CH, CH3 CH3 CH3 \V \/ C CH3 CH3 CH3 CH3 C y I I I I y\ CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ CH, C-CHj HgC-C CH2 y-Carotene \ y' CH» CH» CH3 CH3 CH3 CH3 \y \y C CH3 CH3 CH3 CH3 c y\ 1 I ! 1 y\ CH. C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj I II i I CHa C-CHj HsC-C CH^ \^ / /3-Carotene \ // CH» CHj CH, CH, CH3 CH3 \y \y C CH3 CH3 CH3 CH3 c y\ 1 1 1 ! y\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH^ CH, C-CH3 HaC-C CH^ a-Carotene \^ y' CH, CH NATU RALLY OCCURRING CAROTENOIDS 31 CH3 CH3 C CH3 CH, /\ \V CH2 C==CH CH, CH, CH3 CH3 C I i M I \ .\ /\ CH2 C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH = C-CH=CH-C CHj HjC-C CH2 Mutatochrome=Citroxanthin \ / CH, CH3 CH3 CH3 CH3 \V \V 0 CH, CH, CH, CH, C CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH^ CH, C/^ H,C-C CH, ^ a-Carotene epoxide X / CH, CH, CH CH3 CH, CH, CH, y I I I I V CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CH2 C-CHg HjC-C CHOH \ y" Lycoxanthin \ / CH, CH» CH3 CH, CH3 CH, \V \V y 1 ' ! I I \ CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH HOCH C-CHa HjC-C CHOH \/ Lycophyll \/ CHo CHo CHq OxIq \V \V CH CH3 CH3 CH3 CH3 CH y i I I I \ CH2 CH-CH=CH-C = CHCH=CH-C=CHCH = CHCH=C-CH=CHCH=C-CH=CH-CH CH^ H3COC C-CH3 H3C-C COCH3 \/ Rhodoviolascin(?) \ /" CH CH 32 CHEMICAL CONSTITUTION IV CH, CH, CH3 CH3 \V • \V C CH3 CH3 CH3 CH3 c CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH HOCH C-CH3 H3C-C CHj \ / Rubixanthin \\ / CH, CH„ CH3 CH3 \V C CH3 CH3 CH2 C=^CH CH3 CH3 CH3 CH3 C HOCH C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH HsC-C CH2 Rubichrome \^ / CH, CH3 CH3 CH3 CH3 \/ \/ C CH, CH, CH3 CH3 C /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH = CHCH = C-CH=CHCH = C-CH = CH-C CHj CH2 C-CHa HjC-C CHOH \ / Cryptoxanthin \ / CH, CHj CH3 CH3 CH3 CH3 \/ \/ C CH3 CH3 CH3 CH3 CH /\ II I I \ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CH, C-CHg HgC-C CH Myxoxanthin \ y/ CH, CO CH3 CH3 CH3 CH3 C CH3 CH3 CH3 CH3 C /\ II II /\ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH = CHCH=C-CH=CH-C CH^ I 1 II I CH, C-CHj H3C-C CO Aphanin(?) \^y^ CH, CHa NATURALLY OCCURRING CAROT ENOIDS 33 CH3 CH3 CH, CH3 \V \/ C CH3 CH3 CH3 CH3 c /\ II II /\ CH2 C-CH=CH-C = CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ HOCH C-CH3 H3C-C CHOH \ / Zeaxanthin \ / CH, CH^ CH3 CH3 CH3 CH3 C CH3 CH3 CH3 CH3 c CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CHj HOCH C-CHj HjC-C CHOH \ / Xanthophyll \ / CH, CH CH3 CH3 CH3 CH3 \/ \/ C CH3 CH3 CH3 CH3 C /\ II I I /\ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ I l\o i I HOCH Cr H3C-C CHOH \ / \ Antheraxanthin \ y/ CH„ CH, CH2 CH3 CH3 CH3 CH3 C CH, CH, CH3 CH3 C CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CHj I |\o II HOCH Cr HgC-C CHOH \ / \ Xanthophyll epoxide ^ /" CH, CH, CH CH3 CHj C CH3 CH3 CHj C— CH CH3 CH3 CH3 CH3 C HOCH C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH, CH,| 0 C CHOH CH, Flavoxanthin, Chrysanthemaxanthin X ^\ X H3C CH Carotenoids 3 34 CHEMICAL CONSTITUTION IV CH3 CH3 CH3 CH3 A r r r r- a CHg C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH2 HOCH Cr \C CHOH \ / \ Violaxanthin / A A CH, CH, H3C CHsi CH, CH, CH3 CH3 \A \/ c c A\ ■ /\ CH„ C=^=CH CH, CH3 CH3 H3C CH^C CH^ I I I I I I I I I I HOCH C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH C CHOH CHjI 0 Auroxanthin 0 | CHj CH, H3C CH3 CH3 CH3 CH3 AA \/ C CH, CH, CHq CH, C /A I I I I /\ CH„ C=CHCH=C-CH=CHCH=C-CH=CHCH=CH-C=CHCH=CH-C=CHCH=C CH, CO C-CH3 H3C-C CO A A Rhodoxanthin \ // CH CH CH, CH3 CH3 CH3 AA \/ C CH, CH3 CH3 CH3 C A\ I I I \ /\ CHa C-CH = CH-C=CHCH=CH-C=CHCH = CHCH = C-CH = CHCH=C-CH=CH-CO CH^ HOCH C-CH, Capsanthin HaC-CH, CHOH \A \/ CH» CH- CH3 CH3 CH3 CH3 VA \/ C CH, CH, CH, CH, C CH2 CO-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj HOCH CHsj-CHj HjC-CHj CHOH A A Capsorubin A A CH» CH, NATURALLY OCCURRING CAROTENOIDS 35 CHo CXlo ^Ho CHq \V \V C CH3 CH3 CH3 CH3 c /\ II II /\ CH„ C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, I II 11 I HOCH C-CHs HjC-C CHOH \ / Astaxanthin \^ / CO CO CH3 CH3 CH3 CH3 X f- r r r X CH„ C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, I II II I CO C-CH3 HjC-C CO \/^ .. ^/ CO Astacene CO CHo CHo \V C CH3 CH3 CH3 CH3 dn, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH COOH I II II I CHa C-CHj HgC-C CH \ y Torularhodin (?) \ /' CH, CH \V C OH CH, CH, CH3 y\/ I I I CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH = C-CH=CH-COOH I I CHg C' C'H3 ^ Azafrin Ch„ oh CHo CH, \/ C Clio CXI3 CHj CH3 /\ I I II CHj C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO HOCH C-CHj /3-Citraurin CH, 36 CHEMICAL CONSTITUTION CH3 CH3 CH3 CH3 HOOC- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- COOH Crocetin IV CH3 CH3 CH3 CH3 HOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- COOCH3 Bixin TABLE 8 CLASSIFICATION OF SOME CAROTENOIDS AS DERIVATIVES OF LYCOPENE AND THE CAROTENES Lycopene Lycoxanthin = 3-Hydroxylycopene Lycophyll = 3 : 3'-Dihydroxylycopene Rhodoviolascin ( ? ) y-Carotene ^-Carotene Rubixanthin = 3-Hydroxy-y-carotene Rubichrome = Furanoid oxide of rubixanthin Eschscholtzxanthin = Dihydroxy-y-carotene( ?) Cryptoxanthin = 3-Hydroxy-/5-carotene Citroxanthin = Furanoid monoxide of /^-carotene Zeaxanthin = 3 : 3'-Dihydroxy-/J-carotene Antheraxanthin = Zeaxanthin mono-epoxide Violaxanthin = Zeaxanthin di-epoxide Auroxanthin = Furanoid zeaxanthin di-oxide Aphanin = 3-Keto-/3-carotene( ?) Rhodoxanthin = 3 : 3'-Diketo-/3-carotene Astacene = 3:4:3': 4'-Tetraketo-/5-carotene Astaxanthin = 3:3'-Dihydroxy-4: 4'-diketo-/3-carotene Capsanthin Capsorubin a-Carotene a-Carotene epoxide Xanthophyll = 3 : 3'-Dihydroxy-a-carotene Xanthoph^dl epoxide Flavoxanthin = Furanoid xanthophyll oxide Chrysanthemaxanthin = Furanoid xanthophyll oxide It is not known with certainty to what extent carotenoids are intercon- vertible in nature. It seems very probable, however, that carotenoid epoxides, such as a-carotene epoxide and xanthophyll epoxide, are formed in the plant by oxidation of the corresponding carotenoids (a-carotene, xanthophyll), and REFERENCES 37 that they can be converted into the furanoid oxides (e.g. flavoxanthin, etc.^) under the influence of acids present in the plant. REFERENCES 1. R. WiLLSTATTER and W. MiEG, Ann. 355 (1907) i. 2. P. Karrer, a. Helfenstein, H. Wehrli and A. Wettstein, Helv. Chim. Acta 13 (1930) 1084. 3. P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 304. CHAPTER V Cis-trans isomerism of carotenoids Cis-trans isomerism of the type exhibited by simple ethylenic compounds is also observed in the carotenoid series. Since a carotenoid contains several carbon-carbon double bonds, a considerable number of geometrically isomeric forms are possible. Thus a polyene of the formula R(CH==CH)9R' containing 9 double bonds can theoretically exist in 512 different cis-trans isomeric forms. The fact that bixin occurs in two isomeric forms was discovered by Herzig and Faltis^ in 1923. It was proved by Karrer and co-workers^ in 1929 that these two forms were cis-trans isomers. Bixin is the more labile form. It is easily converted into /so-bixin which in view of its greater stability is regarded as the trans-iorm. Later, Kuhn and Winterstein^ found that crocetin, the chief pigment of saffron, is accompanied by a small quantity of a geometrical isomer, /50-crocetin. /so-crocetin is a labile compound which can readily be converted into the more stable crocetin by means of iodine or other catalytic agents. In 1935, GiLLAM and El Ridi'* found that on repeated chromatographic adsorption of homogeneous ^-carotene, two zones are obtained. The upper zone consists of j3-carotene while the lower zone contains a new pigment, pseudo-a-carotene. It is possible that this isomer of j3-carotene is not formed during adsorption, as assumed by Gillam and El Ridi, but spontaneously in the solution of the pigment^. In recent years, the reversible interconversion of carotenoids believed to be due to cis-trans isomerism has been investigated in detail, particularly by Zechmeister and his collaborators. Although a considerable body of experi- mental material is already available, especially with regard to the spectro- scopic and chromatographic properties of the different isomers, this field of work awaits further development, since the majority of interconversion products have not yet been isolated in crystalline form. Only a brief summary of these investigations will be given here. The properties of the different isomers are described in later chapters dealing with the parent carotenoids. On the basis of recent studies employing x-ray analysis^, spectral analysis and chromatographic analysis, it may be assumed that with a very few ex- References p. 4.2. CIS-TRANS ISOMERISM 39 ceptions (e.g. bixin, pro-y-carotene, pro-lycopene) the natural carotenoids all possess entirely /^-ans-configurations. This is not surprising since the trans- configurations are those with the smallest energy content and the greatest stability. Thus natural j5-carotene is beheved to have the following structure. CH3 CH3 H H C 2C 3C /\ y\y^ CH, C— C C I l|| ' ' CH, C H CH3 CH2 CH3 On the basis of theoretical considerations, Zechmeister, Pauling and collaborators^ concluded that not all ethylenic groups of a carotenoid molecule are capable of taking part in cis-trans isomerisation, but only those of the type -C(CH3) = CH- and the double bond in the centre of the molecule. In the case of ^-carotene only the 3-, 5-, 6-, 7-, and 9-double bonds could thus be involved in cis-trans isomerism. The remaining ethylenic groups are beheved always to assume a ^ra«s-configuration owing to steric hindrance. Isomerisation can be brought about in the following ways.- a) refluxing of a solution of the carotenoid in an organic solvent, b) melting of the crystals, c) treatment with iodine^, d) treatment with acids, and e) illumination. The separation of the isomerisation products is carried out by means of chromato- graphic analysis. All the transformation products of natural /raws-carotenoids so far obtained exhibit the following common features^": 1. The colour intensity of the pigment solution decreases as a result of isomerisation. 2. The isomerisation products are more soluble than the starting materials. 3. The melting points of cis-isomers are lower than those of the pigments with complete /r^ws-configurations. 4. The isomerisation products often revert to the parent pigment with complete /raws-configuration on crystallisation. Others crystallise inhomo- geneously as shown by the fact that fresh solutions of the crystals give rise to several zones on chromatography. This is the case for instance, with pseudo-a- carotene, neo-carotene and neo-a-carotene^i. 5. If the molecule contains one or more asymmetric carbon atoms, isomeri- sation is often accompanied by large changes in optical rotation. 6. The strength of adsorption of the isomerised carotenoids on the chro- matogram column differs considerably from that of the /raws-pigments. 7. The isomerisation products always absorb at shorter wavelengths in the References p. /).2. 4° CIS-TRANS ISOMERISM V visible region of the spectrum than the parent carotenoids with complete trans-conhguvsition. If the labile isomerisation products are treated with iodine, the absorption maxima are again displaced to longer wavelengths, but not as far as the location of the absorption maxima of the original /mws-carotenoids. This is explained by Zechmeister and his coworkers as due to the fact that in these isomerisations an equilibrium is always established so that the reverse change to the original trans-pigment is never complete. 8. The extinction coefficients of the isomerisation products are lower than those of the parent carotenoids with complete ^raws-configurations. 9. The isomerisation products are often characterised by the appearance of a new maximum in the ultra-violet spectrum. Following Zechmeister and PoLGAR these new maxima are termed "cis-peaks". This phenomenon is illustrated in the schematic figure below : ca. 3-^mu. Fig. 2. Carotenoid with complete ^raws-configuration Isomerisation product With all the isomerisation products so far examined, the centre of the cis- peak lies at a distance of 142 (±2) m// from the long-wave maximum (in hexane solution). For a theoretical interpretation of the "cis-peak", compare the review by Zechmeisteri*^. A more detailed discussion of this subject is beyond the scope of this mono- graph. The properties of the different isomers are briefly described in later sections dealing with the corresponding parent carotenoids. As a guide to the relevant literature all the carotenoids so far examined for czs-^yaws isomerisation are summarised in the following table. References p. 42. CIS-TRANS ISOMERISM 41 TABLE 9 CAROTENOIDS EXAMINED FOR CIS-TRANS ISOMERISM Pigment References Capsanthin L. Zechmeister and co-workers, Ann. 530 (1937) 291; ^43 (1940) 248; /. Atn. Chem. Soc. 66 (1944) 186. Capsorubin L. Zechmeister, L. v. Cholnoky, Ann. 543 (1940) 248; A. PoLGAR and L. Zechmeister, /. Am. Chem. Soc. 66 (1944) 186. a-Carotene L. Zechmeister and co-workers, /. Am. Chem. Soc. 6^ (1943) 1522; 66 (1944) 137; Arch. Biochem. 6 (1945) 157. — A. E. GiLLAM, M. S. El Ridi and S. K. Kon, Biochem. J. 31 (1937) 1605. — F. Zscheile and co-workers. Arch. Biochem. 5 (1944) 77, 211. /3-Carotene A. E. Gillam and M. S. El Ridi, Nature (London) 136 (1935) 914; Biochem. J. 30 (1936) 1735; 31 (1937) 251. — L. Zech- meister and co-workers, /. Am. Chem. Soc. 64 (1942) 1856; 65 (1943) 1528; Arch. Biochem. 5 (1944) 107; Arch. Biochem. 7 (1945) 247; Ber. J2 (1939) 1340; Nature (London) 141 (1938) 249; Biochem. J. 32 (1938) 1305;/. Am. Chem. Soc. 66(1944) 137. y-Carotene L. Zechmeister and A. PolgAr, /. Am. Chem. Soc. 67 (1945) 108. — L. Zechmeister and co-workers. Arch. Biochem. 5 (1944) 365. — Cf. also: R. F. Hunter and A. D. Scott, Biochem. J. 35 (1941) 31. — L. Zechmeister and co-workers. Plant Phvsiol. 17 (1942) 91, footnote 2. — L. Zechmeister, L. Pauling and co-workers, /. Am. Chem. Soc. 65 (1943) 1940. A. L. le Rosen and L. Zechmeister, Arch. Biochem. i (1942) 17. H. H. Strain and W. M. Manning, /. Am. Chem. Soc. 64 (1942) 1235. L. Zechmeister and W. A. Schroeder, /. Am. Chem. Soc. 65 (1943) 1535. L. Zechmeister and co-workers. Nature (London) 141 (1938) 249; Biochem. J. 32 (1938) 1305; Ber. y2 (1939) 1340; /. Am. Chem. Soc. 66 (1944) 317. L. Zechmeister and co-workers. Nature (London) 141 (1938) 249; Ber. 72 (1939) 1340; Biochem. J. 32 (1938) 1305; /. Am. Chem. Soc. 65 (1943) 1942; 66 (1944) 137. L. Zechmeister and co-workers, Ber. J2 (1939) 1340, 1678, 2039; /. Am. Chem. Soc. 66 (1944) 317. L. Zechmeister and co-workers, /. Am. Chem. Soc. 64 (1942) 1173; 63 (1943) 1940. L. Zechmeister and co-workers, /. Am. Chem. Soc. 65(1943) 1940. L. Zechmeister and co-workers. Arch. Biochem. 5 (1944) 243. L. Zechmeister and P. Tuzson, Ber. 72 (1939) 1340. H. H. Strain, /. Biol. Chem. i2y (1938) 191. — L. Zechmeister and co-workers, Ber. y2 (1939) 1340; /. Am. Chem. Soc. 65 (1943) 1951; 66 (1944) 137. L. Zechmeister and co-workers, Ber. 72 (1939) 1340, 1678, 2039; /. Am. Chem. Soc. 66 (1944) 317. Celaxanthin Fucoxanthin Gazaniaxanthin CrN^ptoxanthin Lycopene Physalien Pro-y-carotene Pro-lycopene Spirilloxanthin Taraxanthin Xanthophyll Zeaxanthin 42 CIS-TRANS ISOMERISM V REFERENCES 1. J. Herzig and F. Faltis, Ann. 431 (1923) 40. 2. P. Karrer and co-workers, Helv. Chim. Acta 12 {1929) 741. 3. R. KuHN and A. Winterstein, Ber. 66 (1933) 209. 4. A. E. GiLLAM and M. S. El Ridi, Nature 136 (1935) 914; Biochem. J. 30 (1936) 1735; 31 (1937) 251. 5. A. E. Gillam and co-workers, Ann. 530 (1937) 291; Nature 141 (1938) 249; L. Zech- MEiSTER and co-workers, Biochem. J. 32 (1938) 1305. 6. J. Hengstenberg and R. Kuhn, Z. Krist. Mineral, y^ (1930) 301; y6 (1930) 174. — G. MacKinney, /. Am. Chem. Soc. 56 (1934) 488. 7. R. S. MuLLiKEN, /. Chem. Phys. 7 (1939) 364; Rev. Modern Phys. 14 (1942) 265. 8. L. Zechmeister, L. Pauling and co-workers, /. Am. Chem. Soc. 65 {1943) 1940; L. Zechmeister, Chem. Revs. 34 (1944) 267. . 9. P. Karrer and co-workers, Helv. Chim. Acta 12 (1929) 741. 10. L. Zechmeister, Chem. Revs. 34 {1944) 267, 11. L. Zechmeister, Chem. Revs. 34 (1944) 293. — Cf. A. E. Gillam, M. S. El Ridi and S. K. KoN, Biochem. J. 31 (1937) 1605. CHAPTER VI Methods of elucidating the constitution of carotenoids The elucidation of the constitution of the carotenoids is no easy task, and although carotene and some of its congeners have been known for a very long time, it is only within the last 20 years that it has been possible to obtain some insight into the chemical structure of these compounds. The following sections are meant to provide a short summary of the principal methods which have been used for this purpose. For detailed descriptions of the experimental methods the original literature should be consulted. I. DETERMINATION OF THE NUMBER OF DOUBLE BONDS The most characteristic structural feature of the polyene pigments is the large number of double bonds in the molecule. In determining the constitution of a carotenoid it is important to be able to establish the number of carbon- carbon double bonds with small amounts of material (about 5 mg). Useful information can be obtained by first determining the absorption spectrum of the carotenoid. The relationships between the number of double bonds and the absorption spectrum have been fully investigated and a knowledge of one of these properties enables one to make predictions about the other. More exact information is provided by quantitative measurements of the addition of hydrogen, halogen, or iodine chloride, or of oxygen. The most accurate values are obtained from catalytic hydrogenation, but the other methods have some- times been used to confirm the results obtained. Catalytic hydrogenation can be carried out on the macro- or microscale. In either case, all the double bonds in the molecule react including the carbonyl group. Epoxide groups are also reduced during catalytic hydrogenation with the formation of hydroxyl groups^. Colloidal platinum^, platinum oxide^, palladium oxide^, or platinum ad- sorbed on Kieselguhr* can be used as catalysts. The following are suitable solvents: acetic acid (free from higher homo- logues), ethyl acetate, acetic acid-ethanol mixtures, cvc/ohexane, hexane, decalin, etc. Carotenoids are often so sparingly soluble that they have to be References p. 51-52. 44 METHODS OF ELUCIDATING CONSTITUTION VI hydrogenated in suspension; under these conditions the reaction takes con- siderably longer than usual. Another characteristic feature of the hydrogenation of carotenoids is that a relatively large amount of catalyst is required for complete reduction. The microhydrogenation of polyene pigments described by KuHN and MoELLER^ is extremely valuable in view of the small amounts of material required. Using this method, numerous carotenoids which occur in only very small quantities in nature have been examined for the number of double bonds present in the molecule. Details regarding the apparatus and reagents required will be found in the memoir already cited*. It was shown by Zechmeister and Tuzson^ that polyenes add bromine in chloroform solution. The titration method based on this reaction has the dis- advantage, however, that not all double bonds participate. Thus, according to Zechmeister, carotene and xanthophyll absorb only 8 molecules of bromine instead of ii. A more suitable reagent, which in most cases reacts with all the double bonds present, is iodine chloride as described by Pummerer and Reb- MANN, and Pummerer, Rebmann and Reindel^. According to the last-named authors^, the double bonds can also be saturated by the addition of oxygen. In practice, this method consists of reacting the carotenoid with perbenzoic acid in chloroform solution and back-titrating the excess perbenzoic acid after oxidation is complete. However, oxidation with perbenzoic acid again does not always result in the reaction of all double bonds present. Thus, the only completely reliable method of determining the number of double bonds in a carotenoid is catalytic hydrogenation. 2. DETERMINATION OF SIDE-CHAIN METHYL GROUPS The first method employed for the determination of side-chain methyl groups is due to Kuhn, Winterstein and Karlovitz'' and consists of oxidation by means of potassium permanganate in alkaline solution. Under these condi- tions a side-chain methyl group and the carbon atom to which it is attached give rise to one molecule of acetic acid. This method was later replaced by the oxidation with chromic acid, which was found to be more reliable^. Karrer, Helfenstein, Wehrli and Wettstein^ showed that oxidation by means of alkaline permanganate only degrades unsaturated groupings of the type =CH-C= CH3 whereas more saturated groupings such as — CH2— C= CH3 References p. §1-^2. 4 DETERMIN ATION OF HYDROXYL GROUPS 45 are only incompletely oxidised to acetic acid or not attacked. A micro-method for the determination of side-chain methyl groups has been described by KuHN and RothI"^. 3. DETERMIXATIOX OF 7S0PR0P YLIDENE GROUPS For the determination of zsopropylidene groups (CHaJgC^C . . , a method introduced by Karrer, Helfenstein, Pieper and Wettsteix^^ and somewhat modified by Kuhn and Roth^^^ fg used. The z'sopropylidene grouping is degraded by ozonisation to acetone which is determined iodometrically. The micro-determination of Kuhn and Roth depends on the same principle, but ozonisation is followed by oxidation with permanganate, to improve the yield of acetone. 4. determination of HYDROXYL GROUPS By determination of active hydrogen by the method of Zerewitinoff, it was established by Karrer, Helfenstein and Wehrli^^ that the oxygen atoms present in xanthophyll are not present in the form of ether groupings as had previously been assumed, but as hydroxyl groups. An apparatus for the Zerewitinoff determination has been developed by Flaschentrager^^ and a somewhat modified procedure has been described by Roth^^. The following empirical facts must be taken into account in interpreting the results of Zerewitinoff determinations : If several (4-6) hydroxyl groups are present in the polyene molecule, they may not all react. In the presence of two hydroxyl groups, on the other hand, somewhat high values are sometimes obtained^®. Ketones which have a tendency to enolise also give rise to the production of methane, and thus simulate hydroxyl groups^'. The determination of the position of the hydroxyl groups in the polyene molecule is more difficult than the determination of their number. In this connection, compare the investigations discussed on page 201. A clue to the position of the hydroxyl groups is often provided by the absence of certain oxidation products. The question as to whether two hydroxyl groups are present in neighbouring positions can sometimes be decided by the method of Criegee^^, e.g. in the case of azafrin (cf. page 282). 5. determination of methoxyl groups Up to the present time, rhodoviolascin is the only known naturally occurring carotenoid containing methoxyl groups. These can be determined in the usual way by the method of Zeisel^^. References p. 51-52. 46 METHODS OF ELUCIDATING CONSTITUTION VI 6. DETECTION AND ESTIMATION OF CARBONYL GROUPS The detection of carbonyl groups in polyene pigments often presents difficul- ties as the usual carbonyl reagents (hydroxy lamine, semicarbazide, etc.) do not always react. With some carotenoids, special methods of oximation have to be used*. Others (e.g. capsanthin) cannot be oximated at all by the methods at present available. In such cases the hydroxyl groups are first determined and the Zerewitinoff determination is repeated after complete reduction of the pigment. If the number of hydroxyl groups has increased, the presence of a carbonyl group which has been reduced to a secondary or primary alcohol grouping, is indicated. If the carbonyl group is conjugated with the system of conjugated ethylenic bonds, it can be recognised by a strong red shift of the absorption maxima (cf. p. 56). TABLE 10 NATURALLY OCCURRING CAROTENOIDS CONTAINING CARBONYL GROUPS* Number of Carotenoid carbonyl groups Method of determining carbonyl groups Aphanin 1 Formation of oxime Astacene 4 Preparation of dioxime and bis-phenazine deri- vative Astaxanthin 2 Analogy with astacene, but 2 hydroxyl groups present Capsanthin 1 Absorption spectrum, Meerwein-Ponndorf reduc- tion to capsanthol Capsorubin 2 Analogy with capsanthin (not yet definitely proved) /?-Citraurin 1 Formation of oxime from aldehyde group Myxoxanthin 1 Formation of oxime Rhodoxanthin 2 Formation of dioxime * Only those carotenoids the structure of which has been completely or largely de- termined, are mentioned in this table. Bixin, crocetin and azafrin which contain carboxyl groups have not been included. 7. determination of CARBOXYL GROUPS Carboxyl groups are determined by titration with alkalis. The carotenoid is best hydrogenated before the titration which is then easier to carry out. Details will be found in the papers by Kuhn and co-workers^". * The oximation of polyene pigments containing carbonyl groups is usually effected with hydroxylamine acetate. Sometimes it is necessary, however, to employ free hydroxyl- amine. Cf. R. Kuhn and H. Brockmann, Ber. 66 (1933) 828. References p. 51-52. 9 OXIDATIVE DEGRADATION 47 8. OXIDATION WITH PERMANGANATE AND OZONE The introduction of oxidative degradation with potassium permanganate was of decisive importance for the elucidation of the structure of the caro- tenoids. By this means it was possible for the first time to obtain large fragments (dicarboxylic acids, etc.) which gave a clue to the constitution of these pigments. Details of these oxidations will be given in the description of individual carotenoids (cf. p. 132). The reader is also referred to the original hterature^^. The results of degradative oxidation are so reliable that it is possible to draw conclusions regarding the structure of a carotenoid from the absence of certain degradation products (cf. xanthophyll, p. 201). Thus, potassium permanganate degradation of an unsubstituted j8-ionone ring yields dimethylmalonic acid, a : a-dimethylsuccinic acid and a : a-dimethyl- glutaric acid. The same degradation products are formed from an a-ionone ring. Degradation by means of ozone is also an important method for elucidating the constitution of carotenoids. By this means, it is possible for instance, to show that the end groups of lycopene are zsopropylidene groups^^. Similarly, an fsopropylidene group can be shown to be present in y-carotene. Furthermore, by the controlled ozonisation of j3-carotene and /S-ionone, Pummerer, Rebmann and Reindel^^ succeeded in isolating large degradation fragments identical with those obtained from permanganate oxidations. An observation which was of considerable importance in the elucidation of the constitution of carotenoids was made by Karrer and co-workers^*. They showed that, in addition to the degradation products obtained by means of permanganate, the ozonisation of /3-carotene gives rise to geronicacid (a:a-di- methyl-S-acetylvaleric acid), while a-carotene yields geronic as well as iso- geronic acid (y:y-dimethyl-S-acetyl valeric acid) 2^. 9. partial degradation of carotenoids with permanganate AND chromic acid Another important contribution to the elucidation of the constitution of carotenoids was the introduction of step-wise degradation with alkaline permanganate (Karrer and co-workers)^® and of partial oxidation with chromic acid (KuHN and Brockmann^'^). Both methods allow the isolation of large degradation fragments from the structure of which it is possible to draw conclusions regarding the constitution of the parent pigments. Thus Karrer and co-workers^® succeeded in preparing j3-apo-2-carotenal, ^-apo-3-carotenal and /3-apo-4-carotenal by the stepwise degradation of jS-carotene (cf. p. 144). KuHN and Brockmann obtained various ketonic products, e.g. jS-carotenone and semi-j3-carotenone by the mild chromic acid oxidation of /S-carotene, References p. §1—32. 48 METHODS OF ELUCIDATING CONSTITUTION VI depending on the quantity of oxidising agent employed. These partial oxidations are also of interest because they make it possible to interconvert different carotenoids and thus to prove the close relationships which exist between these compounds. TABLE 11 CAROTENOIDS WHICH HAVE BEEN PARTIALLY OXIDISED* Carotenoid Oxidising agent Degradation products Azafrin Cr03 Azafrinone, "Azafrinal I" methyl ester KMnOi Apo-1-azafrinal, "Azafrinal II" methyl ester Bixin KMnO^ Apo-1-norbixinal methyl ester (stable), (Stable form) apo-2-norbixinal methyl ester (stable), apo-3-norbixinal methyl ester (stable) Bixin KMnOi Apo-1-norbixinal methyl ester (labile), (labile form) apo-2-norbixinal methyl ester (labile) apo-3-norbixinal methyl ester (stable) Capsanthin CrOj Capsanthinone, capsanthylal, capsylaldehyde, 4-hydroxy-/?-carotenone aldehyde a-Carotene CrOj Hydroxy-a-carotene, semi-a-carotenone, a-carotone KMnO^ a-Apo-2-carotenal ^-Carotene CrOg Hydroxy-^-carotene, semi-^-carotenone, hydroxy- semi-^-carotenone, /?-carotenone, /3-carotenone- aldehyde, hydroxy-jS-neo-carotene KMnO^ ^-Apo-2-carotenal, ^-apo-3-carotenal, /5-apo-4- carotenal Lycopene KMn04 Apo-3-lycopenal Cr03 Bixin dialdehyde, apo-2-lycopenal, apo-3:12-lyco- penedial, apo-2:12dycopenedial Physalien Cr03 Physalienone Rhodoviolascin KMnOi Complex dialdehyde Xanthophyll KMnO^ a-Citraurin Zeaxanthin KMn04 /3-Citraurin * Details concerning the various degradation products and literature references will be found in the relevant sections of the special part of this monograph. 10. THERMAL DEGRADATION The thermal degradation of carotenoids is a method no longer employed to any extent. The yields of identifiable degradation products are only very small and, in any case, no certain conclusions can be drawn from them regarding References p. 51-^2. 12 CHEMICAL CONSTITUTION AND BIOLOGICAL PROPERTIES 49 the structure of the carotenoid. One advantage of the method of thermal degradation, however, is that the products formed give some indication of the relative position of the side-chain methyl groups. Thus Van Hasselt^^ obtained w-xylene from bixin. According to Kuhn and Winterstein^*, the xylene is derived from a part of the aliphatic chain. •••=CH-C=CHCH=CH-C=--- CH=C-CH3 II / \ CH, CH, > CH3— C CH CH-CH A similar observation was made by Zechmeister and von Cholnoky^" with capsanthin. Later, Kuhn and Winterstein^^ re-investigated thermal decompositions and isolated 2 : 6-dimethylnaphthalene from different caro- tenoids. This must be derived from the central part of the polyene chain. •••CH CH CH CH y yv y\/v CH CH C-CHj CH C C-CHj HoC-C CH CH HoC^C C CH CH CH--^ CH CH II. DETERMINATION OF OPTICAL ROTATION Optical activity can be used to decide the question as to whether the carotenoid molecule has a symmetrical or unsymmetrical structure. The C-line of mercury (656.3 mfi) is often employed as light source as proposed by Zechmeister and Tuzson^^^ Kuhn, Winterstein and Lederer, on the other hand, recommend a quartz cadmium lamp as a more powerful Hght source^^. 12. relationships between chemical constitution and biological properties As was explained on p. 13, the relationship between the vitamin A potency of a carotenoid and its structure appears to be governed by the principle that high vitamin A activity depends on the presence of an unsubstituted jS-ionone ring. In this way, conclusions can be drawn from the physiological activity of a polyene pigment regarding its content of jS-ionone rings. It must be re- membered, however, that some carotenoids (e.g. ^-carotene di-epoxide), which do not contain an unsubstituted /3-ionone ring, nevertheless possess vitamin A-activity because they undergo certain transformations in the animal organ- ism (cf . p. 148) 34 and that some growth-promoting properties are also exhibited by partly demethylated and acetylenic analogues of vitamin A (cf. p. 14, footnote). References p. 51-52. Carotenoids 4 50 METHODS OF ELUCIDATING CONSTITUTION VI 13. RELATIONSHIPS BETWEEN CHEMICAL CONSTITUTION AND COLOUR It has been mentioned before that the colour of a carotenoid is one of its most important characteristics, and that it is often possible to make deductions regarding the structure of a polyene pigment from its absorption spectrum. A great deal of material is available in this field and many useful relation- ships have been established^^. The shortest wavelength selective absorption in the visible region so far observed with a natural carotenoid is shown by auroxanthin (maxima at 454 and 423 m^ in carbon disulphide solution, cf. p. 196). The longest wavelength selective absorption in the visible region is shown by torularhodin (maxima at 582, 541, and 502 m// in carbon disulphide solution*, cf. p. 330). The light absorption properties are determined by the following factors: a) Number and type of double bonds, b) Number and type of carbonyl groups, c) Number of epoxide groups, d) Number and position of carboxyl groups, e) Number of hydroxyl groups, f) Steric configuration of the carotenoid. In recent years the detailed relationships between the constitution and colour of the carotenoids have been investigated particularly by Kuhn and co- workers^^, by Hausser and Smakula^^ and by Karrer and co-workers^. These investigations are discussed in more detail in the following chapter. 14. COMPARISON WITH PARTIALLY SYNTHETIC POLYENE PIGMENTS Recently, comparison with partially synthetic carotenoids has often been used successfully for elucidating the structure of carotenoids of unknown constitution. An example is provided by the comparison of j3-citraurin (p. 219) with ;S-apo-2-carotenal (p. 144) which, according to Karrer and Solmssen^* differ only by the presence of an extra hydroxyl group in |8-citraurin. This comparison lead to the complete elucidation of the constitution of ^-citraurin. Karrer and Jucker have recently succeeded in elucidating the structure of numerous carotenoids by comparison with partially synthetic pigments. Thus the following pairs of natural and partially synthetic pigments were proved to be identical : flavoxanthin and xanthophyll epoxide, antheraxanthin and zeaxanthin mono-epoxide, violaxanthin and zeaxanthin di-epoxide, citro- xanthin and mutatochrome, etc. * In this connection tlie partially synthetic dehydrolycopene is of interest. It exhibits a long wave absorption maximum in CSj at 601 m/z (p. 122). References p. 51-52. REFERENCES 15. DETERMINATION OF THE MOLECULAR WEIGHT 51 The molecular weight of carotenoids can be determined by R.\st's method^*' as well as by other cryoscopic and ebuUioscopic methods. X-ray analysis has also been employ ed^^. REFERENCES 1. P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 300. 2. R. WiLLSTATTER and E. Waldschmidt-Leitz, Ber. 54 (1921) 113. 3. R. Adams and R. L. Shriner, J. Am. Chem. Soc. 45 (1923) 2171. — R. L. Shriner and R. Adams, J. Am. Chem. Soc. 46 (1924) 1683. — M. Frankel, Abderhaldens Handbuch der biologischen Arbeitsmethoden, part 12. — Cf. also H. Gilman and A. H- Blatt, Organic Syntheses, Coll. Vol. I, and ed., 1941, p. 463. 4. R. KuHN and E. F. Moller, Angew. Chem. 4j (1934) i45- 5. L. Zechmeister and P. Tuzson, Ber. 62 (1929) 2226. 6. R. Pummerer and L. Rebmann, Ber. 61 (192S) 1099; R. Pummerer, L. Rebmann and W. Reindel, Ber. 62 (1929) 141 1. 7. R. KuHN, A. WiNTERSTEiN and L. Karlovitz, Helv. Chim. Acta 12 (1929) 64. 8. R. KuHN and L. Ehmann, Helv. Chim. Acta 12 {1929) 907; P. Karrer, A. Helfen- STEiN, H. Wehrli and A. Wettstein, Helv. Chim. Acta 13 (1930) 1084. — R. Kuhn and F. L'Orsa, Ber. 64 (1931) 1732; Angew. Chem. 44 (1931) 847. 9. P. Karrer, A. Helfenstein, H. Wehrli and A. Wettstein, Helv. Chim. Acta 13 (1930) 1084. 10. R. KuHN and H. Roth, Ber. 66 (1933) 1274. 11. P. Karrer, A. Helfenstein, B. Pieper and A. Wettstein, Helv. Chim. Acta 14 (1931) 435. 12. R. KuHN and H. Roth, Ber. 65 (1932) 1285. 13. P. Karrer, A. Helfenstein and H. Wehrli, Helv. Chim. Acta 13 (1930) 87; 13 (1930) 268. 14. B. Flaschentrager, Z. Physiol. Chem. 146 (1925) 219. 15. H. Roth, Mikrochem. 11 (1932) 140. ■ — Cf. also Pregl-Roth, Quantitative organische Mikro-analyse, 5th edition. Springer- Verlag, Vienna, 1947. 16. P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 302. 17. R. Kuhn and H. Brockmann, Ber. 66 (1933) 828. 18. R. Criegee, Ber. 64 (1931) 260. 19. Pregl-Roth, Quantitative organische Mikroanalyse, 5th edition. Springer- Verlag, Menna, 1947. 20. R. Kuhn and co-workers, Helv. Chim. Acta 11 (1928) 716; Ber. 64 (1931) 333. 21. P. Karrer and A. Helfenstein, Helv. Chim. Acta 12 (1929) 1142. — P. Karrer, A. Helfenstein, H. Wehrli and A. Wettstein, Helv. Chim. Acta 13 (1930) 1084. — Cf. also R. Kuhn and A. Deutsch, Ber. 66 (1933) 8S3. — P. Karrer, Helv. 12 (1929) 558. 22. P. Karrer and W. E. Bachmann, Helv. Chim. Acta 12 (1929) 285. — P. Karrer, A. Helfenstein, B. Pieper and A. Wettstein, Helv. Chim. Acta 14 (1931) 435. 23. R. Pummerer, L. Rebmann and W. Reindel, Ber. 64 (1931) 492. 24. P. Karrer, A. Helfenstein, H. Wehrli and A. Wettstein, Helv. Chim. Acta 13 (1930) 1084. 25. P. Karrer, R. Morf and O. Walker, Helv. Chim. Acta 16 (1933) 975. 26. P. Karrer and co-workers, Helv. Chim. Acta 20 {1937) 682; 20 (1937) 1020, 1312; 21 {1938) 1171- 27. R. Kuhn and H. Brockmann, cf. p. 139. 52 METHODS OF ELUCIDATING CONSTITUTION VI 28. J. F. B. VAN Hasselt, Rec. trav. Chim. Pays-Bas et Belg. 30 (1911) i; 33 (1914) 192. Cf. J. Herzig and F. Faltis, Monatsh. 35 (1914) 997. 29. R. KuHN and A. Winterstein, Helv. Chim. Acta 11 (1928) 427. 30. L. Zechmeister and L. v. Cholnoky, Ann. 478 {1930) 95. 31. R. KuHN and A. Winterstein, Ber. 65 (1932) 1873; 66 (1933) 429; 66 (1933) i733- 32. L. Zechmeister and P. Tuzson, Ber. 62 (1929) 2226. 33. R. Kuhn, a. Winterstein and E. Lederer, Z. Physiol. Chem. igj (1931) 141. — Cf. also P. Karrer and O. Walker, Helv. Chim. Acta 16 (1933) 641. 34. Cf. P. Karrer and J. Rutschmann, Helv. Chim. Acta 2g (1946) 355. 35. Cf. P. Karrer, Die bisher bekannten natilrlichen Carotinoide und ihre Absorptions- spektren, Vierteljschr. Naturf. Ges., XV, Zurich 1945. 36. R. Kuhn and H. Brockmann, Ber. 65 (1932) 894; 66 (1933) 407; 66 (1933) 828; 66 {1933) 1319- — R- Kuhn and Ch. Grundmann, Ber. 65 (1932) 898; 65 (1932) 1880. — R. Kuhn and A. Deutsch, Ber. 66 (1933) 883. 37. K. W. Hausser and A. Smakula, Angew. Chem. 4y (1934) 663; 48 (1935) 152. 38. P. Karrer and E. Wurgler, Helv. Chim. Acta 23 (1940) 955; 26 (1943) 116. — E. Wurgler, Dissertation, Zurich 1943. 39. P. Karrer and U. Solmssen, Helv. Chim. Acta 20 (1937) 682. 40. Cf. Pregl-Roth, Die quantitative Mikroanalyse, Vienna, 1947. 41. Cf. e.g. G. Mackinney, /. Am. Chem. Soc. 56 (1934) 4^8. — H. Waldmann and E. Brandenberger, Z. Krist. 82 (1932) 77. CHAPTER VII Relationships between the colour and constitution of carotenoids It has already been stressed that the yellow to violet colour of carotenoids is one of their most important characteristics. It is thus not surprising that repeated attempts have been made to elucidate the relationships between the colour and constitution of carotenoids and to employ absorption spectra for the characterisation and identification of polyene pigments. Many advances in this field have been made during the last 20 years and it is possible to-day to draw definite conclusions regarding the constitution of a carotenoid from its absorption spectrum. Conversely, certain changes in the spectrum can be predicted from a given change in structure^. Although carotenoids possess a relatively complex structure, the absorption spectra of these pigments are comparatively simple in character. In the visible region the spectrum usually consists of three, or occasionally four, absorption maxima and the position of the maxima is related in a relatively simple manner to the constitution of the pigments. In the ultra-violet region, the relationships between constitution and spectral properties are more complicated and it is 2W0 2500 3000 3500 UOOO 4500 Fig. 3. Light absorption of xanthophyll in hexane solution 5000 XinS References p. sg. 54 RELATIONSHIPS BETWEEN COLOUR AND CONSTITUTION VII not yet possible at the present time to predict the ultra-violet absorption of a carotenoid from its structure. As an example of a typical carotenoid spectrum the absorption curve of xanthophyll is shown above. Most natural carotenoids, and also their degradation products such as ^-apo-2-carotenal, /5-apo-2-carotenol, ^S-citraurin, /5-carotenone, etc., exhibit absorption curves similar to the one shown here. Investigations by different workers have shown that the following empirical relationships exist between the constitution and absorption spectrum of polyenes : 1. The addition of a conjugated double bond without other changes in structure results in a displacement of the visible absorption bands towards longer wavelengths by 20-22 m^ (in carbon disulphide solution). The removal of a conjugated double bond has the opposite effect. Example: Crocetin, 7 conjugated ethylenic bonds, longest wavelength maximum in CSj at 482 mjx. Bixin, 9 conjugated ethylenic bonds, longest wavelength maximum in CSj at 523.5 m/i. 2. If an ethylenic bond in a six-membered ring is moved from a conjugated to an isolated position the maxima are displaced by 9-1 1 m[x towards shorter wavelengths. Examples: y3-Carotene, 11 conjugated ethylenic bonds, longest wavelength maximum in CSg at 520 m/z. a-Carotene, 10 conjugated and 1 isolated ethylenic bond, longest wavelength maximum in CSj at 509 m//. Zeaxanthin, 11 conjugated ethylenic bonds, longest wavelength maximum in CSg at 517 vsif.1. Xanthophyll, 10 conjugated and 1 isolated ethylenic bond, longest wavelength maximum in CSg at 508 m/t. 3. If a terminal conjugated double bond is replaced by an epoxide group, the maxima are displaced by 6-9 m^ towards the blue end of the spectrum, Examples: a-Carotene, 10 conjugated and 1 isolated ethylenic bond, longest wavelength maximum in CSj at 509 m/<. a-Carotene epoxide, 9 conjugated and 1 isolated ethylenic bond and 1 epoxide group, longest wavelength maximum in CSj at 503 m/^. Xanthophyll, 10 conjugated and 1 isolated ethylenic bond, longest wavelength maximum in CSj at 508 m//. Xanthophyll epoxide, 9 conjugated and 1 isolated double bond and 1 epoxide group, longest wavelength maximum in CSj at 501.5 m^. /3-Carotene, 11 conjugated ethylenic bonds, longest wavelength maximum in CSg at 520 Ta.fl. References p. 59. RELATIONSHIPS BETWEEN COLOUR AND CONSTITUTION 55 /9-Carotene mono-epoxide, 10 conjugated double bonds and 1 epoxide group, longest wavelength maximum in CSg at 511 m/x. Conversion, of a mono-epoxide to a di-epoxide results in a further displace- ment of the absorption maxima by 6-9 mij, towards shorter wavelengths. 4. Conversion of a carotenoid mono-epoxide to the isomeric furanoid oxide results in a displacement of the absorption maxima towards the blue end of the spectrum. For the longest wavelength bands this displacement amounts to 19-22 niju. Examples: ^-Carotene mono-epoxide Mutatochrome a-Carotene mono-epoxide Flavochrome Capsanthin mono-epoxide Capsochrome longest wavelength maximum in CSj at 511 m//. longest wavelength maximum in CSg at 489 m/i. longest wavelength maximum in CSg at 503 m^. longest wavelength maximum in CSj at 482 m/n. longest wavelength maximum in CSg at 534 mfi. longest wavelength maximum in CSj at 515 m^. Conversion of a di-epoxide into the di-furanoid isomer results in a hypso- chromic displacement approximately twice as great, i.e. ca 40 mfz. 5. Some carotenoids have a completely (e.g. lycopene) or partly (e.g. y-carotene) open-chain structure. If such an open chain undergoes one ring- closure, the absorption maxima are displaced by 4-5 m^ towards the blue end of the spectrum. If both ends of the chain undergo ring closure, the longest wavelength maximum is displaced by ca. 10 m/^. Examples: y-Carotene contains 11 conjugated and 1 isolated double bond. Taking ^-carotene (11 conjugated double bonds), longest wavelength maximum in CSg at 520 m/j., as a basis, the position of the longest wavelength absorption maximum of y-carotene is calculated to be 529-530 m/i. The observed longest wavelength maximum of y-carotene is at 533.5 mfi. Lycopene, contains 11 conjugated and 2 isolated double bonds. Taking /5-carotene as a basis, the position of the longest wavelength maximum is calculated to be at 538-540 m/.i. The observed longest wavelength maximum of lycopene lies at 548 m/t. 6. Introduction of a hydroxyl group only results in a small hypsochromic displacement (1-2 m/^). Examples: /3-Carotene longest wavelength maximum in CSj at 520 m/i. a-Carotene longest wavelength maximum in CSj at 509 m/x. Lycopene longest wavelength maximum in CSj at 548 mfi. Zeaxanthin(Dihydroxy-/3-carotene)longest wavelength maximum in CS2 at 517 m/j.. Xanthophyll (Dihydroxy-a-carotene) longest wavelength maximum in CSg at 508 m/n. Lycophyll (Dihydroxy-lycopene) longest wavelength maximum in CSg at 546 rxifi. References p. 5g. 56 RELATIONSHIPS BETWEEN COLOUR AND CONSTITUTION VII 7. Carbonyl groups (ketone, aldehyde or carboxyl groups) conjugated with the system of conjugated double bonds have a pronounced effect on the ab- sorption spectrum, and produce bathochromic displacements, the magnitude of which varies from case to case. If the introduction of the carbonyl group, involves the opening of a ring, the effects of the two changes are, of course superimposed. Introduction of a second conjugated carbonyl group has a smaller effect on the position of the absorption maxima than the introduction of the first carbonyl group. Examples: /3-Apo-2-carotenal. The system of 9 conjugated ethylenic bonds should exhibit an absorption maximum at about 480-485 m^u. The observed longest wavelength absorption maximum lies at 525 m/i and the difference of 40-45 m// is to be ascribed to the carbonyl group. Capsanthin. The system of 10 conjugated ethylenic bonds should exhibit an ab- sorption maximum at 500-505 m/^. The longest wavelength absorption maximum actually observed lies at 542 m/i and the difference is to be ascribed to the carbonyl group. 8. Cis-trans configuration also has a definite, though small, influence on the position of the absorption maxima. Thus, the longest wavelength maximum of a compound containing one c/s-ethylenic bond, is displaced by ca. 3-4 m/z towards shorter wavelengths as compared with the isomer with complete ^mws-configuration. Examples: Stable (/rans-) bixin longest wavelength maximum in CSg at 526.5 m^u. Labile (cf5-) bixin longest wavelength maximum in CSg at 523.5 m^a. Stable {trans-) crocetin .... longest wavelength maximum in CSj at 463 vci/x. Labile {cis-) crocetin longest wavelength maximum in CSg at 458 m^. After dealing with the effect of different atomic groups on the position of the absorption maxima, the influence of the solvent must be briefly discussed. Experiments have shown that the wavelength locations and fine structure of the absorption maxima are considerably dependent on the solvent. The solvent also has some influence on the absorption coefficients. The interaction between polar solvent such as alcohol and carotenoids with carboxyl groups, (e.g. capsanthin) is further described below. The table below shows that the absorption maxima of most carotenoids are displaced by about 30-40 m^ towards shorter wavelengths in hexane and alcohol as compared with carbon disulphide. In chloroform, the hypsochromic displacement amounts to about 24 m//. The differences in the positions of the maxima in hexane and carbon disulphide increase with the wavelengths at which the carotenoids absorb. In carotenoids containing carbonyl groups (e.g. capsanthin, /5-apo-2- References p. 59. RELATIONSHIPS BETWEEN COLOUR AND CONSTITUTION 57 TABLE 12 DISPLACEMENT OF THE POSITION OF MAXIMA BY DIFFERENT SOLVENTS* Maxima in Pigment carbon disulphide Hexane Ethanol Chloroform a-Carotene 509 Tcifi 478 m/ii 485 m// Xanthophyll 508 m/i 476 m/Li 479 m/n 487 m^u /9-Carotene 520 m/ii 482 nifi 497 m/j, Cryptoxanthin 519 m/z 484 m/i 486 m/i 497 m/x Zeaxanthin 517 m/j, 482.5 m/n 483 m/i 495 m^ y-Carotene 533.5 m// 494 m// 508.5 myu Rubixanthin 533 mfi 494 rrifi 496 m^ 509 m/t Lycopene 548 m/< 506 m/z 517 m/i * Only the longest wavelength maxima are given. Some of the data in the third column were obtained using petroleum ether instead of hexane as a solvent. carotenal, etc.), relationships are more complicated, probably owing to the interaction which can occur between the pigment and certain solvents, e.g. alcohol. Thus, the spectrum of capsanthin in alcohol is completely blurred. The same behaviour is exhibited by y5-apo-2-carotenal and other polyene ketones containing carbonyl groups conjugated with the system of ethylenic bonds. If the conjugation between the ethylenic bonds and the carbonyl group is broken, however, the absorption maxima are as sharp as usual^. The empirical relationships here described between the constitution of carotenoids and their light absorption properties have general validity. The exceptions which are occasionally encountered may often be ascribed to a lack of stability of the pigments involved and do not detract from the great value of spectroscopy in carotenoid research. The theoretical interpretation of the visible and ultraviolet absorption spectra of natural and synthetic polyenes has also attracted considerable attention within recent years. The absorption of light in this region of the spectrum is thought to give rise to electronic oscillations along the axis of the polyene chain, and it can be predicted, on this basis, that the wavelengths and intensities of the maxima will increase with the number of conjugated ethylenic bonds^. References p. sg. 58 RELATIONSHIPS BETWEEN COLOUR AND CONSTITUTION VII TABLE 13 ABSORPTION SPECTRA OF NATURAL CAROTENOIDS Formula Absorption maxima in CS2 Number of con- jugated ethylenic bonds Number of hydroxy] groups Number of carbonyl ist band 2nd band 3rd band groups (Violerythrin) * 625 576 540 ? ? ? Torularhodin ^37^48^2 582 541 502 12 0 1 (Actinioerythrin) * 574 533 495 ? ? ? Rhodoviolascin *^42^60^2 573.5 534 496 13 0 0 Bacterioruberin 571 532 498 ? ? Oscillaxanthin • 568 528 494 ? ? Torulin 565 525 491 ? ? Rhodoxanthin C40H50O2 564 525 491 12 0 2 Celaxanthin C,oH3eO(-H,?) 562 521 487 13? Rhodovibrin 556 517 ? Rhodopurpurin 550 511 479 ? Astacene ^-40^4804 ca. 550-450, Maximum 510 11 0 4 Astaxanthin ^40^52^4 ? ? ? 11 2 2 Lycopene ^40^56 548 507.5 477 13 0 0 Rhodopin C4oH530(-H2?) 547 508 478 12 1 ? Lycoxanthin QoHseO 547 507 473 13 1 0 Aphanizophyll 547 506 474 ? ? ? Lycophyll ^40^56*^2 546 506 472 13 2 0 Myxoxanthophyll ^40"^56^7 544 508 479 10 6 1 Capsanthin *-^40^58^3 542 503 10 2 1 Capsorubin ^40^60^4 541 503 468 9 2 2 Eschscholtzxanthin C,,U,fi,[±U,) 536 502 475 12 2 0 y-Carotene ^40^^56 533.5 496 463 12 0 0 Rubixanthin C40H56O 533 494 461 12 1 0 Aphanin * * * C40H54O 533 494 11 0 1 Aphanicin * * * 533 494 ? ? ? Gazaniaxanthin C,oH,eO(±H2) 531 494.5 461 11? 1 0 /3-Citraurin ^30"^40^2 525 490 457 9 1 1 Bixin (labile) ^25^30^4 523.5 489 457 9 0 2 yS-Carotene C40H56 520 485 450 11 0 0 Echinenone C,„H,eO(±H,) (520) 488 (450) ? 0? 1? Cryptoxanthin QqHsbO 519 483 452 11 1 0 Pectenoxanthin C4oH3403(±H,) 518 486 452 11 2 ? Zeaxanthin QoHseOj 517 482 450 11 2 0 Cynthiaxanthin 517 483 451 ? ? ? Leprotin ^40^54 517 479 447 12 0 0 It is not certain whether this compound belongs to the carotenoid series. ** The data given refer to the esterified pigment. *** The pigments exhibit wide maxima (cf. p. 302 and 305). RELATIONSHIPS B ETW E EN COLOU R AND CONSTITUTION 59 TABLE 13 (continued) Formula Absorption maxima in CS. Number of con- jugated ethylenic bonds Number of hydroxy] groups Number of carbonyl ist band 2nd band 3rd band groups Sulcatoxanthin C40H52O8 516 482 450 ? ? Petaloxanthin C,,U,,0,{ + U,}) 514.5 481 ? 2? Haematoxanthin One band, max. 513 ? ? Fucoxanthin QoHseOg 510 477 445 10? ? Antheraxanthin C40H56O3 510 478 445 10 2 0 a-Carotene ^40^56 509 477 11 0 0 Xanthophyll Qo^^seOa 508 475 445 11 2 0 Pentaxanthin QoH3e05(±H,) 506 474 444 ? 3? ? Rubichrome QqHssOo 506 476 11 1 0 a-Carotene epoxide C40H56O 503 471 10 0 0 Flavorhodin 502 472 ? ? ? Xanthophyll epoxide ^40^56^3 501.5 472 10 2 0 Taraxanthin C40H56O4 501 469 440 ? ? ? Violaxanthin ^40^5604 500.5 469 440 9 2 0 Trollixanthin C4„H5sO,(?) 501 473 ? 3? ? Prolycopene Qo"^56 500.5 469.5 11 0 0 Mytiloxanthin One band, m a.x.500 ? ? Sarcinaxanthin 499 466.5 436 ? ? Sarcinin * ? ? ? ? ? Glycymerin One ban d , m ax. 495 ? ? Pro-y-carotene C40H56 493.5 460.5 12 0 0 Flavacin 490 457 424 ? ? ? Mutatochrome C40H56O 489.5 459 10 0 0 (Citroxanthin) Myxoxanthin C40H54O One ban d , m ax. 488 12 0 1 Azafrin C27H38O4 486 457 7 2 1 Crocetin (stable) C20H24O4 482 453 7 0 2 Chrysanthema- ^40^56^3 480 451 10 2 0 xanthin Flavoxanthin ^40^56^3 478 447.5 420 10 2 0 Auroxanthin C-io^i^Oi 454 423 9 2 0 * In petroleum ether, the absorption maxima are located at 469 and 440 mu. REFERENCES 1. K. W. Hausser and A. Sm.\kula, Angew. Chem. 4j (1934) ^57- — J^^- -^- Morton, The Application of Absorption Spectra to the Study of Vitamins, Hormones and Co-enzymes, London, 1942. — L. Zechmeister, Chem. Reviews 34 (1944) 267. — . P. Karrer, Die bisher bekannten natiirlichen Carotinoide und ihre Absorptionsspektren, Vj so hr. Naturf. Ges., Zurich, 1945. — E. A. Braude, Ann. Reports Chem. Soc. London 42 (1946) 105. 2. G. N. Lewis and M. Calvin, Chem. Reviews 25 (1939) 73. — - L. Pauling, Proc. Nat. Acad. Sci. 25 (1939) 577. — R. S. Mulliken, /. Chem. Physics 7 (1939) 121. — N. S. Bayliss, ibid. 16 (1948) 287. — W. Kuhn, Helv. Chim. Acta 31 (1948) 1780. — ■ E. A. Braude, Nature 155 (1945) 753; /. Chem. Soc. 1950, 379. CHAPTER VIII The synthesis of carotenoids In spite of many attempts, no total synthesis of a natural carotenoid has been achieved up to the present time. Kuhn and his co-workers^ have synthe- sized many carotenoid-like diphenylpolyenes and polyene dicarboxylic acids and thus provided valuable material for the comparison of structure and colour in polyenes. Karrer and his co-workers have synthesized the perhydro- derivatives of three natural carotenoids and thus established the constitution of the natural pigments. The compounds involved are perhydro-lycopene^, perhydro-norbixin^ and perhydro-crocetin*. The first conversion of one natural carotenoid into another was achieved by Karrer and Solmssen^ who reduced dihydrorhodoxanthin to zeaxanthin by means of aluminium zsopropoxide and zsopropyl alcohol (p. 182). Partial syntheses of natural carotenoids are also represented by the oxidative degra- dation of zeaxanthin and xanthophyll to /?-citraurin* and by the conversion of lycopene into norbixin^. By the action of N-bromsuccinimide on lycopene, Karrer and Rutschmann obtained dehydrolycopene, a carotenoid pigment with 15 conjugated double bonds (cf. p. 121). Recently, Karrer and Jucker" have succeeded in converting natural carotenoids containing isolated double bonds into other naturally occurring pigments. Thus, a-carotene can be converted into /5-carotene by the action of sodium ethoxide at elevated temperatures, and similarly, xanthophyll can be converted into zeaxanthin. These conversions are of interest in showing that the isolated double bonds can be brought into conjugation. Thus, until recently, only very few natural carotenoids had been partially synthesized. Within the last few years, however, Karrer and Jucker^ succeeded in preparing about 20 carotenoids by the introduction of oxygen into different carotenoid pigments by means of monoperphthalic acid. Some of these carotenoids were known to occur in nature, but their constitution had previously been unknown. The addition of oxygen by means of perbenzoic acid was employed by * Cf. p. 184. Later L. Zechmeister and L. v. Cholnoky obtained |3-citraurin by the hydrol3rtic fission of capsanthin, cf. p. 248. References p. 64-65. SYNTHESIS 6i PuMMERER and co-workers^ for the determination of the number of double bonds in polyene molecules (cf. p. 44). By the action of perbenzoic acid on /S-carotene, Karrer and Walker^" prepared /^-carotene oxide which has been shown by Karrer and Jucker^^ to be identical with mutatochrome. Numerous investigations^^ by the two last-named authors have also shown that by using small quantities of monoperphthalic acid it is possible to oxidise individual double bonds in a carotenoid molecule. Well-defined crystalline compounds are obtained, which from their method of formation and their properties are regarded as i : 2-epoxides. Their properties show that only the double bonds in CH« CHo \V c CHj C-CH=CH- IV CH. C/ 0 CHq CHo \/ c /\ CH=CH-C CHjs II I C CH, /\/ HoC CHg CH« CHo \V C /\ CH, C-CH=CH- I \)o CH, cr •CH=CH-C CH, CH^ H3C CHj CHj C \ CH^ CHj CH, II the /5-ionone rings have been oxidised, and mono-epoxides or di-epoxides are obtained, depending on the number of /5-ionone rings in the carotenoid mole- cule. No example of the oxidation of the isolated double bond in an a-ionone ring has so far been encountered. Thus, a mono-epoxide I and a di-epoxide II have been obtained from /5-carotene, whereas a-carotene only yields a mono- epoxide. CH, CH, CH, CH, C CH3 /\ I CH. .C-CH=CH-C=CH- I II X-CH C-CH, CH, perphthalic CH- C-CH=CH-C=CH- acid X-CH C/" CHjj Carotenoid, X = H or OH CHj CH3 Epoxide CHjI 0 CH3 Furanoid oxide References p. 64-63. 62 SYNTHESIS VIII The most characteristic property of the epoxides is their extreme sensitivity to dilute mineral acids. Even traces of hydrogen chloride such as are present in chloroform after standing, are sufficient to rupture the epoxide ring^^. The isomeric furanoid oxide is obtained, together with the original carotenoid which is formed as by-product by the loss of oxygen*. The fact that the oxygen atom of these epoxides is readily lost, suggests that it is bound in an unusual way not well represented by the epoxide formu- lation. Karrer^^ has suggested a polar structure which explains the facile conversion into the furanoid oxide as well as the ready loss of oxygen. C CH3 ' / \( + )2 3 I CH, ^C-CH = CH-C=CH- X-CH C— 0' ^ CH, CH, CH2I 0 CH2I 0 CH3 III CH3 IV According to this formulation, hydrogen chloride adds to the polar oxide I with the formation of II. This is transformed into III, converted in turn to the furanoid oxide IV by the loss of hydrogen chloride. The conversion of the polar oxide I into the furanoid oxide IV can also be interpreted in the following way: an electron pair shared by carbon atoms 2 and 3 (formula I) is displaced towards carbon atom i under the influence of the positive charge, and simultaneously the oxygen atom and its electron pair adds to carbon atom 3. The investigations of Karrer and Jucker^^ showed that some natural carotenoids, the constitution of which had not previously been established, were the furanoid oxides of known carotenoids. Thus fiavoxanthin proved to be identical with furanoid xanthophyll oxide, and auroxanthin with furanoid zeaxanthin dioxide. The action of alkyl magnesium salts on carotenoid epoxides gives rise to the same products as the reaction with hydrogen chloride, namely the furanoid * This applies to all partially synthetic epoxides so far prepared, cf. table 14. References p. 64-65. SYNTHESIS 63 oxide, together with the parent carotenoid formed as a by-product by the loss of oxygen^^. TABLE 14 PARTIALLY SYNTHETIC CAROTENOID EPOXIDES AND THEIR PROPERTIES Absorption Colour Epoxide maximum M.P. reaction with in CS, concentrated HCl a-Carotene niono-epoxide 503 471 m/Li 175° very faint, unstable Xanthophyll mono-epoxide 501.5 472 nifi 192° blue, fairly stable /3-Carotene mono-epoxide 511 479 m/i 160° faint blue, unstable Cryptoxanthin mono-epoxide 512 479 m/n 154° blue, unstable Zeaxanthin mono-epoxide * 510 478 m/Li 205° blue, unstable Rubixanthin mono-epoxide 526 491 m/< 171° blue, stable Capsanthin mono-epoxide 534 499 m/x 189° blue, unstable /9-Carotene di-epoxide 502 470 m/j. 184° deep blue, stable Cryptoxanthin di-epoxide 503 473 rcifi 194° deep blue, stable Zeaxanthiin di-epoxide 500 469 TCifj, 200° deep blue, stable Identical with Antheraxanthin. ** Identical with Violaxanthin. With regard to the elucidation of the constitution of the epoxides and the furanoid oxides, references should be made to the original literature^^. TABLE 15 PARTIALLY SYNTHETIC FURANOID CAROTENOID OXIDES AND THEIR PROPERTIES Absorption Colour Oxide maximum M.P. reaction with in CSo concentrated HCl Flavochrome 482 451 m/i 189° very faint, unstable Flavoxanthin 479 449 m^M 180° blue, fairly stable Chrysanthemaxanthin 479 449 m/t 185° blue, fairly stable Mutatochrome 489 459 m/Li 164° faint blue, unstable Cryptoflavin 490 459 mfi 171° blue, unstable Mutatoxanthin 488 459 m/j. 177° blue, unstable Rubichrome 506 476 m/^ 154° blue, stable Capsochrome 515 482 m/< 195° blue, unstable Aurochrome 457 426 mn 185° deep blue, stable Cryptochrome 456 424 m/t ? deep blue, stable Auroxanthin 454 423 m/< 203° deep blue, stable Luteochrome 482 451 m/< 176° deep blue, stable References p. 64—63. 64 SYNTHESIS VIII In the investigation of carotenoid epoxides and their isomeric furanoid oxides, the spectral properties of these compounds are of much importance. The following are some empirically recognised regularities: Conversion of a carotenoid pigment into a mono-epoxide results in a displacement in the absorption bands towards the violet. The displacement of the longest wavelength band amounts, on the average, to 8 m// in carbon disulphide. The formation of a di-epoxide results in a displacement of the bands by about 17 m/n. A rather larger displacement (about 21 mpi) of the absorption bands towards shorter wavelength accompanies the change of a mono-epoxide into the isomeric furanoid oxide. For a di-epoxide, the difference is about twice as great. These regularities allow a fairly certain prediction of the ab- soiption spectra of carotenoid epoxides and their furanoid isomers. Of the carotenoid epoxides and furanoid oxides so far prepared, the following have up to now been found in nature : a-Carotene mono-epoxide Flavochrome (/5-Carotene mono-epoxide)* Citroxanthin = Mutatochrome Zeaxanthin mono-epoxide = Antheraxanthin Zeaxanthin di-epoxide = Violaxanthin Xanthophyll mono-epoxide Flavoxanthin Chrysanthemaxanthin Auroxanthin Rubichrome Trollixanthin", a pigment recently isolated from the blossoms of trollius europaeus, also possesses an epoxide structure. The wide distribution of carotenoid epoxides in plants raises the question as to their physiological significance. No definite answer can be given, but the ready loss of oxygen suggests the possibility that these epoxides play a part in biological oxidation processes in the plant. Further investigations are required to elucidate this problem. REFERENCES 1. Cf. R. KuHN and co-workers, Angew. Chem. 50 (1937) 7°3; ^^^- ^9 (^93^) I757. 19791 71 (1938) 1889. 2. P. Karrer, a. Helfenstein and R. Widmer, Helv. Chim. Acta 11 (1928) 1201. 3. P. Karrer and co-workers, Helv. Chim. Acta 15 (1932) 1218, 1399. * /3-Carotene mono-epoxide has not yet been found in nature, but as mutatochrome (^ citroxanthin) is a natural pigment and almost certainly formed from /3-carotene mono- epoxide, it is very probable that the latter also occurs in plants. REFERENCES 65 4. P. Karrer, F. Benz and M. Stoll, Helv. Chim. Acta 16 (1933) 297. 5. P. Karrer and U. Solmssen, Helv. Chim. Acta 18 (1935) 477. 6. R. KuHN and C. Grundmann, Ber. 65 (1932) 898, 1880. 7. P. Karrer and E. Jucker, Helv. Chim. Acta 30 (1947) 266. 8. P. Karrer and E. Jucker and co-workers, Helv. Chim. Acta 28 (1945) 300, 427, 471, 474, 717, 1143, 1146, 1156; 30 (1947) 531. 9. R. Pummerer and co-workers, Ber. 61 (1928) 1099; 62 (1929) 141 1. 10. H. V. Euler, p. Karrer and O. Walker, Helv. Chim. Acta 15 (1932) 1507. 11. P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 427. 12. P. Karrer and E. Jucker and co-workers, Helv. Chim,. Acta 28 (1945) 300, 427, 471, 474, 717, 1143. 1146, 1156; 30 (1947) 531. 13. P. Karrer, Helv. Chim. Acta 28 (1945) 474. 14. P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 300. 15. P. Karrer, E. Jucker and K. Steinlin, Helv. Chim. Acta 28 (1945) 233. 16. P. Karrer and E. Jucker, Helv. Chim. Acta 28 (1945) 300. 17. P. Karrer and E. Jucker, Helv. Chim. Acta 2g (1946) 1539. Carotenoids 5 CHAPTERIX The distribution of carotenoids in nature Since the discovery of carotene by Wackenroder in 183 1, the distribution of carotenoids in nature has been much investigated. Extensive studies have shown that polyene pigments are present in the whole of the vegetable and animal kingdoms. In the following sections the occurrence of polyene pigments is summarised in tabular form. The following arrangement is used: A. Carotenoids in plants : 1. Phanerogams (a) Unexposed parts of plants (b) Exposed parts of plants (c) Blossoms (d) Fruit 2. Cryptogams B. Carotenoids in amimals: 1. Invertebrates (a) Arthropods (b) Molluscs (c) Echinoderms (d) Worms (e) Coelenterates and sponges (f) Chordata 2. Vertebrates (a) Mammals (b) Birds (c) Fish (d) Amphibia (e) Reptiles (f) Miscellaneous It should be mentioned that only limited significance can be attached to some of the older investigations which were carried out without the use of Tswett's chromatography. In most cases, they merely indicate that the A CAROTENOIDS IN PLANTS 67 presence of carotenoids may be assumed in the materials examined. Definite conclusions regarding the nature of the individual pigments can only be drawn after repeated chromatographic analysis. For this reason no attempt has been made to identify the pigments reported in these older investigations. A. CAROTENOIDS IN PLANTS*' ** I. PHANEROGAMS a) Carotenoids in unexposed parts of plants TABLE 16 (References see p. 99-107) CAROTENOIDS IN ROOTS Beta vulgaris''-. Escobedia scabrifolia^: Azafrin. Brassica campestris^. Ipomoea Batatas': a-Carotene, /5-caro- Brassica Rapa^. tene. Cetastrus scandens^: ^-Carotene {}). Jaundiced potatoes^: Taraxanthin or Daucus Carota^: a-Carotene, /3-carotene, violaxanthin, xanthophyll, a-caro- y-carotene, a hydrocarbon of un- tene(?). known constitution (absorption maxi- Sweet potatoes^: Carotene. ma in CSgi 482, 453 m/^), a second Pastinaca sativa^'^. hydrocarbon of unknown constitution (absorption maxima in CSg: 499, 469 mix), xanthophyll. b) Carotenoids in exposed parts of plants Green parts of plants It has long been known that besides chlorophyll, all green parts of plants contain carotenoids^. These are mainly /9-carotene, xanthophyll and, according to the most recent investigations^, xanthophyll epoxide. Small amounts of a-carotene are also usually present. The carotenoids are found together with chlorophyll in the chromatophores and are present in either an amorphous or crystalline state. The ratio of carotene to xanthophyll in green leaves has been investigated by WiLLSTATTER and Stoll^ and by Karrer and co-workers*. Whereas the former found a ratio of 1.7, the latter obtained values tending to unity. In all the older investigations the fact that the so-called xanthophyll fraction consists * The following abreviations are employed in the tables in this chapter : ■*" = isolated in the crystalline state ; ■*"■*" = definitely present; "^+"'" = probably present. ** The literature references for the tables in this chapter will be found at the end of the general part (p. 99-107). References p. 108. 68 DISTRIBUTION IN NATURE IX of tie.'o main pigments, xanthophyll itself and xanthophyll epoxide, was neglected. Literature references regarding the occurrence of carotenoids in green plants, especially green leaves, are so numerous that no attempt will be made to summarise them. Only the most important investigations in this field can, therefore, be mentioned^. Non-green leaves Numerous investigations have been carried out concerning the pigments which are responsible for the yellow colour of etiolated leaves^. However, most of these studies belong to the early period of carotenoid research. More recent investigations^ show that besides water-soluble pigments, carotenoids, especially xanthophyll, are present in etiolated leaves. Our knowledge concerning the pigments of yellow leaves (aurea varieties) is rather deficient. The last investigation of these pigments was made by WiLLSTATTER and Stoll^ and showed the presence of carotenoids and of water-soluble pigments. As the quantity of carotenoids was very small, however it appears doubtful whether they are, in fact, responsible for the yellow colour of the leaves. As a result of further studies, our knowledge regarding the pigments of yellow attt'umn leaves is somewhat better, and it has been possible to recognise different stages in the rather complicated pigment metabolism. The carotenoids (possibly also the anthocyanins'^) remain in the leaves after the degradation of the chlorophyll in the autumn and produce the well-known, striking colour- ations. Gradually the polyene pigments are ako degraded and carotene seems to be decomposed more rapidly than xa:nthophyll. In the last phases of the necrobiosis, the well-known brown pigments are produced, to which fallen leaves owe their brown colouration. These brown pigments appear to be oxidation and decomposition products; they are soluble in water and produce dark yellow to brown colourations with alkalis. An important question is whether only carotenoids already present in the green leaf are involved in the course of necrobiosis or whether new pigments are formed. According to Willstatter and Stoll^, the total quantity of pigments in autumnal leaves is about equal to that in green leaves. These findings are in agreement with the investigations of Goerrig**. It is interesting that whereas the amount of carotene decreases in the course of necrobiosis, the quantity of xanthoph^/ll is said to increase^". The yellow colouration of dead leaves is due, according to Tswett^^ to epiphasic carotenoid pigments which, in contrast to carotene, can be adsorbed on calcium carbonate from petroleum ether solution. Tswett named these carotenoids "autumn xanthophylls". According to Kuhn References p. io8. A CAROTENOIDS IN PLANTS 69 and Brockmann^", who confirmed the disappearance of carotene in the course of necrobiosis, these autumn xanthophylls are phytoxanthin esters, which are formed only in the autumn by the esterification of free phytoxanthins. The matter has not been completely elucidated, however, as with one exception no crystalline pigments could be obtained^^. Karrer and Walker isolated the carotenoids by precipitation in the form of sparingly soluble iodides and regeneration with sodium thiosulphate^*. The main results of their investigations are as follows: as the leaves decay, the content of carotene and xanthophyll decreases, but the former decreases more rapidly. Xanthophyll can still be isolated in the crystalline state long after no carotene can be detected. Eventually the xanthophyll also disappears com- pletely. The autumn xanthophylls observed by Tswett were also found. These compounds first appear at the beginning of necrobiosis and their con- centration steadily increases at the expense of the carotenoids, so that they become mainly responsible for the colouration of the leaves shortly before the postmortal phase. Nothing definite is yet known regarding the nature 'of these pigments, but they appear to consist of oxidation and degradation products of xanthophyll. Other pigments which absorb more strongly in the ultraviolet are also observed. Further investigations of these autumn xanthophylls would be of interest. The pigments of red winter leaves have also been much studied. It has been found that some leaves owe their red colouration to anthocyanins, while others contain rhodoxanthin. c) Carotenoids in Blossoms Nature has been lavish in the distribution of carotenoids in blossoms. About 35 different carotenoids, i.e. about half of all the known polyene pigments, have been isolated from blossoms up to the present time. This variety is the more remarkable since nothing is at present known regarding the function of carotenoids in blossoms. The following is a summary of the blossoms examined in this respect up to the end of 1948. Many of the older investigations, e.g. those of Courchet, Tammes and Van Wisselingh were carried out using inadequate techniques and only limited significance can be attached to them. References p. 108. 70 DISTRIBUTION IN NATURE TABLE 17 (References see p. 99-107) CAROTENOIDS IN BLOSSOMS (i). Monocotyledoneae IX Gramineae Hungarian wheat blossoms*^'': Xantho- phyll, carotene. Bromeliaceae Tillandsia splendens'^^. Liliaceae Allium siculuw}^. Aloe vera^*: Rhodoxanthin+"'"+. Asphodelus cerasiferus^^ . Bulbina semibarbata^^ . Fritillaria unperialis^^' ^*. Hemerocallis Middendorffii^^. Knipliofia aloides'^^. Lilium bulbiferum'^'' . Lilium bulbiferum ssp. croceum'^^. Liliiifn candidum^^: Antheraxan- thin++(?), violaxanthin++, cis- antheraxanthin . Lilium tigrinum (anther)^*: Anthera- xanthin+, capsanthin+. Tulipa Gesneriana^^. Tulipa horiensis^^. Tulips, yellow variety^": Viola- xanthin++. Uvularia grandiflora^^. Amaryllidaceae Clivia niiniata^^. Narcissus poeticus^^' ^®' ^^. Narcissus Pseudonarcissus^^: Xantho- phyll++. Narcissus Tazetia^^. Iridaceae Crocus luteus^^: Crocetin+. Crocus neapolitanus^^: Crocetin+. Crocus reticulatus^^' ": Crocetin++. Crocus sativus^^: Crocetin+. Crocus variegatus^^' ^': Crocetin++. Iris Pseudacorus^^' ^*: /5-Carotene, xanthophyll, violaxanthin. Saffron (Crocus sativiis)^^: a-Caro- tene++, ;8-carotene++, y-carotene++, lycopene++, zeaxanthin++, cro- cetin+. Tritonia aurea = Ixia crocata^^' ^''■. Crocetin++. Musaceae Strelitzia Reginae'^^' ^^. Orchidaceae Cypripedium Argus^^. Cypripediuni Boxallii^^. Cypripedium insigne^^. Dendrobium thyrsiflorum^^. Gongora galeata^^. Lycaste aromatica^^. Masdevallia V eitchiana^^ . Odontoglossum species^^. Oncidium species^^. (ii). Dicotyledoneae Proteaceae Grevillea robusta^^: /3-Carotene, cryptoxanthin+, xanthophyll+, carotenoid of unknown constitu- tion+ (cf. p. 340). Nymphaceae Nuphar luteuni^^. Ranunculaceae Adonis vernalis^^. Caltha palustris^^' *^^: Xanthophyll+' xanthophyllepoxide++, troUixan- thin++, j5-carotene++, a-carotene++. Eranthis hiemalis^^' ^®. Ranunculus acer (R. Steveni ?)^^'^^'^°: Violaxanthin++, xanthophyll+, flavoxanthin+, chrysanthema- xanthin+, fiavochrome+, xantho- phyll epoxide++, a-carotene epo- xide++, a-carotene++, ^-carotene++, taraxanthin+. CAROTENOIDS IN PLANTS IT- Ranunculus arvensis'^y Carotene++, xanthophyll+. Ranunculus auricomus'^^' ^^. Ranunculus Ficaria^^' ^^. Ranunculus gramineus^^. Ranunculus repens^^' ^*. Ranunculus Steveni^^: Xanthophyll+. Trollius asiaticus'^^. Trollius europaeus^^' ^^■. /3-Carotene++, xanthophyll+, xanthophyll epo- xide++, trollixanthin+, epoxide of unknown constitution++. Berberidaceae Epimediuni tnacranthuni^^. Magnoliaceae Liriodendron tulipifera^^. Papaveraceae Chelidonium majus'^^' ^*. Corydalis lutea^^. Eschscholtzia californica^^' ^^' ^®: Eschscholtzxanthin+. Glaucium luteuni^''. Meconopsis cambrica'^^. Cruciferae Alyssum saxatile^^. Cheiranthus Cheiri'^^' ^^. CheiranthusSenoner i^^'-'Kanthophyll^. Erysimum Perofskianum^^. I satis tinctoria^^ . Nasturtium species^^. Raphanus Raphanistrum^^ . Sinapis officinalis^'^: Carotene++, violaxanthin+. Sisymbrium Sophia^^. Saxifragaceae Ribes aureuni^^. Rosaceae Geum coccineum^^. Geum montanum^^. Kerria japonica^^' ^^' ^^' ^®' ^®' ^': Xanthophyll+, xanthophyll epo- xide"'"'", jS-carotene''"'". Potentilla erecta (TormentillaP^: ^-Carotene, xanthophyll, zeaxan- thin { ? ) , fla voxanthin { ? ) . Rosa, yellow species^*' ^^. Waldsteinia geoides^-. Leguminosae Acacia decurrens var. mollis^'^' '*<': Carotene++, xanthophyll++. Acacia discoloy^°: Carotene+, xantho- phyll+. Acacia linifolia''-'^: Carotene+, xantho- phyll+. Acacia longifolia^": Carotene+, xan- thophyll+. Colutea media^^. Cytisus sagittalis^^. Genista racemosa^^. Genista tinctorid^^. Genista tridentata^'^: a-Carotene"'", ^-carotene+, xanthophyll+. Laburnum anagyroides^^' ^^: Caro- tene++, violaxanthin++, /3-caro- tene++, xanthophyll++, xantho- phyll epoxide++. Lotus corniculatus*^: a-Carotene++, |S-carotene++, xanthophyll++, xan- thophyll epoxide++, violaxan- thin++, carotenoid of unknown constitution++. Melilotus officinalis^^. Sarothamnus scoparius*^' ^": a-Caro- tene++, ^-carotene++, xantho- phyll+, xanthophyll epoxide+, chrysanthemaxanthin+, fiavo- xanthin+. Spartiuni junceutn^^. Thermopsis lanceolata^^. Ulex europaeus^^' *^: a-Carotene"'', ^-carotene+, violaxanthin+, tara- xanthin+, xanthophyll isonier'"(?) and a carotenoid of unknown con- stitution with spectral properties similar to those of flavoxanthin. Ulex Gfl//u*^:a-Carotene+,/3-carotene"'", violaxanthin+, taraxanthin+, xan- thophyll isomer+(?), flavoxan- thin+++(?). Vicia, violet-blue varieties^^: Lyco- pene. Mehaceae Cedrela Toona'^^: Crocetin''". Tropaeolaceae Tropaeolum majus^^' ^^: Xantho- phyll++. 72 DISTRIBUTION IN NATURE IX Malvaceae Abutilon Darwini^^. Abuiilon megapotamicum^^. Abutilon nervosum'''^. Balsaminaceae Impatiens noli xanthin+. ': Tara- Violaceae Viola biflora^^. Viola cornuta var. Daldowie^^. Viola lutea^^' ^^. Viola odorata^^' *^. Viola tricolor'^''' **: Violaxanthin+, zeaxanthin++, fiavoxanthin++, xanthophyll+, auroxanthin+, caro- tene++. Loasaceae Loasa (Cajophova) lateritia^^. Oenotheraceae Oenothera biennis^^' ^^. Umbelliferae Ferula species^'. Primulaceae Primula acaulis^^. Primula officinalis^'^' ^^. Plumbaginaceae Armeria vulgaris''-^. Oleaceae Forsythia Fortunei^^. Forsythia viridissima^^' ^^' ^*. Jasminum Sambac^'': Crocetin+++. Nyctanthes Arbor-tristis**' **• ^O; Crocetin+. Asclepiadaceae Asclepias curassavica^^. Boraginaceae Nonnea lutea^^. Labiatae Ladanum hybriduni^^ . Solanaceae Atropa Belladonna^^. Fabiana indica^^' ^^: Crocetin+. Scrophulariaceae Calceolaria species^^. Calceolaria rugosa^^. Calceolaria scabios&folid^^. Mimulus longiflorus^^: /3-Carotene+, y-carotene++, lycopene+, crypto- xanthin++, zeaxanthin+, pro-y-ca- rotene, prolycopene^*. Mimulus moschatus^^. Verbascum species^^. Verbascum Thapsus^^: Crocetin+. Rubiaceae Manettia bicolor'^^. Cucurbitaceae Cucurbita foetidissinia^^. Cucurbita melanosperma^^ . Cucurbita Pepo^^: Carotene+, crj^pto- xanthin+, xanthophyll+, zeaxan- thin+, petaloxanthin+. Momordica Balsamina^'^. Campanulaceae Siphocampylus bicolor"^^. Compositae Arnica montana'^^' *^: Xanthophyll+, xanthophyll epoxide++, zeaxan- thin+. Aster species^^. Buphthalmum salicifolium^^. Cacalia coccinea^^. Calendula arvensis^^. Calendula officinalis^^, dark variety; Carotene+, lycopene+, xantho- phyll+, violaxanthin+, y-caro- tene+++(?); light yellow variety; lycopene absent. Chrysanthemum frutescens^^' ^*. Chrysanthemum segetum^^. Chrysanthemum^^: Xanthophyll+, xanthophyll epoxide+, chrysanthe- maxanthin+ (carotene++). Crepis species^^*. Crepis aurea*^: a-Carotene++, /5-Caro- tene++, xanthophyll++, violaxan- thin++, pigment of unknown con- stitution, absorption maxima in CSg 501, 470 m/z. CAROTENOIDS IN PLANTS 73 Dahlias (anther) i^; Lycopene+. Dimorphotheca aurantiaca^'^: Lyco- pene+. Doronicnm Colnninae^^' •^^' ^*. Doronicmn Pardalianches^^: Xanto- phyll++. Doyonicuni plantagineittn^^. Doronicuni excelsum^^. GaiUardia splendens^. Gazania rigens, a) Portuguese origin*^; Xanthophyll+, rubixanthin+, gaza- niaxanthin+, carotenoid of un- known constitution, /3-carotene+, y-carotene. b) Californian origin*^: Xantho- phyll+, gazaniaxanthin+, crypto- xanthin+, lycopene+, /5-carotene+, y-carotene+. Gazania splendens^^' ^®. Helenium autumnale*": Xanthophyll. Heleniuni autumnale var. grandice- phalum^^: Xanthophyll. Helianthus annuus^'^' ®^: Xantho- phyll+, taraxanthin+, crj'ptoxan- thin+, carotene+. Heliopsis scabrae cinniaeflorae-^: Xan- thophyll. Heliopsis scabramajor'^" : Xanthophyll. Hieracium aurantiacmn^^ . Hieraciuni murorum^^. Hieracium Pilosella^^. Inula Helenium'^^. Kleinia Galpinii^^. Leontodoyi autumnalis*^: Xantho- phyll+, taraxanthin+. Rudbeckia Neumannii^^: Xanthophyll. Senecio Doronicuni^''-: Zeaxanthin+. Senecio vernalis^^: Flavoxanthin+. Silphium perfoliatuni-^: Xanthophyll. Tagetes aurea^^: Xanthophyll++. Tagetes erecta*^: Xanthophyll+. Tagetes grandiflora*": Xanthophyll+, violaxanthin++. Tagetes 7iana*°: Xanthophyll+. Tagetes patula*'^' ^*: Xanthophyll+, xanthophyll epoxide+, rubixan- thin++, rubichrome+, a-carotene++, /3-carotene++. Taraxacum officinales^' ^*' ®": Xantho- phyll+, flavoxanthin+, thin++, taraxanthin+( ?) Telekia speciosissima^^. Tragopogon pratensis^*^' ^^ tene++, ^-carotene++, epoxide++, xanthophyll+, xantho- phyll epoxide++, violaxanthin+, fla voxanthin+ . Tussilago Farfara^^: Taraxanthin+ violaxanthin+. violaxan- a-Caro- a-carotene d) Carotenoids in Fruit and Seeds TABLE 18 (References see p. 99-107) CAROTENOIDS IN FRUIT AND SEEDS (i). Gymnospermae Taxaceae Taxus baccata (Arillus)'": Rhodoxanthin. (ii) . A ngiospermae a] Monocotvledoneae Pandanaceae Pandanus polycephalus^: Lycopene+. Gramineae Avena sativa''^. Barley gerni'^. Hordeum sativum''^. Oryza sativa'''^. Rye-seed oiP°: a-Carotene++, ^-caro- tene++, y-carotene+++, xantho- phvll++, zeaxanthin++. Triticum vulgare'''^: ^-Carotene++, xanthophyll++. Wheat gerni'^''^^: Xanthophyll, caro- tene(?). Zea Mays"''"'''': y-Carotene++, crypto- 74 DISTRIBUTION IN NATURE IX xanthin+, xanthophyll++, zeaxan- thin+. '* Hydroxy-a-carotene*3*. /S-Carotene, a-carotene, K-caro- tene(?), neo-cryptoxanthin. Palmae Actinop/iloeus angustifolia^: Lyco- pene+'*". Actinophloeus Macarthurii^: Lyco- pene+^^. Archontophoenix Alexandrae^: Lyco- pene++. Areca Alicae^: Lycopene++. Attalea gomphococca^^: Carotene. Calyptrocalyx spicatus^: Lycopene. Elaeis guineensis^^: /5-Carotene+++, lycopene+++^*. Elaeis melanococca^^: /3-Carotene+++, lycopene+++. Nenga Polycephalus^: Lycopene++. Palm fruit^'^: Zeaxanthin, carotene. Palm oil^^: a-Carotene+, /5-carotene+, y-carotene++, lycopene+, neolyco- pene+, neo-y-carotene( ?), pigments of unknown constitution. Palm oil from different species^'' '^^'^^: a-Carotene+, /?-carotene, y-caro- tene++, lycopene++. Ptychandra elegans^: Lycopene++. Ptychandra glauca^: Lycopene. Sabal (serenaea) serrulatum^^: /3-Caro- tene+. Synaspadix petrichiana^: Lycopene++. Araceae Aglaonema com,mutatum^^' ^^. Aglaonema nitidum^: Lycopene. Aglaonema oblongifolium^: Lyco- pene++. Aglaonema oblongifolium var. Cur- tisii^: Lycopene++. Aglaonema simplex^: Lycopene++. Arum, italicum^' '^' '^: Lycopene++. Arum maculatum^^: Lycopene++. Arum orientale^^: Lycopene++, /?-caro- tene++, xanthophyll. Bromeliaceae Ananas sativus"^^: Carotene, xantho- phyll. Liliaceae Asparagus officinalis^^: Zeaxanthin++. Convallavia inafalis^^' ": a-Carotene++, /3-carotene+, y-carotene+, lyco- pene+, xanthophyll+. Dioscoraceae Tamus communis^^' ^^: Lycopene+, lycoxanthin+, lycophyll+. Musaceae Musa paradisiacal"'': Carotene+, xanthophyll+. Amaryllidaceae Clivia species^^. Eriobotyra japonica Lindl.*^^: Crypto- xanthin, /3-carotene, neo-^-caro- tene U and neo-^-carotene B. ^) Dicotyledoneae Moraceae Cannabis saliva''''-. Polygonaceae Fagopyrum esculentum^^ Berberidaceae Berberis vulgaris'-^. Anonaceae Polyalthia species*^. Myristicaceae Myristica fragrans^^' ^^. Cruciferae Brassica campesiris^"^. Brassica 7iigra^"'-. Rosaceae "Apricot-peach" ''-°^: Carotene++, lycopene++, xanthophyll++. Cotoneaster species^^. Cotoneaster occidentalism"'^: Viola- xanthin++, xanthophyll. Crataegus Cms gallim"'^. Prunus armeniaca^"^: j3-Carotene+, y-carotene++, lycopene+. CAROTENOIDS IN PLANTS 75 Primus persica'^: /3-Carotene, crypto- xanthin, xanthophyll, zeaxanthin, carotenoid of unknown constitu- tion. Rosa canina^^' ^°®' ^"'i Lycopene+, /3-carotene+, y-carotene++, rubixan- thin+, zeaxanthin++, xantho- phyll++, taraxanthin++. Rosa damascena^°'^: As for Rosa canina. Rosa ruhiginosa^^'': As for Rosa canina. Rosa rugosa Thumb}'^^: a-Carotene+, ^-carotene+, y-carotene+, lycope- ne+, rubixanthin+. Rubus Chaniaemorus^'^^: a-Carotene, /3-carotene, y-carotene( ?), lycopene, rubixanthin, zeaxanthin. Sorbus Aria^^' ^®. Sorbus aucKparia^'^^: a-Carotene+, )S-carotene+. Sorbus aucuparia dulcis^^^: Carotene. Sorbus siiecica^^. Leguminosae Afzelia cunazensis^^ . Soya beans^'^^' *^^' *^": a-Carotene, /3-carotene. Vigna sinensis'^^^: /S-Carotene+, xan- thophyll++. Linaceae Linum usitatissmium?-'^^ . Erythroxylaceae Erythroxylon coca^*: Lycopene++. Erythroxylon novogranatens^^' ^*: Lycopene+. Rutaceae Citrus aurantium'^''-^' ^^^' ^^'' i^^: /3-Ca- rotene+, lycopene+++, cryptoxan- thin+, xanthophvll+, violaxan- thin++, zeaxanthin+, /3-citraurin+, citroxanthin+ (= mutatochrome). Citrus grandis^^^: /S-Carotin, lycopene. Citrus grand is Osbeck'^^^: Lycopene. Citrus Limonum^^' ^^. Citrus niadurensis^-'^: j8-Carotene+, xanthophyll+, cryptoxanthin+, violaxanthin+++( ?), zeaxan- thin+++(?). Citrus poonensis hort.^^^: /3-Carotene+, cryptoxanthin+,violaxanthin+++( ?) cryptoxanthin+, violaxan- thin+++( ? ) . xanthophyll++. Anacardiaceae Mangifera indica^^^' ^24- a-Carotene+, /5-carotene+, xanthophyll+, carote- noid of unknown constitution. Celastraceae Celastrus scandens''-^^. Evonymus europaeus^'^^: Zeaxan- thin++. Evonymus japonicus'^^' ^*: Lyco- pene++. Evonymus latifolius'^^' ^^. Icacinaceae Gonocaryum obovatuyn^^'^"^: a-Caro- tene+, /5-carotene+, y-carotene++, lycopene+. Gonocaryum pyriforme^*' *^; a-Caro- tene+, /3-carotene+, y-carotene++, <5-carotene++( ?), lycopene+. Vitaceae Ampelopsis hederacea^''' ^^. Malvaceae Gossypium hirsutum''-^''' 'i. Gossy/^iwrn-species^^^' ^2*: Carotene++, xanthophyll++. Bixaceae Bixa orellana (cf. p. 256) : Bixin+. Passifioraceae Passiflora coerulea^^' ^^°: Lycopene+. Caricaceae Carica Papaya^^'^' ^^^i cryptoxan- thin+, violaxanthin+. Myrtaceae Eugenia uniflora^^. Elaeagnaceae Hippophae rhamnoides^^: Zeaxan- thin+. Ericaceae Vaccinium Vitis idaea^^'^: Lyco- pene++, /5-carotene++( ?), zeaxan- thin++, xanthophyll++. 76 DISTRIBUTION IN NATURE IX Arbutus Unedo'^^^: a-Carotene++, /S-carotene+, h'Copene++, crypto- xanthin+, xanthophyll"'", zeaxan- thin+, violaxanthin+. Cap- Ebenaceae Capsicum friitecens japM'^' ^^^: santhin+, carotene+. Diospyros costata^^^: a-Carotene++, /3-carotene+, lycopene+, cryptoxan- thin+. Diospyros Kaki^^'^: Lycopene++, zea- xanthin++. Apocynaceae T ahernaemontana pentasticta^: Lyco- pene++. Solanaceae Lycium barbaruni^^: Zeaxanthin+. Lyciurn carolinianum^". Lycium halimifoliuni^'^^: Zeaxan- thin+. Lycium ovatuni^^. Lycopersicum ceraciforme^''-. Lycopersicum esculentimi^^^' ^*^: Ly- copene+. Physalis Alkekengi'^*^: Cryptoxan- thin+, zeaxanthin+. Physalis Franchetii'^'^^: Cryptoxan- thin+, zeaxanthin+. Solanum Balbisii^^. Solanum corymbosum^^ . Solanum decasepalum^*: Lycopene+. Solanum Dulcamara^^: Lycopene+, lycophyll+, lycoxanthin+. Solanum Hendersonii^^: Zeaxanthin+. Solanum Lycopersicum'^*'^: Carotene, lycopene, xanthophyll. Solanum Pseudocapsicum^^. Pedaliaceae Sesamufn indicum^^'^. Rubiaceae Gardenia grandiflora^^: Crocetin+. Gardenia jasminoides''-^'^' ^^^: Croce- tin+++. Gardenia lucida^'': Crocetin++. Neriera depressa^*: Lycopene++. Caprifoliaceae Lonicera tatarica^^^. Lonicera Xylosteum'^^°' **' ^5' ^*^. Sambucus nigra^*^. V iburnum Opulus^^' ^*^' i**. V iburnum Lantana^'^^' ^^' i*^. Cucurbitaceae Bryonia dioica^^' ^^: Lycopene+. Citrullus vulgaris'^^^' ^*^: Lycopene+, a-carotene+, ^-carotene+, y-caro- tene+. Cucumis Melo^^. Cucurbita maxima^^*' ^^^: a-Caro- tene+, yS-carotene+, xanthophyll+, violaxanthin+. Cucurbita Pepo''-^^. Luffa species^": ^-Carotene++, xan- thophyll+. Momordica Balsamina^^^' ^^^: Xantho- phyll++, lycopene++. Momordica Charantia^^^: jS-Caro- tene++, lycopene++. Trichosanthes species^*": Lycopene++. Compositae Helianthus annuus'^^'^. Sunflower oil^^'^: carotenoid of un- known constitution. 2. CRYPTOGAMS Much less is known about the carotenoids of cryptogams than about the carotenoids of phanerogams, no doubt partly due to the greater difficulty of collecting experimental material. Recent investigations, however, have yielded such interesting results that further studies in this field appear very desirable. * The arrangement of the groups follows Engler's system. The species belonging to one group are given in alphabetical order. CAROTENOIDS IN PLANTS 77 Investigations on bacterial pigments are particularly difficult because of the shortage of materials. Recent observations show that bacteria produce carotenoids {e.g. rhodoviolascin, sarcinin, sarcinaxanthin, leprotin, rhodopin, etc.), not found in the higher plants. Further interesting results can be expected from a renewed study of bacterial carotenoids. The carotenoids of fungi have been the subject of several investigations, some of recent date. In some mushrooms, certain other pigments (e.g. torulin, torularhodin) have been found besides carotene, and the question arises whether mushrooms produce these pigments by the degradation of other caro- tenoids or by independent synthesis. A large number of data are to be found in the literature regarding carotenoids in algae. Many of these are of an early date and possess only relatively small significance, but others have been obtained more recently and provide some interesting results. In this connection the comprehensive studies of Kylin^^ and Heilbron^^ may be particularly mentioned. We are least well informed regarding carotenoids in archegoniates, for which practically no data are available. It is probable, however, that the green organs of these plants also contain carotene, xanthophyll and xanthophyll epoxide besides chlorophyll. In the discussion of rhodoxanthin (p. 221) it will be mentioned that this pigment has been found in the intemodes of equisetum and selaginella. TABLE 19 (References see p. 99-107) CAROTENOIDS IN CRYPTOGAMS (i). Schizophyta a) Bacteria Bacillus Grasberger^^^: /3-Carotene, Micrococcus erythromyxa^^^: Asta- y-carotene, lycopene and a pigment cene( ?). resembling capsanthin. Micrococcus rhodochrous^'^^: Asta- Bacillus Lombardo Pellegrini^^'^) ;/J-Ca- cene( ?). rotene, y-carotene and a phyto- liTycobacteriumlacticola^^^: ^-Carotene, xanthin of unknown constitution. two similar pigments, astacene. Bacteriutn chrysogloea^*^ . Mycobacterium leprae^^^' 2^*' ^so; Bacterium egregium^'^^. yellow lipochromes, leprotin. Bacterium halobium^^^' 2": a-Bacte- Mycobacterium phlei^^^' 2i6> 247. ^-Ca- rioruberin, ^-bacterioruberin, hypo- rotene, y-carotene, cryptoxanthin, phasic pigment, absorption-maxi- xanthophyll, zeaxanthin, azafrin ma in CS2: 571, 532 m/^ (demethyl- ester, leprotin, azafrin, a-carotene ated rhodoviolascin(?). Purple bacteria (sulphur-free)^^*: Corynebacterium'^^^: ^-Carotene. Hydrocarbon similar to lycopene Corynebacteriu}n carotenum^^^' ^56, 257. g^j-^^ phytoxanthin of unknown /3-Carotene, yellow pigment with constitution, vitamin A activity. Rhodobacillus palustris^^^. References p. 108. 78 DISTRIBUTION IN NATURE IX Rhodovibrio Bacteria^^^: Rhodoviolas- cin, rhodopin, rhodovibrin, rhodo- purpurin, flavorhodin, ^-caro- tene(?). Sarcina aurantiaca^^^: /5-Carotene, lycopene(?), zeaxanthin^^*. Sarcina lutea^^°' "»' 2"' 2*2. Sarcinin, new hypophasic pigment, yellow phytoxanthin ester, sarcinaxan- thin. Sphaerotilus roseus^''^: pigment of un- known constitution, absorption maxima in petroleum ether: 494, 449 m^. Spirillum rubruni Esmarch^^^' ^^^: Bacteriochlorophyll, rhodoviolascin = spirilloxanthin and two pigments of unknown constitution. Staphylococcus pyrogenes aureus^'^^' ^*°: Zeaxanthin. Strepotothrix coraliinus^^^: Coralin (absorption maxima in ether; 495, 457 m/u). Thiocysiis Bacteria^*^' 250. 251; Lyco- pene, a-carotene, ^-carotene, flavo- rhodin, rhodoviolascin, rhodopin, rhodovibrin, rhodopurpurin, bac- teriochlorophyll, bacteriopurpurin . Timoiheegras Bacteria'^^^: ^-Carotene, pigments of unknown constitution. Torula rubra^^^' ^^*: ^-Carotene, toru- lin, torularhodin. Cyanophyceae Anabaena flos-aquae'^^' ^*. Aphanizomenon flos-aquae^^'^: /S-Caro- tene, aphanin, aphanicin, aphanizo- phyll, fiavacin. Calothrix species 222, 194 Calothrix-scopuloriim^^^' 231; Xantho- phyll, myxoxanthin, myxoxantho- phyll. Microcystis flos aquae^^^. Nodularia species^^. Nostoc species232, is Oscillator ia^^^' ^ss- 233 Oscillatoria limosa^^^' 234 Oscillator ia lapotricha'^^^. Oscillatoria Froelichii^'^' ^^. Oscillatoria rubescens^^^' 237; j3-Caro- tene, myxoxanthin, myxoxantho- phyll, oscillaxanthin, zeaxanthin, xanthophyll. Phormidiutn vulgare^^^' ^^. Rivularia species^*. Rivularia nitida^^^: Carotene, myxo- xanthin, xanthophyll. Rivularia atra^^^: Carotene, myxo- xanthin, myxoxanthophyll. Tolypothrix species^^. (ii). Myxomycetes Lycogala epidendron'^^*: Torulin(?), rhodoviolascin ( ?), ^-carotene (^-ca- rotene in spores^*^). Lycogala flavofuscuni'-^*. Siemonitis ferruginea ^**. Stemonitis fusca^^^. (Hi). Flagellatae Chrysomonadales Apistonema Carteri^^^: Carotene, xan- thophyll, fucoxanthin. Chromulina Rosanoffi^^*' 225. Chrysomonadina^^^. Glenochrysis maritima^^^: Carotene, xanthophyll, fucoxanthin. Hydrurus penicillatus^^^ . Thallochrysis litoralis'^^^: Carotene, xanthophyll, fucoxanthin. P) Euglenales Euglena heiiorubescens^^^: Astacene. Etiglena sanguinea^^^' ^*°. y) Dinoflagellatae Ceratium tripos^^^. Ceraiium fusus^^^. Ceratium furco^^^ Dinophysis acuta^^^. Dinophysis laevis^^^. d) Heterocontae Botvydium granulatum^^^: Carotene, fucoxanthin. CAROTENOIDS IN PLANTS Euglena viridis^^^' ^"' ^°®. 79 Glenodinium species^^^. Gymnodiniuni helix^^^. Peridinium diver gens^^^. Prorocentruni micans^^^. Botrydium species^ (iv). Bacillariophyta Diatomeae Achnantidium lanceolata'^'^^ . Cymatopleura solea^^^. Eunotia pectinalis^'^^. Fragilaria species^^. Gomphoncma species^^i, i5_ Melosira species^'^. Navicula species^^' i*^: Carotene, fuco- xanthin, zeaxanthin. Navicula torquatum*^^: /5-Carotene, e-carotene. Nitzschia closterium}^^' ^°^' ^^^' *^^ /3-Carotene, cryptoxanthin, xantho- phyll, isoxanthophyll(?), fucoxan- thin, pigment of unknown con- stitution. Nitzschia Palea^^^. Nitzschia sigmoidea^^'^. Nitzschia species^^*' *^®: "Diatoxan- thin", "diadinoxanthin", fucoxan- thin, "neo-fucoxanthin A", "neo- fucoxanthin B". (v). Conjugatae Prasiola species^^*. Zygnema cruciatum^^. Spirogyra crassa^°^' ^°*' ^^' ^^' ^**. Zygnema pectinatuni^^^: Carotene, Spirogyra maxima'^^'^' ^^' ^'*. xanthophyll, fucoxanthin. t (vi) . Chlorophyceae Protococcales Chlorella protothecoides'^^ . Chlorella variegata^^. Haeniatococcus pluvialis = Sphaerella pluvialis^^^' 202. 214. 215, is, u, 216, 217. 218 (cf. Z. physiol. Ghent. 267 (1941) 281): a-Carotene, ^-carotene, xan- thophyll, zeaxanthin, haematoxan- thin, astacene. (In earlier investig- ations, J. TiscHLER reported a new pigment, euglenarhodon, later shown to be identical with astacene.) Hydrodictyon utriculatum^^. Palmellococcus miniatus^^'^. Phyllobium dimorphum^^^. Phyllobium incertum^''-^ . Phyllobium Naegelii'^^^: Carotene, xanthophyll. Protococcus pluvialis = Pleurococcus pluvialis'^^^' 2i2_ Protococcus vulgaris^^. Scotinosphaera paradoxa^^^. Volvox species2"i. 8o DISTRIBUTION IN NATURE IX /?) Ulothrichales Cephaleurus laevis^'^^' ^'". Cephaleurus sohitus^°^' 2<". Cephaleurus albidus-^^' "^^"^ . Cephaleurus parasiticus'^^^' ^°^. Cephaleurus minimus^^^' ^°''. Phycopeltis epiphyton^"^' 2"^. Phycopeltis aurea^"^' '^°^. Phycopeltis amboinemis^^^' 2"''. Phycopeltis Treubii^"^' 2"'. Phycopeltis maritima^^^' ^''^. Trentepohlia aurea^^^' 202. 172, 208, les- a-Carotene, /3-carotene, xantho- phyll, zeaxanthin. y) Ulvales Enteromorpha compressa^^^: Carotene, xanthophyll. Enteromorpha intestinal is''-"' ^^^' ^^^: a-Carotene, ^-carotene, xantho- phyll, violaxanthm( ?). 6) Oedogoniales Bulbochaete setigera'-^. Bulbochaete species^"''' 2°^. Oedogonium species^^' ^^' ^^' ^^^' ^^*; e) Cladophorales Cladophora glomerata''-^^' i^' 1^' 1*. Cladophora rupestris'-^^: /S-Carotene, xanthophyll, violaxanthin. ^) Siphonales Vaucheria hamata'-^^: Carotene, xan- thophyll, violaxanthin. Trentepohlia aureum tomentosum^^^' 211 Trentepohlia bisporangiata'^°^' ^o'. Trentepohlia crassiaepta'^^^' 20'. Trentepohlia Cyania^^^' 2"''. Trentepohlia jolithus^"^' 2°^' ^^: a-Caro- tene, ^-carotene, xanthophyll, zeaxanthin. Trentepohlia tnonilifortnis^'^^' ^^''. Trentepohlia umbrina^^^' '^^^' ^^^' "^i a-Carotene, )3-carotene, xantho- phyll, zeaxanthin. Ulva lactnca'-^^' i*^' ^^^r Carotene xanthophyll. a-Carotene, ^-carotene, xantho- phyll, taraxanthin. Cladophora Sauteri^^^' ^^^: /^-Carotene, xanthophyll, taraxanthin. Spaeroplea species-"^. Vaucheria species^^^. (vii). Charophyta Chara ceratophylla Wallr.^^': ^-Caro- tene, )'-carotene, lycopene. Chara fragilis''-^' ^^. Niiella opaca'-^^: Carotene, xantho- phyll. Nitella spores^®. Nitella syncarpa (Thuill.)"': /3-Caro- tene, y-carotene, lycopene. (viii). Phaeophyceae Ascophyllum nodosum^^' ^^°: Caro- tene+, ^^' "^ fucoxanthin. Chorda Filum'-^^: Carotene, fucoxan- thin. Cladostephus spongiosus^^^: Carotene, fucoxanthin. Cutleria miiltifida^^^' ^^^. Cystosira abrontanifolia^^''-. Desmaresiia aculeata^^^. Dictyota dichotoma^^'-' ^^^' ^^*' ^^^: Carotene, fucoxanthin, xantho- phyll. CAROTENOIDS IN PLANTS Dictyota polypodioides^^' i*^' "". Ectocarpaceae species^^-. Ectocarpus siliculosus^^^: /3-Carotene, fucoxanthin, xanthophyll, viola- xanthin, zeaxanthin. Ectocarpus tomentosus^^^: Carotene, xanthophyll, fucoxanthin. Elachistea species^*^' i*^. Fucus cerayioides^^^: Carotene, fuco- xanthin. Fucus nodosus'^^^ . Fucus serraius^^^' "". iss. 12. ise, isi, i87, 180, 16, 188; Fucoxanthin+, caro- tene++, xanthophyll. Fucus species^*^. Fucus versoides^^'^. Fucus vesiculosus^^^' i^s. 12, i»o, iso, i«, 191, 198, 199. ^-Carotene, fucoxanthin, xanthophyll, violaxanthin, zea- xanthin. Halydrys siliquosa^^^' i^^. lai, iso, i98. Carotene, fucoxanthin. Haliseris polypodioides'^^^' "^' ^^S; ^-Carotene, zeaxanthin. Lammaria digitata^^' i^i- !*"> i«' i^^, i9s. Carotene, fucoxanthin, xantho- phyll. Laminar ia saccharina^^^' ^*^' ^^' "i.iso, 187, 16 Leathesia niarina^^^' ^*^. Padina Pavonia^^'^. PilayMa littoralis^^°' ^^^' i»«: Carotene, fucoxanthin, xanthophyll, viola- xanthin. Sphacellaria cirrhosa^^^: Carotene, fucoxanthin. Stypocaulon scopariuni^^^: Carotene, fucoxanthin. (ix). Rhodophyceae Ahnfeltia plicata^^^: Carotene, xantho- phyll. Bangia species^^' ^^^. Batrachospermum moniliforme^^^' ^^^' 15 Callithatnnion hiemale'^^^ . Ceramium diaphanuni^^^ . Ceramium rubrtini^^^' ^®*' ^^*' ^^*: Caro- tene, xanthophyll, a little taraxan- thin. Chantransia species^^' ^®*. Chdndrus crispus^^^' ^®; Carotene, xanthophyll. Corallina officinalis''-^^' "^: Carotene, xanthophyll. Cystoclonium purpurascens^^^ . Delesseria sanguinea'^^^. Dilsea edulis'-^'^' ^^^■. Carotene, xantho- phy 11198. Dumontia filiformis^^'^. Fiircellaria fastigiata'^^^' "*. Gelidium corneuni'-^^: Carotene, xan- thophyll. Gigartina stdlatd^^^: Carotene, xantho- phyll. Laurencia pinnatifida^^^ . Lemania fluviatilis^^^' 1^. Lemania niamillosa''-^^: Carotene, xanthophyll. Nemalion niultifidum'^^'^. Odonthalia dentata^^^. Phyllophora Brodiaei'^^°. Phyllophora membranifolia^^°. Phyllophora memhranifolid^^^: Caro- tene, xanthophyll. Plocamium coccineum''-^^: Carotene, xanthophyll. Poly ides rotundus'-^^' ^^^' ^^^■. Carotene, xanthophyll. Polysiphonia fastigiata^^^: Carotene, xanthophyll. Polysiphonia nigrescens'-^^' i^*' i^s- Carotene, xanthophyll, fucoxanthin. Polysiphonia species^^. Porphyra hiemalis'-^^. Porphyra laciniata''-^' 1®*. Porphyra umbilicalis^^^: Carotene, xanthophyll. Porphyra vulgaris^^*. Rhodomela subfusca^^^. Rhodomela virgata^^^. Rhodymenia palmata^^^' ^^S; ^-Caro- tene, xanthophyll, taraxanthin. Spermothamnion roseoluni^^^. Carotenoids 6 82 DISTRIBUTION IN NATURE IX (x). Fungi a) Phycomycetes Chytridium species^^. Mucor flavus^^' ^®. Phycomyces^^'' . Pilobolus crystallinns^^' ^*®: Xantho- phyll(?). j3) Eumycetes 1. Ascomycetes Ascobolus species^^^' ^'2. Leotia lubrica^''^' ^'^. Neciria cinnabarina^''*' ^'^' ^^' ^®. Peziza aurantia^''^. Peziza (Lachnum) bicolor'^''*. Peziza (Lachnea) sciitellata^''^. Polystigma ochracemn = Polystignia fulvuni^''^. Polystignia rubruni^^''' ^'^: Lycoxan- thin and an acidic pigment. Saccharomyces species^®^: Carotene. Spathiddria flavida^^' ^"■^. SphaerostUbe coccaphili^^ . Sporobolomyces roseiis'^^^: Toruliii, acidic pigments. Sporobolomyces sahnonicolor'^^^: To- rulin, acidic pigments. Torula ruhra^^^' ^'°: ^-Carotene, toni- lin, torularhodin. 2. Basidiomycetes Aecidio- and Basidio spores^^. Aleuria aurantiaca^^^: a-Carotene, |S-carotene, rubixanthin( ?). Allomyces'^''^: /S-Carotene, occasionally y-carotene. Calocera cornea''-^. Calocera palmata^^. Pilobolus Kleinii^^' ^^^' ^®': Carotene, xanthophyll( ?). Pilobolus Oedipiis^^^: Xanthoph^dl. Pleotrachelus fulgens'^^^. Calocera viscosa^^' ^'i. Cayitharellus cibarius'^'^^: a-Carotene, ^-carotene, lycopene and two caro- tenoids of unknown constitution. Cantharellus infundibuliformis^''^: Same pigments as in Cantharellus lutescens (below). Cantharellus lutescens^''^: Lycopene, carotenoid of unknown constitu- tion. Coleosporium pulsatilla^''^' ^''. Coleosporium senecio7iis^^^: a-Caro- tene, /5-carotene, acidic pigment. Dacryomyces stillatus^'''^. Ditiola radicata^''^. Gymnosporangiuni juniperi-virgi- nianae'^^^: a-, ^-, y-Carotene. Gymnosporangiutn juniper inuni^''*. Melampsora aecidioides'^''^' ^''. Melampsora salicis capreae'^''*. Neurospora crassa*^°: Neurospores. Phragmidium violaceum}"^^ . Puccinia coronata^''*. Puccinia coronifera^^^: /3-Carotene and acidic pigments. Tremella mesenterica^^^: ^-Carotene. Triphragmium tilmariae^'''^. Uredo (Coleosporium) eiiphrasie^'^'' . Uromyces alchern ille'^'^^. B. CAROTENOIDS IN ANIMALS I. INVERTEBRATES Many investigations, some of which are now out of date, deal with the distribution of carotenoids in invertebrates. More recent studies in this field have shown that carotenoids are present in a variety of invertebrates, though it is not known with certainty whether the pigments are synthesized by the B CAROTENOIDS IN ANIMALS 83 animal or contained in the food. It would be of interest to examine the lower plants which serve as feeding stuffs for these classes of animals in more detail from this point of view. a) Arthropods TABLE 20 (References see p. 99-107) CAROTENOIDS IX ARTHROPODS I Insects Bombyx mori'^''*' ^^s. 276, 266. Carotene, xanthophyll. Carausius morosus^''^: Protein-bound carotenoids. Caterpillar of the cabbage butterfly"""^: a-Carotene, taraxanthin. Coccinella septempunctata-^^: a-Caro- tene, ^-carotene, lycopene. Clythra qnadripunciata^''-: Carotene. Coleoptera coccinella^^^: a-Carotene, ^-carotene, lycopene. Locusia viridissima^''^: Protein-bound carotenoids. Locustiden^'^^: Protein-bound caro- tenoids. Oedipoda coeridescens^^^: Traces of carotenoids. Oedipoda miniata^^^: ^-Carotene and an unknown pigment. Perilhis biociilatus'^''^: Carotene. Pieris brassicae-'''': Carotene, xantho- phyll. Pyrrhocoris apteriis^^^' ^®*: Lycopene. Rhynchota^^'^ . Schizoneura lanigera^'"'. Sphinx ligustri^''^: Protein-bound carotenoids. Tritogenaphis rudbeckiaf^'^'^. (ii) Crustacea*' ** Ampelisca tenuicornis''^^: Carotene, xanthophyll. Anapagnrus chiroacanthus-^^: Asta- cene( ?). Astacus fluviatilis^^'': Astacene. Astacus gammarus^^^' ^^': Astacene, carotene. Astams'^^^: Astacene. Balanus balanus^^^. Balaniis crenatus^^^: Carotene, xantho- phyll. Calanus finmarchianiis^^^: Astacene, carotene. Calocaris macandreae^^-: Carotene, xanthophyll. Cancer pagurns^^^' ^**: Astacene, Caro- tene. Carciniis maenas^^^: Carotene. Crangon allmani-^^: Carotene, xantho- phyll. Diaptomus bacilli fer^''^: Carotene. Ebalia tumefacta^^^: Carotene, xantho- phyll. Eupagurus prideauxii^^'^: Astacene. Eurynome aspera^^'^. Galathea inter media'^^^: Astacene, caro- tene, xanthophyll. Haploops tubicula^^'^. Hyas araneus^^^. Idothea baltica^^'^. Idothea emarginata^^^: Carotene, xan- thophyll. Idothea neglecta^^-: Carotene, xantho- phyll. * This section is based on the summary given by O. Walker, Dissertation, Zurich, 1935. revised and completed up to 1946. Data concerning the occurrence of carotene and xanthophyll should not be regarded as conclusive as the pigments were only rarely isolated in the crystalline state. 84 DISTRIBUTION IN NATURE IX Idya furcata^^°. Leander serratus^^^: Astacene. Maja squinado^^^' ^^^: Astacene. Munida banffia^^^: Astacene, carotene, xanthophyll. Mysis flexuosa^^^: Carotene. Neohela monstrosa'^^^ . Nephrops norvegicus'^^^' ^^^i Astacene, carotene, xanthophyll. Pagurus hernhaydus^^'^: Carotene, xanthophyll. Pagurus riibescens'^^: Carotene. Palaemon fabricii-^^' -^^i Astacene, ca- rotene, xanthophyll. Palaemon serratus^^^: Astacene. Palinurus vulgaris^^^: Astacene. Pandalus brevirostris^^^: Astacene. carotene, xanthophyll. Pandalus borealis^^^: Astacene(?). Pandalus montagui^^'^: Astacene, caro- tene. Pontophilus spinosus^^'^. Porcellana longicornis^^^: Carotene. Portunus depuvator^^^: Carotene. Portunus longicornis^^^: Carotene, xanthophyll( ?). Portunus pusillus^^'^: Carotene, xan- thophyll(?). Portunus puber^^^: Astacene. Potamobius astacus = Astacus fluvia- tilis^^^: Astacene. Scalpelluni scalpellum^^'^. Spirontocaris lilljeborgii^^^. Stenorhynchus species^^"-. For lurther information regarding carotenoids in Crustacea refererice should be made to the investigations of Lonnberg (Ref. 17, p. 108). b) Molluscs* The carotenoids of molluscs have been repeatedly studied. Recent investig- ations in this field are mainly due to Lonnberg and Lederer. TABLE 21 (References see p. 99-107) carotenoids in molluscs (i) Amphineures Lepidopleurus cancellatus"^^^: Carotene, xanthophyll. Tonicella marmorea^^^: Carotene(?), xanthophyll. Chaetoderma nitidulum'^^^: Carotene. (ii) Lamellibranchia Anomia ephippium-^^: Carotene. A starts banksi^^^. Astarte sulcata^^^: Carotene. Axinus flexuosiis^^^. Cardiuni echinatum^^^: Carotene. xanthophyll( ?). Cardiuni norvegicutn^^^. Cardiuni tuberculatuni^^^: Xantho- phyll(?). Cochleodesnia praetenue^^^: Carotene. Corbula gibba^^^. Cultellus pelbicidus"^^: Carotene, xanthophyll. Cyprina islandica-^^' ^"' ^°^: Carotene. * Crystalline pigments were obtained only in very few cases and most of the data are therefore not conclusive. CAROTENOIDS IN ANIMALS 85 Dosina exolata^^^' 297. Xanthophyll( ?). Leda parvula^^^: Carotene, xantho- phyll(?). Lima loscombei^^^: Carotene, xantho- phyll(?). Lima hians^°'^. Lucina horealis^^^: Carotene(?). Lyonsia norvegica^^^' ^^*. Modiolaria rnarmorata^^^: Carotene ( ?). xanthophyll( ?). Mya trimcata^^^: Carotene(?), xantho- phyll(?). Mytilus californianus^"''-: Zeaxanthin, mytiloxanthin . Mytilus edulis^^''' ^oo; Carotene (cryst.), xanthophyllf ?). Nucula sulcata^^^' ^^^■. Carotene(?), xanthophyll. Pecteyi jacobaciis^^^: Pectenoxan- thin(?). Pecten maximns^^*: Pectenoxanthin. Pecten opercularis^^^: Carotene, xan- thophyll(?). Pecten septemradiatus^^^ . Pecten striatus^^^: Carotene. (iii) Scaphopoda Dentalium entale^^^: Xanthophyll. (iv) Gastropoda a) Opisthobranchia: Aeolis papillosa^^'. Acer a bullata^^^' ^^^: Carotene. Ap'lysia rosea^^^. Dendrovotus frondosus^^^: Xantho- phyll. Doris repanda-^^. Pecten tigrinus^^^. Pectunculus glycimeris^^"^' ^^^: Glycy- merin, carotenoids. Psammohia ferroensis^^^: Carotene, xanthophyll. Saxicava rugosa^^^: Carotene, xantho- phyll. Solen ensis^^^: Carotene, xanthophyll. Spisula solida^^^: Carotene^?), xan- thophyllf?). Spisula subtruncata^^^: Carotene, xan- thophyllf?). Syndosmia alba^^^^ ^^^■. Carotene(?). Syndosmia nitida^^^. Tapes pullastra^^^: Carotene, xantho- phyll. Tellina crassa"^^^: Carotene. Thracia convexa^^^: Carotene. Venus fasciata^^^: Carotene. Venus gallina^^^: Carotene. Venus ovata"^^^: Carotene. Vulsella barbata^^^: Carotene, xantho- phyllf?). Vulsella modiolus^^^' ^''°: Carotene, xanthophyllf ?). Doto coronata^^^: Carotene. Philline aperta^^^: Carotenef?), xa thophyll. Pleurobranchus species^^'' ^'®: Asta- cenef ?). Tritoma hombergi^^^' "^*. ^) Prosobranchia: Acmaea virginea^^^. Aporrhais pes pelecani^^^: Carotene, xanthophyll. Buccinum undatum"^^: Carotene, xanthophyll. Calliostoma miliare-^^: Carotene, xanthophyll. Capulus hungaricus^^^: Carotene, xanthophyll. Emarginula crassa^^^. Emarginida fissura^^^. Gibbula cineraria-^^: Carotene, xanthophyll. Gibbula tumida^^^: Carotene, xantho- phyllf?). Lacuna divaricata-^^: Carotene, xan- thophyll. Littorina littorea-^^' ^°°: Carotene, xanthophyll. Nacella pellucida. 86 DISTRIBUTION IN NATURE IX Nassa incrassata^^^: Carotene. Nassa reticulaia^^^: Carotene. Natica nitida^^^: Carotene, xantho- phylK?). Neptunea antiqua^^^' ^°''. Patella vulgaris^^^: Carotene, xantho- phyll. Purpurea lapillus^^^: Carotene, xan- thophyll. Rissoa species^**: Carotene, xantho- phyll. Scalaria elatior^^^' 299 Stylifer stylifera'^^^. Trivia europaea^^^: Carotene. Trochus zizyphiniis^^^: Carotene, xanthophyll( ?). Turritella coniniunis'^^: Carotene, xanthophyll(?). Velutina velutina^^^: Carotene, xan- thophyll(?). c) Echinoderms Numerous species of echinoderms contain carotenoids. Here, too, specific polyene pigments are sometimes associated with particular animals. It is not known whether the carotenoids are derived from the food or whether they are synthesized by the animal. Most of the data regarding the distribution of carotenoids in echinoderms are of a qualitative nature, and cannot, therefore, be regarded as conclusive. TABLE 22 (References see p. 99-107) CAROTENOIDS I Asteroidea Aster ias glacialis^^^: Carotene, xan- thophyll(?), 296. Asterias muelleri^^^' ^^^. Asterias rubens-^^' ^°*' ^°5: Asterin- acid(?)30'. Asterina gibbosa^^^' ^^^: Xanthophyll. Astropecten irregularis^^^' ^"'': Xantho- phyll( ?), carotene. Astropecten aurantiaciis^"^: Xantho- phyll. Asteracanthion glacialis'^^^: Xantho- phyll. Cribella oculata"^^^: Carotene. Crossaster papposus^^^' ^°*: Carotene, xanthophyll( ?). IN ECHINODERMS Goniaster equestris^^^: Carotene. Henricia sanguinolenta^^^: Carotene, xanthophylP°*. Hippasteria phrygiana^^^: Astacene, carotene, xanthophyll. Luidia sarsii^^^' ^°*: Carotene. Ophidiaster ophidianus^"^: Astacene. Porania pulvillns^^^: Carotene, xan- thophyll. Solaster papposa'^^^: Carotene. Stichastrella endeca^°^. Stichastrella rosea^^^. (ii) Ophiuroidea Amphiura chiajei"^^^' ^''^■. Carotene(?) xanthophyll. A mphiura filiformis^'^^. Ophiocoma nigra^^^' ^^'^: Carotene(?), xanthophyll. Ophiopholis aculeata^^^' ^"*. Ophiothrix fragilis^^^' ^°^: Astacene, carotene ( ?). Ophiura affinis^"*. Ophiura texturata^^^' ^°*: Carotene ( ?) xanthophyll. B CAROTENOIDS IN ANIMALS 87 (iii) Crinoidea Antedon petasus^^^' ^o*: Xanthophyll. (iv) Echinoidea Brissopsis lyrifera^^^: Xanthophyll. Echinaster sepositus^^^: Astacene. Echinus esculenius^^^: Xantho- phyll(?)305_ Psaimnechinus niiliaris^^^: Xantho- phyll. Spatangus purpureus^^^: Carotene, xanthophyll. Strongylocentrotus drobachiensis-^^: Carotene, xanthophyll. Strongylocentrotus lividus^"^: a-Caro- tene, /3-carotene, echinenone, penta- xanthin. Holothurioidea Cucumaria elongata^^^: Carotene, xanthophyll( ?). Cucumaria lactea-^^' ^o*. Holothuria brunnea^^^: Astacene(?). Holothuria nigra ^^*: Astacene(?). Holothuria poli^°^: Astacene(?). Holothuria tubulosa^^^: Astacene(?). Mesothuria intestinalis^^^' ^''*: Xan- thophyll. Phyllophorus lucidus-^^' ^°*: Carotene. Psolus phantapus^^^' ^°*. Thy one fusus^^^' ^*. d) Carotenoids in Worms Carotenoids in worms have recently been investigated, particularly by LoNNBERG and by Lonnberg and Hellstrom. x\lthough these studies are mainly based on spectroscopic data, it appears very probable that carotene and xanthophyll occur in worms. The red carotenoids with the single absorption band previously reported by Krukenberg were not observed by the first- named authois. The following is a summary of carotenoids in worms which has been mostly taken from the dissertation of Walker (Ref. 18, p. 108) and brought up to date. TABLE 23 (References see p. 99-107) CAROTENOIDS IN WORMS AND RELATED SPECIES (i) Nemertini Amphiporus pulcher^^^. Carinella annulata^^^ . Cerebratulus fuscus^^^. Cerebratulus marginatus^^^. Malacobdella grossa^^^: Xanthophyll. (ii) Polychaeta Amphitrite affinis^^^. Aphrodite aculeata^^^. Arenicola niarina^^^. Arenicola piscatorum^^^. Aricia norvegica^^^. Chaetopterus variopedatus^'^^: Caro- tene. Cirratulus cirratus-^^: Carotene. Cirratulus tentaculatus^^^: Carotene. Euwienia crassa^^^. Glycera goesii^^^: Carotene, xantho- phyll(?). Harmothoe sarsii^^^: Carotene. Laetmonice filicornis^^^: Carotene. 88 DISTRIBUTION IN NATURE IX Lepidonotus squmnatus^^^. Lumbrinereis fragilis^^^: Carotene. xanthophyll( ?). Neoamphitrite figulus^^^: Carotene, xanthophyll. Nephihys caeca^^^: Carotene. Nephthys ciliata^^^' 2'^: Carotene. Nereis virens^^^' ^^^: Carotene. Nereis pelagica*^^. Pectinaria belgica^^^. Polymnia nebulosa^^^: Carotene(?), xanthophyll. Polynoe spinifera^^^. Sabella penicillus^^^: Carotene, xan- thophyll. Siphonostoma diplochaitos^^: Asta- cene( ?). Stylarioides plumosus^^^: Carotene, xanthophyll. Terebella species^®^. Terebella stroemii^^^: Carotene, xan- thophyll(?). Thelepus cincinnatus^^^: Carotene, xanthophyll. (iii) Gephyrea Phascolosoma elongatum^^^: Carotene. Priapulus caudatus^^^. (iv) Bryozoa Alcyonidium gelafinosum^^^: Caro- tene, xanthophyll( ?). Bugula neriiina^"^: Carotene. Flustra foliacea^^^: Astacene. Flustra securifrons^^^: Carotene, xan- thophyll. Lepralia foliacea^^^: Astacene. (v) Brachiopoda Crania anomala^^^: Carotene(?) thophyll. Tarebratulina caput serpentis^^^: Ca- rotene. e) Coelenterates and Sponges Carotenoids in coelenterates and sponges have been repeatedly studied in recent years, especially by Lonnberg. In contrast to Krukenberg, and MacMunn, this author did not observe any single-banded red carotenoids and describes only the presence of pigments of the carotene and xanthophyll type. Most studies in this field are again confined to spectroscopic observations, so that the data are not conclusive. TABLE 24 (References see p. 99-107) CAROTENOIDS IN COELENTERATES AND SPONGES Actinia equina^^'^: Actinioerythrin, 314, 293 Alcyonium digitaium^^^: Carotene, xanthophyll. Anemonia sulcata^'^^: Sulcatoxanthin. Aplysina aerophoba^^'^. Axinea rugosa-^^. Axinella crista-galli^^^: Astacene. Caryophyllia sniiihi^^^: Astacene(?), carotene. Chondrosia reniformis^^''-. Coccospongia species'^^. Dysidea fvagilis^^^. Epiactis prolifera^'^^: a carotenoid acid. Esperia foliata^^^: Carotene. CAROTENOIDS IN ANIMALS Halcampa duodecirrhata^^^: Carotene. xanthophyll. Halichondria albescens^''-^: Astacene( ?). Halichondria caruncula^'^^: Carotene. Halichondria incrustans^^^: Carotene. xanthophyll. Halichondria panicea^^^: Carotene. Halichondria rosea^'^-: Carotene. Halichondria seriata^^-: Carotene, xanthoph^'ll. Halma Bucklandi^^^: Astacene(?). Hircinia spinosula^'^^: Carotene. Hymeniacidon sangiiineum^^^: Echi- nenone, a-carotene, y-carotene. Leuconia fossei'^''-^: Astacene(?). Lucernaria quadricornis^^^: Carotene, xanthophyll( ?). Metridium dianthus^^^: Carotene, xanthophyll( ?). Metridiuin senile^'^^: Astacene, caro- tene, phytoxanthins. Papillina suberea^''-^ . Permatnla phosphorea^^^: Carotene, xanthophyll. Protanthea sitnplex^^^: Carotene(?), xanthophyll. Radiella spinolaria^^^: Carotene. Reniera aquaeductus^'^'^ . Sagartia undata^^^: Xanthophyll. Sagartia viduaia^^^: Carotene, xantho- phyll(?). Stenogorgia rosea^^^: Carotene. Suberites domuncula^^^' ^i^: Asta- cene( ?). Suberites ficus^^^: Carotene. Suberites flavus^^^: Carotene, xantho- phyll. Suberites massa^^^' ^^i; Carotene. Tedania muggiana^^''-: Carotene. Tentorium semisuberiies^^^: Carotene. Tealina felina^''-*: Actinioerythrin. Tethya lyncurium^'^'^: Carotene. Tubularia indivisa'^'^'^: Astacene{?). Tubularia larynx^^^: Carotene. Urticina felina^^^: Carotene, xantho- phyll. f) Chordata TABLE 24a (References see p. 99-107) CAROTENOIDS IN CHORDATA (i) Hemichordata : Enteropneusta Harrimania kupferi^^^. (ii) Tunicata Ascidia virginea-^^. Botryllus schlosseri pallus^^^' ^^°. Xanthophyll, capsanthin, capso- rubin, pectenoxanthin. Ciona intestinal is^^^: Xanthophvll. Clavellina lepadiformis^^^: Carotene, xanthophyll. Corella parallelogranima^^^: Caro- tene(?), xanthophyll. Cynthia papillosa^^^' ^^^: Astacene, cynthiaxanthin, a-carotene, /3-ca- rotene. Dendrodoa grossularia^'^^: a-Carotene, j3-carotene, astacene. Microcosmus sulcatus^^''-: Phyto- xanthins. Molgula occulta^^^: Carotene{?), xanthophyll( ?). Muxilla matnmillaris^^^: Carotene(?), xanthophyll( ?). Styela rustica^^^: Carotene, xantho- phyll(?). Synoicum pulmonaria^^^ . go DISTRIBUTION IN NATURE IX 2. VERTEBRATES a) Mammals A great deal of information is available in the literature concerning the occurrence of carotenoids in mammals. Carotenoids have been found in nearly every part of the mammalian organism. It is almost certain that these caro- tenoids are derived from the vegetable feeding stuffs. It is not known, however, whether the pigments undergo chemical changes in the animal body, or whether they are stored unchanged. In the following sections the occurrence of polyene pigments in mammals is described. The treatment given is not complete ; it is merely meant to illustrate the manifold distribution of carotenoids in mammalian organisms. (i) Faeces Recent investigations have shown that part of the carotenoids taken with the food leave the animal body unchanged^, while the rest is absorbed in the intestine and then reaches the various organs. Cow dung^^^: Carotene, xanthophyll. Sheep dung^^'': Xanthophyll( ?). It is noteworthy that on feeding a-carotene or /9-carotene to experimental animals, only the same pigment is found in the faeces^^^. (ii) Blood serum^^^ A variety of carotenoids have been found in the blood serum of human beings, horses, cows, calves, oxen, sheep, pigs, goats, cats and rats. The com- position of the pigments depends on the diet fed to the animals. On the other hand, no polyene pigments have been found in the sera of dogs, guinea pigs and rabbits. Blood of pregnant ivomen^^'^: Carotene. Serum {human)^^^: ^-Carotene, lyco- Serum (cattlej^^i- Carotene, xantho- pene, ;5-hydroxycarotene( ?), phyll, cryptoxanthin. ^-hydroxyseniicarotenone( ?) xan- Seruni (human) ^^^i Carotene, lyco- thophyll, occasionally zeaxanthin. pene, xanthophyll. (iii) Fat tissues Bone marrow (humanj^^s, 329. Caro- Fat tissues (horses)^25- Carotene. tenoid. Fat tissues (human)^'": Carotene, lyco- Fat tissues (cats)^-*: Carotene, xan- pene, xanthophyll, capsanthin. thophyll. Intestine and subcutaneous fat (horses Fat tissues (cows)^". a-Carotene, ^-ca- and cattlej^ss; Xanthophyll. rotene. References p. gg-io^. B CAROTENOIDS IN ANIMALS 91 (iv) Nerve tissues Nerves of humans and cows^^^: Caro- Peripheral nerves^^^: Carotenoids. tenoids. (v) Inner Organs, Glands, Secretions^^^ Corpora rubra of cows^^®: ^-Carotene. Corpus luteum of cows^"' ^^°' ^^^' ^*^' 353, 354, 355- a-Carotene, /5-carotene and traces of xanthophyll. Gallstones (cattle)'"' '*': Carotene, xanthophyll. Heart tissue s^^^: Carotenoids. Hypophyses of cattle^^^: Carotenoids. Kidneys (human beings, horses, guinea pigs, cats, dogs, pigs)^'*' ^'^^' 338,337,338,326; Carotcnc, xanthophyll. Liver^^^' ^^^' ^^^: Carotenoids. Liver (human)'^'; Carotene, lycopene, zeaxanthin, xanthophyll, violaxan- thin(?), 2 pigments of unknown constitution (degradation products of /3-carotene) . Liver (human)'*': Carotene, xantho- phyll, lycopene. Liver (pigs)'**: Carotene. Milk fats (all mammals)'", seo, 36i, 362, 363, 364. Carotene, a little xantho- phyll, pseudo-a-carotene( ?)'^*. Placenta (cow)'®^' '^2; /9-Carotene. Placenta (human)'^^. 357, 355, 322, 358. Carotene and xanthophyll. Skin^^^: Carotenoids'*^. Spleen^^^: Carotene. Testes (various mammals)'^': Caro- tenoids. Yellow skin of diabetic subfects^^^' ^^^' "": Carotenoids. Yellow skin produced by special diet '*': Carotenoid'*®. (vi) Other parts Blood of umbilical cord^''^: Carotene. Colostrum (women)"^: Carotene. Retina^''^: jS-Carotene, retinene. b) Birds The numerous investigations on the pigments of the plumage of birds bear witness to the great interest shown by chemists and zoologists in these natural compounds. Other parts of birds, such as the skin, the inner organs, and the fat tissues, have also been repeatedly examined. The majority of these studies, have been carried out, however, with very small amounts of material, and have therefore yielded merely qualitative data. Crystalline pigments have only been obtained very rarely. Most of the available information consists of spectroscopic data or even colour reactions with concentrated sulphuric acid or antimony trichloride and cannot, therefore, be regarded as conclusive. The following parts of birds have been examined: (i) Feathers ; (ii) Fat tissues; (iii) Foot skin, body skin, beaks; (iv) Blood serum and inner organs ; (v) Egg yolk. References p. gg-ioy. 92 DISTRIBUTION IN NATURE IX As a result of these studies, certain regularities have emerged which are briefly summarised in the following paragraphs : (i) Carotenoids in Feathers The colours of the feathers of birds, particularly of sub-tropical and tro- pical birds, are often of extraordinary brilliance. Apart from basic blue and white pigments and melanin pigments, various red and yellow lipochromes are present, some of which possess carotenoid character. No example of the isolation of a crystalline carotenoid from feathers seems to have been recorded and the available information is wholly restricted to spectroscopic data. Nevertheless it appears certain that bird feathers contain only phytoxanthins (and their transformation products) , especially those with two hydroxyl groups (xanthophyll, zeaxanthin, capsanthin^^) . By suitable feeding experiments Brockmann and Volker^^ showed that canaries which have white feathers as a result of a xanthpphyll-free diet are unable to absorb /^-carotene, lycopene or violaxanthin and to deposit these in the feathers, even though the birds were in an otherwise healthy condition . The feathers only assume their original yellow colour after feeding xanthophyll or zeaxanthin. It was shown by Sauermann^" that paprica pigments are ad- sorbed in the same way as xanthophyll and zeaxanthin and are deposited in the feathers which, in this case, assume a red colouration. Examination of the feathers of xanthophyll-fed birds showed that, apart from xanthophyll, a transformation product of the latter was present, which has been termed canaryxanthophyll (p. 337). In some birds yet another carotenoid, picofulvin, has been found. The nature of canaryxanthophyll and picofulvin is not yet known. The former is not identical with xanthophyll epoxide (p. 206) as might be assumed in view of the similar absorption spectra (unpublished observation by Karrer and Jucker). Apart from well-defined pigments such as xanthophyll and zeaxanthin and pigments of unknown structure such as canaryxanthophyll and picofulvin, some birds also contain carotenoids which appear to be related to astacene. Even these more recent investigations are of a purely qualitative nature, while some of the older observations, e.g. those of Krukenberg, are now only of historical interest. Thus Krukenberg distinguished between 5 red and 5 yellow lipo- chrome-type pigments. Of the red pigments, zoonerythrin^^ and rhodophan^^ were most widely distributed. The behaviour of these two pigments is strongly reminiscent of polyene pigments and their spectral properties and possible combination with protein suggests a certain resemblance with astacene or astaxanthin. The latest investigations by Lonnberg-^ and by Brockmann and VoLKER confirm the probable relation to astacene. The nature of the yellow pigments mentioned by Krukenberg has been References p. 108. B CAROTENOIDS IN ANIMALS 93 elucidated in one case: zoofulvin has been identified with xanthophyll. The structure of picofulvin, on the other hand, is still unknown. (ii) Carotenoids from fat tissues, foot skin, body skin and beaks It was mentioned above that no carotenoid, or other polyene pigment of hydrocarbon nature, has been found in bird feathers. By contrast, the beaks and skin of various animals contain mixtures of carotene and xanthophyll. At an early date, Krukenberg^^ established the presence of lipochromes in the fat tissues and foot skin of many animals. In 1930, Lonnberg^^ showed that chloriosulfurin consisted of a mixture of xanthophyll and carotene, and zoofulvin of almost pure xanthophyll. Further investigations in this field have been carried out by KuHN and Brockmann^^ and by Capper, McKibbin and Prentice^^. With two exceptions {phasianus colchicus, "Rosen" and Anser domesticus) , all these investigations are of a purely qualitative nature. (iii) Carotenoids from blood serum and inner organs It has long been known that a lipochrome pigment is present in the blood serum of pigeons and chicken. It was later identified by spectroscopic means as xanthophyll by Schunck^. The same pigment has been found in the livers of chicken^^. It appears that the carotene and cryptoxanthin contained in the diet (maize) are quickly degraded, while xanthophyll, which has no vitamin A activity, is stored. (iv) Carotenoids from egg yolk The pigments from egg yolk have long aroused the interest of chemists. Stadeler^" was the first to isolate a crystalline pigment with well-defined properties from chicken egg yolk. Thudichum^^ classified this pigment as one of the "luteins" (lipochromes) and it was related to xanthophyll by Schunck^^. In 1912, Willstatter and Escher^^ proved the xanthophyll nature of the pigment isolated by Stadeler and in 1931 Kuhn and co-workers showed it to be a mixture of xanthophyll and zeaxanthin^*. The composition of the egg yolk pigments can be altered by varying the diet^". Carotene has also been shown to be present in chicken egg yolk^^. TABLE 25 (References see p. 99-107) CAROTENOIDS IN BIRDS* Acanthis flammea (red forehead)^'*: Anser dornesticus [retma)'^'^^: Kst?LcenQ, Astacene(?), carotenoids. astaxanthin^''®. Anipelis garrula^''*: Astacene(?), Anas penelope (foot skin)'''*. carotenoids. A nas platyrhyncha (red foot skin, beak * Tbe above table is taken from O. Walker, Dissertation, Zurich, 1935. References p. 108. 94 DISTRIBUTION IN NATURE IX skin)"*: Carotene, xanthophyll, and decomposition products*. Anas platyrhyncha domestical'' ^ (yellow foot skin and beak skin): Carotene, xanthophyll and decom- position products. Anser domesticus (beak skin)^^®: Caro- tene, xanthophyll and decompo- sition products. Aprosmictus melanurus (yellow feathers)"^: Astacene( ?) carotenoids of unknown constitution. AsUir gentilis (yellow feathers)"*." Carotene, xanthophyll. Cacatua roseicapilla (red feathers)"'." Carotenoids with single absorption bands. Calurus aurictps (red feathers) ." Ca- rotenoids with single absorption bands. Campethera niibica (feathers) ." Caro- tenoids of unknown constitution"''. Cardinalis virginianus (feathers)"'." Carotenoids with single absorption bands. Cavduelis spinus (feathers)"^." Xantho- phyll and transformation products. Carduelis carduelis (feathers)"*." Xanthophyll and transformation products. Cerihiola mexicana (feathers)." Caro- tenoids of unknown constitution"'. Chloris Moris (feathers)"*." Xantho- phyll and transformation products. Chloronerpes auridentus (feathers)^"." unknown pigments. Chloronerpes kirki (feathers); Caro- tenoids of unknown constitution"'. Chloronerpes yucatensis (feathers)^'*." Picofulvin (violaxanthin and tara- xanthin( ?). Chlorophanes atricapilla (feathers)"'; unknown pigments. Chrysoptilus punctigula (feathers)"'; Carotenoids of unknown consti- tution. Colaptes auratus (feathers)^". Colaptes olivaceus (feathers)"'. Cotinga coeriilea (feathers)"'; Carote- noids with single absorption bands. Cymbirhynchus macrorhynchus^'''' : Ca- rotenoids with single absorption bands. Dendropicos cardinalis (feathers)"'. Diphyllodes magnijica (yellow neck feathers)"'. Dryocopus auratus (feathers)"'. Dryohates major (black feathers)"*; Picofulvin. Eclectus polychlorus (feathers)"'; Ca- rotenoids with single absorption bands and carotenoids of unknown constitution with two or three ab- sorption bands. Emberiza citrinella (feathers) ; Xan- thophyll and decomposition pro- ducts"*' "*. Emberiza icterica (feathers)"*; Xan- thophyll and decomposition pro- ducts. Euphone nigricollis (feathers)"'; Ca- rotenoids of unknown constitution. Fringilla canaria (feathers)"'* ^'*. Callus bankiva domesticus'^'''' ' "* (yellow footskin) ; Carotenoids. Hypoxanthus rivolii (feathers)"*; Picofulvin. Ithaginis cruentatus (feathers)"'; Ca- rotenoids with single absorption bands. Loxia curvirostra (feathers)"*' "*; Carotenoids with single absorption bands, xanthophyll and decompo- sition products. Lyrurus tetrix^''''' "*; Carotenoids with single absorption bands. Megaloprepia magnifica (feathers)"'.' Carotenoids with single absorption bands. Milvus (foot skin)"'; Carotenoids with single absorption bands. Motacilla cinerea (feathers)"*; Xan- thophyll and decomposition pro- ducts. phyll. Canaryxanthophyll predominates amongst the transformation products of xantho- CAROTENOIDS IN ANIMALS 95 Oriolus galbida (feathers)^". Oriohis oriolus (feathers)^''*; Xantho- phyll and transformation products. Oriolus xanthonotus (feathers)^'*; Xanthophyll and transformation products. Paradisea papuana (feathers) ^"^; Ca- rotenoids with single absorption bands. Paradisea rubra (feathers) ^'^^^ Caro- tenoids with single absorption bands. Paroaria cucullata (feathers) ^''^.' Ca- rotenoids with single absorption bands. Parus coeriileus (f eathers) ^'^ .• Xantho- phyll and transformation products. Parus major (feathers) ^'^^.^ Xantho- phyll and transformation products. Phasianus colchicus x torquatus (feathers) =''*.• Astacin(?). Phasianus colchicus ("Rosen")^'^' ^'^.• Astacin (crystallised) . Phlogoena cruenta (feathers)^'''.' Ca- rotenoids with single absorption bands. Phoenicopterus antiquoruin (feathers) ^'^'.' Carotenoids with single absorption bands. Phylloscopus sibilatrix^''^: Xantho- phyll and transformation products. Picidae species (feathers) ^'^^." Caro- tenoids with single absorption bands and picofulvin. Picus canus (feathers) ^^*." Picofulvin. Piciis major (feathers)^''; Carotenoids with single absorption bands. Picus viridis (feathers) ^'^.^ Picofulvin. Pinicola species (feathers)^''*." un- known pigments. Ploceus cucullatus (feathers)^'*." Xan- thophyll and transformation products. Pyrocephalus rubineus (feathers) ^'^.* Carotenoids with single absorption bands. Pyromelana franciscana (feathers) ^^^." Carotenoids with single absorption bands. Pyrrhula pyrrhula^''^' ^'^: Carotenoids with single absorption bands, xan- thophyll and transformation pro- ducts. Pyrrhula vulgaris (feathers) ^^^.' Caro- tenoids with single absorption bands. Regulus regulus (feathers) ^^^." Caro- tenoids with single absorption bands. Seleucides alba (feathers)^". Serinus canaria (feathers)^'*; Xantho- phyll and transformation products. Serinus canaria serinus (feathers) ^'^.' Xanthophyll and transformation products. Sittace macao (feathers) ^''^.- Caro- tenoids with single absorption bands and other unknown pigments. Somateria mollissima (yellow foot skin)^^'*.- Carotenoids of unknown constitution. Tetrao tetrix (" Rosen ")^^*.- Carotenoids \\'ith single absorption bands. Tiga tridactyla (feathers)^''.* Caro- tenoids of unknown constitution. Trogon massena (feathers) ^^'." Caro- tenoids with single absorption bands. Turdus morula (Beak skin and throat skin)^'*: unknown pigments. c) Fish The carotenoids of fish have been repeatedly investigated, but the isolation of the crystalline pigments has only rarely been achieved. It can be regarded as certain, however, that fish (skin, flesh, inner organs, eyes) almost invariably contain carotenoids. Xanthophyll, carotene, astacin, and according to Lonn- 96 DISTRIBUTION IN NATURE IX BERG*, taraxanthin, appear to predominate. It should be mentioned, however, that most of the available data merely show that the occurrence of carotenoids is probable. The designation of individual pigments appears unjustified in the majority of cases since the pigments were neither isolated nor chromato- graphically separated. It is probable that a carotenoid mixture was generally obtained in which one component predominated and that only the absorption bands of this component were observed in the spectroscopic determination. A careful chromatographic analysis would probably have shown the presence of other pigments as well. The carotenoids present in the eyes of fish mostly include xanthophyll and taraxanthin{?) (cf. Lonnberg, Ref. 37, p. 109). TABLE 26 (References see p. 99-107) CAROTENOIDS IN FISH Ahramis brania^^". Agonus cataphractus^^" . Ammodytes lanceolaius^^'^. Anguilla anguilla^'"*: XanthophylP^^^ carotene. Anther ina presbyter^'^: Xanthophyll. Aphiga minuia^''^: Taraxanthin. Arnoglossus megastoma^^^: Xanthophyll. Barbus fhwiatilis^^^. Belone belone^''^. Belone rostrata^^^. Beryx decadactylus^^*: Astacene^^^. Bothus maximus^''^: Taraxanthin. Bothus rhonibus^''^: Xanthophyll, tara- xanthin. Callionymus lyra^^^' ^^^: Xanthophyll, carotene. Carassius auratus^^'^' ^^^: Lycopene( ?)^**, astacene, carotene. Caranx irachurus^''^: Xanthophyll. Centrolabrus exoletus^^'*: Astacene^'^, xan- thophyll, taraxanthin. Clupea harengus^''^' ^*^: Xanthophyll, taraxanthin. Coregonus albula^^'' (eggs) ; Asterm acid. Cottus bubalis^'^^' ^^^' ^^^: Carotene, xan- thophyll, taraxanthin. Cottus scorpius (skin and muscles)^^'' ^^''.• Xanthophyll, taraxanthin. Crenilabrus melops'^'^^' ^^°: Xanthophyll or taraxanthin. Crenilabrus suillus^''^' ^^"i Taraxanthin or xanthophyll. Cyclopterus lumpiis (fins)^^".' Taraxan- thin or xanthophyll. Cyclopterus lumpus^'^^: Taraxanthin. Cyclopterus lumpus (liver)^*.- Astacene. Cyprinus auratus^^^ . Cyprinus carpio^^^. Eleginus navaga (Ovaries)^*^." ^-Caro- tene, 3 phytoxanthins. Embiotocidae^^°: Carotene, a carotenoid acid and a phytoxanthin. Esox anguilla^^°. Esox liicms^^°. Esox lucius (fins)^^^.' 2 Phytoxanthins, similar to taraxanthin or eloxanthin = xanthophyll epoxide. Esox lucius (liver) 3^^.- Xanthophyll. Esox lucius (roe)^^^." Carotene, xantho- phyll. Esox lucius (spermatozoa) ^^^.' Carotene, xanthophyll. Fundulus parvipinnis^^^: Taraxan- thin ( ?), phytoxanthins. Gadiis aeglefinus^''^: Taraxanthin. Gadus caUarias^''^' ^^'^' ^^^\ Carotene, xanthophyll, taraxanthin. * It is doubtful whetJaer the pigment tound by E. Lonnberg, Arkiv. Zool. 31 A (1938) No. I is really taraxanthin as the latter is hypophasic and not epiphasic as described by Lonnberg. ** Unless otherwise stated, the data'^-^fer to skin. B CAROTENOIDS IN ANIMALS 97 Gadus callarias (roe) ; Carotene, xantho- phylPss. Gadus esniarkii^^°. Gadus merlangus^^°' ^'^: Taraxanthin. Gadus ■minutus^'^^' ^^^■. Carotene, xantho- phyll, taraxanthin. Gadus pollachhis^'^' ^^°: Taraxanthin. Gadus virens^''^' ^^°: Taraxanthin. Gaidropsarus cimbrius'^'^: Xanthophyll. Gaidropsarus niustela'^'^: Xanthophyll, carotene. Gasterosteus aculeatus^''^: Taraxanthin. Gasterosteus spinachia^^^: Xanthophyll. Gobius niger^^^: Carotene, xanthophyll. Hippoglossus hippoglossus^''^' ^^^ (roe) .• Xanthophyll, carotene, zeaxanthin. Hippoglossus platessoides^''^' ^*°. Labriis bergsnyltrus (scales, fins)^*^; Caro- tene, xanthophyll, taraxanthin. Labnts bergsnyltrus^''^: Xanthophyll. Labrus nielops^^^: Carotene, xanthophyll Labrus ossifagus^^"' ^'®: Carotene, xan- thophyll. Leuciscus rutilus (liver)^^^' ^'^ : Xantho- phyll, taraxanthin. Lophius piscatorius^^"^ (liver) ; Astacene, taraxanthin^*^' "^ and a carotenoid similar to eloxanthin. Lota vulgaris (roe)^^*.- Carotene, xan- thophyll. Molva iitolva^''^' ^^°. Muraena helena^^*. Mullus barbatus^^^. Nerophis aequoreus^^^ : Carotene, xan- thophyll. Nerophis ophidion^''^: Taraxanthin. Orthagoriscus mola^^''-: Carotene. Osmerus eperlanus^^''-: Carotene. Perca fluviatilis (fins)^*^: Astacene, tara- xanthin. Perca fluviatilis'^^ (fins) .' Astacene. Pholis gunellus^''^' ^®°: Carotene, xantho- phyll, taraxanthin. Pleuronectes flesus^^'^''-: Carotene, xan- thophjdl. Pleuronectes kitt^''^' ^^*'' ^^^i Carotene, xanthophyll, taraxanthin. Pleuronectes limanda^''^' ^^^: Carotene, xanthophyll, taraxanthin. Pleuronectes niicrocephalus^^'^: Carotene. Pleuronectes platessa ^*^: Carotene. Raja clavata^''^: Xanthophyll. Raja batis^''^: Xanthophyll. Raniceps raninus^''^' ^*°: Xanthophyll. Regalecus glesne (liver)^^®.- Astacene. Salmo salar (meat)^".' Carotene, salmon acid (astacene ?)33^. Salmo salar (liver)^^®.' Xanthophyll. Salmo trutta^''^: Taraxanthin. Salmo irideiis*^^: )3-Carotene, astaxan- thin. Scomber scombrus^^-' ^^^' ^'^^: Carotene, xanthophyll, taraxanthin. Scophthalmus norvegicus^''^' ^^^: Caro- tene, xanthophyll, taraxanthin. Scorpaena scrofa^^*: Astacene. Sebastes marinus^^^: Astacene. Shark (embrj^o)^^'^; Carotene, xantho- phyll, zeaxanthin. Siphostoma typhle^''^' ^^i; Xanthophyll, taraxanthin. Solea solea^''^' ^^o. Solea variegata^^^: Carotene. Solea vulgaris (roe)^^*: Carotene. Spinachia spinachia^^^. Syngnatus acus^''^' ^^^: Xanthophyll, taraxanthin. Trachiniis draco^''^: Taraxanthin. Trigla gurnardus^''^' ^*^: Taraxanthin. Trigla hirundo^^^: Xanthophyll. Zeus faber^^"^: Xanthophyll. Zoarces viviparus^''^' ^^^: Carotene, xan- thophyll, taraxanthin. Carotenoids 7 DISTRIBUTION IN NATURE IX d) Amphibia TABLE 27 (References see p. 99-107) CAROTENOIDS IN AMPHIBIA Batrachier species*"^. Bombinator igneus*'^*: Lacertofulvin (xanthophyll?). Bufo calamita'^^^' *": Xanthophyll. Bufo calamita (ovaries) ; Xantho- phyll(?)*03. Bufo viridis*°^. Bufo vulgaris'^^^ . Frogs (retina)**"*; Carotenoids of un- known constitution. Frogs (retina, fat tissues, skin)*"i.- xan- thophyll(?). Hyla arborea and Rana esculenta'^^^. Rana esculenta (skin, ovaries, fat tis- sues)*"® .• Carotene, xanthophyll, zea- xanthin. Rana esculenta (liver) .• a-Carotene, )3-ca- rotene, xanthophyll, zeaxanthin*"®. Rana temporaria, rana esculenta, rana bufo (skin, liver, ovaries, ovarian ducts, eggs, fat tissues, kidneys, tes- tes)'*°''' *"8: Carotene, xanthophyll. Salamandra maculosa^'^: Xantho- phyll(?). Triton cristatus*°^. Triton cristatus (fat tissues)*"^.' Xantho- phylK?). e) Reptiles Our knowledge of reptile pigments is even more deficient than that of amphibia pigments. The investigations of Kuhne and Krukenberg show that the pigments of snakes are not carotenoids. Salamanders and tortoises, on the other hand, appear to contain carotenoids (Ref. 38 and 39, p. log). TABLE 28 (References see p. 99-107) CAROTENOIDS IN REPTILES Chamaeleon vulgaris^^'^: Lacertofulvin (xanthophyll?). Chry semis scripta elegans [eye)^^^ : y-Ca- rotene; (back).- a-carotene( ?) ; (in- testine) .■ /5-carotene and a phyto- xanthin. Clemmys insculpata (retina)*^" .' Astacene. Lacerta agilis^^^. Lacerta muralis'^^*. Tortoise (blood serum, fat tissues)*"® .• Carotenoids. f) Miscellaneous Alfalfa (after acid treatment )*24; five new carotenoids. Bees wax*^''' *2*: Xanthophyll derivative, xanthophyll, carotene. Carrot leaves^^'^: a-Carotene, ^-carotene, y-carotene. Egg yolk*^^: /S-Carotene, cryptoxanthin, xanthophyll. Elodea canadensis'^'^^' *^^: Carotene, elo- xanthin = xanthophyll epoxide, xanthophyll. Marine so;7*^*: a-Carotene, ^-carotene, xanthophyll. Oil from acacia acimiinata^^": ^-Caro- tene and pigments of unknown con- stitution. REFERENCES TO TABLES 99 Plankton^^^: Carotenoids. Pyvacantha coccinia*''-'^: a-Carotene, /J-ca- rotene, y-carotene, lycopene, xantho- phyll epoxide, xanthophyll (flavo- xanthin). Sardine oil*^^: Carotene, xanthophyll and (sometimes) fucoxanthin. Seaweed*^^: Carotene, xanthophyll. Seatang*'^^: Carotene. Swanip'^'^^: Carotene, xanthophyll. LITERATURE REFERENCES 1. Wackenroder, cf. p. 128. — p. Kar- RER, O. Walker, Helv. Chim. Acta 16 (1933) 641. — P. Karrer, K. Schopp and R. More, Helv. Chim. Acta 15 (1932) II58. D. VAN Stolk, J. GuiL- BERG and H. Penau, Chimie & Indus- trie 2y (1932) 550. — R. KuHN and E. 'L'E.T)-ERE'R,N aturwissenschaften ig (1931) 306; Ber. 64 (1931) 1349. — R. Kuhn and H. Brockmann, N aturwissenschaf- ten 21 (1933) 44; Ber. 66 (i933) 407- — H. V. Euler and E. Nordenson, Z. physiol. Chem. 56 (1908) 223, etc. 2. Wakemann, Am. J. Pharm. 23 (1935) 873. 3. R. Kuhn, A. Winterstein and H. Roth, Ber. 64 (1931) 333. — C. Lie- bermann, Ber. 44 (191 1) 850. 4. J. Formanek, /. prakt. Chem. 62 (1900) 310. 5. L. S. Palmer, Carotenoids and related Pigments, New York 1922. 6. M. W. LuBiMENKO, Rev. gen. botan. 25 (1914) 475. — H. Kylin, Z. physiol. Chem. 163 (1927) 229. 7. L. S. Palmer, Carotenoids and related Pigments, New York 1922. 8. L. S. Palmer, cf. footnote 7. — J. C. Lanzing and A. G. van Veen, Chem. Centr. 1938, I, 2081. 9. L. ScHMiD and R. Lang, Monatsh. 72 (1939) 322. 10. J.M. Petrie, BiocAem. J. 18 (1924) 957. 11. J. C. Miller and H. M. Corington, Proc. Am. Soc. Hort. Sci. 40 (1942) 519. 12. T. Tammes, Flora 87 (1900) 205. 13. CouRCHET, Ann. sci. nat. Botan. Ser. VII, 7 (1888) 263. 14. H. Kylin, Z. physiol. Chem. 163 (1927) 229. 15. F. G. Kohl, Untersuchungen iiber das Carotin und seine physiologische Bedeu- tunginden Pfianzen, 1-165, Berlin 1902. 16. C. VAN WissELiNGH, Flora 107 (1914) 371- 17- 19- 22. 23- 24. 25- 26. 27. 28. 29. 30- 31- 32. 33- 34- 35- 36. 37- A. Hansen, Dissertation Wiirzburg, Botan. Centr. 20 (1884) 36. P. Karrer and A.* Oswald, Helv. Chim. Acta 18 (1935) 1303. W. Gugelmann, Dissertation Ziirich 1938; G. Tappi and P. Karrer, Helv. Chim.. Acta 32 (1949) 50. BiDGOOD, J. Roy. Hort. Soc. 29 (1905) 463. — C. A. ScHUNCK, Proc. Roy. Soc. (London) 72 (1903) 165. BiDGOOD, /. Roy. Hort. Soc. 2g (1905) 463- R. Kuhn and A. Winterstein, Natur- wissenschaften 18 (1930) 754. BiDGOOD, /. Roy. Hort. Soc. 2g (1905) 463- W. F. O'Connor, P. J. Drumm, Nature (London) 147 (1941) 58. R. Kuhn, A. Winterstein and W. WiEGAND, Helv. Chim. Acta 11 (1928) 716. R. Kuhn and A. Winterstein, Natur- wissenschaften 18 (1930) 754. Wehmer, Pflanzenstoffe (1931) 2nd Ed., Vol. II, p. 957. C. A. ScHUNCK, Proc. Roy. Soc. (Lon- don), 72 (1903) 165. R. Kuhn and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. P. Karrer, E. Jucker, J. Rutsch- mann and K. Steinlin, Helv. Chim. Ada 28 (1945) 1 146. P. Karrer and A. Notthafft, Helv. Chim. Acta 15 (1932) 1195. H. H. Escher, Helv. Chim. Acta ir (1928) 752. P. Karrer and E. Jucker, Helv. Chim. Acta 2g (1946) 1539- H. Schrotter-Kristelli, Botan. Centr. 61 (1895) 33. L. DippEL, Flora 61 (1878) 17. H. H. Strain, /. Biol. Chem. 123 (1938) 425- H. ScHMALFUSS, Z. physiol. Chem. 131 (1923) 166. — H. ScHMALFUSS and K. DISTRIBUTION IN NATURE IX Keitel, Z. physiol. Chem. 138 (1924) 156. 38. A. TscHiRCH, Ber. deut. botan. Ges. 22 (1904) 414. 39. T. Ito, H. Suginome, K. Ueno and Sh. Watanabe, Bull. Soc. Chem. Japan 11 (1936) 770. 40. R. KuHN, A. WiNTERSTEiN and E. Lederer, Z. physiol. Chem. igy (1931) 141. 41. K. ScHON, Biochem. J. 30 (1936) i960. 42. P. Karrer, E. Jucker and E. Krause- VoiTH, Helv. Chim. Acta 30 (1947) 53S. 43. P. Karrer and E. Jucker, Helv. Chim. Acta 2y (1944) 1585- 44. A. G. Perkin, /. Chem. Soc. loi (1912) 1538. 45. R. KuHN and E. Lederer, Z. physiol. Chem. 213 (1932) 188. 46. H. MoLiscH, Ber. deut. botan. Ges. 14 (1896) 18. 47. R. KuHN and A. Winterstein, Ber. 64 (1931) 326. 48. P. Karrer and J. Rutschmann, Helv. Chim. Acta 2j (1944) 1684; 25 (1942) 1624. 49. E. G. Hill and A. P. Sikkar, /. Chem. Soc. gi (1907) 1501- 50. R. KuHN and A. Winterstein, Helv. Chim. Acta 12 (1929) 899. 51. Keegan, C/zem. iVezws JJJ (1916) 85,114. 52. M. E. FiLHOL, Compt. rend. 39 (1854) 194- 53. L. Zechmeister and W. A. Schroe- DER, Arch. Biochem. i (1942) 231. 54. W. A. ScHROEDER, /. Am. Chem. Soc. 64 (1942) 2510. 55. L. ScHMiD and E. Kotter, Monatsh. 59 (1932) 341- 56. L. Zechmeister, T. Beres and E. UjHELYi, Ber. 68 (i935) 1321 ; 69 (1936) 573- 57. G. and F. Tobler, Ber. deut. botan. Ges. 28 (1910) 365. 58. L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. 208 (1932) 27. 59. H. C. SoRBY. Proc. Roy. Soc. (London) 21 (1873) 442. 60. P. Karrer and E. Jucker, Helv. Chim. Acta 26 (1943) 626. — Cf. P. Karrer and co-workers, Helv. Chim. Acta 28 (1935) II55- 61. K. Schon, Biochem. J. 32 (1938) 1566. 62. L. Zechmeister and W. A. Schroe- der, J. Am. Chem. Soc. 65 (1943) i535- 63. L. Zechmeister and P. Tuzson, Ber. 63 (1930) 3203; 67 (1934) 170- 64. P. Karrer, E. Jucker and K. Stein- LiN, Helv. Chim. Acta 30 (1947) 531. 65. P. Karrer and H. Salomon, Helv. Chim. Acta 13 (1930) 1063. 66. R. Kuhn and E. Lederer, Z. physiol. Chem. 200 (1931) 108. 67. P. Karrer and J. Rutschmann, Helv. Chim. Acta 25 (1942) 1144. 68. P. Karrer and R. More, Helv. Chim. Acta 15 (1932) 863. 69. L. Zechmeister and A. PolgAr, /. Biol. Chem. 140 (1941) i. 70. R. Kuhn and H. Brockmann, Ber. 66 (1933) 828. Who also give references to the earlier literature. 71. L. S. Palmer and H. L. Kempster, /. Biol. Chem. 39 (1919) 299, 331. 72. G. Mackinney, Plant Physiol. 10 (1935) 365. 73. O. C. M agist AD, Plant Physiol. 10 (1935) 187. 74. L. ScHMiD and A. Polaczek-Wittek, Mikrochem. 27 (1939) 42. 75. P. Karrer, H. Salomon and H. WEHRLi,His/y. Chim. Acta 12 (1929) 790. 76. J. W. White, F. P. Zscheile and A. M. Brunson, /. Am. Chem. Soc. 64 (1942) 2603. 77. R. Kuhn and Ch. Grundmann, Ber. 67 (1934) 593- 78. B. Sullivan, C. H. Bailey, /. Am. Chem. Soc. 58 (1936) 383. 79. H. V. EuLER and M. Malmberg, Chem. 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Zool. 32, A (1939) No. 8. 38. W. KuHNE, /. Physiol. (London) i (1878) 109. — C. F. W. Krukenberg, cf. Physiol. Stud. 1882 Ser. II part 2, 50. 39. C. F. W. Krukenberg, loc. cit. — W. D. Halliburton, /. Physiol. 7 (1886) 324, SPECIAL PART CHAPTER X Carotenoid hydrocarbons of known constitution I. LYCOPENE C^qH^q History 1873 Hartsen^ isolates a dark-red crystalline pigment, later identified as lycopene, from Tamus communis L. 1875 MiLLARDET^ obtains impure lycopene, which he terms solanorubin, from tomatoes. 1903 ScHUNCK^ shows that the pigment from tomatoes, which he terms lyco- pene, has an absorption spectrum different from that of carotene. 1910 WiLLSTATTER and EscHER^ make a detailed investigation of lycopene. They determine the correct molecular formula C^oHgg, and recognise that lycopene is an isomer of carotene. 1928-31 Karrer and co-workers^ elucidate the constitution of lycopene. 1932 KuHN and Grundmann^ carry out the chromic acid oxidation of lyco- pene and obtain long-chain degradation products, the constitution of which confirms the formula of lycopene. Occurrence Recent investigations employing the highly refined chromatographic method of separation have shown that the tomato pigment is much more widely distributed in nature than was formerly believed. The frequent occurrence of lycopene in ripe fruit is especially striking (cf. p. 119 concerning the formation of the pigment during the ripening process). Lycopene is also found in other parts of plants and in animal sources, though often only in small quantities. TABLE 29 VEGETABLE AND ANIMAL SOURCES FROM WHICH LYCOPENE HAS BEEN ISOLATED Source ^ References a) Fruit of: Actinophloeus Macarthurii J. Zimmerman, Rec. Trav. chini. 51 (1932) 1001. Bryonia dioica Jacq. A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 207 (1932) 25, 32. References p. 165-ijo. Carotenoids 8 114 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Source Citrullus vulgaris Schrd. Citrus deciiniana L. Citrus grandis Osb. Convallaria majalis L. Diospyros cost at a Diospyros Kaki L. Erythroxylon novogranatense Palm oil Passi flora coerulea Prunus armeniaca L. Ptychospernia elegans Rosa canina Rosa riibiginosa Rosa rugosa Thumb. Rubits Chaniaemorus Solanum Balbisii L. Solanum Dtilcaniara L. Solanum Lycopersicum Tamus communis L. References L. Zechmeister and P. Tuzson, Ber. 63 (1931) 2881. — L. Zechmeister and A. Polgar, /. biol. Chem. 139 (1941) 193. M. B. Matlack, Chem. Centr. 1928, I, 2948. M. B. Matlack, Chem. Centr. 1935, I, 95; /. biol. Chem. 110 (1935) 249. A. WiNTERSTEiN and U. Ehrenberg, Z. physiol. Chem. 207 (1932) 25, 32. K. ScHON, Biochem. J. 29 (1935) 1779. P. Karrer and co-workers, Helv. Chim. Acta 15 (1932) 490. J. Zimmerman, Rec. Trav. chim. 51 (1932) 1001. R. F. Hunter and A. D. Scott, Biochem. J. 35 (1941) 31. P. Karrer and co-workers, Helv. Chim. Acta 19 (1936) 28. H. Brockmann, Z. physiol. Chem. 216 (1933) 47. J". Zimmerman, Rec. Trav. chim. 51 (1932) 1001. H. H. Escher, Helv. Chim. Acta 11 (1928) 753. — P. Karrer and R. Widmer, Helv. Chim. Acta 11 (1928) 751. R. KuHN and C. Grundmann, Ber. 67 (1934) 339. H. Willstaedt, Chem. Centr. 1935, II, 707; Svensk Kem. Tidskr. 47 (1935) 112. H. Willstaedt, Skand. Arch. Physiol. 75 (1936) 155. H. Kylin, Z. physiol. Chem. 163 (1927) 229. — L. Zechmeister and L. v. Cholnoky, Ber. 63 (1930) 787. do. C. A. ScHUNCK, Proc. Roy. Soc. (London) 72 (1903) 165. — R. Willstatter and H. H. Escher, Z. physiol. Chem. 64 (1910) 47. — R. Kuhn and C. Grundmann, Ber. 65 a 932) 1886. — H. v. Euler, P. Karrer, E. v. Krauss and O. Walker, Helv. Chim. Acta 14 (1931) 154. L. Zechmeister and L. v. Cholnoky, Ber. 63 (1930) 423. b) Other vegetable and animal sources: Bacillus Grasberger E. Chargaff and E. Lederer, Chem. Centr. 1936, I, 3159. Bacillus Lombardo Pellegrini E. Chargaff and E. Lederer, Chem. Centr. 1936, I, 3159. V. Reader, Biochem. J. 19 (192G) 1039. L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. 208 (1932) 28. — P. Karrer and A. Notthaft, Helv. Chim. Acta 15 (1932) 1196. P. Karrer and co-workers, Helv. Chim. Acta 26 (1943) 2121. Bad. Sarcina aurantica Calendula officinalis Chara (Anther) LYCOPENE 115 Source Cuscuta salina Cuscuta subinclusa Diniorphotheca aurantiaca Gazania rigens Gonocaryum ohovatuni and Gonocaryum pyri forme Human liver Mimulus longiflorus Saffron Thiocystis-hdiCteridi Vicia References G. Mackinney, /. biol. Chem. 112 (1935/36) 421. G. Mackinney, /. biol. Chem. 112 (1935/36) 421. P. Karrer and A. Notthafft, Helv. Chim. Acta 15 (1932) 1196. L. Zechmeister and W. A. Schroeder, /. Am. Chem. Soc. 65 (1943) 1535. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. — A. WiNTERSTEiN, Z. physiol. Chem. 215 (1933) 51 ; 219 (1933) 249. L. Zechmeister and P. Tuz.son, Z. physiol. Chem. 234 (1935) 241. — H. Willstaedt and T. Lindqvist, Z. physiol. Chem. 240 (1936) 10. L. Zechmeister and W. A. Schroeder, Arch. Biochem. 1 (1943) 231. R. KuHN and A. Winterstein, Ber. 67 (1934) 344. P. Karrer and U. Solmssen, Helv. Chim. Acta 19 (1936) 1019; 18 (1935) 25. P. Karrer and A. Notthafft, Helv. Chim. Acta 15 (1932) 1196. vegetable and animal Source a) Fruit of: A ctinophloens angustifolia Aglaonema nitidwtn Aglaonema oblongifolium Aglaonema oblongifolium, Var. Curtisii Aglaonema simplex Arbutus Unedo Archontophoenix Alexandrae Areca Alicae Arum italicum Arutn maciilatum Arum orientate Calyprocalyx spicatus Citrus aurantiiim ( ?) Cranberries Elaeis giiineensis ( ?) Elaeis melanococca Erythroxylon coca TABLE 30 sources in which LYCOPENE HAS BEEN DETECTED References V. N. LuBiMENKO, Rev. gen. botan. 23 (1914) 474. V. X. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKo, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. K. ScHON, Biochem. J. 2g (1935) 1779. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LUBIMENKO, Chem. Abstracts 14 (1920) 1697. H. Kylin, Z. physiol. Chem. 163 (1927) 229. V. M. LuBiMENKo, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKO, Rev. gen. botan. 23 (1914) 474. L. Zechmeister and P. Tuzson, N aturwissenschaften 19 (1931) 307. H. Willstaedt, Chem. Centr. 1937, I 3658. A. H. Gill, /. Ind. Eng. Chem. 9 (1917) 136; 10 (1918) 612. A. H. Gill, /. Ind. Eng. Chem. 9 (1917) 136; 10 (1918) 612. V. M. LuBiMENKO, Rev. gen. botan. 23 (1914) 474. ii6 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Source Evonymus japonicus (Arillus) Momordica Balsamina (Arillus) Momordica Charantia (Arillus) Nertera depressa Palm oil Pandanus polycephahis Solanum decasepalum Synaspadix petrichiana Tabernaemontana penta- sticta Taxus baccata Trichosanihes-species References V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. G. and F. Tobler, Ber. deutsch. botan. Ges. 28 (1910) 365, 496. — B. M. Duggar, Washington Univ. Stud, i (1913) 22. G. and F. Tobler, Ber. deutsch. botan. Ges. 28 (1910) 365, 496. — B. M. Duggar, Washington Univ. Stud. 1 (1913) 22. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. P. Karrer, H. v. Euler and H. Hellstrom, Chem. Centr. 1932, I, 1800. — R. Kuhn and H. Brockmann, Z. physiol. Chem. 200 (1931) 255. V. N. LUBIMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LuBiMENKO, Rev. gen. botan. 25 (1914) 474. V. N. LUBIMENKO, Rev. gen. botan. 23 (1914) 474. R. Kuhn and H. Brockmann, Chem. Centr. 1933, II, 553. N. A. Monteverde and N. V. Lubimenko, Bull. Acad. Sci. Petrograd. Ser. 6, 7, II, 1105. b) Other vegetable and animal sources: Butter Cantharellus cibarius Cantharellus httescens Cantharellus infundib ili- formis Coccinella septem punctata Human serum Vaccinium vitis idaea A. E. Gillam and I. M. Heilbron, Biochcm. J. 2g (1935) 834. H. WiLLSTAEDT, Chem. Centr. 1938, II, 2272. H. WiLLSTAEDT, Chem. Centr. 1938, II, 2272. H. WiLLSTAEDT, Chem. Centr. 1938, II, 2272. E. Lederer, Chem. C^ntr. 1936, I, 3853. E. V. Daniel and G. J. Scheff, Proc. Soc. Exp. Biol. Med. 33 (1935) 26. — E. v. Daniel and T. Beres, Z. physiol. Chem. 238 (1936) 160. H. WiLLSTAEDT, Svcnsk. Chem. Tid. 48 (1936) 212. Isolation For the isolation of lycopene, it is convenient to start from tomato pre- serves rather than from the fresh fruit which contain about 97 % water . WiLLSTATTER and Escher' worked up 75 kg of "Puree di pomidori con- centrata" and obtained 11 g of once recrystallised lycopene. The preserves are shaken in bottles in portions of about 8 kg with 4 1 (or preferably more) 95% ethanol. The mixture is then pressed through a fine cloth under slight pressure. The operation is repeatedj|iLising about 3 1 of ethanol. The red References p. 165-iyo. I LYCOPENE 117 residue is dried at 40-50°, finely ground and continuously extracted with carbon disulphide. The solvent is removed by distillation, finally under reduced pressure at 40° in an atmosphere of dry carbon dioxide, which is passed in through a capillary. The dark reddish brown paste which remains is diluted with 3 1 ethanol when crystallisation immediately sets in. After a short time the crystalline mass is filtered and the residue washed with cold petroleum ether. The crude lycopene can be purified either by dissolution in carbon disulphide and precipitation with ethanol, or bv recrystalUsation from a large volume of petroleum ether (b.p. 50-80°, 4-5 1 per 1 g of lycopene). For analysis, the material is first crystallised from petroleum ether, any sparingly soluble components being rejected, and then recrystallised from carbon disulphide, or a mixture of carbon disulphide and ethanol, without further fractionation. Using this procedure, 11 g of once recr\'stallised lycopene can be obtained from 75 kg of preserves, whereas Willstatter and Escher obtained 2.7 g of pigment from 135 kg of fresh tomatoes. Thus 1 kg of fresh fruit yields 0.02 g of lycopene, while 1 kg of preserves yields 0.15 g of lycopene. Some commercial preserves contain acidic materials which have a deleterious effect during the iso- lation of Ivcopene. By addition of potassium carbonate to the tomato puree, Kuhn and Grundmann^ were able to improve the yield of lycopene considerably. From 1 kg of preserves they obtained 0.2G5 g of pigment. According to Zechmeister and vox Cholxoky^, lycopene can also be isolated from Tamils communis berries. Chemical Constitution CH, CH, CH3 CH3 C ' CH3 CH3 CH3 CH3 c y I I I I \ •CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CHj C-CH3 H3C-C CHj \^ / Lycopene \ y^ CH2 CH2 Willstatter and Escher^*' recognised that lycopene is an isomer of caro- tene and determined the correct empirical formula, C40H56. The constitution of the pigment was elucidated by Karrer and his co-workers^. The results of these investigations can be briefly summarised as follows. The deep red colour of lycopene suggested the presence of numerous double bonds. Karrer and Rose Widmer^^ found that 13 mols of hydrogen were absorbed during the catalytic hydrogenation of the pigment. The empirical formula of perhydrolycopene was C40H82, which showed that lycopene must have an open-chain structure (cf. the corresponding discussion of carotene). According to Pummerer, Rebmann and Reindel^^^ lycopene absorbs 13 mols of iodine chloride, thus confirming the presence of thirteen ethylenic bonds. Karrer and Bachmann^^ ^nd Karrer, Helfenstein, Pieper and Wettstein^* found that ozonisation affords considerable quantities of acetone. One mol of the pigment gave 1.6 mols of ketone, suggesting the presence of References p. id^-iyo. ii8 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X 2 zsopropylidene groupings (CH3)2C = . Ozonisation also furnished succinic acid, but no higher fatty acids were obtained. The succinic acid is derived from the ^^°^P^"^ =CH.CH,CH,CH = - By means of oxidative degradation of lycopene with permanganate and chromic acid, Karrer, Helfenstein, Wehrli and Wettstein^^ proved the presence of 6 side-chain methyl groups. Finally, Karrer, Helfenstein and WidmerI' synthesized perhydrolycopene from dihydrophytol and showed that it was identical in all respects with the product obtained by the catalytic hydrogenation of lycopene. As the result of these investigations the formula for lycopene shown above was put forward by Karrer, Helfenstein, Pieper and Wettstein^^. This formula has been confirmed by later investigations by Kuhn and co- workers. KuHN and Winterstein^^ isolated toluene and m-xylene from the products of the thermal decomposition of the pigment and Kuhn and Grund- mann^'' obtained lycopenal, a complex degradation product, together with methyl heptenone from the oxidation of the pigment with chromic acid. Further oxidation of lycopenal with chromic acid gave bixin dialdehyde and methyl heptenone. The structure of lycopenal was established by the conversion of bixin dialdehyde into norboxin, the constitution of which had been elucidated earlier by Karrer and co-workers. CH, CH, CH, - CH, C CHo Clio CHo CH3 C y I I I I V CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CH2 C*CH3 H3C"C CHg \ / Lycopene \^ / CH2 CH2 CHo CHo CHo CHo 0 Cxin CHo OHo OHo \j^ / I I I I V CH OHC-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CHj C=0 HjC-C CH2 \/^| . Lycopenal \^y HgC CH3 CHj Methylheptenone CHo CHo \V CH3 CH3 CH, CH3 C, I II I V OHC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- CHO CH 0=C CHj Bixin dialdehyde l\/ H3C CHg References p. 165-iyo. Methylheptenone I LYCOPENE 119 NHaOH CH3 CH3 CH3 CH3 HON=CH- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- CH=NOH Bixindialdehyde dioxime Dehydration CH3 CH3 CH3 CH3 I I I I NC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- CN Hydrolysis CH3 CH3 CH3 CH3 I I I I HOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- COOH Norbixin The observation of Strain^i, ^j^^^ ^j^g ozonisation of lycopene also gives rise to levulinic aldehyde and levulinic acid, is also in accord with the formula given above. CHo CHo c / CH CH--- 1 II CHj C-CH3 CH2 r\iir\ CHj CO \/\ CHj CH3 Formation Numerous early investigations deal with the formation of carotenoids in the plant during the ripening process. Thus Duggar^^ showed in 1913 that the red tomato pigment is no longer formed at temperatures above 30°, a yellow pigment, probably a flavone or fiavanol, being produced instead. The agents responsible for the formation of lycopene are not, however, destroyed at 30° since yellow tomatoes ripened at 30° again acquire a red colour due to lycopene on being restored to a lower temperature. Some authors consider light to be a necessary agent for the ripening process^^ ; according to Duggar, however, the ripening process is indepen- dent of light but requires the presence of oxygen. The investigations of Duggar were repeated and confirmed by Karrer and co-workers^*. KuHN and Grundmann^s, in 1932, examined the tomato pigment at different stages of ripening by means of adsorption analysis. They obtained the following figures for fresh fruit grown in the open. References p. 165-iyo. I20 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X TABLE 31 COMPOSITION OF THE TOMATO PIGMENTS (kUHN AND GRUNDMANN) Mg of pigment in 100 g of fresh fruit green half ripened fully ripened Lycopene /3-Carotene (isolated) Xanthophylls, free Xanthophylls, esterified 0.11 0.16 0.02 0.00 0.84 0.43 0.03 0.02 7.85 0.73 0.06 0.10 Properties and physical constants Crystalline form: Lycopene crystallises from a mixture of carbon disulphide and ethanol in long red needles. From petroleum ether it crystallises in charac- teristic felted hair-like needles, or occasionally in long dark red-violet prisms. In powder form, it is a dark reddish-brown. In contrast to most carotenoids, crystals of lycopene show little metallic lustre. (For X-ray diffraction pattern, see Mackinney^*'.) Melting point: 170° (uncorr.)^?; 173° (uncorr.)28; 174° (corr.)^^; 175° (corr.)^*'. Solubility: Ethanol, cold almost insoluble Ethanol, hot very sparingly soluble Methanol almost insoluble Benzene, cold fairly soluble Benzene, hot very soluble Chloroform, cold easily soluble Chloroform, hot very easily soluble Carbon disulphide very easily soluble 1 g of lycopene is soluble in 50 ml of cold carbon disulphide, 3 1 of boiling ether, 10-12 1 of boiling petroleum ether, or 14 1 of hexane of 0°3i. Spectral properties: Solvent: Absorption maxima: Carbon disulphide 548 507.5 477 m/^ Chloroform 517 480 453 m/i Benzene 522 487 455 m/t Petroleum ether 506 475.5 447 m/x Ethanol 503 472 443 m^ Hexane 504 472 443 m^ (cf. Fig. 4, p. 349, and Fig. 31, p. 361) Colour of solutions: In carbon disulphide blue with a red tinge In ether (saturated solution) . . . bluish-red In ethanol (hot saturated solution) dark yellow References p. 165-iyo. I LYCOPENE 121 Optical activity: Lycopene is optically inactive. Colour reactions: Lycopene dissolves in concentrated sulphuric acid with an indigo blue colour. With fuming nitric acid, it yields a purple colouration which rapidly disappears^^. On adding a solution of antimony trichloride in chloroform to a solution of lycopene in chloroform, an intense unstable blue colour is produced^^. Partition test: Lycopene is completely epiphasic. Chromatographic behaviour: Lycopene is adsorbed six times more strongly than carotene on alumina from petroleum ether solution. Similar behaviour is shown on adsorption on calcium oxide or calcium hydroxide. Petroleum ether containing a little methanol is used for elution^*. Like other carotenoid hydro- carbons, lycopene is only weakly adsorbed on calcium or zinc carbonate and can thus be separated from phytoxanthins^^. Detection and estimation: After saponification of the whole extract, lycopene, together with the carotenes, is present in the epiphasic fraction. It is best identified by chromatographic separation on calcium hydroxide, followed by a determination of the absorption maxima. CoNNELL^^ recommends a solution of potassium dichromate and cobalt sulphate as standard for colorimetric determinations. According to Kuhn and Brockmann^^, an alcoholic solution of azobenzene can also be employed. Physiological properties: As would be expected from its structure, lycopene possesses no vitamin A potency. Derivatives Perhydrolycopene C^^Hg^ Perhydrolycopene is formed by the catalytic hydrogenation of lycopene^. It has also been synthesized by treating dihydrophytol with phosphorus penta- bromide and heating the dihydrophytyl bromide {i6-bromo-2:6:io:i4:-tetra- methylhexadecane) with potassium at 130-140°^^. Perhydrolycopene is a colourless oil. B.p. 238-24070.3 mm.; 212-214°/0.02 mm". dl^ 0.822 from lycopene, 0.824 from phytol*o, n^^ 1.4560 from lycopene; 1.4567 from phytol. Dehydrolycopene C^qH^2 Lycopene is dehydrogenated by the action of bromsuccinimide (2 mols) and converted into dehydrolycopene (Karrer and Rutschmann*^) : References p. 165-ijo. 122 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X CH, CH, CH, CH3 C CH3 CH3 CH3 CH3 C CH CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CHCH CH CH C-CH3 H3C-C CH \v / Dedydrolycopene \ ^ \h CH Dehydrolycopene contains 15 conjugated double bonds. It can only be crystallised from pyridine, being almost insoluble in most other solvents. The crystals appear dark violet to black in direct light and are slowly discoloured and decomposed on heating in an evacuated tube above 200°, no definite melting point being observed. With a solution of antimony trichloride in chloroform, dehydrolycopene gives a fairly stable blue colouration, the ab- sorption spectrum of which exhibits a sharp band with a maximum at 472 m/i and continuous absorption above 640 m//. Solvent: Absorption maxima: Carbon disulphide 601 557 520 mn Hexane 542 504 476 m// Benzene 570 531 493 mfx Pyridine 574 535 498 m^ Chloroform 567 528 493 m/z Apo-2-lycopenal (lycopenal*) C3,H420: OHC-CH=CH-C=CHCH=CH-C=CHCH=CHCH = C-CH=CHCH=C-CH=CHCH=C-CH2CH2CH=C-CH, II I I I I CH3 CH3 CH3 CH3 CH3 CH3 Lycopenal is obtained by the chromic acid oxidation (3 atoms O) of lyco- pene in a mixture of acetic acid and benzene*-. It crystalUses from a mixture of benzene and ethanol in deep-red plates, melting point 147° (corr.). It is easily soluble in chloroform, carbon disulphide and benzene, but only sparingly in ethanol. It is entirely epiphasic in the partition test. Solvent: Absorption maxima: ,. ' .. Carbon disulphide 569 528.5 493.5 m/t Petrol 525.5 490.5 455.5 m^ Lycopenal is very easily oxidised in the solid state or in solution. On treat- ment with chromic acid (3 atoms O), 2-methylhept-2-en-6-one and apo-2:i2- lycopene dialdehyde (bixin dialdehyde)*^ are formed. With free hydroxylamine, apo-2-lycopenal gives apo-2-lycopenal oxime wich crystallises from pyridine in lustrous blue- violet prisms, 198° (corr., evacuated capillary). The old name for apo-2-lycopenal is lycopenal. References p. 165-iyo. I LYCOPENE 123 Apo-3-lycopenal C30H4PO: OHC- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CHCH=C- CH2CHi,CH=C- CH, 11 I I I I CH3 CH3 CH3 CH3 CH3 CH3 Apo-3-lycopenal was obtained by Karrer and Jaffe*^ by the mild oxid- ation of lycopene with potassium permanganate. It separates from petroleum ether in brown-black crystals, m.p. 138°. Solvent: Absorption maxima: Carbon disulphide 545 508 ca. 478 van Petrol 502 473 m/z Benzene 518 488 m/z Apo-2:i2-lycopenedial (bixindialdehyde) C^JA^^O^'. OHC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- CHO Cri3 ^H3 VXI3 ^^13 Bixindialdehyde is formed by the oxidation of lycopene or lycopenal (apo-2- lycopenal) with chromic acid*^. It crystallises from pyridine in lustrous blue prisms, m.p. 220° (corr.). On heating in air, it decomposes at 180° ^vithout melting. Bixindialdehyde is appreciably soluble in pyridine and chloroform, and sparingly soluble in hot benzene, but only dissolves with great difficulty in petrol, alcohols, carbon disulphide, ether, acetone, or dioxan. Solvent: Absorption maxima: Carbon disulphide 539.5 502 467.5 m/x Petroleum ether 502 468 437.5 m^i Pyridine 534.5 494 m^u Chloroform 528 490 m/i (cf. Fig. 17, p. 355) Bixindialdehyde dioxime crystallises from pyridine in needles which de- compose above 250° without melting*®. It is soluble only in pyridine. Solvent: Absorption maxima: Pyridine 514 482 452 m^ Apo-3:i2-lycopenedial (apo-1-bixindialdehyde) CgaHjeO.^,: OHC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO CH3 CH3 CH3 CH3 Apo-i-bixindialdehyde was obtained by Karrer and Jaffe by the chromic oxidation of lycopene*'. It separates from methanol in dark crystals, m.p. 168° (uncorr.) . References p. 165-iyo. 124 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Solvent: Absorption maxima: Carbon disulphide 517 484 453 m/z Petroleum ether 480 452 m/f With free hydroxylamine, apo-3:i2-lycopenedial forms a dioxime which separates in lustrous red crystals which sinter above 210°. Solvent: Absorption maxima: Carbon disulphide 510 480 m/i Ethanol 481 449 m/i Neo-lycopene C^qH^^: In the presence of small quantities of iodine, on standing at elevated temperatures, or merely on standing in solution at room temperature for i or 2 days, lycopene is partly converted into an isomer, neolycopene (Zechmeister and TuzsoN*^) . It has not yet been possible to obtain this pigment in a crystal- line state. During chromatography on calcium hydroxide it gives rise to a brown-red loosely adsorbed zone below that of lycopene. A cis-trans change appears to be involved in this isomerisation. Solvent: Absorption maxima: Carbon disulphide 536 498 466 mfx Benzene 512 479 450 m// Chloroform 512 478 447.5 m/i Acetone 499.5 468 439 m/i Petroleum ether 499.5 468 439 m/z Ethanol 500 469 439 m/i Neolycopene is more easily soluble in organic solvents than lycopene. Lycopersene C4QH66: CH, CH, CH, CH3 C CHo CHo CHq CHo Kj / I I I I V CH CH-CH2CH2C=CHCH2CH2C=CH-CH2CH2CH=C-CH2CH2CH=C-CH2CH2CH CH CH2 C-CHa HsC-C CH2 \ / Lycopersene \^ CH2 ' CHj Lycopersene has been prepared by Karrer and Kramer*^ by reacting geranyl-geranyl bromide with sodium, a method analogous to that employed in the synthesis of squalene^". Lycopersene is a rather viscous oil, which can be distilled at 0.02 mm pressure in an air bath at 225-228°. It is a colourless compound. It absorbs 8 molecules of hydrogen chloride, forming a crystalline octa-hydrochloride C40H74CI8, which after recrystallisation from acetone melts at 126°. References p. 16^-170. 2 PROLYCOPENE 125 2. PROLYCOPENE C4oH5e In 1941, Zechmeister and collaborators^^ discovered a new polyene pigment in the "tangerine tomato", a variety of lycopersicum escuUntum. They proposed the name prolycopene for this new pigment which has also been found in several other plants including Butia capitata^'^, Butia eriospatha Becc.^^, Pyrocantha angustifoUa^^ , Evonymus fortunei^^ and Mimulus longiflorus Grant^^. According to Zechmeister and collaborators^^'^^ prolycopene is to be regarded as a naturally occurring stereoisomer of lycopene. The chromophoric system of this pigment is assumed to contain 5 to 7 czs-double bonds in contrast to lycopene which is believed to have an exclusively ^raws-arrangement of double bonds. Under the catalytic influence of iodine, prolycopene is converted into a complicated mixture of stereoisomers which includes the natural [trans)- lycopene. Prolycopene crystallises from petroleum ether or ethanol in plates, m.p. 111°. In other solvents, this pigment is more easily soluble than lycopene. On chromatography on calcium hydroxide it gives rise to a zone below that of lycopene. Solvent: Absoyption maxima: Carbon disulphide 500.5 469.5 m^ Benzene 485 455.5 m[x Chloroform 484 453.5 m/t Ethanol (471) (445) m^ Petroleum ether 470 443.5 m^ Zechmeister and Pinckard^' discovered 6 new lycopene isomers, believed to contain 4 to 7 cis-double bonds, in ripe berries oipyracantha angnstifolia (Schneid). They were designated according to decreasing strength of adsorption as poly-cfs- lycopenes I-VI. Three of these compounds could be crystallised. M.p. Absorption nia.xim,a in hexane solution Poly-cis-h'-copene I . . . . 93-95° 444-445 m/i Poly-cis-lycopene II . . . 85-87° 441-442 m/z Poly-cis-lycopene III . . . 105-106° 443-446 m^ Poly-cis-lycopene IV 426 m/^ Poly-cis-lycopene V 431-432 m/z Poly-cis-lycopene VI 433 m^ References p. 16^-iyo. 126 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X 3. ^-CAROTENE C^pHge History 1831 Wackenroder discovers carotene* in the roots of the carrot {Daucus Carota) . 1847 Zeise^^ describes the new pigment in more detail and determines the empirical formula CgHg. 1866 Arnaud^^ establishes that carotene is a hydrocarbon. 1907 Willstatter and Mieg®" prove the identity of carotene from leaves and carrots. They establish the correct molecular formula C4oH5g. 1928 Zechmeister, von Cholnoky and Vrabely establish the presence of II double bonds and 2 ring systems in carotene^^. 1929-31 Karrer and collaborators®^ elucidate the constitution of ^-carotene. 1932-35 KuHN and Brockmann®^ carry out extensive investigations on j3-carotene and obtain long-chain degradation products which confirm the formula assigned to /3-carotene. Occurrence |5-Carotene is very widely distributed in nature. All green parts of plants (leaves®*, stalks, etc.) contain this pigment which invariably accompanies chlorophyll together with xanthophyll, xanthophyll epoxide and frequently a-carotene®^. Autumnal leaves also contain ^-carotene®®. In fact, numerous investigations have shown that ^-carotene is to be found almost throughout the whole of the vegetable and animal kingdoms. The following summary will give some in- dication of the variety of j8-carotene sources. TABLE 32 VEGETABLE SOURCES FROM WHICH /3-CAROTENE HAS BEEN ISOLATED Source References a) Fruit: Arbutus K. Schon, Bwcheni. J. 2g (1935) 1779. Capsicum frutescens jap. [skin)!^. Zechmeister and L. v. Cholnoky, Ann. 489 (1931) 1. Capsicum japonicum (skin) L. Zechmeister and L. v. Cholnoky, Ann. 454 (1927) 54; 455 (1927) 70; 509 (1934) 269. Wackenroder, Geigers Magazin Pharm. 33 (1831) 141. For nearly 100 years, caro- tene as isolated from the carrot was regarded as a homogenous compound. It was only by means of modern chemical and physical methods of separation (particularly chromato- graphy) that the complex nature of carotene was established. It was recognised by several investigators simultaneously that the carrot pigment is a mixture of several isomers in which ^-carotene predominates. Investigations in which several times recrystallised caro- tene were employed therefore relate to materials consisting mainly of ^-carotene. References p. id^-i^o. ^-CAROTENE 127 Source Citrullus vulgaris Schrad. Citrus aurantiuni Risso. Citrus niadurensis Lour. Citrus poonensis hort. Convallaria majalis Cucurbita maxima Duch. Diospyros costata Gonocaryum pyriforme (skin) Mangifera indica Pirus aucuparia Prunus armeniaca Rosa canina Rosa daniasccna Rosa rubiginosa Solanum Lycopersicuni Taxiis baccata b) Blossoms: Acacia decurrens Acacia discolour, Acacia linifolia, Acacia longi folia Calendula officinalis Caltha palustris Gazania rigens Genista tridentata Kerria japonica DC Laburnum anagyroides Ranunculus Sarothamnus scoparius Tragopogon pvatensis References L. Zechmeister and P. Tuzson, Ber. 63 (1930) 2883. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 221 (1934) 279. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 221 (1934) 279. R. Yamamoto and S. Tin, Chem. Centr. 1934, I, 1660. A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 207 (1932) 31. H. Suginome and K. Ueno, Chem. Centr. ig3i, II, 2892. — L. Zechmeister and P. Tuzson, Ber. 67 (1934) 824. K. ScHON, Biochem. J. 29 (1935) 1779. A. Winterstein, Z. physiol. Chem. 21 ^ (1933) 52; 2ig (1933) 249. R. Yamamoto, Y. Osima and T. Goma, Chem. Centr. 1933, I, 441. R. KuHN and E. Lederer, Ber. 64 (1931) 1354. H. Brockmann, Z. physiol. Chem. 216 (1933) 45. R. Kuhn and C. Grundmann, Ber. 6y (1934) 341. R. Kuhn and C. Grundmann, Ber. 6y (1934) 341. R. Kuhn and C. Grundmann, Ber. 67 (1934) 341. R. Willstatter and H. H. Escher, Z. physiol. Chem. 6^(1910) 49. R. Kuhn and H. Brockmann, Ber. 66 (1933) 834. J. M. Petrie, Biochem. J. 18 (1924) 957. do. L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. 208 (1932) 29. P. Karrer and A. Notthafft, Helv. chim. Acta 1$ (1932) 1195. K. Schon, Biochem. J. 32 (1938) 1566. — L. Zech- meister and W. A. Schroeder, /. Am. Chem. Soc. 63 (1943) 1535. K. Schon and B. Mesquita, Biochem. J. 30 (1936) 1966. P. Karrer and E. Jucker, Helv. chim. Acta 29 (1946) 1539. do. P. Karrer and A. Notthofft, Helv. chim. Acta 15 (1932) 1195. — P. Karrer, E. Jucker, J. Rutsch- mann and K. Steinlin, Helv. chim. Acta 28 (1945) 1146. P. Karrer and E. Jucker, Helv. chim. Acta 27 (1944) 1585. see Ranunculus . 128 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Source Trollius enropaeus Ulex europaeus Ulex Gallii References P. Karrer and E. Jucker, Helv. chim. Acta 2g (1946) 1539. K. ScHON, Biochem. J. 30 (1936) 1960. do. c) Other vegetable soui Oil from Acacia acuminaia Aphanizomenon jlosaquae Bacillus Grasberger Bacillus Lonibardo Pellegrini Brown algae Cladophora Sauteri Crocus sativus Cuscuta salina Cuscuta subinclusa Diatomea Fucus vesiculosus Haematococcus pluvialis Lycogala epidendro7i Nitella opaca Oedogonium Palm oil Rhodymenia palmata Torula rubra Trentepohlia aurea Yellow maize T. M. Trikoyus and J. C. Drummond, Nature ijg (1937) 1105. J. TisCHER, Z. physiol. Chem. 2^1 (1938) 109. E. Chargaff and E. Lederer, Chem. Centr. 1936, I, 3159. do. H. Kylin, Z. Physiol. Chem. 82 (1912) 224. — R. Willstatter and H. J. Page, Ann. 404 (1914) 251; P. W. Carter, L. C. Cross, I. M. Heilbron and E. R. H. Jones, Biochem. J. 43 (1948) 349. I. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 2g (1935) 1376. R. KuHN and A. Winterstein, Ber. 6y (1934) 349. G. Mackinney, /. biol. Chem. 112 (1935) 421. do. F. G. Kohl, Chem. Centr. igo6, I, 1669. I. M. Heilbron and R. F. Phipers, Biochem. J. 2g (1935) 1369. J. TiscHER, Z. physiol. Chem. 250 (1937) 147; 252 (1938) 225. E. Lederer, Chem. Centr. ig3g, I, 2991. I. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 2g (1935) 1376. do. R. Kuhn and H. Brockmann, Z. physiol. Chem. 200 (1931) 255. I. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 2g (1935) 1376. E. Lederer, Compt. rend, igy (1933) 1694. E. Lederer, Chem. Centr. ig3g, I, 2991. R. Kuhn and C. Grundmann, Ber. 6j (1934) 593. Source Bloodserum ^-CAROTENE 129 TABLE 33 OCCURRENCE OF /3-CAROTENE IN THE ANIMAL ORGANISM References H. V. EuLER, B. V. EuLER and H. Hellstrom, Biochem. Z. 202 (1930) 370. — H. Willstaedt and T. LiNDQVisT, Z. physiol. Chem. 240 (1936) 10. Corpus luteum of cows and sheep Corpora rubra of cows Faeces of sheep and cows Fat tissues of mammals Fish roe Gallstones of oxen Human milk Human placenta Integuments of insects Kidneys of mammals Livers of mammals Milk fat Salmon flesh H. H. Escher, Z. physiol. Chem. 83 (1913) 198. — R. KuHN and E. Lederer, Z. physiol. Chem. 200 (1931) 246. — P. Karrer and W. Schlientz, Helv. chim. Acta ly (1934) 55. R. KuHN and H. Brockmann, Z. physiol. Chem. 206 (1932) 41. P. Karrer and A. Helfenstein, Helv. chim. Acta 13 (1930) 86. L. S. Palmer and C. H. Eckles, /. Biol. chem. ly (1914) 211. — H. van den Bergh, P. Muller and J. Broekmeyer, Biochem. Z. 108 (1920) 279. — C. L. Connor, /. biol. Chem. yy (1928) 619. — L. Zech- meister and P. Tuzson, Ber. 6y (1934) 154. H. V. Euler, U. Gard and H. Hellstrom, Svensk. Kem. Tidskr. 44 (1932) 191; Chem. Centr. 1932, I, 1504. G. Fischer and H. Rose, Z. physiol. Chem. 88 (1913) 331. — H. Fischer and R. Hess, Z. physiol. Chem. i8y (1930) 133. L. S. Palmer and C. H. Eckles, /. biol. Chem. ly (1914) 237. R. KuHN and H. Brockmann, Z. physiol. Chem. 206 (1932) 41. E. Lederer, Chem. Centr. 1936, I, 3853. H. van den Bergh, P. Muller and J. Broekmeyer, Biochem. Z. 108 (1920) 279. — O. Bailly, Chem. Centr. 1935, I, 3806. H. V. Euler and E. Virgin, Biochem. Z. 245 (1932) 252. — H. V. Euler and E. Klussmann, Biochem. Z. 2^6 (1932) 11. — H. Willstaedt and T. Lindqvist, Z. physiol. Chem. 240 (1936) 10. A. E. GiLLAM and M. S. El Ridi, Biochem. J. 31 (1937) 251. — L. S. Palmer and C. H. Eckles, /. biol. Chem. ly (1914) 191. H. V. Euler, H. Hellstrom and M. Malmberg, Svensk. Kem. Tidskr. 45 (1933) 151. Carotenoids 9 I30 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X TABLE 34 SOURCES FOR THE PREPARATION OF |8-CAROTENE Yield of carotene Source from 1 kg of material Literature references Carrots 1 g (max.) R. WiLLSTATTER and H. H. Escher, Z. physiol. Chem. 64 (1910) 47. — R. Kuhn and E. Lederer, Ber. 64 (1931) 1349. — H. N. Holmes and H. M. Leicester, /. Am. Chem. Soc. 54 (1932) 716. — N. T. Deleano and J. Dick, Biochem. Z. 23g (1933) 110. Cucurbita maxima Duch. (Fruit) 0.1 g L. Zechmeister and P. Tuzson, Ber. 6y (1934) 824. Palm oil 1.5-2.0 g P. Karrer, H. V, Euler and H. Hell- STROM, Ark. Kemi. B. 10 (1931) No. 15. — 0. Ungnade, Chem. Ztg. 63 (1939) 9. Paprica skin 0.3 g L. Zechmeister and L. v. Cholnoky, Ann. 455 (1927) 70. Stinging nettles (ground) 0.15-0.2 g R. WiLLSTATTER and A. Stole, Unttr- suchungen iiber Chlorophyll, Berlin 1934, Julius Springer, p. 237. — R. Willstatter and W. MiEG, Ann. 355 (1907) 12. TABLE 35 a-CAROTENE CONTENT OF DIFFERENT CAROTENE PREPARATIONS (kuhn and lederer*', KUHN AND BROCKMANN'^) Palm oil Green chestnut leaves Red berries Large pumpkin®' Green stinging nettles Paprica Spinach Grass traces traces traces * None P. Karrer and W. Schlientz"*'. Preparation Following WiLLSTATTER and Escher''^, finely cut carrots are dried, ground and continuously extracted with petroleum ether at room temperature. The combined extracts are concentrated as far as possible at 30-40° under reduced pressure and diluted with an equal volume of carbon disulphide. From this solution the crude References p. 16^-iyo. 3 ^-CAROTENE 131 carotene is precipitated with ethanol. Spirits of wine are added in small portions every 2 to 5 minutes. At first only colourless materials separate. As soon as the first carotene crystals are formed, the colourless materials are separated by rapid filtration. The mother liquors are diluted with the remainder of the alcohol (about 3_6 volumes of the original carotene solution are required) and allowed to stand for 20 hours at -10°. After this time the crude carotene is filtered off, dissolved in carbon disulphide, precipitated with ethanol, extracted with a little warm petroleum ether to remove remaining impurities, and finally recrystallised from a large volume of petroleum ether. In order to obtain the pure /3-isomer from crude carotene, the latter is chromato- graphed from petroleum ether on calcium hydroxide'^ Karrer and Walker'^ obtained 17 g of pure /S-carotene and 2.5 g of pure a-carotene from 35 g of crude carotene. In contrast to Willstatter and Escher'^, Kuhn and Lederer'* submit the shredded carrots to a preliminary extraction with methanol. Chemical Constitution CH, CH, CH, CH, C CH, CH, CH3 CH3 C /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj CH2 C-CH3 HsC-C CH2 \^ / /5-Carotene ' \ / CHa CH2 In 1907, Willstatter and Mieg" established the correct molecular formula C40H56 for carotene. Zechmeister, von Cholnoky and Vrabely'^^ showed that carotene contains 11 double bonds which can be saturated by hydrogenation. The formula of perhydrocarotene, C40H78 proves the presence of 2 ring systems. According to Pummerer and Rebmann'^^ carotene absorbs 11 molecules of iodine chloride, thus confirming the presence of 11 carbon-carbon double bonds. Karrer and co-workers'* oxidised jS-carotene with permanganate and with ozone and obtained a:a-dimethylglutaric acid, a:a-dimethylsuccinic acid, dimethylmalonic acid and geronic acid (a :a-dimethyl-6-acetylvaleric acid), the latter being a particularly characteristic degradation product. All these compounds are also formed by the oxidation of /5-ionone, in comparable yield. Thus by the oxidation of pure /^-carotene, Karrer and More obtained geronic acid in 16 % yield, while ^-ionone afforded the acid in 19.4 % yield. It was thus concluded that jS-carotene contains two j8-ionone groupings. CH, CH, C /\ CHg C I II \V CH2 |3-ionone group References p. 165-iyo. 132 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X CH3 CH3 V /\ lOOC COOH CH3 CH3 C CH2 COOH GHo CHo \V C CHj COOH CH3 CH3 V /\ CH2 COOH COOH CH2 COOH CH2 CO-CH CH2 Dimethyl- malonic acid aa-Dimethyl- succinic acid aa-Dimethyl- glutaric acid Geronic acid Karrer and Helfenstein'^^ quantitatively estimated the acetic acid formed by the permanganate oxidation of carotene and thus estabhshed the presence of 4 groupings of the type =CH-C=CH- CH, It was therefore concluded that the two /?-ionone rings are joined by a chain of 4 isoprene residues. Furthermore, according to Kuhn and Ehmann^" and Kuhn and L'Orsa^^, the results of chromic acid oxidation indicated the presence of 2 groupings of the type — CH2-C=C I CH3 These results led Karrer, Helfenstein, Wehrli and Wettstein^^ and Karrer and Morf^^ to propose the now accepted formula for jS-carotene. The structure of j3-carotene was confirmed by the later investigations of Pummerer, Rebmann and Reindel^*, Strain^^ and Kuhn and Winterstein^^. The latter authors showed that thermal decomposition of the pigment gives rise to 2 :6-dimethylnaphthalene. CH3 CHj X i"' CHj C-CH=CH-C=CH 1 II 1 1 ii CHj CH3 CH CH CH3 CH CH C CH CH CH CH C C C CH CH CH3 CH3 /\/ / \/ H3C CH CH C \ /\ CH=C-CH=CH-C CH2 C C CH y\/\y H3C CH CH CXI3 1130' 0 0x12 /3-Carotene \/ CH2 2 :6-Dimethylnaphthale References p. 165-1^0. p-CAROTENE 133 Further confirmation of the /3-carotene formula was provided by the in- vestigations of KuHN and Brockmann^^ in which /3-carotene was related to azafrin through mild stepwise degradation reactions. CH3 CH, CH3 CH3 \V \/ A r r r r a CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj CH2 C'CHj a^Kj' Kj vyH2 \ / /3-Carotene A / CH2 CH2 CrO, CH3 CH3 \/ C I CH, CH, CH3 CH, CH3 CH3 \/ C CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ I II KO^I I CH2 C-CHj H0\| I A A Dihydroxy-)9-carotene HaC-C CHj CHj, \A Pb(OCOCH3)4 CH3 CH3 CH3 CH3 \A ^ \A C CH3 CH3 CH3 CHs C A\ I I I I A\ CHa C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj CHj C-CH3 HjC-CO CHa \ / Semi-)3-carotenone A A CHa CH, CrO, CH, CH, \/ C ^ CH, CH, CH, CH, CH3 CH, \/ C CHa C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHa I |\0H I I I/OH CHo C' CH, CHa CH3 CH, C Dihydroxysemi-^-carotenone Pb(OCOCH3)4 CH, CH, HaC-CO CHa CHa CH3 CH, \/ C Clio CHq /\ r I I I ■ A\ CHa CO-CH = CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHa CHa CO-CHs H3C-C0 CHj A A /5-Carotenone A A CHa CHa References p. 165-170. 134 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X CrO, CHo CH \V C CH, CH, CH3 CH3 CH3 C /\ I I I /\ CH. CO-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCHO+HOOC CH, CH, CO-CH, \/ CHj CH3 CHa c /3-Carotenone aldehyde NH2OH i HjC-CO CH, CH, CH, /\ r 1 I CH2 CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CH=NOH I CH, CO-CH, CH2 CH, CHo \/ C /3-Carotenone aldehyde monoxime (CH3C0)20 CH, CH, CH, /\ I I I CH2 CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CN CH, CO-CH, Nitrile CH2 CH, CH, \/ C KOH CH, CHo CHo /\ r I 1 CH2 C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CONH2 CH2— C- CO- CH3 Anhydroazafrinone amide As anhydroazafrinone amide can also be obtained from azafrin via azafri- none amide the relation between /5-carotene and azafrin is established. (For the individual compounds involved in these reactions see p. 284). Formation P. Karrer and E. Jucker^^ obtained /5-carotene by the action of sodium ethoxide on a-carotene. Up to the present time this is the only way in which this pigment has been obtained synthetically*. P. Karrer and S. Schwyzer, Helv. Chim. Acta 31 (1948) 1055, have recently described the formation of traces of a carotenoid pigment, probably /3-carotene, in the reaction of vitamin A ^-toluenesulphonic ester and sodium iodide in acetone. The main product is anhydro-vitamin A. References p. 165-iyo. 3 ^-CAROTENE 135 Properties Crystalline form: Dark violet hexagonal prisms from benzene-methanol. Red, rhombic, almost quadratic plates from petroleum ether. Melting point: 181-182° (corr.)^^; 181-182° (uncorr., Karrer and co- workers^"); 183° (corr., evacuated capillary, Kuhn and Brockmann^^) ; 187.5° (Miller92)_ Solubility: j8-Carotene is less soluble than the a-isomer, so that the latter is concentrated in the mother liquors during the crystallisation of carotene. ^-Carotene is easily soluble in carbon disulphide, benzene and chloroform, and fairly easily soluble in ether and petroleum ether. 100 ml of w-hexane dissolve 109 mg of ^-carotene at 0°. The pigment is almost insoluble in ethanol and methanol. Spectral properties: Solvent: Absorption maxima: Carbon disulphide 520 485 450 m/z Chloroform 497 466 m/x Petrol 483.5 452 426 m/x Hexane 477 450 425 m^ (cf. Fig. 6, p. 350 and Fig. 31, p. 361) Quantitative extinction measurements: Hausser and Smakula'^. Raman spectrum: von Euler and Hellstrom^*. Colour reactions: On dissolving 1-2 mg of ^-carotene in 2 ml of chloroform and adding concentrated sulphuric acid, the acid layer is coloured blue. On dissolving the pigment in chloroform and adding one drop of fuming nitric acid, an immediate blue colouration is first produced which then turns green and finally dirty yellow. On dissolving 1-2 mg of /3-carotene in chloroform and adding a solution of antimony trichloride in chloroform a dark blue colouration is produced which has an ab- sorption maximum at 590 m/z. a-Carotene behaves differently; cf. von Euler, Karrer and Rybdom®^. Hydrogen chloride in ether or methanol solution produces no colouration. (For further data, see Zechmeister^^)^ Optical activity: j3-Carotene has a symmetrical structure and is optically inactive. Partition test: On partition between petroleum ether and 90% aqueous methanol, the concentration of j3-carotene in the former is 660 times as great as in the latter (Kuhn and Brockmann^'). Chromatographic properties: ^-Carotene is fairly strongly adsorbed on cal- cium hydroxide from petroleum ether solution. It is found below y-carotene and above a-carotene on the chromatographic column^^. Elution can be effected References p. 165-ijo. 136 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X by means of ether containing about 5 % methanol. jS-Carotene is only very weakly adsorbed on zinc carbonate and calcium carbonate, and is washed through during the development of a chromatogram on these adsorbents. Behaviour towards oxygen: On standing in air, carotene absorbs oxygen with increasing rate with the formation of colourless products®^. According to VON EuLER etc.^°° the autoxidation of very pure preparations only begins after several days' contact with air, formaldehyde being formed^*'^. On shaking a solution of j3-carotene in carbon tetrachloride in oxygen, a little glyoxal is formed^°2. Detection and estimation: j3-carotene can be separated from the other caro- tenoid hydrocarbons by chromatographic adsorption on calcium hydroxide from petroleum ether. It is identified by its absorption maxima. According to KuHN and Brockmann^"^ an alcoholic solution of azobenzene can be used as standard for colourimetric determinations. Physiological behaviour: /3-Carotene possesses high vitamin A potency which has been studied in detail by von Euler, Karrer and co-workers^"* (cf. p. II). Derivatives ^-Dihydrocarotene C40H58; CH, CH, CH3 CH3 C CH3 CH3 CH3 CH3 C /\ II II /\ CH2 C-CH2CH=C-CH=CHCH=C-CH=CHCH=CH-C=CHCH=CH-C=CH-CH2-C CH^ CHj C-CH3 HgC-C CHj \ / /5-Dihydrocarotene \ y^ CHj CH2 )3-Dihydrocarotene is formed together with other products by the reduction of jS-carotene with aluminium amalgam^"^. Pure j8-dihydrocarotene was isolated by Karrer and Ruegger^"^ using the refined chromatographic method of separation. The constitution shown above was derived by these authors. Dihydrocarotene crystallises from petroleum ether in salmon-red plates, m.p. 182°. It is vitamin A-inactive even in high doses. Solvent: Absorption maxima Carbon disulphide 461 432 m/j, (cf. Fig. 30, p. 360 and Fig. 31, p. 361) Perhydrocarotene C40H78 : Perhydrocarotene is obtained by the hydrogenation of jS-carotene in the presence of colloidal platinum as catalyst^ "''. Perhydrocarotene is a very viscous, distillable oil. It is very soluble in cyclohexane, easily soluble in ben- References p. 165-ijo. 3 p-CAROTENE 137 zene and ether, but only sparingly soluble in cold methanol and ethanol. Perhydrocarotene is optically inactive. According to von Euler, Demole, Karrer and Walker^*^ it possesses no biological activity. Dehydro-^ -carotene (Isocarotene) C40H54: This hydrocarbon is formed by the decomposition of iodine addition products of j6-carotene with thiosulphate, acetone, mercury or finely divided silver^ *'^. According to Karrer and Schwab^^", dehydro-^-caiotene has the following constitution* : CH3 CH3 CH3 CH, C CH3 CH3 CH3 CH3 C /\ II II /\ CH~ C=CHCH=C-CH=CHCH=C-CH=CHCH=CH-C=CHCH=CH-C=CHCH=C CHg 11 II CHj C-CH3 HgC-C CH, \ ^ Dehydro-^-carotene (Isocarotene) %^ y^ CH CH Dehydro-j8-carotene crystallises from petroleum ether in glistening violet- blue needles or plates, and from a mixture of benzene and methanol in violet prisms, m.p. 192-193° (corr., Karrer, Schopp and Morf)!^^ It is very sparingly soluble in petroleum ether, but easily soluble in benzene and chlo- roform. It is practically insoluble in alcohols. Solvent: Absorption maxima: Carbon disulphide 543 504 472 m/i Petroleum ether 504 475 447 m/z Chloroform 518 485 455 m/z (of. Fig. 31, p. 361) Quantitative extinction measurements have been recorded by Hausser and Smakula^i^. With antimony trichloride in chloroform, isocarotene gives a stable blue colouration. It exhibits no vitamin A activity. 'p-Carotene oxide' C4oH5gO: This compound was obtained by von Euler, Karrer and Walker^^^ by the oxidation of ^-carotene with perbenzoic acid. It is not an epoxide as was at first assumed, but a furanoid oxide of the following constitution (Karrer and JUCKER^^*). * According to H. v. Euler, P. Karrer and O. Walker, Helv. chim. Acta 15 (1932) 1507, small amounts of isocarotene are formed by the oxidation of /5-carotene with per- benzoic acid. References p. 16^-iyo. 138 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X CHq CHo \V -CH CH3 CH3 CH3 CH3 C II I I I /\ CH-C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- C CHj HoC* C CHo \/ CHj For the properties and reactions of mutatochrome see p. 147. '^-Carotene oxide' = Mutatochrome Dihydroxy-^-carotene C4oH5g02* : CHo CHo CHo CHo C OH CH3 CH3 CH3 CH, C /\/ I I I r /\ CHa C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHjj CH, C-OH H,C-C CH, CH2I Dihydroxy-j5-carotene ? CHj CH3 Dihydroxy-j3-carotene is formed during the careful oxidation of j3-carotene with aqueous o.i N-chromic acid (1.5 atoms O). It crystalhses from a mixture of petrol and methanol in orange-red needles, m.p. 184° (Kuhn and Brock- MANN^^^). Dihydroxy-j3-carotene is easily adsorbed on aluminium oxide from benzene solution, but it is not adsorbed on calcium carbonate. (The lack of adsorption on calcium carbonate is unexpected for a compound assumed to contain two hydroxyl groups). Dihydroxy-^-carotene is easily soluble in benzene, chloroform and carbon disulphide, sparingly soluble in petrol, and insoluble in alcohols. On partition between petroleum ether and 90% methanol, it is found almost entirely in the upper layer. (The insolubility in alcohols and the epiphasic character of the compound are irreconcilable with the proposed formula). Solvent: Absorption maxima: Carbon disulphide 508 475 446 m/^ Chloroform 487 456 429 m^i Petrol 478 448 420 m^ Hexane 476 446 419 m/i Benzene 489 457 428 m/i Dihydroxy-j3-carotene shows vitamin A activity. Concerning the molecular formula of dihydroxy-^-carotene see R. Kuhn and H. Brockmann, Ber. 6y (1934) 1408 and Ann. 516 (1935) 99. References p. 165-ijo. 3 ^-CAROTENE 139 DiJiydroxy semi-P-carotenone C^^Yi^^O^ : C OH CH, CH3 CHg CH3 C /\/ II I I /\ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH. II I CHj C-OH HgC-CO CHg \ /\ Dihydroxysemi-j5-carotenone \^ / CH2I CHj CH3 Dihydroxysemi-^-carotenone is prepared by the oxidation of dihydroxy- /3-carotene with o.i N chromic acid^^®. The pigment ciystalUses from a mixture of benzene and petroleum ether in dark red prisms with a bluish lustre, m.p. 172°. It is readily soluble in chloroform, somewhat less soluble in benzene and ethanol and hardly soluble in petroleum ether. Dihydroxysemi-jS-carotenone is hypophasic in the partition test. Solvent: Absorption maxima: Carbon disulphide 534 495 464 m// Petroleum ether 497 468 440 m;^ Benzene 512 481 452 m/i Ethanol (498) (471) m^u Chloroform 510 479 452 m/i Sem,i-^-carotenone C40H56O2 : CHo CHo CHo CHo C CHo CHo CHo CHo C /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH, I Hg C'GH3 H3C'C0 CH2 \ / Semi-^-carotenone \^ / CH2 CH2 Semi-j8-carotenone was prepared by Kuhn and Brockmann^" by the oxidation of ^-carotene with o.i N chromic acid. It is also formed on treating a solution of dihydroxy-j3-carotene in benzene with lead tetra-acetate in glacial acetic acid^^*. It crystallises from methanol in square, scarlet plates, m.p. ii8-iig° (corr., evacuated capillary). Semi-^-carotenone is fairly soluble in petroleum ether and less soluble in ethanol. It is epiphasic in the partition test. For further information, cf. Kuhn and Brockmann^^'. Solvent: Absorption maxima: " Carbon disulphide 538 499 m/z Chloroform (519) (487) m^ (diffuse) Petroleum ether 501 470 446 m^ Benzene 518 486 458 van Hexane 500 469 443 m/< References p. 165-iyo. I40 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Semi-;8-carotenone oxime crystallises from 90 % ethanol in scarlet needles, m.p. 134-135° (evacuated capillary). The absorption maxima of the oxime are almost identical in wavelength location with those of the parent compound. Neosemi-^-carotenone C40H56O2 : Karrer and Solmssen^^^ observed that instead of semi-jS-carotenone a different compound, neosemi-j3-carotenone, can be formed by the oxidation of j3-carotene with aqueous o.i N chromic acid. This compound separates from methanol in almost black crystals, m.p. 143°. Solvent: Absorption maxima: Carbon disulphide 510 479 m/i. Anhydrosemi-^-carotenone C40H54O : CH, CHo CH, CH, /\ r r I r /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHg CH2— C-CO-CHs HjC-C CHj Anhydrosemi-^-carotenone \^ y^ CHjs This compound is formed by splitting off a molecule of water from semi- jS-carotenone by treatment with methanolic potassium hydroxide^-". The pig- ment crystallises from a mixture of benzene and methanol in almost black prisms, with a green lustre, m.p. 177°. It is readily soluble in carbon disulphide, chloroform and benzene, and sparingly soluble in petroleum ether and absolute ethanol. It is entirely epiphasic in the partition test. Solvent: Absorption maxiina: Carbon disulphide 547 509 481 m^ Chloroform . . ' 524 489 459 m/i Benzene 528 490 459 m// Petroleum ether 512 480 452 m^ ^-Carotenone C40H55O4 : C CHo CHo CHo CHo C /\ I I I \ /\ CH2 CO-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH^ CH2 CO-CHg HgC-CO CHg \ / /9-Carotenone \ / CH2 CHj KuHN and Brockmann^^^ prepared jS-carotenone by the oxidation of j3-carotene with chromic acid. It is also formed by the oxidation of semi- References p. 16^-1 jo. 3 ^-CAROTENE 141 j8-carotenone with chromic acid^^^. ;S-Carotenone crystaUises from a mixture of benzene and hght petroleum in scarlet, hexagonal flakes with a blue lustre, m.p. 174-175° (corr., evacuated capillary^-^) . The pigment can be adsorbed on aluminium oxide or calcium carbonate from petroleum ether solution. On partition between petroleum ether and 90 °o methanol it is found mainly in the lower layer. ^-Carotenone is easily soluble in chloroform, carbon disulphide and benzene, sparingly soluble in cold methanol and ethanol, and very sparingly soluble in petroleum ether. Solvent: Absorption maxima: Carbon disulphide 538 499 466 m,u Chloroform 527 489 454 mn Petroleum ether 502 468 440 m// Benzene 522 486 453 m/z Hexane 500 466 436 m^ (cf. Fig. 29, p. 360) Quantitative extinction measurements in hexane have been recorded by HAUSSERand Smakula^24_ ^-Carotenone iorm.sa.dioxime, m.p. 198° (corr., evacuated capillary) . Bis-anhydro-^-carotenone C40H52O2 : CH, CH, CH, CH, C CH, CH, CH3 CH3 C /\ \ \ \ \ /\ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj I i . II I CHj — C • CO • CH3 Bis-anhydro-/3-carotenone H3C • OC • C CH2 This derivative was obtained by Kuhn and Brockmann^^s ^y treating ^-carotenone with methanolic potassium hydroxide. It crystallises from a mixture of benzene and methanol in steel-blue prisms or plates, m.p. 209°. It is almost entirely epiphasic in the partition test. Bis-anhydro-^-carotenone is soluble in chloroform, benzene and carbon disulphide, but almost insoluble in petrol, methanol and petroleum ether. Solvent: Absorption maxima: Carbon disulphide (567) (525) (477) m/z Chloroform (545) (506) (481) m^ Benzene (545) (505) (478) m/z Petrol 530 494 462 m/x Dihydro-bis-anhydro-^-carotenone C4(,H5402 : CH, CH, CH3 CH3 C CH3 CH3 CH3 CH3 C /\ I I I I /\ OH. C=CH-CH=C-CH=CHCH=C-CH=CHCH=CH-C=CHCH=CH-C=CH-CH=C CH^ II . II CHa — CH-C0-CH3 Dihydro-bis-anhydro-^-carotenone H3C-0C-CH — CHg (Keto form) References p. i6§-iyo. 142 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X This compound is formed by shaking bis-anhydro-/3-carotenone with zinc dust in a mixture of pyridine and glacial acetic acid^^e^ -pj^g pigment crystallises from aqueous pyridine in brilliant red needles, m.p. 217° (corr., evacuated capillary) . Dihydro-bis-anhydro-/3-carotenone is readily soluble in carbon di- sulphide, benzene, chloroform and pyridine, but only sparingly soluble in petrol and petroleum ether. It is rapidly oxidised by air in alkaline alcoholic solution to bis-anhydro-/S-carotenone. Solvent: Absorption maxima: Carbon disulphide 510 478 448 m/^ Chloroform 490 459 430 mfi Benzene 492 460 430 m/i Petrol 479 448 421 m/< Dihydro-^-carotenone C^f^^fii'. CH, CH, CH, CH, C CH3 CH3 CH3 CH3 C /\ I I I I /\ CHa CO-CH2CH=C-CH=CHCH=C-CH=CHCH=CH-C=CHCH=CH-C=CH-CH2-CO CHj CHg CO-CHs H3C-C0 CHj \ / Dihydro-;S-carotenone \ / CHa CHa Dihydro-j5-carotenone is formed by the reduction of /9-carotenone with zinc dust in pyridine and glacial acetic acid^^'. The pigment crystallises from a mixture of petrol and benzene in golden-yellow needles, m.p. 130° (corr. evacuated capillary). It is readily soluble in pyridine, chloroform and benzene, but only sparingly soluble in petroleum ether and alcohols. It is almost entirely hypophasic in the partition test. Solvent: Absorption maxima: Carbon disulphide 454.5 426 m/i Chloroform 435 411 m/i Hexane 426 m/t Petrol 429 m/^ Benzene 436 411 m/i Petroleum ether 424 m/t On treatment with hydroxylamine, dihydro-/5-carotenone forms a dioxime C4oHgo04N2, which crystallises from hot benzene in golden-yellow plates, m.p. 151° (corr., evacuated capillary). The dioxime is less readily soluble in petrol and benzene and more readily soluble in alcohol than dihydro-/5-carotenone. Solvent: Absorption maxima: Carbon disulphide 454.5 426 m/i Petrol 429 m/i Ethanol 426 m/i References p. 165-iyo. 3 ^-CAROTENE 143 P-Carotenone aldehyde C27H35O3: CHo CHo /\ ! I I CH2 CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCHO CHa CO-CHj \ ^ ^-Carotenone aldehyde CHjj This aldehyde is formed by the oxidation of /9-carotene or j5-carotenone with chromic acid^^^. It crystalhses from a mixture of benzene and petrol in yellow-red needles with a bluish lustre, m.p. 146-147° (corr. evacuated capil- lary). The compound is readily soluble in chloroform, carbon disulphide, ben- zene and hot methanol, but only sparingly soluble in cold petrol and petroleum ether. Solvent: Absorption maxima: Carbon disulphide 491 459 430 m/^ Chloroform 482 450 423 m/x Petroleum ether 457 430 404 m^ Petrol 461 432 406 m/x Benzene 476 446 420 m[i, Hexane 458 431 405 mn Ethanol (473) (442) m/^ On prolonged treatment with excess hydroxylamine, /5-carotenone aldehyde forms a dioxime. With regard to its constitution, compare Kuhn and Brock- MANN^^^. It crystallises from dilute methanol in yellow-red plates, m.p. 183- 184°. It is sparingly soluble in petroleum ether, petrol and cold benzene, but more easily soluble in ethanol. Solvent: Absorption maxima: Carbon disulphide 492 Chloroform 478 Petrol 462 Benzene 477 Using molecular proportions of hydroxylamine and |5-carotenone aldehyde^ a monoxime is obtained which is a mixture of aldoxime and ketoxime. The monoxime crystallises from methanol in yellow-red plates or needles, m.p. 174°. (Concerning the conversion of the aldoxime into anhydroazafrinone amide, see p. 134 and Kuhn and Brockmann loc. cit.). References p. 165-iyo. 460 431 m^ 448 420 mfi 435 mfi 447 419 m/i 144 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X ^-Apo-2-carotenal C30H40O: \V C CH3 CH3 ^113 CH3 /\ I I I I CHa C- CH=CH- C=CHCH=CH- C=CHCH = CHCH=C- CH=CHCH=C- CHO CH2 C*CH3 \^ / yS-Apo-2-carotenal CH2 This aldehyde is formed by the potassium permanganate oxidation of /9-carotene^^^. j8-Apo-2-carotenal crystaUises from methanol in violet plates, m.p. 139°. On addition of concentrated hydrochloric acid to an ethereal solution of the pigment an intense stable blue colouration is produced. The aldehyde shows strong vitamin A activity. Solvent: Absorption maxima: Carbon disulphide 525 490 m^u Petroleum ether 484 454 m/z Ethanol (498 . . . 447)m/i /3-Apo-2-carotenal oxime crystallises in glistening violet rhombs or prisms, m.p. 180°. Solvent: Absorption maxima: Carbon disulphide 507 473 m/i Petroleum ether 471 441 m/z Ethanol 475 445 m^ )3-Apo-2-carotenal semicarbazone melts at 212° (sinters above 205°). ^-Apo-2-carotenol C30H42O: CH3 CH3 C CH3 CH3 CH3 CH3 CH2 C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHjOH I II CHj C" CH3 \ / /3-Apo-2-carotenol CH3 This compound was obtained by voN Euler, Karrer and Solmssen by the reduction of /5-apo-2-carotenal with isopropyl alcohol and aluminium iso- propoxide^^". It crystallises from a mixture of benzene and petroleum ether in yellow plates, m.p. 145°. Solvent: Absorption maxima: Carbon disulphide 486 456 m/x Petroleum ether 453 423 m/i Ethanol 456 426 m// (cf. Fig. 29, p. 360) References p. 165-iyo. 3 p-CAROTENE 145 P-Apo-4-carotenal C25H34O: CH3 CH3 C CH3 CH3 CH3 /\ I I I CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CHO CHj C'CH3 \ / /?-Apo-4-carotenal This compound is obtained together with /3-apo-2-carotenal by the oxidation of /5-carotene with potassium permanganate^^^. It has not been obtained in the crystalhne state, but forms a crystalhne oxime and semicarbazone. Solvent: Absorption maxima: Carbon disulphide about 460 m/z (diffuse) Petroleum ether about 442 m// (diffuse) y5-Apo-4-carotenal oxime crj-'stallises from methanol in rhombic plates and clusters, m.p. 165°. Solvent: Absorption maxima: Petroleum ether 408 m/^ Ethanol 409 m^ Carbon disulphide 456 m/< (slightly diffuse) ^-Apo-4-carotenal semicarbazone separates from ethanol as a scarlet-red powder, m.p. 217° (with decomposition), sintering above 214°. Solvent: Absorption maxima: Carbon disulphide 474 m/^ Ethanol 445 m/i (broad band) P-Apo-4-carotenol CjsHjgO: CHo CH3 C CH3 CH3 ^^3 /\ I I I CH2 C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH2OH I i . CHj C*CH3 \ / /9-Apo-4-carotenol CH2 This polyene alcohol is formed by the reduction of /5-apo-4-carotenal with isopropyl alcohol and aluminium isopropoxide^^^^ ^-Apo-4-carotenal has so far only been obtained as a viscous oil. Oxides of ^-Carotene By the oxidation of /^-carotene with monoperphthalic acid, Karrer and JuCKER^^^ obtained a number of oxidation products which are epoxides or References p. 16^-iyo. Carotenoids 10 146 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X furanoid oxides. (Cf . p. 61 concerning the constitution of these compounds). The oxides of ^-carotene are very similar to the corresponding derivatives of crypto- xanthin (p. 178) and zeaxanthin (p. 189), from which they differ only by the absence of the hydroxyl groups. ^-Carotene mono-epoxide C40H55O : CH, CH, CH, CH, C CH3 C/H3 CH3 CHo C /\ I I I I /\ CHa C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHg )0 ' ' C CH2 CH, C /\/ \^ / ^ /3-Carotene mono-epoxide HjC CHj CH2 CH3 This compound crystallises from a mixture of benzene and methanol, or ether and methanol, in lustrous orange leaflets, m.p. 160° (uncorr., in vacuum). On shaking an ethereal solution of the pigment with concentrated aqueous hydrochloric acid, the acid layer slowly develops a pale blue colouration which is not very stable. Solvent: Absorption maxima: Carbon disulphide 511 479 m// Benzene 492 460 m^ Petroleum ether 478 447 m/^ Chloroform 492 459 m/f ^-Carotene di-epoxide C40H55O2: CHo CHo CHo CHo C CHo CHo CHo CHo 0 CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ )0 0( CH2 0 C CH2 \^ /\ /S-Carotene di-epoxide /\/ CH2 CH3 H3C CHjj j5-Carotene di-epoxide crystallises from a mixture of benzene and methanol in yellow-orange leaflets, m.p. 184° (uncorr., evacuated capillary). On shaking an ethereal solution of the pigment with concentrated aqueous hydrochloric acid, the acid layer is coloured dark blue. The colouration is stable for several days. /5-Carotene di-epoxide exhibits entirely epiphasic properties. Solvent: Absorption maxima: Carbon disulphide 502 472 m/i Benzene 485 456 m/< Petroleum ether 470.5 443 mpi Chloroform 484 456 m/x References p. 16^-iyo. ^-CAROTENE 147 Mutaiochrome C40H55O' CHo CHo CHo CHo \V CH CH3 CH3 CH3 CH3 C CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- C CHj Mutatochrome = Citroxanthin C CH, /\/ H3G CH2 By the action of hydrogen chloride on /5-carotene mono-epoxide, the corresponding furanoid oxide, mutatochrome is formed, together with a small proportion of |5-carotene. Mutatochrome crystallises from a mixture of benzene and methanol in yellow-orange leaflets, m.p. 163-164° (uncorr., in vacuum), Mutatochrome behaves in the same way as /S-carotene mono-epoxide in the hydrochloric reaction and partition test. Solvent: Absorption maxima: Carbon disulphide 489.5 459 m/i Benzene 470 440 m/i Petroleum ether 456 427 m/z Chloroform 469 438 m/z Aurochrome CioHjgOj: CH, CH, CH3 CH, C C CHa C=CH CH3 CH3 CH3 H3C CH=C CH2 CHo C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH C CH, .CH2I 0 • Aurochrome 0 \ CHg CH3 H3C Aurochrome is formed by the action of dilute hydrochloric acid on /5-caro- tene di-epoxide. It crystallises from a mixture of benzene and methanol in beautiful yellow leaflets, m.p. 185° (uncorr., in vacuum). On adding con- centrated aqueous hydrochloric acid to an ethereal solution of this pigment, a very stable dark blue colouration is formed. On partitioning aurochrome By the action of perbenzoic acid on ^-carotene, H. v. Euler, P. Karrer and O. Walker, {Helv. chim. Acta 15 (1932) 1507) obtained an oxide which they formulated as /5-carotene mono-epoxide. These authors were unaware of the great sensitivy of this com- pound to acids and later comparison has shown that they had in fact obtained the furanoid oxide, mutatochrome, and not the mono-epoxide formed primarily. P. Karrer and E. JucKER, Helv. chim. Acta 30 (1947) 536 established the identity of mutatochrome with the pigment citroxanthin which they had isolated from orange peel {Helv. chim. Acta 2j (1944) 1695). Mutatochrome is thus a naturally occurring pigment. References p. 16^—iyo. 148 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X between methanol and petroleum ether, the pigment is found almost quantita- tively in the upper layer. Solvent: Absorption maxima: Carbon disulphide 457 428 ran Benzene 440 m/x Petroleum ether 428 m^ Chloroform 437 m/z Luteochronie CioHggOj: CH, CH3 C \V /\ C CH3 CH3 CH3 H3C CH:=C CHj /\ I I I I I I I CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH C CH, )0 0 I CH, CH, C H3C \ / \ Luteochrome CH, CH3 , Luteochrome is formed together with /^-carotene mono-epoxide and /5-carotene di-epoxide during the oxidation of /3-carotene with monoper- phthalic acid^^*. The pigment crystalhses from a mixture of benzene and methanol in thin yellow-orange leaflets, m.p. 176° (uncorr., in vacuum). Con- centrated aqueous hydrochloric acid has the same effect as with ^^-carotene di-epoxide and aurochrome. On partitioning between methanol and petroleum ether, luteochrome is found mainly in the upper layer. Solvent: Absorption maxima: Carbon disulphide 482 451 m/x Properties of the ^-Carotene Oxides /5-Carotene epoxides and furanoid oxides can only be separated with difficulty by chromatographic analysis, using petroleum ether as solvent and calcium hydroxide as adsorbent. The solubihties of these compounds do not differ appreciably from those of /9-carotene. They are easily soluble in carbon disulphide, chloroform, benzene and ether, somewhat less easily in petroleum ether, and only very sparingly soluble in methanol and ethanol. Physiological Properties The vitamin A activity of the various /5-carotene oxides has been examined by VON EuLER^^^. It has been found that 177-doses of /3-carotene di-epoxide and 187-doses of luteochrome produce full vitamin A activity in rats. It may References p. 165-170. 3 p-CAROTENE 149 be concluded that they are partly deoxygenated to /?-caroteneormutatochrome* in the animal organism, since according to all previous experience, vitamin A activity requires the presence of an unsubstituted /5-ionone ring in the caro- tenoid**. Cis-trans-Isomers of ^-Carotene^^^' ^^' In 1935, GiLLAM and El Ridi^^^ observed that several zones are developed during the chromatographic adsorption of pure /S-carotene. They ascribed this phenomenon to a change of the pigment during adsorption, and were able to isolate a transformation product which they termed pseudo-a-carotene. This compound exhibited the characteristic properties of a carotenoid, melted at 166°, and showed epiphasic behaviour, no optical activity and vitamin A potency. Solvent: Absorption maxima: Carbon disulphide 507 477 m// Chloroform 486 456 m/t Petroleum ether 477 446 m/t Ethanol 478 447 m/^ More recently, Zechmeister and co-workers^^'' have investigated these changes and have shown that they are not produced by adsorption, but that polyene pigments generally can be isomerised by dissolution, melting of the crystals, or certain other operations (cf . p. 39) . From a number of considerations, including the fact that these transformations are reversible, Zechmeister concluded that m-^rans-isomerism is involved. About 10 zones are observed in the transformation chromatogram and the individual pigments are regarded as stereoisomers of /5-carotene. Only one of these pigments has so far been obtained in the crystalline state, and has been termed neo-/5-carotene U. It is rather more strongly adsorbed chromatographically than /3-carotene, is somewhat more soluble in organic solvents, and exhibits epiphasic behaviour on partition between petroleum ether and methanol. Thus 250 mg of /9-carotene yielded 41 mg of neo-/5-carotene U, m.p. 122-123° (corr., block). According to Zechmeister and collaborators^^, neo-^-carotene U contains one cis-double bond whereas the pseudo-a-carotene obtained by Gillam and El Ridi^^^ may contain two czs-double bonds. Solvent: Absorption maxima of neo-^-carotene U: Carbon disulphide 512.5 478.5 m/^ Benzene 494 461 m^ Petroleum ether 481 450 m/i Chloroform 493.5 461 m/j, Ethanol 482 450.5 m/t * Early investigations showed that mutatochrome exhibits vitamin A activity. See, however, footnote p. 14. References p. 16^-ijo. I50 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Neo-^-carotene U shows vitamin A activity (cf. p. 15), but of a lower order than that of natural /S-carotene. The other isomers could not be prepared in the crystalline state. They were characterised by their absorption maxima in petroleum ether solution, and by their positions in the chromatogram. In the following table, the compounds are enumerated in the sequence in which they occur in the chromatogram. Designation Absorption petrolev maxima in im ether Neo-)3-carotene U 481 450 m/i Neo-^-carotene V 472.5 441.5 m/i Neo-^-carotene A 469 437.5 m/t Neo-^-carotene B 475.5 444.5 m/z Neo-^-carotene C 465.5 433 m/i Neo-^-carotene D 474.5 441.5 vapL Neo-/3-carotene E 477.5 445 ran These are followed by a few other transformation products to which no names have been assigned. Natural |S-carotene with complete ^rans-configuration occurs between neo-/9-carotene V and neo-/S-carotene A in the chromatogram. According to Polgar and Zechmeister^**', neo-y5-carotene B is identical with pseudo-a-carotene. , 4. a-CAROTENE C4oHg6 History 1931 a-Carotene is discovered simultaneously by Kuhn and Lederer^*^ and Karrer and co-workers^^^. The new pigment is an isomer of /5-carotene and generally accompanies the latter in vegetable and animal materials. 1933 Karrer and Walker^^^ introduce calcium hydroxide and calcium oxide as new adsorbents for the chromatographic separation of epiphasic polyene pigments. Pure a-carotene is thus prepared for the first time. 1933 Karrer, More and Walker^** elucidate the constitution of a-carotene. Occurrence a-Carotene is almost as widely distributed in the vegetable kingdom as the /5-isomer. It is present in varying amounts in most carotene preparations and is concentrated in the mother liquors during recrystallisation. Mackinney^*^ examined the presence of a-carotene in green parts of plants, especially leaves, and found that the following plants contain a-carotene: References p. 165-1^0. * a-CAROTENE 151 Coprosma haueri Endlicher; Daucus Carota L., Petroselinum hortense, Hedera helix L., Quercus agrifolia, Aesculus calif ornica Nuttall; Parthenocissus quinque- folia, Amsinckia douglasiana De Candolle, Cuscuta salina Engelman, Solanum tuberosum, Lycopersicum esculentem Miller, Citrus maxima, Malva parviflora L., Urtica urens L., Ficus carica L., Thea sp.. Camellia sp., Sedum acre L., Dracaena draco L., Washingtonia filijera Wendland; Pinus radiata Don; Libocedrus de- currens Torrey; Moss sp., Chlorella vulgaris, Ranunculus californicus Bentham, Magnolia grandiflora L., Phoenix, Sequoia sempervirens Engelmann. Further information regarding the occurrence of a-carotene in plant leaves is given by Strain^*^. {Data regarding the a-carotene content of carotene preparations are given on p. 130). Source Arbutus (fruit) Cantharellus species Citrullus vulgaris Cladophora Saiiteri Coleosporium senecionis Convallaria majalis Cow fat Cucurbita maxima (fruit) Cuscuta subinclusa and Cuscuta salina Oil from Cyclopterus Dendrodoa grossularia (Sty clops is) Diospyros costata (fruit) Formosa tea-leaves TABLE 36 OCCURRENCE OF a-CAROTENE * References K. ScHON, Biochem.J. 2g (1935) 1779. H. WiLLSTAEDT, Chem. Centr. igjS, II, 2272. L. Zechmeister and A. PolgAr, /. biol. Chem. ijg (1941) 193. I. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 2g (1935) 1376. E. Lederer, Chem. Centr. igjO, I, 3852. A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 2oy (1932) 25. L. Zechmeister and P. Tuzson, Bcr. 67 (1934) 154. L. Zechmeister and P. Tuzson, Ber. 6j (1934) 824. G. Mackinney, /. biol. Chem. 112 (1935) 421. N. A. S0RENSEN, Chem. Centr. ig34, I, 3817. E. Lederer, Chem. Centr. ig36, I, 3853. K. ScHON, Biochem.J. 2g (1935) 1779. R. Yamamoto and T. Muraoka, Chem. Centr. ig33, I, 441. K. ScHON and G. Mesquita, Biochem. J. 30 (1936) 1966. Gonocaryum obovatum (skin) A. Winterstein, Z. physiol. Chem. 215 (1933) 51. Gonocaryum pyriforme (skin) do. Genista tridentata Gorse Haematococcus pluvialis Hymeniacidon sanguineum P. Karrer and E. Jucker, Helv. chim. Acta 2j (1944) 1585. J. TiscHER, Z. Physiol. Chem. 252 (1938) 225. P. J. Drumm and W. F. O'Connor, Nature 145 {ig4o) 425. Only references to investigations resulting i n the isolation and unambiguous identific- ation of the pigment are included. References p. 165-170. 152 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Source Ipomoea Batatas Mangifera indica (fruit) Marine and deep-sea soil Oedogoniuni Oil irom Orthagoriscus mola Palm oil Paprica Oil from Regalecus Red Euglene Rhodymenia palmata Rye-germ oil Saffron Sorhus aucuparia (fruit) Soya beans Ulex Gallii Yellow maize References J. C. Lanzing and A. G. van Veen, Chem. Centr. igjS, I, 2081. R. Yamamoto, Y. Osima and T. Goma, Chem. Centr. 1933, I, 441. D. L. Fox, Chem. Centr. 1937, II, 3899. I. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 29 (1935) 1376. N. A. S0RENSEN, Chem. Centr. J934, I, 3817. P. Karrer, H. v. Euler and H. Hellstrom, Chem. Centr. 1932, I, 1800. — R. Kuhn and H. Brockmann Z. physiol. Chem. 200 (1931) 255. — P. Karrer and 0. Walker, Helv. chim. Acta 16 (1933) 641. — R. F. Hunter and A. D. Scott, Biochem. J. 55 (1941) 31. L. Zechmeister and L. v. Cholnoky, Ann. 509 (1934) 269. N. A. S0RENSEN, Chem. Centr. 1934. I, 3817. H. TiscHER, Z. physiol. Chem. 259 (1939) 163. 1. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 29 (1935) 1376. H. A. Schuette and R. C. Palmer, Chem. Centr. 1938, I, 1898. R. Kuhn and A. Winterstein, Ber. 6y (1934) 344. R. Kuhn and E. Lederer, Ber. 64 (1931) 1349. W. C. Scherman, Chem. Centr. 1941, I, 2673. K. Schon, Biochem. J. 30 (1936) 1960. G. S. Fraps and A. R. Kemmerer, Chem. Centr. 1942, II, 1641. Preparation a-Carotene is widely distributed in plants but never occurs in large concentra- tions. According to Karrer and Walker^*' it is best prepared from commercial carotene (carrot carotene). The pigment can be separated in good yield from /S-carotene by chromatographic adsorption on calcium hydroxide. Glass tubes about 70 cm in length and 5 cm in diameter are filled with air-dry calcium hydroxide (cf. p. 27) and the columns are wetted with a little ligroin (b.p. 60-70° C). 200 mg of carotene (a mixture of ^, a, and a little y-carotene) are dissolved in about 100 ml of ligroin and the solution is poured on the calcium hydroxide column. The chromatogram is developed with petroleum ether, b.p. 70-80°. As soon as the yellow, a-carotene -containing zone has reached the lower end of the tube, the development is interrupted and the pigment is eluted with a mixture of ether and methanol (10:1). After evaporating the solvent, the pigment remains as a dark -red crystalline mass. For further purification, the a-carotene is crystallised 2-3 times from a mixture of benzene and methanol or from petroleum ether. From 1 g of carotene an average of 80 mg of pure a-carotene are obtained in this way. Other methods^*^ of preparation of a-carotene are hardly used, as they are relatively cumbersome and do not furnish pure products. References p. 165-iyo. a-CAROTENE Chemical Constitution 153 CH, CHj CH, CH, C CH, CH, CH, CH, C /\ I I I I /\ CH, C-CH=CH-C=CHCH=CH-C«CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH, CH» C'CHa H,C-C CH, a-Carotene CH, CH The elucidation of the constitution of a-carotene has been carried out mainly by Karrer and co-workers^*^. The hydrocarbon contains 11 double bonds^^". The absorption spectrum, the maxima of which are displaced by 12 mix towards shorter wavelengths as compared with /9-carotene, suggests that not all double bonds are in conjugation. A definite proof for the formula of a-carotene was given by Karrer, More and Walker^^i ^^]^Q showed that ozonisation of the pure pigment (chromatographed on calcium hydroxide) furnishes small quantities of isogeronic acid, besides geronic acid which is also formed in the oxidation of /^-carotene. CH, CH, CH, CH» CH, CH. CH, C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH = C- CH=CH- CH CH2 C' CH3 CH, a-Carotene H,C-( \ / CH3 CH, X CH, COOH CHo CHo V /\ 0x12 ^H2 CO, CH, CH; C /\ HOOC-CH CHi CH, CO-CH, CH, HjC-CO CH, HOOC HgC-CO CH. HOOC Geronic acid Isogeronic acid (yydimethyl- <5-acetylvaleric acid) CH This formula is in agreement with the fact that a-carotene is optically active. Further confirmation of this constitution is provided by the complex oxidation products which have recently been prepared by Karrer and co- workers (cf. p. 155). Properties Crystalline form: a-Carotene separates from a mixture of benzene and methanol in violet prisms and clusters and from petroleum ether in dark violet prisms or polygons. References p. 165-170. 154 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Melting point: 187-188° (corr.)i52_ Solubility: a-Carotene is considerably more soluble than the /^-isomer. It is very soluble in carbon disulphide and chloroform, easily soluble in benzene and ether, sparingly soluble in petroleum ether and almost insoluble in alcohols. 100 Ml of hexane at 0° dissolve 294 mg of a-carotene^^^. Spectral properties: Solvent: Absorption ynaxiina: Carbon disulphide . , 509 477 m/i Petrol 478 447.5 m^ Chloroform 485 454 m/i Hexane 475 445 420 395 m^ (cf. Fig. 7, p. 350 and Fig. 31, p. 361) For quantitative extinction measurements, see Hausser and Smakula^^*. For Raman spectra, see von Euler and Hellstrom^^^. Colour reactions: a-Carotene in chloroform solution gives a blue colouration with concentrated sulphuric acid. On adding antimony trichloride to a chloroform solution, a deep blue colouration is produced with an absorption maximum near 542 m/i (Karrer and WalkerI^^j Optical activity: The specific rotation in benzene is +385° (643.85 m/^ cadmium hne) (KuHN and Lederer^^'). The rotatory dispersion was determined by Karrer and Walker152 • [a]j8 = 315° (±7°) [a]45 = +385° (±5°) Partition test: a-Carotene exhibits entirely epiphasic properties on partition between petroleum ether and 90 % methanol. Chromatographic properties: a-Carotene is adsorbed less strongly than j8-carotene on calcium hydroxide from petroleum ether solution, and is found below /3-carotene on the column. (Karrer and Walker^^^). It can be eluted by means of ether containing about 5 % methanol. Behaviour toivards oxygen: The oxidation of a-carotene in light is autocata- lytic (BaurIS'). Detection and estimation: The separation of a-carotene from other caro- tenoid hydrocarbons is effected by chromatographic adsorption on calcium hydroxide. Its presence can be established by the determination of the ab- sorption maxima. For the colorimetric estimation of the pigment, Kuhn and Brockmann^^^ recommend an alcoholic solution of azobenzene as a standard. Physiological behaviour: a-Carotene exhibits strong vitamin A activity^^^. References p. 165-ijo. a-CAROTENE 155 Derivatives "Dihydro-a-caroiene": By the reduction of a-carotene with aluminium amalgam, Karrer and Morf^**' obtained a dihydro-a-carotene as a light yellow oil. The homogeneity and con- stitution of this product is still uncertain. Degradation by means of ozone yields geronic acid while on oxidation with potassium permanganate a : a-dimethyl- glutaric acid is formed. Dihydroxy-a-carotene C4oH5g02 CH, CH3 CH, CHo OHo CH, CH, CH, C CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH = C-CH=CH-CH CHj I ^OH I I I I/OH C CH2 CH3 c /\/ \/'\ Dihydroxy-a-carotene(?) H3C CH CHj CH3 Dihydroxy-a-carotene was obtained by Karrer and collaborators together with a-semicarotenone and a-carotone during the oxidation of a-carotene with chromic acid^^^. With regard to the constitution of this derivative, cf. Karrer, VON EuLER and Solmssen^^^. The compound crystallises from a mixture of methanol and petroleum ether in needles, m.p. 183° (uncorr.). Dihydroxy- a-carotene is sparingly soluble in petroleum ether. It is dextrorotatory. Ac- cording to Karrer, von Euler and Solmssen it has no vitamin A activity. Solvent: Carbon disulphide Senii-a-carotenone C40H58O2 : OHo GH^ A bsorption maxima: 502 471 440 m/f CH, CH, \/ 0 ' CHo CHi CHo CHo C /\ I I I \ /\ CH, CO-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH. CH2 CO-CH, \V CH, Semi-a-carotenone H,C-C CH, \ CH Semi-a-carotenone is formed by the oxidation of a-carotene with chromic acid^^^ Semi-a-carotenone crystallises from methanol in needles, m.p. 135° (uncorr.). The question of its constitution is dealt with in detail in the original communication^^*. The fact that semi-a-carotenone has no vitamin A activity^^^ is in agreement with the formula shown. Solvent: Absorption maxima: Carbon disulphide 533 499 mfj, Semi-a-carotenone mono-oxime C4oH5702Ni^® forms red crystals, m.p. 132". References p. 165— lyo. 156 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X a-Carotone C4QH56O5: a-Carotone is formed by the oxidation of a-carotene with chromic oxide^'''. It crystalHses from methanol in glittering steel-blue prisms, m.p. 148° [a]g44 = +341° (± 15°). a-Carotone has no vitamin A activity. Solvent: Absorption maxima: Carbon disulphide (535) 502 471 vafx Chloroform 484 454 rtifx a-Apo-2-carotenal C30H40O: CHo CHo \/ C CH3 CH, CH, CH, /\ I I I I CH2 CH- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO CH2 C* CH3 \ ^ a-Apo-2-carotenal CH * This aldehyde is formed by the potassium permanganate oxidation of a-carotene. a-Apo-2-carotenal crystallises from petroleum ether in clustered light red prisms, m.p. 158°. It is less easily soluble in the common solvents than the y?-isomer. Solvent: Absorption maxima: Carbon disulphide 519 484 454 m^ Petroleum ether 479 450 m/j, (cf. Fig. 28, p. 359) a-Apo-2-carotenal oxime separates from absolute methanol in clustered red leaflets, m.p. 178°. Md ~ +692° (± 35°) (for further data compare the original communication). Solvent: Absorption maxima: Carbon disulphide 499 469 m/n Petroleum ether 466 438 m/t (diffuse) Ethanol 469 439 m// By the oxidation of chromatographically unpurified carotene, von Euler, Karrer and Solmssen obtained a compound which, was spectroscopically indistinguishable from a-apo-2-carotenal. The analytical figures for this com- pound as well as those for the oxime agreed with the calculated values for a-apo-2-carotenal. This new degradation product had m.p. 174°, however, and the oxime had m.p. 185°. The melting points are considerably higher than for a-apo-2-carotenal. For details, compare the original communication^^^. References p. 165-iyo. a-Apo-2-carotenol C3QH42O: a-CAROTENE 157 C CH, CH3 CH3 CH3 /\ I I I ■ I CH, CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH,OH I I CHg C" CH3 \ ^ a-Apo-2-carotenol CH This alcohol is obtained by the reduction of a-apo-2-carotenal (lower melting form) with isopropyl alcohol and aluminium isopropoxide. It crystallises from a mixture of benzene and petroleum ether in spherical golden-yellow clusters, m.p. 157° (sinters at 150°). Solvent: Absorption maxima: Carbon disulphide 478 448 vn.pL Petroleum ether 446 420 m/z Ethanol 448 423 m// On treating a solution of a-apo-2-carotenol in chloroform with antimony trichloride, a fairly stable blue colouration with an absorption maximum at 562 vafi, is observed. a-Carotene iodide: a-Carotene adds two atoms of iodine with the formation of a crystalline di-iodide C40H56I2 (Karrer, Solmssen and Walker)i®^. This iodide exhibits vitamin A activity. With regard to another iodide of ^-carotene, cf. Kuhn and Brockmann^''". Neo-a-carotene U and neo-a-carotene W: The question of stereoisomerism in a-carotene was examined several years ago by GiLLAM, El Ridi and Kon^'^^. These investigations have been renewed by Zechmeister and Polgar who succeeded in isolating two cis-trans isomers of a-carotene in the crystalline state* ^'^^. Neo-a-carotene U is formed from a-carotene under the influence of heat, or illumination, or treatment with iodine or acids, or by melting the crystals. It is found above a-carotene in the chro- matogram on calcium hydroxide. The pigment crystallises from a mixture of benzene and methanol in orange prisms, m.p. 65° (corr.). Neo-a-carotene U is more soluble in the usual solvents than a-carotene. On treatment with iodine it is partly' transformed into a-carotene. Solvent: Absorption maxirna: Carbon disulphide 503 470.5 m/i Benzene 485.5 453.5 m^ Chloroform 485 453 m^ Petroleum ether (B.p. 60-70° C) . . . 471.5 441.5 m^i The work of L. Zechmeister has recently been confirmed in detail by F. P. Zscheile and co-workers, Arch. Biochem. 5 (1944) 77, 211. References p. 165-170. 158 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Further information can be found in the original communication^'^. Data regarding vitamin A activity are given by Zechmeister and co-workers^'^. Neo-a-carotene U possesses weaker growth-promoting properties than a-carotene. Neo-a-carotene W is formed together with neo-a-carotene U, as well as several other stereoisomers not obtained in the crystalline state"^. Neo-a-caro- tene W is found below neo-a-carotene U but above a-carotene in the chromato- gram on calcium hydroxide and crystallises from a mixture of benzene and methanol in small prisms, m.p. 97° (corr.). Its solubility is similar to that of the U-isomer. Solvent: Absorption maxima: Carbon disulphide 502 469.5 m/< Benzene 484 453.5 ran Chloroform 484 453 m/x Petroleum ether (B.p. 60-70° C) . . . 470.5 441 m/z The absorption maxima of the crystalline and non-crystalline transformation products in ligroin are shown below : Neo-a-carotene U (crystallised) . . . 471.5 441.5 m/i Neo-a-carotene V 465.5 437 m^ Neo-a-carotene W (crystallised) . . . 470.5 441 m/i Neo-a-carotene X 463.5 435 m/x Neo-a-carotene Y 467.5 437 vcifi a-Carotene (natural) 477 446.5 m^ Neo-a-carotene A 468.5 439 van Neo-a-carotene B 466.5 437 m^ Neo-a-carotene C 472.5 442.5 m/z Neo-a-carotene D 460 432 van Neo-a-carotene E 461.5 433.5 m/i (The sequence of isomers given is that in which they occur on the chromato- gram). a-Carotene mono-epoxide and flavochrome C4oH5gO: CH3 CH, CH3 CH3 /\ \ \ \ I /\ CH, C— CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CHg )0 ' ' C CH2 CH, C • /\/ \/\ H3C CH CH2 CH3 a-Carotene mono-epoxide a-Carotene mono-epoxide is formed by the oxidation of a-carotene with monoperphthalic acid (Karrer and Jucker^'*). It crystalHses from a mixture of benzene and methanol in thin, reddish-yellow plates, m.p. 175° (uncorr., in vacuum). On treating an ethereal solution of the pigment with concentrated References p. i6§-iyo. 4 a-CAROTENE 159 aqueous hydrochloric acid, the acid layer assumes a very weak, unstable blue colouration. Solvent: Absorption maxima: Carbon disulphide 503 471 m^u Benzene 484 455 m/x Petroleum ether 471 442 mfj, Chloroform 483 454 m/z a-Carotene mono-epoxide occurs in the blossoms of various plants {Trago- -pogon pratensis, Ranunculus acery^^. According to von Euler, a-carotene mono- epoxide possesses vitamin A potency"^. CH3 CH3 (] , CHo CHo A \V CHa C=CH CH3 CH3 CH3 CH3 C CH, C CH— C=CHCH=CH-C=CHCH = CHCH=C-CH=CHCH=C-CH=CH-CH CHj Flavochrome H3C-C CHj CH Flavochrome is formed by the action of dilute acids (e.g. hydrogen chloride in chloroform) on a-carotene mono-epoxide. It crystalhses from a mixture of benzene and methanol in thin lustruous yellow plates, m.p. 189° (uncorr., in vacuum). It exhibits a colour reaction with hydrochloric acid, similar to that of a-carotene mono-epoxide. Solvent: Absorption maxima: Carbon disulphide 482 451 m^ Benzene 462 434 m/i Petroleum ether 450 422 m/x Chloroform 461 433 m/z Flavochrome does not exhibit growth-promoting properties. Both a-caro- tene mono-epoxide and flavochrome are epiphasic in the partition test. Flavo- chrome occurs in Ranunculus acer and Tragopogon pratensis. ^:6-Dihydro-a-carotene and ^:6'dihydro-^-carotene C^^r,^: PoLGAR and Zechmeister"' treated solutions of a-carotene or /5-carotene in petroleum ether with cold concentrated hydrogen iodide, and in both cases obtained several chromatographically separable reduction products, from which two pigments could be isolated in the crystalline state. From the analytical data, the results of catalytic hydrogenation, the ab- sorption maxima, and the absence of isopropylidene groupings, these compounds References p. 165-170. i6o CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X are formulated as 5 : 6-dihydro-a-carotene and 5 : 6-dihydro-/3-carotene, re- spectively. It is of interest that both these hydrogenation products are obtained from a-carotene as well as /3-carotene. Under the usual conditions of isomerisation (cf. p. 39) both dihydrocaro- tenes undergo reversible cis-trans isomerisation. The absorption spectra exhibit the characteristic "as-peak", \V \V C CHo CHo CHo CHo C /\ I I I \ /\ CH2 CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, I I II I " CHj CH"CH3 HsC'C CHj \ / 5 :6-Dihydro-^-carotene \ / CH2 CHj CHo CHo CHo CHo \V . \V C GHo CHo CHo CHo C /\ r I I I /\ CH2 CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CHj CH2 CH'CH3 H3C*0 CH2 \ / 5:6-Dihydro-a-carotene "%/ CHj CH 5 : 6-Dihydro-/?-carotene crystallises from mixtures of carbon disulphide and ethanol, benzene and methanol, or chloroform and methanol, in characteristic plates or wedges. The highest m.p. which has been recorded is 164°, but in a number of cases it was considerably lower, e.g. 155 and 160°. The solubility of dihydro-y5-carotene and its behaviour in the partition test corresponds to that of /3-carotene. In the chromatogram the pigment is adsorbed below /9-caro- tene, but above 5 : 6-dihydro-a-carotene. Solvent: Absorption maxima: Carbon disulphide 509.5 476 m/n Benzene 489 458 m/j. Chloroform 489 457 m/i Petroleum ether 477.5 447.5 m^ Ethanol 477.5 448 m/n 5 : 6-Dihydro-a-carotene crystallises from a mixture of carbon disulphide and ethanol in microscopic rectangular yellow leaflets (cf . the original communi- cation). From a mixture of benzene and methanol it is obtained in crystals which resemble those of natural a-carotene, and melt at 202-203° (with previous sintering). 5 : 6-Dihydro-a-carotene is somewhat more soluble than 5 : 6-dihydro-jS-carotene. References p. iG^-ijo. y-CAROTENE Solvent: Absorption ynaxima: Carbon disulphide 501 486.5 m/i Benzene 483.5 453.5 m/i Chloroform 482.5 452.5 m/i Petroleum ether 470.5 442.5 m^ Ethanol 471 443 m/^ i6i A o. I % solution of the pigment in benzene shows no optical rotation in a 10 cm tube. Dihydro-a-carotene is entirely epiphasic in the partition test. 5. y-CAROTENE C40H56 History 1933 KuHN and Brockmann discover a third carotene-isomer, y-carotene, by means of chromatographic adsorption analysis*' i'^. They elucidate the constitution of the new pigment. Occurrence y-Carotene is one of the rarest carotenoids. In carotene from carrots it occurs only to the extent of about o.i % of /^-carotene. Source Aleuria aurantiaca Allomyces Bacillus Lombardo Pelle- grini, Bacillus Grasberger Butia capitata Carotene from carrots Chara ceratophylla Wallr. Chrysemis scripta elegans (Japanese tortoise) TABLE 37 OCCURRENCE OF y-CAROTENE References E. Lederer, Cham. Centr. igsg, I, 2991. Bl. Soc. Chim. biol. 20 (1938) 611. R. Emerson and D. L. Fox, Pvoc. Roy. Soc. (London) (B) 128 (1940) 275. E. Chargaff and E. Lederer, Chem. Centr. 1936, I, 3159. L. Zechmeister and W. A. Schroeder, /. Am. Chem. Soc. 64 (1942) 1173. R. KuHN and H. Brockmann, Ber. 66 (1933) 407. P. Karrer, W. Fatzer, M. Favarger and E. Jucker, Helv. chim. Acta 26 (1943) 2121. E. Lederer, Chem. Centr. 1939, I, 2990. — Bl. Soc. Chim. biol. 20 (1938) 554. V. N. LuBiMENKO observed a pigment with properties intermediate to those of lycopene and ^-carotene in fruit of the Gonocaryum species. This was probably y-carotene {Rev. gen. bat. 25 (1914) 474). A. Winterstein isolated impure y-carotene from Gonocaryum pyriforme (Z. physiol. Chem. 215 (1933) 51), and shortly afterwards established the identity of this pigment with the y-carotene of R. Kuhn and H. Brockmann (Z. physiol. Chem. 219 (1933) 249). References p. 165-ijo. Carotenoids ii i62 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Source Citrullus vulgaris Schrad. Crocus sativus Cuscuta subinclusa and Cuscuta salina Gazania rigens Gonocaryum pyriforme Mimulus longiflorus Mycobacterium phlei Nitella syncarpa (Thuill.) Palm oil Prunus armeniaca Pyracantha coccinia Red Sponge ( Hymeniacedon Sanguineum) Rhodotorula Sanniei Rosa rubiginosa L. Rosa rugosa Thumb. Rubus Chamaemorus L. References L. Zechmeister and A. PolgAr, /. biol. Chem. ijg (1941) 193. R. KuHN and A. Winterstein, Ber. 6y (1934) 344. G. Mackinney, /. biol. Chem. ii2 (1935) 421. K. Sch5n, Biochem. J. 32 (1938) 1566. — L. Zech- meister and W. A. Schroeder, /. Am. Chem. Soc. 65 (1943) 1535. A. Winterstein, Z. physiol. Chem. 215 (1933) 51; 219 (1933) 249. W. A. Schroeder, /. Am. Chem. Soc. 64 (1942) 2510. E. Chargaff, Chem. Centr. 1934, I, 1662. — Y. Takeda and T. Ohta, Z. physiol. Chem. 263 (1940) 233. P. Karrer, W. Fatzer, M. Favarger and E. Jucker, Helv. chim. Acta 26 (1943) 2121. R. F. Hunter and A. D. Scott, Biochem. J. 35 (1941) 31. H. Brockmann, Z. physiol. Chem. 216 (1933) 45. P. Karrer and J. Rutschmann, Helv. chim. Acta 28 (1945) 1528. P. J. Drumm and W. O'Connor, Nature (London) 145 (1940) 425. C. Fromageot and J. Leon Tchang, Chem. Centr. 1930, I, 1580; Arch. Mikrobiol. 9 (1938) 424. R. Kuhn and C. Grundmann, Ber. 67 (1934) 342. H. WiLLSTAEDT, Chem. Centr. 1935, II, 707; Svensk Kern. Tidskr. 47 (1935) 112. H. WiLLSTAEDT, Chem. Centr. 1937, I, 2620. Preparation According to Kuhn and Brockmann^'* crude carotene is used for the preparation of y-carotene. The crude carotene is crystallised three times from a mixture of benzene and methanol, the pigment being extracted with pure boiling methanol after each crystallisation. 300 Mg of pigment purified in this way are dissolved in 300 ml of benzene, the solution is diluted with 900 ml of petroleum ether and poured on a column of alumina (17x5 cm). The chromatogram is washed with a benzene- petrol mixture (1:4) until the uppermost zone containing y-carotene is separated from the next lower zone by a colourless strip. After elution of the pigment with methanolic petroleum ether, the methanol is removed by washing and the carmine- red solution is dried and the solvent distilled off. The residue is repeatedly extracted with boiling pure methanol and recrystallised several times from a mixture of benzene and methanol (2:1). The yield of analytically pure material is about 1 %, based on carotene. Winterstein^^" prepared y-carotene from Gonocaryum pyriforme. 300 Fruit skins yield 3 mg of pigment. References p. 165-170. y-CAROTENE 163 Chemical Constitution CHo CH, CH3 CH, C CH3 CH3 CH3 CH3 c^ /\ II I I \ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH I II II I CHj C-CHg ^ HaC-C CH, \ / y-Carotene \ y^ CH, CH, The elucidation of the constitution of this pigment was especially difficult in view of the small amount of material available. After establishing the carbon and hydrogen content of y-carotene, Kuhn and Brockmann^^^ proved the presence of 12 double bonds by means of catalytic hydrogenation. y-Carotene therefore contains one isocyclic ring. The absorption spectrum indicates that only II of the 12 double bonds are conjugated. By ozonisation of the pigment Kuhn and Brockmann obtained 0.85 mol of acetone, and concluded that one end of the molecule must have an open-chain structure. No geronic acid could be isolated, so that the presence of a /5-ionone ring has not been finally proved. However, the vitamin A activity (cf. p. 15) of y-carotene supports the proposed formula since it is known that only compounds containing an unsubstituted j5-ionone ring show growth-promoting properties. Properties Crystalline form: y-Carotene crystallises from a mixture of benzene and methanol in microscopic dark red prisms with a blue lustre. On rapid crystal- lisation, the pigment is obtained in more lightly coloured needles. Melting point^^^: 178° (corr., in vacuum)!^^; 176.5° (corr.)^^*. Solubility: y-Carotene is less soluble in the usual solvents than the ^S-isomer. Spectral properties: Solvent: Absorption maxima: Carbon disulphide 533.5 496 463 m// Chloroform 508.5 475 446 mfi Benzene 510 477 447 m// Petrol . .' 495 462 431 m^ Hexane 494 462 431 ran (cf. Fig. 5, p. 349) Quantitative extinction measurements: Kuhn and Brockmanni*^, A. WiNTERSTEiN and U. Ehrenberg, {Z. physiol. Chem. 20J (1932) 25) ascribed the above formula to a pigment which they had isolated from Convallaria majaiis. Subsequent investigations showed, however, that this pigment was a mixture and not identical with y-carotene. References p. 165-170. i64 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X Optical activity: y-Carotene is optically inactive. Partition test: On partition between petroleum ether *and 90% methanol, y-carotene is entirely epiphasic. Chromatographic behaviour: y-Carotene is more strongly adsorbed from petroleum ether solution than /5-carotene. In the chromatogram on calcium hydroxide or alumina it is found above /9-carotene and below lycopene. Detection and estimation: The separation of y-carotene from other carotenoid hydrocarbons is achieved by chromatographic adsorption on alumina or calcium hydroxide. Its presence can be established by the determination of the absorption maxima. Physiological properties: y-Carotene exhibits strong vitamin A potency^^^. Several investigators have reported on the supposed function of y-carotene in the sexual metabolism of various plants^^^. Stereoisomers of y-carotene: By the treatment of y-carotene with heat, light, or iodine, or fusion of the crystals, Zechmeister and PolgAr^^' partly con- verted y-carotene into various cis-trans isomers. None of these compounds has so far been obtained in the crystalline state. The different isomers are separated by means of chromatographic adsorption on calcium hydroxide and can be distinguished by their absorption spectra. A bsorption maxima (in petroleum ether) Neo-y-carotene U 489 457 m/x (Natural y-Carotene) 494 461.5 m/< Neo-y-carotene A 486 455.5 m/x Neo-y-carotene B 486 455.5 m/^ Neo-y-carotene H 489 457.5 m/i Neo-y-carotene A' 485.5 455 m/i Neo-y-carotene B' 486 455 m/t Neo-y-carotene G' 483 452 mix (y-Carotene with all-c?s configuration) 456 m// The vitamin A activities of y-carotene and pro-y-carotene have recently been investigated by Zechmeister and co-workers^^'^. 6. PRO-y-CAROTENE C4oH5g In 1941, Zechmeister and Schroeder^^^ found a new polyene pigment in the fruit of Butia capitata which they termed pro-y-carotene. The new pigment has also been observed in the following plants: Pyracantha angustifolia Schneid^^", Evonymus fortunei L.^^^ and Mimulus longiflorus Grant^^^. References p. 165-iyo. REFERENCES 165 Pro-y-carotene is a naturally occurring stereoisomer of y-carotene. According to Zechmeister and Schroeder^^^ 6 or 7 of the double bonds have a trans, and 4 or 5 a cis configuration. By the fusion of pro-y-carotene crystals, by heating solutions of the pigment, or by treatment with concentrated hydro- chloric acid or iodine, a mixture of stereoisomers is obtained which contains y-carotene. Pro-y-carotene crystallises from a mixture of benzene and methanol in glistening redplates,m.p. 118-119° (corr.) (cf. Zechmeister and Schroeder)^^^^ It is easily soluble in benzene, petroleum ether and other organic solvents, with the exception of alcohols. Solvent: Absorption maxima Carbon disulphide 493.5 460.5 m/x Benzene 477 447.5 m/j. Chloroform 473 (444) m/i Ethanol (465) (437) m/x Petroleum ether 464 (435) mfi Pro-y-carotene is adsorbed a little more weakly than y-carotene on calcium hydroxide from petroleum ether solution. REFERENCES 1. Hartsen, Chem. Centr. 1S73, 204; Compt. rend 76 (1873) 385; Courchet, Ann. Sci. nat. (7) 7 (1888) 320, 356. 2. MiLLARDET, Bull. Soc. Sci. Nuncy (2) / (1875) p. 21. 3. C. A. ScHUNCK, Proc. Roy. Soc. (London) 72 (1903) 165 (cf. Zopf, Biol. 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Willstatter and H. H. Escher, Z. physiol. Chem. 64 (1910) 52. 33. B. V. EuLER, H. V. EuLER and P. Karrer, Helv. chim. Acta 12 (1929) 279. — H. v. EuLER, P. Karrer and M. Rydbom, Ber. 62 (1929) 2446. — R. Kuhn and E. Lederer, Ber. 65 (1932) 638. — B. v. Euler and P. Karrer, Helv. chim. Acta 15 (1932) 496. — H. V. Euler, P. Karrer, E. Klussmann and R. More, Helv. chim. Acta 15 (1932) 502. 34. A. Winterstein, Z. physiol. Chem. 215 (1933) 52. 35. A. Winterstein, and G. Stein, Z. physiol. Chem. 220 (1933) 250. 36. S. J. B. Connell, Biochem. J. 18 (1924) 1127. 37. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 45. 38. P. Karrer and R. Widmer, Helv. chim. Acta 11 (1928) 751. 39. P. Karrer, A. Helfenstein and R. Widmer, Helv. chim. Acta 11 (1928) 1201. 40. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 436. 41. P. Karrer and J. Rutschmann, Helv. chim. Acta 28 (1945) 793. 42. R. Kuhn and C. Grundmann, Ber. 65 (1932) 900. 43. R. Kuhn and C. Grundmann, Ber. 65 (1932) 1887. 44. P. Karrer and W. Jaffe, Helv. chim. Acta 22 (1939) 69. 45. R. Kuhn and C. Grundmann, Ber. 65 (1932) 1882 (cf. P. Karrer and W. Jaff6, Helv. 22 (1939) 69. 46. R. Kuhn and C. Grundmann, Ber. 65 (1932) 1888. 47. P. Karrer and W. Jaffe, Helv. chim. Acta 22 (1939) 69. 48. L. Zechmeister and P. Tuzson, Nature 141 (1938) 249; Biochem. J. 32 (1938) 1305; Ber. 72 (1939) 1340- 49. P. Karrer and H. Kramer, Helv. chim. Acta 2j (1944) 1301. 50. P. Karrer and A. Helfenstein, Helv. chim. Acta 14 (1931) 78. 51. L. Zechmeister and co-workers, Proc. Nat. Acad. Sci. 27 (1941) 468. 52. L. Zechmeister and co-workers. Science 94 (1941) 609. 53. L. Zechmeister and co-workers, /. biol. Chem. 144 (1942) 315. 54. L. Zechmeister and co-workers, /. biol. Chem. 144 (1942) 321. 55. L. Zechmeister and co-workers. Arch. Biochem. i (1943) 231. 56. A. L. LE RosEN and L. Zechmeister, /. Am. Chem. Soc. 64 (1942 )io75. 57. L. Zechmeister and J. H. Pinckard, /. Am. Chem. Soc. 6g (1947) 1930. 58. Zeise, Ann. 62 (1947) 380; /. prakt. Chem. 40 (1847) 297. 59. A. Arnaud, Compt. rend. 102 (1886) 11 19. 60. R. Willstatter and W. Mieg, Ann. 355 (1907) I. 61. L. Zechmeister, L. v. Cholnoky and V. Vrabely, Ber. 61 (1928) 566; Ber. 66 (1933) 123. 62. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 1142; 13 (1930) 1084; 14 (1931) 1033- 63. R. Kuhn and H. Brockmann, Ber. 65 (1932) 894; 66 (1933) 1319; 67 (1934) 1408; Ann. 516 (1935) 95- 64. cf. Strain, /. biol. Chem. iii (1935) ^5 for the occurrence of carotene in green leaves. REFERENCES 167 65. R. WiLLSTATTER and A. Stoll, Untersuckungen iiber Chlorophyll, Berlin 191 3. — R. WiLLSTATTERandA. Stoll, Untersuchnngen iiber die Assimilation der Kohlensaure, Berlin 191 8. 66. L. S. Palmer, Carotenoids and related Pigments, New York, 1922. — P. Karrer and O. Walker, Helv. chim. Acta 17 (1934) 43- — R- Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 41. 67. R. KuHN and E. Lederer, Z. physiol. Chem. 200 (1931) 246. 68. R. KuHN and H. Brockmann, Z. physiol. Chem. 200 (1931) 255. 69. L. Zechmeister and P. TuzsoN, Ber. 67 (1935) 824. 70. P. Karrer and W. Schlientz, Helv. chim. Acta ij (1934) 7- 71. R. WiLLSTATTER and H. H. Escher, Z. physiol. Chem. 64 (1910) 47. 72. P. Karrer and O. Walker, Helv. chim. Acta 16 (1933) 641. — Cf. also H. H. Strain, /. biol. Chem. 105 (1934) 524; iii (1935) 86. 73. R. WiLLSTATTER and H. H. Escher, Z. physiol. Chem. 64 (1910) 47- 74. R. KuHN and E. Lederer, Ber. 64 (1931) 1349. 75. R. WiLLST.\TTER and W. MiEG, Ann. 355 (1907) i. 76. L. Zechmeister, L. v. Cholnoky and V. VRABi;LY, Ber. 61 (1928) 566; 66 (1933) 123. yj. R. PuMMERER and L. Rebmann, Ber. 61 (1928) 1099. 78. P. Karrer and A. Helfenstein, Helv. chim. Acta 12 (1929) 1142. — P. Karrer, A. Helfenstein, H. Wehrli and A. Wettstein, Helv. chim. Acta 13 (1930) 1084. — P. Karrer and R. More, Helv. chim. Acta 14 {1931) 1033. 79. P. Karrer and A. Helfenstein, Helv. chim. Acta 12 (1929) 1142. — cf. R. Kuhn and A. Winterstein and L. Karlovitz, Helv. chim. Acta 12 (1929) 64. 80. R. Kuhn and L. Ehmann, Helv. chim. Acta 12 (1929) 904. 81. R. Kuhn and F. l'Orsa, Ber. 64 (1931) 1732. 82. P. Karrer, A. Helfenstein, H. Wehrli and A. Wettstein, Helv. chim. Acta 13 (1930) 1084. 83. P. Karrer and R. More, Helv. chim. Acta 14 (1931) 1033. 84. R. Pummerer, L. Rebmann and W. Reindel, Ber. 64 (1931) 492. 85. H. H. Strain, /. biol. Chem. 102 (1933) 137. 86. R. Kuhn and A. Winterstein, Ber. 66 (1933) 429- 87. R. Kuhn and H. Brockmann, Ann. 516 (1935) 95; Ber. 65 (1932) 894; Z. physiol. Chem. 213 (1932) i; Ber. 66 (i933) 1319; 67 (1934) 1408. 88. P. Karrer and E. Jucker, Helv. chim. Acta 30 (1947) 266. 89. R. Kuhn and E. Lederer, Ber. 64 (1931) 1352; Z. physiol. Chem. 200 (1931) 247. 90. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 615. 91. R. Kuhn and H. Brockmann, Ber. 66 (1933) 408. 92. E. S. Miller, Chem. Centr. ig35, I, 3545. 93. K. W. Hausser and A. Smakula, Z. angew. Chem. 47 (1934) ^^3; 48 (1935) 152. 94. H. v. Euler and H. Hellstrom, Z. physik. Chem. (B) 15 (1932) 343. 95. H. V. Euler, P. Karrer and M. Rydbom, Ber. 62 (1929) 2445; Helv. chim. Acta 14 (1931) 1428. 96. L. Zechmeister, Carotinoide, Berlin 1934, p. 127. 97. R. Kuhn and K. Brockmann, Z. physiol. Chem. 206 (1932) 46. 98. P. Karrer and O. Walker, Helv. chim. Acta 16 (1933) 641. 99. R. WiLLSTATTER and W. MiEG, Ann. 355 (1907) 19. — R. Willstatter and H. H. Escher, Z. physiol. Chem. 64 (1910) 57. 100. H. V. Euler, P. Karrer and M. Rydbom, Ber. 62 (1929) 2445. loi. C. H. Warner, Proc. Roy. Soc. (London) (B) 87 (1914) 384; Chem. Centr. 1914. 1, 21 10. 102. R. Pummerer, L. Rebmann and W. Reindel, Ber. 64 (1931) 496, 500. 103. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 43. 104. B. V. Euler, H. v. Euler and H. Hellstrom, Biochem. Z. 203 (1928) 370. — B. v. Euler, H. v. Euler and P. Karrer, Helv. chim. Acta 12 (1929) 278. — H. v. Euler, P. Karrer and M. Rydbom, Ber. 62 (1929) 2445. 105. For a review of the literature see Helv. chim. Acta 23 (1940) 955. i68 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X io6. P. Karrek and A. Ruegger, Helv. chim. Ada 23 (1940) 955. 107. L. Zechmeister, L. v. Cholnoky and V. Vrab^ly, Ber. 61 (1928) 566; 66 (1933) 123. — cf. J. H. C. Smith, /. biol. Chem. go (1931) 597; 96 (1932) 35. 108. H. V. EuLER, V. Demole, p. Karrer and O. Walker, Helv. chim. Acta 13 (1930) 1078. 109. R. KuHN and E. Lederer, Naturwissenschaften 19, (1931) 306; Ber. 65 (1932) 639. — cf. A. E. GiLLAM, I. M. Heilbron, R. a. Morton and J. C. Drummond, Biochem. J. 26 (1932) 1 174. — P. Karrer, K. Schopp and R. More, Helv. chim. Acta 15 (1932) 1158. no. P. Karrer and G. Schwab, Helv. chim. Ada 23 (1940) 578. 111. P. Karrer, K. Schopp and R. More, Helv. chim. Ada 15 (1932) 1158. — cf. R. Kuhn and E. Lederer, Ber. 65 (1932) 639. — A. E. Gillam, I. M. Heilbron, R. A. Morton and J. C. Drummond, Biochem. J. 26 (1932) 1174. 112. K. W. Hausser and A. Smakula, Angew. Chem. 47 (1934) 663; 48 (1935) 152. 113. H. V. Euler, p. Karrer and O. Walker, Helv. chim. Ada 15 (1932) 1507. 114. P. Karrer and E. Jucker, Helv. chim. Ada 28 (1945) 427. 115. R. Kuhn and H. Brockmann, Ber. 65 (1932) 896; Ber. 6j (1934) 1408; cf. Ann. 516 (1935) 95. 116. R. Kuhn and H. Brockmann, Ann. 516 (1935) 98, 120. 117. R. Kuhn and H. Brockmann, Ber. 66 (1933) 1319. 118. R. Kuhn and H. Brockmann, Ann. 516 (1935) 120. 119. P. Karrer and U. Solmssen, Helv. chim. Ada 18 (1935) 25. 120. R. Kuhn and H. Brockmann, Ann. 516 (1935) 113, 122. 121. R. Kuhn and H. Brockmann, Ber. 65 (1932) 894; 6y (1934) 885; Ann. 5/6(1935) 123. 122. R. Kuhn and H. Brockmann, Ber. 66 (1933) 1324. 123. R. Kuhn and H. Brockmann, Z. physiol. Chem. 213 ^1932) 3. 124. K. W. Hausser and A. Smakula, Angew. Chem. 4y (1934) 663; 48 (1935) 152. 125. R. Kuhn and H. Brockmann, Ann. 516 (1935) 113; 516 (1935) 123. 126. R. Kuhn and H. Brockmann, Ann. 516 (1935) 113, 124. 127. R. Kuhn and H. Brockmann, Ber. 66 (1933) 1324. 128. R. Kuhn and H. Brockmann, Ber. 6y (1934) 886; Ann. 516 (1935) 98, 125, 127; cf. Z. physiol. Chem. 215 (1932) 5. 129. P. Karrer and U. Solmssen, Helv. chim. Ada 20 (1937) 682. — P. Karrer, U. Solmssen and W. Gugelmann, Helv. chim. Ada 20 (1937) 1020. 130. H. V. Euler, P. Karrer and U. Solmssen, Helv. chim. Ada 21 (1938) 211. 131. P. Karrer and U. Solmssen, Helv. chim. Ada 20 (1937) 688. — P. Karrer, U. Solmssen and W. Gugelmann, Helv. chim. Ada 20 (1937) 1023. 132. H. V. Euler, P. Karrer and U. Solmssen, Helv. chim. Ada 21 (1938) 211. 133. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 427. 134. P. Karrer and E. Jucker, Helv. chim. Ada 28 (1945) 427. 135. P. Karrer, E. Jucker, J. Rutschmann and K. Steinlein, Helv. chim. Ada 28 (1945) 1150- 136. A. E. Gillam and M. S. El Ridi, Nature (London) 136 (1935) 914; Biochem. J. 30 (1936) 1735; 31 (1937) 251- 137. L. Zechmeister and co-workers, Nature (London) 141 (1938) 249; Biochem. J. 32 (1938) 1305; Ber. 72 (1939) 1340;/. Am. Chem. Soc. 64 (1942) 1856; 65 (1943) 1528; 66 (1944) 137; Arch. Biochem. 5 (1944) 107; 7 (1945) 247. — cf. also F. P. Zscheile and co-workers. Arch. Biochem. 5 (1944) 211, and H. H. Strain, /. Am. Chem. Soc. 63 (1941) 3448. 138. L. Zechmeister and co-workers, Nature (London) 141 (1938) 249; Biochem. J. 32 (1938) 1305; Ber. 72 (1939) 1340;/. Am. Chem. Soc. 64 (1942) 1856; 65 (1943) 1528; 66 (1944) 137; Afch. Biochem. 5 (1944) 107; 7 (1945) 247. — cf. also F. P. Zscheile and co-workers. Arch. Biochem. 5 (1944) 211; H. H. Strain, /. Am. Chem. Soc. 63 (1941) 3448. REFERENCES 169 139. A. E. GiLLAM and M. S. El Ridi, Nature (London) 136 (1935) 914; Biochem. J. 30 (1936) 1735; 31 {1937) 251- 140. For further data see/. Am. Chem. Soc. 64 (1942) i860. 141. R. KuHN and E. Lederer, Ber. 64 (1931) 1349; Naturwissenschaften ig (1931) 306. 142. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 614; Ark. Kemi. B. 10 (1931) No. 15. 143. P. Karrer and O. Walker, Helv. chim. Acta 16 (i933) 641- 144. P. Karrer, R. More and O. Walker, Helv. chim. Acta 16 (1933) 975- 145. G. Mackinney, /. biol. Chem. iii (1935) 75. 146. H. H. Strain, /. biol. Chem. iii (1935) 85. 147. P. Karrer and O. Walker, Helv. chim. Acta 16 (1933) 641. 148. R. KuHN and E. Lederer, Ber. 64 (1931) 1349; Z. physiol. Chem. 200 (1931) 246. — R. KuHN and H. Brockmann, Z. physiol. Chem. 200 (1931) 255. — H. H. Strain, J. biol. Chem. 105 (1934) 523; ^^i (i935) 86. 149. P. Karrer, A. Helfenstein, H. Wehrli, B. Pieper and R. More, Helv. chim. Acta 14 (1931) 614. — P. Karrer and R. More, Helv. chim. Acta 14 (1931) 833. 14 (1931) I033- — P- Karrer, R. More, E. v. Krauss and A. Zubrys, Helv. chim. Acta 15 (1932) 490. — P. Karrer, K. Schopp and R. More, Helv. chim. Acta 15 (1932) 1158. R. KuHN and E. Lederer, Ber. 64 (1931) 1349- 150. J. H. C. Smith, /. biol. Chem. 102 (1932) 159. — R. Kuhn and E. F. Moller, Z. angew. Chem. 4j (1934) I45- 151. P. Karrer, R. IMorf and O. Walker, Helv. Chim. Acta 16 (1933) 975- 152. P. Karrer and O. Walker, Hev. chim. Acta 16 (1933) 642. 153. R. Kuhn and E. Lederer, Z. physiol. Chem. 200 (1931) 254. 154. K. W. Hausser and A. Smakula, Angew. Chem. 47 (1934) 663; 48 (1935) 152. 155. H. V. Euler and H. Hellstrom, Z. physik. Chem. (B) 75 (1932) 343. 156. R. Kuhn and E. Lederer, Naturwissenschaften ig (1931) 306; Ber. 64 (1931) 1349. 157. E. Baur, Helv. chim. Acta ig (1936) 1210. 158. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1942) 43. 159. H. V. Euler, P. Karrer, H. Hellstrom and M. Rydbom, Helv. chim. Acta 14 (1931) 839. — R. Kuhn and H. Brockmann, Ber. 64 (1931) 1859; Z. physiol. Chem. 221 (1933) 130. — H. Brockmann and M. L. Tecklenburg, Z. physiol. Chem. 221 (1933) 117. — H. Brockmann, Angew. Chem. 4j (1934) 523- 160. P. Karrer and R. More, Helv. chim. Acta 14 (1931) 836. - — cf. U. Solmssen , Disser- tation, Zurich 1936. 161. P. Karrer, U. Solmssen and O. Walker, Helv. chim. Acta ij (1934) 4i7- — P. Karrer, H. v. Euler and U. Solmssen, Helv. chim. Acta 17 (1934) 1171- 162. P. Karrer, H. v. Euler and U. Solmssen, Helv. chim. Acta ij (1934) 1171. 163. P. Karrer, U. Solmssen and O. Walker, Helv. chim. Acta ly (1934) 4i7- -~ P. Karrer, H. v. Euler and U. Solmssen, Helv. chim. Acta 17 (1934) 1171- 164. P. Karrer, H. v. Euler and U. Solmssen, Helv. chim. 17 (1934) 1171. 165. P. Karrer, H. v. Euler and U. Solmssen, Helv. chim. Acta 17 (1934) 1169. 166. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 25. 167. P. Karrer, U. Solmssen and O. Walker, Helv. chim. Acta 17 (1934) 417. — P. Karrer, H. v. Euler and U. Solmssen, Helv. chim. Acta 17 (1934) 1169. 168. H. V. Euler, B. Karrer and U. Solmssen, Helv. chim. Acta 21 (1938) 211. 169. P. Karrer, U. Solmssen and O. Walker, Helv. chim. Acta 17 (1934) 4i8- 170. R. Kuhn and E. Lederer, Ber. 65 (1932) 639. 171. A. E. Gillam, M. S. El Ridi and S. K. Kon, Biochem. J. 31 (1937) 1605. 172. L. Zechmeister and A. Polgar, /. Am. Chem. Soc. 66 (1944) 137. 173. L. Zechmeister and co-workers. Arch. Biochem. 6 (1945) 157. 174. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 471. 175. P. Karrer, E. Jucker, J. Rutschmann and K. Steinlin, Helv. chim. Acta, 28 (1945) 1146. I70 CAROTENOID HYDROCARBONS OF KNOWN CONSTITUTION X 176. P. Karrer, E. Jucker, J. RuTSCHMANN and K. Steinlin, Helv. chim. Acta 28 (1945) 1150. 177. A. PolgAr and L. Zechmeister, /. Am. Chem. Soc. 65 (1943) 1528. 178. R. KuHN and H. Brockmann, Naiurwissenschaften 21 (1933) 44; Ber. 66 (1933) 407. 179. R. KuHN and H. Brockmann, Ber. 66 (1933) 407- 180. A. Winterstein, Z. physiol. Chem. 2ig (1933) 249. 181. R. KuHN and H. Brockmann, Ber. 66 (1933) 407. 182. Cf. communication of L. Zechmeister and W. A. Schroeder, Arch. Biochem. i (1942) 231. 183. R. KuHN and H. Brockmann, Ber. 66 (1933) 407. 184. H. WiLLSTAEDT, Chem. Cenir. 1935, II, 707. 185. A. Winterstein, Z. physiol. Chem. 215 (1933) 55. — R. Kuhn and H. Brockmann, Chem. Centr. 1933, II, 1205; Z. physiol. Chem. 221 (1933) 131. — H. Brockmann and M. L. Tecklenburg, Z. physiol. Chem. 221 (1933) 117. 186. R. Emerson and D. L. Fox, Proc. Roy. Soc. (London) (B) 128 (1940) 275. — P. Karrer and co-workers, Helv. chim. Acta 26 (1943) 2 121. 187. L. Zechmeister and A. PolgAr, /. Am. Chem. Soc. 6y (1945) 108. 188. L. Zechmeister and co-workers. Arch. Biochem. 5 (1944) 365. 189. L. Zechmeister and W. A. Schroeder, Science 94 (1941) 609. — Cf. /. Am. Chem. Soc. 64 (1942) 1 173. 190. L. Zechmeister and W. A. Schroeder, /. biol. Chem. 144 (1942) 315. 191. L. Zechmeister and R. B. Escue, /. biol. Chem. 144 (1942) 321. 192. L. Zechmeister and W. A. Schroeder, Arch. Biochem. i (1942) 231. 193. L. Zechmeister and W. A. Schroeder, /. Am. Chem. Soc. 64 (1942) 1173. CHAPTER XI Carotenoids of known structure containing hydroxy I groups I. LYCOXANTHIN C^oHggO History and Occurrence 1936 In the course of investigations on lycopene from Solatium Dulcamara, Zechmeister and von Cholnoky^ isolate two new phytoxanthins, lycoxanthin and lycophyll. Lycoxanthin also occurs in Solanum esculen- tum and Tamus communis*' ^. Preparation^ 17 Kg of fresh berries of Solanum Dulcamara are dehydrated with ethanoland extracted with ether at room temperature. After evaporation of the solvent, the pigment mixture is dissolved in benzene and chromatographedoncalciumhydroxide. This procedure is repeated several times and the pigment is finally recrystallised from a mixture of benzene and methanol. The yield was 125 mg lycoxanthin, 920 mg lycopene and 9 mg lycophyll. Chemical Constitution \V \V y I I I I V CH HC-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CHCH CH HOCH C-CHa HaC-C CHg \ / Lycoxanthin \ y^ CHg CH2 Owing to the small amount of material available, the elucidation of the constitution of lycoxanthin presented difficulties and could not be carried through completely. From the similarity of the absorption maxima of lyco- xanthin and lycopene, Zechmeister and von Cholnoky^ concluded that the According to E. Lederer, Bull. Soc. chem. Biol. 20 (1938) 613, Polystigma rubrum also contains lycoxanthin besides an acidic pigment. References p. 214-21J. 172 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI two pigments contain identical chromophoric systems. The oxygen is present in the form of a hydroxyl group which can be acetylated. The position of the hydroxyl group has not been established, but it is probably at carbon atom 3 by analogy with other phytoxanthins (cf. Karrer and co-workers^) . Properties Lycoxanthin crystallises from a mixture of benzene and petroleum ether in jagged or circular reddish-brown plates. From carbon disulphide, the pigment is obtained in violet needles, m.p. i68° (corr.). Lycoxanthin is easily soluble in carbon disulphide and benzene, somewhat less easily in petroleum ether, and only very sparingly in ethanol. On partition between methanol and petroleum ether it behaves in the same way as cryptoxanthin and rubixanthin. It can only be adsorbed on calcium carbonate from petroleum ether solution, whereas it can be adsorbed on calcium hydroxide and aluminium oxide from benzene solution. Solvent: Absorption maxima: Carbon disulphide 546 506 472 m;u Petroleum ether 504 473 444 myw Benzene 521 487 456 m/z Ethanol 505 474 444 m// On shaking an ethereal solution of lycoxanthin with concentrated hydro- chloric acid, no blue colouration is observed. The monoacetate of lycoxanthin is formed by treating lycoxanthin with acetyl chloride in pyridine. The monoacetate crystallised from a mixture of benzene and methanol in violet-red needles, m.p. 137° (corr.). The acetate is easily soluble in carbon disulphide but only sparingly soluble in ethanol and petroleum ether. Its absorption spectrum is identical with that of lycoxanthin. 2. RUBIXANTHIN C4oH5gO History 1934 KuHN and Grundmann^ discover a pigment isomeric with cryptoxanthin amongst the pigments of Rosa rubiginosa. They propose the name rubi- xanthin and propose a constitutional formula. Occurrence Rubixanthin is one of the polyene pigments which are not widely distributed in nature. It occurs mainly in different species of roses. References p. 214—212. RUBIXANTHIN 173 Source References Cuscuta salina G. Mackinney, /. biol. Chem. 112 (1935) 421. Cuscnta subinclusa, do. Gazania rigens K. Schon, Biochem. J. 32 (1938) 1566. — L. Zech- MEiSTER and W. A. Schroeder, /. Am. Chem. Soc. 65 (1943) 1535. Rosa canina, Rosa rubigi- nosa, Rosa damascena R. Kuhn and H. Grundmann, Ber. 6y (1934) 341, 1133. Rosa rugosa H. Willstaedt, Svensk Kemisk Tidskr. 4J (1935) 113. Rubus Chamaemorus H. Willstaedt, Scand. Arch. Physiol. 75 (1936) 155. Preparation 27 Kg of fresh, ripe hips (Rosa riibiginosa) are mashed, dehydrated with pure methanol and dried at 37*^. The kernels are separated from the skins by grinding in a mill and the skins are extracted at room temperature with a mixture of benzene, absolute methanol and petroleum ether. The dark red extract is concentrated (finalh' in vacuum) to a small volume and saponified with ethanolic potassium hydroxide for two hours at 40°, and for a further two hours at room temperature. After this period, the pigments are extracted with a benzene-petrol (1 :4) mixture, the solution is washed free from alkali, dried and adsorbed on alumina. The chro- matogram is developed with the same solvent mixture and the rubixanthin is eluted with petroleum ether containing a little ethanol. The pigment is crystallised from a benzene-petroleum ether (1 :5) mixture. The yield is about 400 mg. For further purification the crude pigment is again saponified by allowing a benzene solution to stand with about 50 ml of 10% ethanolic potassium hydroxide for 4 hours at 40°. The pigment is extracted with petroleum ether and the solvent is removed by distillation. The rubixanthin is recr^^stallised from a mixture of benzene and methanol. The yield of pure pigment amounted to 36 mg. Chemical Constitution CHj CH3 CH, CH, C CHo CHo Clio CHo C, /\ \ I I I V CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH I II II I HO-CH C-CH3 HgC-C CHg \y Rubixanthin \^ CHg CHj The formula for rubixanthin was proposed by Kuhn and Grundmann^. Like y-carotene, the pigment takes up 12 mols of hydrogen on catalytic hydro- genation and therefore contains i isocyclic ring. On ozonisation the pigment yields 0.94 mol of acetone, evidently derived from the open end of the molecule. Rubixanthin exhibits absorption bands of the same wavelength location as y-carotene and the two polyenes may therefore be assumed to have the same References p. 214-217. 174 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI chromophoric systems. Zerewitinoff determinations show that the oxygen atom is present as a hydroxyl group. Since rubixanthin exhibits no vitamin A activity, the hydroxyl group must be substituted in the /?-ionone ring, since compounds containing an unsubstituted ^-ionone ring generally possess growth promoting properties. For reasons of analogy, Kuhn and Grundmann assume that the hydroxyl group is present in position 3. Although the formula of rubi- xanthin has thus not been finally proved, it appears the most probable according to present knowledge, and is in complete agreement with all the properties of the pigment. Properties Crystalline form: Rubixanthin crystallises from a mixture of benzene and petroleum ether in orange-red needles, and from a mixture of benzene and methanol in dark red needles with a copper-like lustre. Melting point: 160°. Solubility: The pigment is easily soluble in benzene and chloroform, but, only sparingly soluble in alcohols and petroleum ether. Spectral properties: Solvent: Absorption maxima: Carbon disulphide 533 494 461 rafi Chloroform 509 474 439 m/x Ethanol 496 463 433 m^ Petroleum ether 495.5 463 432 m/i Hexane 494 462 432 m[i Optical activity: Rubixanthin is optically inactive. Partition test: On partition between petroleum ether and 90 % methanol, the pigment is found in the upper layer. If 95 % methanol is used, however, the pigment is hypophasic. Chromatographic behaviour: Owing to the presence of one hydroxyl group, rubixanthin is very much more strongly adsorbed on calcium hydroxide than carotenoid hydrocarbons. On the other hand, rubixanthin is more weakly adsorbed on zinc carbonate or alumina than phytoxanthins containing 2 hydroxyl groups (zeaxanthin, xanthophyll, etc.). The separation of rubixanthin from cryptoxanthin is very tedious as these two compounds show almost identical adsorption properties. References p. 214-21'j. CRYPTOXANTHIN 175 Detection and estimation: After separation from other phytoxanthins by chromatographic analysis, rubixanthin can be identified by its spectral prop- erties. Physiological properties: Rubixanthin exhibits no vitamin A activity. 3. CRYPTOXANTHIN C40H56O History 1932 Yamamoto and Tin^ isolate a phytoxanthin from Carica papaya and propose the name caricaxanthin. The formula C4oH5g02 is assigned to the new pigment. 1933 KuHN and Grundmann^ discover a new polyene pigment in the red berries of Physalis Alkekengi and Physalis Franchetii. They term the new pigment cryptoxanthin, determine the correct molecular formula, and derive a constitution formula. 1933 Karrer and Schlientz^ establish the identity of caricaxanthin and cryptoxanthin, and correct the formula given by Yamamoto and Tin. Source Arbutus Unedo Blood serum of cattle Butter Capsicum annuum Carica- papaya Celastrus scandens L. Citrus poonensis (fruit) Cucurhita Pepo Diospyros costata (fruit) Egg yolk Grevillae robusta, Cunningham Helianthus annuus References p. 214— 2iy. TABLE 38 OCCURRENCE OF CRYPTOXANTHIN References K. ScHON, Biochem. J. 2g (1935) 1779, 1782. A. E. GiLLAM and M. S. El Ridi, Biochem. /. 29 ( 1 935) 2465. A. E. Gillam and I. M. Heilbron, Biochem. J. 2g (1935) 834. L. Zechmeister and L. v. Cholnoky, Ann. ^og (1934) 269. R. Yamamoto and S. Tin, L. Set. Pap. Inst. phys. chem. Res. 20 (1933) 411. — Chem. Centr. igjj, I. 3090. A. L. Le Rosen and L. Zechmeister, Arch, of Biochem. J (1943) 17. R. Yamamoto and S. Tin, L. Sci. Pap. Inst. phys. chem. Res. 21 (1933) 422/425. — Chem. Centr. 1934, I, 1660. L. Zechmeister, T. BeRes and E. Ujhelyi, Ber. 68 (1935) 1322. K. Schon, Biochem. J. 2g (1935) 1779, 1782. A. E. Gillam and I. M. Heilbron, Biochem. J. 2g (1935) 1064. L. Zechmeister and A. PolgAr, /. biol. Chem. 140 (1941) 1. L. Zechmeister and P. Tuzson, Ber. 67 (1934) 170. 176 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Source References Iris of chicks L. Busch and H. J. Neumann, N atiirwissenschaften 29 (1941) 782. Mycobacterium phlei M. A. Ingraham and H. Steenbock, Biochem. J. 2g (1935) 2553. Nitzschia closierium Nello Pace, /. biol. Chem. 140 (1941) 483. Orange peels L. Zechmeister and P. Tuzson, Ber. 6g (1936) 1878. — P. Karrer and E. Jucker, Helv. chim. Acta 2y (1944) 1695. Phy sails Alkekengi and Physalis Franchetii R. Kuhn and C. Grundmann, Ber. 66 (1933) 1746. Tangerines L. Zechmeister and P. Tuzson, Z. physiol. Chem. 240 (1936) 191. Zea Mays R. Kuhn and C. Grundmann, Ber. 6y (1934) 593. Preparation^ Dried physalis cups are finely ground and extracted with methanol to remove resinous materials. The cup meal is then continuously extracted with benzene at room temperature. Most of the solvent is removed in vacuum and the residue which contains zeaxanthin and cryptoxanthin esters is saponified at room temperature with alcoholic potassium hydroxide. After about 15 hours, petroleum ether is added to the solution, followed by distilled water until the zeaxanthin precipitate begins to become resinous. The petroleum ether-benzene layer contains cryptoxanthin which is isolated by adsorption on alumina. The pigment is purified by crystalli- sation from a mixture of benzene and methanol. The yield amounts to about 100 mg pure cryptoxanthin from 1600 cups. Chemical Constitution CHo CHo CHo CHo 0 01i<. Cxi.j CHo OHo G /\ \ \ \ \ /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj HOCH C-CHj HsC-C CHg \^ / Cryptoxanthin \ / CHj CHg The constitution of cryptoxanthin was elucidated mainly by Kuhn and Grundmann^. On hydrogenation, the pigment takes up 11 mols of hydrogen. It therefore contains 11 double bonds and 2 isocyclic rings. The spectral properties correspond to those of ^-carotene and indicate that all the 11 double bonds are conjugated. Cryptoxanthin gives exactly i mol of methane with methyl magnesium iodide, indicating the presence of i hydroxyl group. This is confirmed by the formation of a monoacetate. The position of the hydroxyl group could not be determined with certainty, but by analogy the 3-position References p. 214— 2iy. 3 CRYPYOXANTHIN i-j-j is the most probable. By oxidation with chromic acid, Kuhn and Grundmann obtained 4.85 mols of acetic acid. The formula for cryptoxanthin is in agreement with the fact that this phytoxanthin possesses vitamin A activity^- ^°' ^^. Properties Crystalline form: Cryptoxanthin crystallises from a mixture of benzene and methanol in lustrous prisms which tenaciously retain some methanol. Melting point: 169° (corr., evacuated capillary). Solubility: As would be expected from its constitution, cryptoxanthin has properties intermediate between those of /^-carotene and zeaxanthin. The pigment is easily soluble in chloroform, benzene and pyridine, and less soluble in ligroin, petroleum ether, methanol and ethanol. Spectral properties: Solvent: Absorption maxima: Carbon disulphide 519 483 452 m/x Chloroform 497 463 433 m/z Ethanol, absolute 486 452 424 m/^i Petrol 485.5 452 424 mpi Hexane 484 451 423 ra/t Optical activity: Cryptoxanthin exhibits no optical acti\'ity. Partition test: On partition between petroleum ether and 90 % methanol, cryptoxanthin is found in the upper layer, and thus behaves like a hydrocarbon. With 95 % methanol, however, it is hypophasic. Chromatographic behaviour: Cryptoxanthin is more strongly adsorbed on calcium hydroxide than the carotenes and can readily be separated from the latter in this way. On zinc carbonate or calcium carbonate, it is held less strongly than phytoxanthins with two hydroxyl groups. The separation of crypto- xanthin from rubixanthin presents some difficulty. Colour reactions: Cryptoxanthin gives a dark blue colouration with anti- mony trichloride in chloroform solution. The solution exhibits a maximum at 590 m/x (cf. /5-carotene) . Detection and estimation: The separation of cryptoxanthin from other carotenoids is achieved by means of chromatographic adsorption. The pigment can be identified by the determination of absorption maxima and by means of the partition test. For the colourimetric estimation, a standard solution of azobenzene in ethanol can be used^^. Physiological properties: Cryptoxanthin exhibits vitamin A activity (cf. p. 14). References p. 214.— 21J. Carotenoids 12 178 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Derivatives Cryptoxanthin monoacetate C42H58O2: This derivative is formed by treating cryptoxanthin with acetic anhydride in pyridine^^. It crystalHses in red needles, m.p. 117-118° (corr.). The absorption maxima are identical with those of cryptoxanthin. The monoacetate is entirely epiphasic. Cryptoxanthin mono-epoxide C4QH5g02: This compound was obtained by Karrer and Jucker^^ by the action of monoperphthalic acid on crypto- xanthin acetate. The biological assay^^ of cryptoflavin derived from crypto- xanthin epoxide showed that it is vitamin A-inactive even in high doses. It thus contains no unsubstituted /5-ionone ring. For this reason the following formula is ascribed to cryptoxanthin epoxide. CH3 CH3 CH, CH, \V \V /\ I I I I /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ HOCH C-CHj \/ C CR CH2 Crj^ptoxanthin epoxide / \/ H3C CHj The solubility of cryptoxanthin epoxide is similar to that of cryptoxanthin. It crystallises from a mixture of benzene and methanol in beautiful needles or plates, m.p. 154° (uncorr. in vacuum). Solvent: Absorption maxima: Carbon disulphide 512 479 m/z Benzene 494 461 m/i Chloroform 488 456 m// Ethanol 481 449 m/i On shaking an ethereal solution of the pigment with concentrated hydro- chlorid acid, the latter assumes a somewhat unstable blue colouration. Cryptoflavin^^: By the action of mineral acids on cryptoxanthin mono- epoxide, the latter is transformed into the furanoid oxide, cryptoflavin, which is biologically inactive^^. CHo CHo \V C CHo CHo CHg C OH CH3 CH3 CH3 CHg C CH, C CH-C=CHCH=CH-C=CHCH=CHCH = C-CH=CHCH=C-CH=CH-C CH» CHJ 0 HaC-C CHOH CH3 Cryptoflavin \^ y/ CHj References p. 214-21'/. 3 CRYPTOXANTHIN 179 Cryptoflavin crystallises from a mixture of benzene and petroleum ether in beautiful lustrous plates, m.p. 171° (uncorr., in vacuum). The pigment exhibits the same behaviour towards aqueous hydrochloric acid as cryptoxanthin mono-epoxide. Solvent: Absorption maxima: Carbon disulphide 490 459 m/^ Benzene 470 439 m/x Chloroform 468 438 van Ethanol 460 430 m/z Cryptoxanthin di-epoxide C4QH5g03^^: This compound is formed by the oxidation of cryptoxanthin acetate with monoperphthalic acid. CH3 CH3 CH, CH, \V \V C CHq CH« CHo CHo c /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, )o 0( CH2 C C CHOH \ /\ Cryptoxanthin di-epoxide y^^\/^ CHa CH3 H3C CHj Cryptoxanthin di-epoxide crystallises from a mixture of benzene and petro- leum ether. M.p. 194° (uncorr., in vacuum). Solvent: Absorption maxima: Carbon disulphide 503 473 m/i. Benzene 486 455 m/n Chloroform 482 453 mfi Ethanol 473 442 m/i With concentrated aqueous hydrochloric acid the di-epoxide gives a dark blue colouration which is stable for several days. Cryptochrome C4(,H5g03 : CH3 CH3 CH, CH, C C CH2 C^=CH CH3 CH3 CH3 H3C CH=C CH2 CH2 C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH C CHOH CH2I 0 Cryptochrome 0 | CHg CHs CH3 Cryptochrome is formed by the action of hydrogen chloride in chloroform on cryptoxanthin di-epoxide, besides cr5rptoflavin and cryptoxanthin. Because References p. 214— 2iy. i8o CAROTENOIDS CONTAINING HYDROXYL GROUPS XI of the small amount of material available it has not yet been obtained in a crystalline state. Solvent: Absorption maxima: Carbon disulphide 456 424 m^ Cis-trans Isomers A number of cis-trans isomers of cryptoxanthin have been prepared by Zechmeister and Lemmon^'. According to these authors, natural crypto- xanthin has an a.\\-trans configuration (cf. p. 38). By standing or boiling a solution of the pigment, by fusion of the crystals, by treatment with iodine or illumination with sunlight, various isomers are formed which are believed to contain a number of cw-double bonds. The sequence of isomers in the table below is that in which they are observed in the chromatogram. A hsorption ntaxima (in petroleum ether) Neo-Cryptoxanthin U 478.5 448 m^ (Cryptoxanthin) M83.5 452.5 m^) Neo-Cryptoxanthin A 477 446 m/j. Neo-Cryptoxanthin B 479.5 449.5 m/i Apart from natural cryptoxanthin, none of these compounds has been obtained in a crystalline state. The absorption curves of the pigments can be found in the original communication. 4. ZEAXANTHIN C4oH5g02 History 1929 Karrer, Salomon and Wehrli^^ isolate a new phytoxanthin, for which they propose the name zeaxanthin, from maize. 1931-32 Karrer and co-workers elucidate the constitution of zeaxanthin^^. Occurrence Zeaxanthin is widely distributed in plants in the free state as well as esterified (physalien). Some plants contain zeaxanthin as the main pigment, so that its isolation in appreciable amounts is relatively easy to achieve. References p. 214—217. ZEAXANTHIN TABLE 39 OCCURRENCE OF ZEAXANTHIN Source Capsicum annuum Capsicum frutescens japoni- cum. Celastrus scandens Citrus aurantium, Crocus sativus Cucurbita Pepo L. Diospyros costata Diospyros Kaki Egg yolk Evonymus europaeus Feathers of Serinus canaria Fucus vesiculosus Halyseris polypodioides Hippophae rhamnoides Human fat Human liver Lycium barharum Lycium halinii folium Phy salts Alkekengi, Physalis Franchetii Prunus persica Rana esculenta (liver) Rosa canina, Rosa rubigi- nosa, Rosa damascena Rubus Chamaemorus References L. Zechmeister and L. v. Cholnoky, Ann. 5og (1934) 269. L. Zechmeister and L. v. Cholnoky, Ann. 48g (1931) 1. A. L. Le Rosen and L. Zechmeister, Arch. Biochem. 1 (1943) 17. L. Zechmeister and P. Tuzson, Ber. 6g (1936) 1878. R. KuHN and A. Winterstein, Ber. 6y (1934) 344. L. Zechmeister and co-workers, Ber. 68 (1935) 1322. K. ScHON, Biochem. J. 2g (1935) 1779. P. Karrer, R. More, E. v. Krauss and A. Zubrys, Helv. chim. Ada 15 (1932) 490. R. Kuhn, a. Winterstein and E. Lederer, Z. physiol. Chem. igy (1931) 141. L. Zechmeister and co-workers, Z. physiol. Chem,. igo (1930) 67; Z. physiol. Chem. ig6 (1931) 199. H. Brockmann and O. Voelker, Z. physiol. Chem. 224 (1934) 193. I. M. Heilbron and R. F. Phipers, Biochem. J. 2g (1935) 1369; (with H. R. Wright) /. Chsm. Soc. ig34 1572. P. Karrer, F. Rubel and F. M. Strong, Helv. chim. Acta ig (1935) 28. P. Karrer and H. Wehrli, Helv. chim. Acta 13 (1930) 1104. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 225 (1934) 189; Z. physiol. Chem. 231 (1935) 259. H. WiLLSTAEDT and T. Lindqvist, Z. physiol. Chem. 240 (1936) 10. A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 207 (1932) 25. L. Zechmeister and L. v. Cholnoky, Ann. 481 (1930) 42. R. Kuhn and W. Wiegand, Helv. chim. Acta. 12, (1929) 499; R. Kuhn, A. Winterstein and W. Kauf- mann, Ber. 63 (1930) 1489. G. Mackinney, Plf.nt. Physiol. 12 (1937) 216. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 238 (1936) 197. R. Kuhn and C. Grundmann, Ber. 6/(1934) 339, 1133. H. WiLLSTAEDT, Skand. Arch. Physiol. 75 (1936) 155. CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Source Rudbeckia Neumannii Sarcina aurantiaca Senecio Doronicum Solanum Hendersonii Solanum Lycopersicum L. Staphylococcus aureus Vaccinium vitis idaea Viola tricolor Zea Mays References P. Karrer and A. Notthafft, Helv. chim. Acta 15 (1932) 1195. E. Chargaff, Compt. rend, igy (1933) 946. P. Karrer and A. Notthafft, Helv. chim. Acta 15 (1932) 1195. A. WiNTERSTEiN and U. Ehrenberg, Z. physiol. Chem. 207 (1932) 25. R. KuHN and C. Grundmann, Ber. 65 (1932) 1880. E. Chargaff, Compt. rend, igj (1933) 946. H. WiLLSTAEDT, Svensk Kemisk Tidskr. 48 (1936 (212). P. Karrer and J. Rutschmann, Helv. chim. Acta 2y (1944) 1684. P. Karrer, H. Salomon and H. Wehrli, Helv. chim. Acta 12 (1929) 790. TABLE 40 zeaxanthin content of various plants Source Quantity Yield of Zeaxanthin References Maize meal Physalis leaves Evonymus europaeus Lycium halimifolium berries Hippophaes rhamnoides berries 100 kg 1 kg (dry) 1 kg seeds 1 kg (fresh) 1 kg (fresh) 100-200 mg 4g 200 mg 400-500 mg 25 mg 20 21 22 23 24 Preparation Zeaxanthin can be prepared either from maize^" or from leaves of physalis cups^^. In physalis, the pigment occurs in the form of the palmitic acid ester, physalien, the preparation of which is described on p. 187. Zeaxanthin is obtained from physalien by saponification. 3 g of physalien are dissolved in ether and saponified by shaking with 10% methanolic potassium hydroxide at room temperature. By dilution with water, the zeaxanthin is transferred to the ether layer, which is concentrated slightly until the pigment crystallises. For further purification, the zeaxanthin is crystallised once from a mixture of chloroform and ether. The yield is just under 1 g. * Formation Karrer and Solmssen^^ succeeded in converting a carotenoid with 40 carbon atoms into another natural pigment with the same number of carbon atoms by converting rhodoxanthin (p. 221) into zeaxanthin by reduction of the dihydroderivative with aluminium wopropoxide. References p. 214-217. 4 ZEAXANTHIN 183 CH, CH, CH3 CH. \V \V C CH, CH3 CH3 CH3 C /\ i I I \ /\ CH2 C=CHCH=C-CH=CHCH=C-CH=CHCH=CH-C=CHCH=CH-C=CHCH=C CHj HjC-C CO Rhodoxanthin ^ / CH CHo CHo \V CH3 CH3 CH3 CH3 C C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- C CHg HaC-C CO Dihvdrorhodoxanthin OC C-CH CH CH3 CH3 C CHj C-CH= OC C-CHg CHa CH3 CH3 C CHj CHo CHg \/ CHo CHo CHo CHo C /\ I I I I /\ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, 1 II II I HOCH C-CH3 HjC-C CHOH \ y^ Zeaxanthin \^ y^ CHj CH2 This transformation of rhodoxanthin into zeaxanthin represents the first partial synthesis of a C40 carotenoid and also confirms the constitution of both pigments. Karrer and Jucker^'' also carried out another partial synthesis of zea- xanthin by treating xanthophyll with sodium ethoxide. This resulted in the dis- placement of the isolated double bond into conjugation and the zeaxanthin thus obtained was identical in spectral properties and melting point with the natural pigment. These experiments show that polyenes containing an isolated double bond readily undergo prototropic rearrangement to the fully conjugated isomer. Chemical Constitution^ •^'^ CH3 CH3 CXI3 CH3 C CHo CHo CM« CHo C /\ r I I I ■ /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj i II II I HOCH C-CHa HjC-C CHOH \ / Zeaxanthin \ / CH2 CH^ The elucidation of the constitution of zeaxanthin is mainly due to Karrer and co-workers, and is similar to the corresponding investigations on xantho- phyll (cf. p. 201). The empirical formula and the number of hydroxyl groups References p. 214-217. i84 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI present indicate that zeaxanthin is an isomer of xanthophyll. The nature of the isomeric relationship was estabHshed by the investigations of Karrer and co-workers^^'^^ who showed that xanthophyll is derived from a-carotene, whereas zeaxanthin is the corresponding derivative of /5-carotene. By the dry distillation of the pigment, Kuhn and Winterstein^" obtained toluene, w-xylene and 2 :6-dimethylnaphthalene. By ozonisation and potassium permanganate oxida- tion of zeaxanthin, Karrer and co-workers^^ obtained a : a-dimethylsuccinic acid. The arguments applied in the case of xanthophyll regarding the position of the hydroxyl groups are therefore also valid for zeaxanthin (cf. p. 201). The number of side-chain methyl groups and double bonds was determined by Kuhn and co-worker s^2. In 1938, Karrer and co-workers provided further confirmation for the constitution of zeaxanthin by preparing /9-citraurin from zeaxanthin by partial oxidative degradation with potassium permanganate^^. CHo CHo C CH3 CH3 CH3 CH3 /\ I I II CH„ C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CHO I II HOCH C-CHg \ / ^-Citraurin CHa Properties and Physical Constants Crystalline form: Zeaxanthin crystallises from methanol in long, yellow plates and clusters which, in contrast to xanthophyll, do not occlude solvent. The pigment crystalHses especially well from ethanol in short thick rombic prisms. From a mixture of carbon disulphide, petroleum ether and ether, zea- xanthin separates in clustered needles. Melting point: 205° (uncorr.)^*, 215.5° (corr.)^^. Solubility: 1 g zeaxanthin dissolves in about 1.5 1 of boiling methanol. The pigment is almost insoluble in petroleum ether and hexane. Its solubility in ether, chloroform, carbon disulphide and pyridine is somewhat greater. A suspension of the pigment in glacial acetic acid becomes homogeneous on addition of hexane. Spectral properties (cf. Fig. 8, p. 350) : Solvent: Absorption maxima: Carbon disulphide 517 482 450 m// Chloroform 495 462 429 m/i Ethanol 483 451 423.5 m/z Petrol 483.5 451.5 423 mn Methanol 480.5 449.5 421.5 m/< Quantitative extinction measurements : In carbon disulphide, Kuhn and Smakula^^; in ethanol and carbon disulphide, Hausser and Smakula^' and Hausser38. References p. 214-217. 4 ZEAXANTHIN 185 Optical activity: According to several workers, zeaxanthin, like ^-carotene, is optically inactive. Recently, however, Zechmeister and co-workers^^ reported that their zeaxanthin preparations have a rotation of [aj^^ = — 40-50° in chloroform. Perhydrocarotene obtained by the reduction of perhydrozeaxanthin dibromide, is optically inactive in contrast to the corresponding products from xanthophyll. Partition test: Zeaxanthin exhibits entirely hypophasic character on parti- tion between methanol and petroleum ether. Chromatographic behaviour: Zeaxanthin is easily adsorbed on calcium car- bonate or zinc carbonate from benzene solution. Detection and estimations: Zeaxanthin can be separated from other phyto- xanthins b}^ adsorption on zinc carbonate. It can be identified by its absorption maxima, in conjunction with the partition test. According to Kuhn and Brockmann the pigment can be determined colorimetrically, using a solution of azobenzene in ethanol as a standard*". Physiological behaviour: Zeaxanthin exhibits no vitamin A activity, but it yields an active product on treatment with phosphorous tribromide*^. Colour reactions: Zeaxanthin dissolves in concentrated sulphuric acid with a fairly stable deep blue colouration. On treating a solution of the pigment in chloroform with antimony trichloride a blue colouration is produced which has been examined spectroscopically*^. Derivatives Perhydrozeaxanthin C4oH7g02.' Colourless, viscous oil*^-* which is leavo- rotatory in contrast to perhydroxanthophyll. [aj^ = — 24.5^*^-* Zeaxanthinhalogenides: Karrer and co-workers** replaced the two hydroxyl groups in perhydrozeaxanthin by bromine, thus obtaining 3:3'-dibromo- perhydrozeaxanthin. On treating a solution of zeaxanthin with bromine, 8 mols of the halogen are absorbed. Zeaxanthin monomethyl ether €4^115802: This compound is formed on treating zeaxanthin with the potassium derivatives of tertiary am.yl alcohol and methyl iodide*^. It crystallises from methanol in needles, m.p. 153°. Zeaxanthin dimethyl ether C42H5QO2: This compound is obtained as by-product in the preparation of the monomethyl ether*^. It crystallises from petroleum ether in dark red needles, m.p. 176°. It is very sparingly soluble in methanol and ethanol. Zeaxanthin diacetate C44H60O4: Karrer and Solmssen obtained this ester by treating a solution of zeaxanthin in pyridine with acetic anhydride*®. The diacetate crystallises from a mixture of benzene and methanol. M.p. 154-155°. According to P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 492, perhydro- zeaxanthin is optically inactive. References p 214— 2iy. i86 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Zeaxanthin dipropionate C46H54O4*' : Crystallises from a mixture of benzene and methanol. M.p. 142°. Zeaxanthin dihutyrate C48H6804^^. Crystallises from a mixture of benzene and methanol. M.p. 132°. Zeaxanthin di-n-valerate C5oH7204^' : Crystallises from a mixture of benzene and methanol. M.p. 125°. Zeaxanthin di-n-capronate C52H7804*^: M.p. 117-118°. Zeaxanthin di-n-caprylate C56H84O4*' : This compound crystallises from benzene. M.p. 107°. Zeaxanthin dilaurate C54Hio„04*^. M.p. 104°. Zeaxanthin monopalmitate CsgHggOg: This compound was obtained by Karrer and ScHLiENTZ*' by partial saponification of physalien. The half-ester crystallises from a mixture of benzene and ethanol in plates, m.p. 148°. Zeaxanthin distearate C76HJ24O4: Prepared by treating a solution of zeaxanthin in pyridine with stearic acid chloride. M.p. 95°. Physalien (zeaxanthin dipalmitate) C^^HhqO^: Kuhn and Wiegand found a caret enoid in Phy salts Alkekengi and Physalis Franchetii^^ which they termed physalien. This pigment was later obtained from numerous other plants. TABLE 41 OCCURRENCE OF PHYSALIEN Source References Asparagus officinalis A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 207 (1932) 26. Hippophae rhamnoides (berries) P. Karrer and H. Wehrli, Helv. chim. Acta 13 (1930) 1104. Lycium barbarum (skin) and Solanum Hendersonii A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 2oy (1932) 26. Lycium halimifolium (skins) L. Zechmeister and L. v. Cholnoky, Ann. 481, (1930) 42. Investigations by Zechmeister and von Cholnoky^^ and Kuhn and co- workers^^ showed that physalien is an ester of zeaxanthin, namely zeaxanthin dipalmitate. CH3 CHo CH3 CH3 C CHo CHq CHo CHo C /\ I I I r /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj I II II I CisHaiCOO-CH C-CHg HaC-C CH-OCOCisHai \ / Physalien \^ / References p. 214—217. 4 ZEAXANTHIN 187 Physalien has been partially synthesized from zeaxanthin and palmitic acid chloride, thus confirming its constitution. PhysaHen is best prepared from physalis cups^^, the pigment content of which is exceptionally large, amounting to 0.9-1.8% of dry weight. A third of the pigment consists of cryptoxanthin (cf. p. 176). Physalis cups are dried at 40-50°, coarsely ground and exhaustively extracted with benzene at room temperature. The combined extracts are concentrated in vacuum to a small volume and the polyene wax is precipitated with acetone. The precipitation is not carried out all at once, but at intervals of several hours, each precipitate being filtered separately. The mother liquors are finally diluted with much ethanol and set aside in the cold. In this way additional amounts of pigment are obtained. The purification of physalien is carried out as follows: the pigment wax is dissolved in hot benzene and fractionally precipitated in the hot with methanol. The first fractions sometimes contain a waxy colourless substance, which must be separated from the solution by filtration through a steam-jacketed filter. The subsequent fractions yield the physalien. It is purified by re-crystalUsation from a mixture of benzene and methanol. The purification of crude physalien can also be achieved more simply by dis- solution in about 60 parts of hot benzene, followed by addition of 160 parts of hot ethanol. On cooling, the pigment wax separates and can be purified by crystalli- sation from a mixture of benzene and methanol. 1 g of the crude products yield about 0.5 to 0.7 g of pure physalien. Physalien can also be isolated from Physalis berries^*. 2 kg of berries yield about 1 g of polyene wax. L. Zechmeister and von Cholnoky^^ also describe the isolation of the pigment from fresh Lycium berries, 5 kg of which yielded 5 g physalien. Proferties Physalien crystallises from a mixture of benzene and methanol in long, flat rods, or in fine needles. It can also be obtained in the form of stout needles. From cyclohexane and ethanol, the pigment separates in flat, dark red prisms, several millimeters long. In larger quantities, physalien has the appearance of a fiery brilliant red powder, with the consistency of a hard wax. It melts at 98.5-99.5°. It is very easily soluble in carbon disulphide, benzene, chloroform, and carbon tetrachloride, and readily soluble in petroleum ether, hexane, tetralin, decalin, ether and pyridine. Cyclohexane, glacial acetic acid and acetic anhydride readily dissolve the pigment in the hot, but only sparingly in the cold. The compound is almost insoluble in ethanol and acetone. According to Kuhn and Brockmann^^, physalien is optically inactive. The positions of the absorption bands are indistinguishable from those of zeaxanthin. Quantitative extinction measurements are reported by Hausser and Smakula^'. On standing in air, physalien slowly absorbs oxygen, which results in a lightening of the colour, lowering of the m.p. and increase in the solubility in ethanol. Physalien dissolves in concentrated sulphuric acid to give a dark blue solution^. Further colour reactions are de scribed by KuH N and Wiegand^^. References p. 214—21'j. i88 CAROTENOIDS CONTAINING HYRDOXYL GROUPS XI Derivatives of Physalien a) Perhydrophysalien C72Hj3g04: A colourless oil, easily soluble in ether, sparingly in ethanol. b) Physalien iodide: This compound is formed by treating an ethereal solution of the pigment with an ethereal solution of iodine. On treating the addition product with thiosulphate, unchanged physalien is regenerated. c) Physalienone: By the treatment of physalien with chromium trioxide, Karrer, Solmssen and Walker^° obtained a tetraketone of the following constitution (cf. Karrer and Gugelmann^^) : CH3 CH3 CH3 CH3 C CHo CHo CHq CHo C /\ 1 11 \ /\ CH2 CO-CH=CHC=CHCH=CHC=CHCH=CHCH=CCH=CHCH=CCH=CH-OC CHj CisHsiCOO-CH CO-CHs HjC-OC CH-OOC-CisHsi \^ / Physalienone \ / CH2 CH2 The tetraketone crystallises in clustered needles, m.p. 144-145°. Its optical properties are very similar to those of ^-carotenone. Solvent: Absorption maxima: Physalienone p-Carotenone Carbon disulphide .... 536 500 463 538 499 466 m/z Petroleum ether 497 464 436 502 468 440 ni/^ Chloroform 525 488 452 527 489 454 m^t With regard to the action of bromine on physalien, cf. Zechmeister and VON Cholnoky^^. With regard to the reaction with iodine, cf. Kuhn and co- workers®^. The assimilation of physalien by rats and chicks has been investi- gated by Kuhn and Brockmann®*. Cis-trans Isomers of Zeaxanthin By a variety of treatments, such as heating in solution, dissolution of the crystals, catalysis with iodine and illumination with sunlight, Zechmeister and co-workers converted zeaxanthin into mixtures of different pigments which they regard as cis-trans isomers®^. Three compounds could be obtained in the crystalline state, namely neo-zeaxanthin A, neo-zeaxanthin B and neo- zeaxanthin C. Another pigment different in character from the other isomers is also formed®^. Treatment of this compound with iodine (except in alcohol solution) results in a displacement of the absorption maxima towards shorter wavelength by 2-4 m.[_i. The three crystalline neo-zeaxanthins have the following properties : References p. 214— 2iy. 4 ZEAXANTHIN 189 Neo-zeaxanthin A: Small plates from methanol, m.p. about 106° (not sharp, corr.). Solvent Absorption maxima: Carbon disulphide 508 475.5 m/i Benzene 489 457.5 m^ Petrol 477 447 m/z Ethanol 478.5 448.5 m/z [a]j. about +120" (in chloroform). Neo-zeaxanthin B: Flat, obliquely cut plates, from dilute methanol, m.p. 92° (not sharp, corr.). Absorption maxima in carbon disulphide, benzene, petrol and ethanol are the same as for neo-zeaxanthin A. The optical rotation shows varying values. Neo-zeaxayithin C: Small crystals from a mixture of carbon disulphide and benzene. M.p. 154° (corr.). Solvent: Absorption maxima: Carbon disulphide 502 470 m/i Benzene 488.5 455 m/i Petrol 473.5 444 m^ Ethanol 473 443.5 m// Epoxides of Zeaxanthin and their Transformation Products By the oxidation of zeaxanthin acetate with monoperphthaUc acid, Karrer and JuCKER^'' obtained various epoxides and furanoid transformation products some of which proved to be identical with natural carotenoids, the structure of which had previously been unknown. As the natural oxides of zeaxanthin are dealt with in detail in the following chapter only a brief description of these compounds will be given here. a) Zeaxanthin mono-epoxide, Antheraxanthin* : CH, CH, CH, CH, CHo CH.> oHo CHo C I I I I /\ CH^ C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHg HsC-C CHOH HOCH C \ / \/\ Antheraxanthin CHj A detailed description of this pigment will be found on p. 191. Antheraxanthin was first isolated by P. Karrer and A. Oswald from anthers of Lilium tigrinum, Helv. chim. Acta 18 (1935) 1303. References p. 214— 21J. igo CAROTENOIDS CONTAINING HYDROXYL GROUPS XI b) Mutatoxanthin: \/ C CH3 CH, CHjj C=CH CH3 CH3 CH3 CH3 C HOCH C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, CH2I 0 HsC-C CHOH CH3 Mutatoxanthin " \ / CH2 Mutatoxanthin was first obtained by Karrer and Rutschmann^^ by the action of dilute hydrochloric acid on natural violaxanthin (cf. p. 195). It has the formula C4oH5g03, and contains 10 double bonds and 2 hydroxyl groups®^. The nature of the third oxygen atom was first established by the partial synthesis^^ of the pigment, in which mutatoxanthin was obtained by the action of acidic chloroform on zeaxanthin mono-epoxide (antheraxanthin) (cf. p. 62). The formula given above for mutatoxanthin is in accord with all the properties of the pigment. This formulation also explains the formation of mutatoxanthin from violaxanthin. By the action of dilute hydrochloric acid on the latter, one of the epoxide groups is converted into a more stable furanoid system, while the oxygen of the other epoxide group is split off. (With regard to the formula of violaxanthin, see below). Mutatoxanthin crystallises from methanol or from a mixture of benzene and methanol. M.p. 177° (uncorr., evacuated capillary). On partition between methanol and petroleum ether, the pigment is found in the lower layer. On treating an ethereal solution of mutatoxanthin with concentrated aqueous hydrochloric acid, a blue colouration is produced which is weaker than that given by auroxanthin (cf. p. 197) and not very stable. Solvent: Absorption maxima: Carbon disulphide 488 459 m/i Ethanol 457 427 m/^ Benzene 468 439 m// Petroleum ether 456 426 m/i Chloroform 468 437 m^u Pyridine 473 443 m/z c) Violaxanthin, Zeaxanthin di-epoxide: CH, CH, CH, CH, C CH3 CH3 CH3 CH3 C /\ II I I /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ )0 0{ HOCH C C CHOH \ y \ Violaxanthin / \ / CHa CH3 H3C CHa References p. 214— 2iy. 5 ANTHERAXANTHIN 191 Violaxanthin is formed together with antheraxanthin, though in somewhat poorer yield. It is a natural pigment of wide occurrence and has been repeatedly investigated within recent years. It will be described in detail on p. 193. d) Auroxanthin: CH, CH, CH3 CH, \/ C CH CH3 CH3 CH3 H3C CH=C CHg CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH C CHOH Auroxanthin The furanoid dioxide of zeaxanthin, auroxanthin, is a carotenoid occurring in blossoms. It can be obtained by the isomerisation of violaxanthin^" in the presence of acids, a reaction which proves its constitution. A detailed description of auroxanthin will be found on p. 196. 5. ANTHERAXANTHIN C40H56O3 In the course of investigations on the carotenoids of the anthers of Liliiim tigrinum, Karrer and Oswald''^ discovered a previously unknown phyto- xanthin which they termed antheraxanthin. Antheraxanthin occurs together with capsanthin and is found below the latter in the chromatogram. It shows an entirely hypophasic character in the partition test and its absorption spectrum differs very little from that of zeaxanthin. Karrer and Oswald deter- mined the molecular formula of the pigment, but were unable to establish the nature of the third oxygen atom. The constitution of antheraxanthin was elucidated later'^, when the pig- ment was identified with zeaxanthin mono-epoxide obtained by the oxidation of zeaxanthin with monoperphthalic acid. The two compounds could not be separated by chromatography on zinc carbonate and gave no mixed melting point depression. This partial synthesis established the constitution of anthera- xanthin (cf. p. 189). The formula is in agreement with the fact that the pigment absorbs 11 mols of hydrogen on catalytic hydrogenation'^. \V \V 0 CHq Cxlq CHq 0 H O \J /\ I I I I /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ HaC-C CHOH HOCH C \ / \^ / \^ Antheraxanthin CHj CH2 CH3 References p. 214— 2iy. 192 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Antheraxanthin crystallises from methanol or a mixture of benzene and methanol in needles or thin plates, m.p. 205°. By the action of acidic chloro- form, mutatoxanthin and a little zeaxanthin are formed from antheraxanthin. On shaking an ethereal solution of the pigment with concentrated aqueous hydrochloric acid, a blue colouration is produced after a period of time. Solvent: Absorption maxima: Carbon disulphide 510 478 mpL Chloroform 490.5 460.5 m/x Recently, Tappi and Karrer'^^ have isolated a cis isomer of anthera- xanthin from Lilium candidum. The formulation of the pigment is based on the following facts. Cis antheraxanthin has a lower melting point than trans antheraxanthin and its absorption maxima are displaced towards shorter wavelengths by 4-6 m/i. Spectral measurements show that on irradiation with ultraviolet light and on treatment with iodine the new isomer is partly isomerised to trans antheraxanthin, though the amount of available material was insufhcient to isolate the latter in a cystalline state. On treatment with chloroform containing traces of hydrochloric acid, cis antheraxanthin is con- verted into mutatoxanthin which is also obtained from trans antheraxanthin under these conditions. Cis antheraxanthin is the first natural carotenoid epoxide with a partial cis configuration. The small difference in the location of the absorption maxima of cis and trans antheraxanthin suggests that only one double bond in the former has a czs-configuration. The large difference in melting points, on the other hand, suggests that there is a considerable difference in molecular shape and that the double bond involved in the geometrical isomerism may perhaps be situated in the centre of the polyene chain. Cis antheraxanthin crystallises from methanol in long, yellow-red needles, m.p. 110° (uncorr., evacuated capillary). It is easily soluble in benzene, carbon disulphide and ether, fairly soluble in warm methanol and very sparingly soluble in petroleum ether. Solvent: Absorption maxima: Carbon disulphide 506 476 m/^ Benzene 487 457 m/z Ethanol 472 445 m^ On shaking an ethereal solution with concentrated hydrochloric acid, the latter assumes a fairly stable blue colouration. References p. 214— 2ij. 6 VIOLAXANTHIN 193 6. VIOLAXANTHIN C4oH5e04 History 1931 KuHN and Winterstein^^ isolate a pigment of the formula C4oH5g04 from the yellow blossoms of pansies {viola tricolor). The pigment is named violaxanthin. 1931-44 Karrer and co-workers''* carry out detailed investigations on the constitution of violaxanthin. 1945 Karrer and Jucker achieve a partial synthesis of violaxanthin by the oxidation of zeaxanthin and thus establish the constitution of the pigment'^. Occurrence Recent investigations have shown that violaxanthin is fairly widely distributed in nature. In view of the formation of violaxanthin from zea- xanthin''^ it was to be expected that plants containing the former would also contain the latter pigment. As the investigations regarding the genetic relation- ship of the two compounds are very recent, this question has so far received comparatively little attention. a) Blossoms: Calendula officinalis Cytisiis laburnum Laburnum anagyroides Ranunculus acer Sinapis officinalis Stinging nettles Tagetes grandiflora Taraxacum officinale Tragopogon pratensis Tulipa (yellow variety) Tussilago Farfara References p. 214— 217. Carotenoids 13 TABLE 42 OCCURRENCE OF VIOLAXANTHIN References L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. 208 (1932) 27. P. Karrer and A. Notthafft, Helv. chim. Acta 15 (1932) 1195. P. Karrer and A. Notthafft, Helv. chim. Acta 13 (1932) 1195. R. KuHN and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. P. Karrer and A. Notthafft, Helv. chim. Acta 15 (1932) 1195. R. KuHN, A. WiNTERSTEiN and E. Lederer, Z. physiol. Chem. igy (1931) 141. — P. Karrer and co- workers, Helv. chim. Acta 28 (1945) 1146. R. KuHN, A. WiNTERSTEiN and E. Lederer, Z. physiol. Chem. igy (1933) 141. R. KuHN and E. Lederer, Z. physiol. Chem. 200 (1931) 108. P. Karrer and A. Notthafft, Helv. chim. Acta 13 (1932) 1195. C. A. ScHUNCK, Proc. Roy. Soc. 72 (1903) 1G5. P. Karrer and R. Morf, Helv. chim. Acta 13 (1932) 863. 194 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Ulex europq^us Viola tricolor b) In fruit: Arbutus Unedo L. Carica Papaya Citrus aurantium Citrus poonensis hort. Cucurbita maxima Diospyros costata Iris Pseiidocorus c) Human liver( ?) References C. A. ScHUNCK, Proc. Roy. Soc. 72 (1903) 165. R. KuHN and A. Winterstein, Ber. 64 (1931) 326. — P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) 1624; Helv. chim. Acta 2y (1944) 1684. K. ScHON, Biochem. J. 2g (1935) 1779. R. Yamamoto and S. Tin, Chem. Centr. ig33, I, 3090. L. Zechmeister and P. Tuzson, N aturwissenschaften ig (1931) 307. — P. G. F. Vermast, Naturwissen- schaften ig (1931) 442. R. Yamamoto and S. Tin, Chem. Centr. ig34, I, 1660. L. Zechmeister and P. Tuzson, Ber. 67 (1934) 824. K. Schon, Biochem. J. 2g (1935) 1779. P. J. Drumm, F. O'Connor, Biochem. J. jg (1945) 211. H. WiLLSTAEDT and T. Lindqvist, Z. physiol. Chem. 240 (1936) 10. Preparation'^ Yellow blossoms of Viola tricolor, containing as few deeply pigmented patches as possible, are dried and extracted at room temperature with petroleum ether. The combined extracts are concentrated in vacuum to a small volume and the pigment esters are saponified with a solution of sodium ethoxide in ethanol. The free phytoxanthins are dissolved in methanol, petroleum ether is added, and the violaxanthin is precipitated by very careful addition of water. The crude pigment is filtered and recrystallised from a mixture of methanol and ether. The yield amounts to 0.05 to 0.07% of the dry blossom powder. CH3 CHa c Chemical Constitution^'^ CH, CH, CH, CH, CH3 CH, \/ C CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ )o o( HOCH C C CHOH Violaxanthin CH» CHo HoC CHp Since 1931, numerous experiments have been made with the object of elucidating the constitution of violaxanthin. There is some disagreement between the results of different workers and a complete proof of the constitution was only obtained by the partial synthesis of the pigment'^. KuHN andWiNTERSTEiN determined the correct molecular formula (C4QHgg04) of violaxanthin'^^. Karrer and Morf^*' subjected the pigment to permanganate degradation and obtained a : a-dimethylsuccinic acid, but no a : a-dimethyl- References p. 214-217. 6 VIOLAXANTHIN 195 glutaric acid. These results established the relationship of violaxanthin to the xanthophyll and zeaxanthin series (of. p. 201). Karrer and co-workers^^ showed that violaxanthin absorbs 10 mols of hydrogen on catalytic hydrogenation. The position of the absorption maxima indicated that all 10 double bonds must be conjugated*. According to Karrer and Rutschmann^^ only two of the four oxygen atoms in the violaxanthin molecule are present as hydroxyl groups; the nature of the other two oxygen atoms could not be established**. Eventually Karrer and JucKER were able to identify violaxanthin with their synthetic zeaxanthin di-epoxide. The constitution of violaxanthin was thus proved (cf. p. 190). Properties Violaxanthin crystallises from methanol in yellow-orange prisms, or from carbon disulphide in reddish-brown spears, m.p. 200°. It is easily soluble in ethanol, methanol, carbon disulphide and ether, but almost insoluble in petroleum ether. Solvent: Absorption maxima: Carbon disulphide 501 470 440 m^ Chloroform 482 451.5 424 m^ Petrol 472 443 417.5 m/i Ethanol 471.5 442.5 417.5 ran Methanol 469 440 415 m/< (cf. Fig. 9, p. 351) For quantitative extinction measurements, cf. Hausser and Smakula^^. Violaxanthin gives a very characteristic, stable deep-blue colouration on shaking an ethereal solution with 20% hydrochloric acid***. The pigment is separated from other phytoxanthins by chromatographic adsorption from benzene solution on zinc carbonate or calcium carbonate. The specific rotation in chloroform is [aj^d = +35°- Violaxanthin is converted into mutatoxanthin, auroxanthin and zeaxanthin under the influence of dilute acids®^. These reactions are discussed on p. 62. Derivatives Perhydroviolaxanthin: This compound is obtained on catalytic reduction of violaxanthin in ethanol. It is a colourless, weakly leavo-rotatory oil^^. In view With regard to apparent contradictions concerning the number of double bonds, cf. Helv. chim. Acta 28 (1945) 300. With regard to the contradictory data concerning the number of hydroxyl groups, cf. Helv. chim. Acta 28 (1945) 300. *** For further colour reactions cf. R. Kuhn and A. Winterstein, Ber. 64 (1931) 326. References p. 214— 21';. 196 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI of the fact that the epoxide nature of violaxanthin was not realized at the time, some of the hydrogenations were carried out in glacial acetic acid solution, resulting in a partial isomerisation of the pigment (cf. p. 6i). Violaxanthin di-p-nitrohenzoate C54H620ioN2^^ : The ester is obtained by treating violaxanthin dissolved in pyridine with p-nitrobenzoyl chloride. M.p. 208° (with decomposition, uncorr., evacuated capillary). Violaxanthin dibenzoate Cg4Hg40g^^ : The dibenzoate is prepared in a manner analogous to that employed for the compound described above. M.p. 217° (uncorr. evacuated capillary). In agreement with the formula of violaxanthin, neither ester contains any active hydrogen atoms. 7. AUROXANTHIN C4oH5g04 History and Occurrence During the chromatographic separation of carotenoids from Viola tricolor, Karrer and Rutschmann^'' isolated a new pigment which they termed auro- xanthin in view of the beautiful golden-yellow colour of its crystals. Up to the present time this phytoxanthin has only been found in the blossoms of Viola tricolor, but it is probable that it occurs more widely together with its isomer, violaxanthin*. Preparation Dried blossoms of Viola tricolor are extracted with petroleum ether, the com- bined extracts are strongly concentrated in vacuum and the phytoxanthin esters are saponified with methanolic potassium hydroxide. After the saponification has been completed, the free phytoxanthins are dissolved in methanol, petroleum ether is added, and the pigments are precipitated by careful addition of water. The crude carotenoid mixture thus obtained is recrystallised once from methanol. Auroxanthin remains in the mother liquors. The pigments from the mother liquors are extracted with benzene and chromatographed on zinc carbonate. Auroxanthin is eluted with a mixture of ether and methanol from the upper part of the absorption column. After two recrystallisations from methanol, the pigment is obtained analytically pure. Chemical Constitution^^ C C CH2 C CH CH3 C1XI3 CH3 HgC CH — C CH2 HOCH C GH-C=CHCH=CH-C=GHCH=CHCH=C-CH=CHCH=C-CH C GHOH Auroxanthin For the relationships between auroxanthin and violaxanthin, cf. below. References p. 214— 21J. 8 XANTHOPHYLL iq-j The empirical formula of auroxanthin was determined by Karrer and RuTSCHMANN^*'. The number of double bonds was determined by catalytic hydrogenation. The position of the absorption bands (454, 423 m^ in carbon disulphide) indicated the presence of seven conjugated double bonds. Zerewiti- NOFF determinations gave values corresponding to two or three active hydrogen atoms. Since the determination of active hydrogen in xanthophyll, however, gave values corresponding to 2.5 active hydrogen atoms, it could be assumed that auroxanthin contains only two hydroxyl groups. The nature of the other two oxygen atoms could not at first be established. However, when auro- xanthin, together with mutatoxanthin and a little zeaxanthin had been obtained by the acid treatment of violaxanthin^^, a connection between these pigments was clearly indicated. The nature of this relationship was elucidated by the investigations of Karrer and Jucker in the course of which violaxanthin was identified as zeaxanthin di-epoxide (cf. p. 195) and auroxanthin as the corresponding di-furanoid oxide^^. The proposed formula of auroxanthin is in complete agree- ment with all the properties of the compound. The configurations of natural and partially synthetic auroxanthin appear to be the same^^. Properties Auroxanthin crystallises from methanol in golden-yellow needles m.p. 203° (uncorr., evacuated capillary). The solubility of the pigment is very similar to that of violaxanthin. The pigment shows no optical rotation in benzene solution. A characteristic test for auroxanthin is the blue colouration which is ob- served on shaking an ethereal solution with 15 % hydrochloric acid. This blue colouration is very stable. In carbon disulphide, auroxanthin shows absorption maxima at 454 and 423 m^ (cf. Fig. 10, p. 351). 8. xanthophyll C4oH5g02* History 1837 Berzelius^^ coins the term "xanthophyll" for the yellow pigment of autumn leaves. 1907 WiLLSTATTER and MiEG^^ isolate xanthophyll from green leaves in the crystalline state and determine its empirical formula and its molecular weight. There is no uniformity in the Uterature regarding the designation of this compound. Some investigators employ the term "lutein" proposed by Kuhn, and use the term xantho- phyll only as a generic term for hydroxyl-containing carotenoids, for which Karrer proposed the term phytoxanthins. In this monograph the name xanthophyll is retained for the yellow leaf pigment C^oHggOg. References p. 214— 2iy. ^ igS CAROTENOIDS CONTAINING HYDROXYL GROUPS XI 1912 WiLLSTATTER and EsCHER^® isolate lutein, later found to be a mixture of xanthophyll and zeaxanthin, from egg yolk. 1930-33 Karrer and co-workers^' elucidate the constitution of xanthophyll. Occurrence Xanthophyll is very widely distributed in nature. It is found together with carotene and chlorophyll in all green parts of plants. It also occurs very frequently in red and yellow blossoms, sometimes as an ester (e.g. as the di- palmitic acid ester, helenien^^). According to a communication by Jungp-^^. xanthophyll occurs in various green insects combined with protein. These findings, however, require further confirmation. a) Blossoms: Acacia decurrens var. mollis Acacia discolor Acacia linifolia A cacia longi folia Calendula officinalis Caltha palustris Ciicurbita Pepo Gazania rigens Genista tridentata Helianthus annuus Heleniwtn autumnale Kerria japonica Lcontodon autumnalis Ranunculus acer References p. 214-217. TABLE 43 OCCURRENCE OF XANTHOPHYLL References R. KuHN, A. WiNTERSTEiN and E. Lederer, Z. physiol. Chem. igy (1931) 141. J. M. Petrie, Biochem. J. 18 (1924) 957. do. do. L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. 208 (1932) 27. P. Karrer and A. Notthafft, Helv. chim. Acta 15 (1932) 1195. L. Zechmeister, T. Beres and E. Ujhelyi, Ber. 68 (1935) 1321; 69 (1936) 573. K. Sch5n, Biochem. J. 32 (1938) 1566. — L. Zech- meister and W. A. Schroder, /. Am. Chem. Soc. 65 (1943) 1535. K. Schon and B. Mesquita, Biochem. J. 30 (1936) 1966. R. Kuhn, A. Winterstein and E. Lederer, Z. physiol. Chem. igy (1931) 141. — L. Zechmeister and P. TuzsoN, Ber. 63 (1930) 3203; 67 (1934) 170. R. KuHN, A. Winterstein and E. Lederer, Z. physiol. Chem. igy (1931) 141. T. Ito, H. Suginome, K. Ueno and S. Watanabe, Bill. chem. Soc. Japan 11 (1936) 770. R. Kuhn and E. Lederer, Z. physiol. Chem. 213 (1932) 188. C. A. Schunck, Proc. Roy. Soc. London 27 (1903) 165. — R. Kuhn and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. — P. Karrer and co-workers, Helv. chim. Acta 28 (1945) 1146. XANTHOPHYLL 199 Ranunculus arvensis Ranunculus Steveni Rudbeckia Neumannii Sarothamnus scoparius Tagetes erecta T. Tagetes grandiflora Tagetes nana Tagetes patiila Taraxacum officinale Tragopogon pratensis Trollius europaeus Viola tricolor Winter asters References P. Karrer and A. Notthafft, Helv. chim. Acta 15 (1932) 1195. H. H. EscHER, Helv. chim. Acta 11 (1928) 752. R. KuHN, A. WiNTERSTEiN and E. Lederer, Z. physiol. Chem. igj (1931) 141. P. Karrer and E. Jucker, Helv. chim. Acta 2^ (1944) 1586. R. KuHN, A. WiNTERSTEiN and E. Lederer, Z. physiol. Chem. igy (1931) 141. do. do. do. P. Karrer and H. Salomon, Helv. chim. Acta 13 (1930) 1063. — P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) 1144. — R. Kuhn and E. Lederer, Z. physiol. Chem. 200 (1931) 108. P. Karrer and A. Notthafft, Helv. chim. Acta 13 (1932) 1195. — P. Karrer and co-workers, Helv. chim. Acta 28 (1945) 1146. P. Karrer and A. Notthafft, Helv. chim. Acta 13 (1932) 1195. — P. Karrer and E. Jucker, Helv. 29 (1946) 1539. R. Kuhn and A. Winterstein, Ber. 64 (1931) 326. — P. Karrer and co-workers, Helv. chim. Acta 25 (1942) 1624; 2j (1944) 1684. P. Karrer and E. Jucker, Helv. chim. Acta 26 (1943) 626. b) Fruit: Ananas sativus Arbutus Capsicum annuum Citrus aurantiutn Citrus madurensis Convallaria majalis Cranberries Cucurbita maxima Luffa-species Mangifera indica Moniordica Balsamina O. C. Magistad, PlayU. Physiol. 10 (1935) 187. K. ScHON, Biochem. J. 2g (1935) 1779. L. Zechmeister and L. v. Cholnoky, Ann. sog (1934) 269. L. Zechmeister and P. Tuzson, Ber. 6g (1936) 1878. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 221 (1933) 278; 240 (1936) 191. A. Winterstein and U. Ehrenberg, Z. physiol. Chem. 2oy (1932) 25. H. Willstaedt, Svensk Kemisk. Tidskr. 48 (1936) 212. H. SuGiNOME and K. Ueno, Chem. Centr. igji, II, 2892. — L. Zechmeister and P. Tuzson, Ber. 6j (1934) 824. T. N. Godnew and S. K. Korschenewsky, Chem. Centr. igji, I, 1299. R. Yamamoto, Y. Osima and T. Goma, Chem. Centr. 1933, I, 441. G. and F. Tobler, Ber. bot. Ges. 28 (1910) 365, 496. — B. M. DuGGAR, Washington Univ. Stud, i (1913) 22. CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Musa pavadisiaca Prunus persica Rosa rubiginosa Wheat germ References H. V. LoESECKE, /. Am. Chem. Soc. 31 (1929) 2439. G. Mackinney, Plant. Physiol. 12 (1937) 216. R. KuHN and C. Grundmann, Ber. 6y (1934) 339. B. Sullivan and C. H. Bailey, /. Am. Chem. Soc. 38 (1936) 383. c) Other vegetable and animal sources: Bombix mori Chicken fat Cladophora Sauteri Egg yolk (chicken) Feathers {Serinus canaria) Feathers (woodpecker) Haemaiococcus pluvialis Human fat Human liver Marsh soil Mycobacterium phlei Nitella opaca Oedogonium, Peat Rana esculenta Rhodymenia palmata Serum (cattle) F. G. DiETEL, Kl. Wschr. 12 (1933) 601. — C. Rand. Biochem. Z. 281 (1935) 200. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 225 (1934) 189. I. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 2g (1935) 1376. A. E. GiLLAM and I. M. Heilbron, Biochem. J. 2g (1935) 1064. H. Brockmann and O. Voelker, Z. physiol. Chem. 224 (1934) 193. do. J. TiscHER, Z. physiol. Chem. 250" (1937) 147. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 225 (1934) 189; 231 (1935) 259. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 234 (1935) 241. — H. Willstaedt and T. Lindqvist, Z. physiol. Chem. 240 (1936) 10. 0. Baudisch and H. v. Euler, Arch. Chem. Mineral. Geol. Ser. All, No. 21, Chem. Centr. J935, II, 1390. M. A. Ingraham and H. Steenbock, Biochem. J. 2g (1935) 2553. 1. M. Heilbron, E. G. Parry and R. F. Phipers, Biochem. J. 2g (1935) 1376. do. R. C. Johnson and R. Tiessen, Chem. Centr. ig34, I, 2686. L. Zechmeister and P. Tuzson, Z. physiol. Chem. 238 (1936) 197. I. M. Heilbron, E. G. Parry and A. F. Phipers, Biochem. J. 2g (1935) 1376. A. E. Gillam and M. S. El Ridi, Biochem. J. 2g (1935) 2465. This summary of the occurrence of xanthophyll, though incomplete, indicates the extraordinarily wide distribution of this phytoxanthin. Preparation^^^ 6 kg of finely ground, dry stinging nettles are extracted with 80% methanol in completely filled bottles. The extraction is then continued with peroxide-free ether. The combined extracts are shaken with water, saponified with methanolic References p. 214-217. 8 XANTHOPHYLL 201 potassium hydroxide and washed free from alkali. The solvent is removed by- distillation in a stream of carbon dioxide until the volume is reduced to 300 ml; on cooling the solution, part of the xanthophyll crystallises out. Further quantities of pigment can be obtained from the mother liquors after further concentration and addition of petroleum ether. The remaining mother liquors are finally taken up in methanol and diluted with water under petroleum ether, when the remainder of the pigment is precipitated. The total yield amounts to up to 6 g. The preparations obtained in this way differ widely with regard to rotation and m.p. The pigment can be purified bv repeated crystallisation from methanol or chromatography on zinc carbonate from benzene solution. For other methods of preparation cf. Kuhn, WiNTERSTEiN and LedererI"!, Miller^°2 and Zechmeister and Tuzson^"^. Chemical Constitution CHo CHo CHj Cxlo \V \V C CHo CHo CHo CHo C /\ I I I r /\ CHg C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH, HOCH C-CHa H,C-C 3'CHOH \4/ Xanthophyll \a'/ CHj CH The constitution of xanthophyll was elucidated mainly by Karrer and co- workers^"*. The empirical formula of xanthophyll was determined by Will- STATTER and MiEG^"^. Zechmeister and Tuzson^"^ established the presence of II double bonds by catalytic hydrogenation. This was confirmed by Pumme- rer and Rebmann^"', who showed that the pigment absorbs 11 mols of iodine chloride. The spectral properties of xanthophyll are similar to those of a-caro- tene, indicating the presence of similar chromophoric systems. According to Karrer, Helfenstein and Wehrli^*'^, the two oxygen atoms are present as hydroxyl groups, which can be quantitatively determined by the method of Zere'witinoff. These findings were confirmed by the preparation of a number of esters and of a monomethyl ether of xanthophyll (Karrer and co- workers^^^'ii"). Furthermore, Karrer, Zubrys and Morf^ were able to oxidise perhydroxanthophyll to a diketone, thus proving that the two hydroxyl groups are secondary and do not below to enol groupings. The number of side- chain methyl groups was determined by means of chromic acid and perman- ganate oxidations^^^. Karrer and co-workers^^^ obtained further evidence for the structure of xanthophyll by the potassium permanganate oxidation of the pigment, which yielded a:a-dimethylsuccinic acid and dimethylmalonic acid. No geronic acid or a:a-dimethylglutaric acid were formed and it was concluded from the result that xanthophyll differs from carotene in the struc- ture of the two carbon rings. The two hydroxyl groups are contained in the rings and the two most likely positions are the carbon atoms 3 or 4, and 3' or References p. 214-217. 202 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI 4', since only these will explain the formation of a:a-dimethylsuccinic acid and the absence of a:a-dimethylglutaric acid on oxidation. CH3 CHg c CH2 COOH CHj CHj 0 /\ HOOC COOH COOH a : a-dimethylsuccinic acid a : a-dimethylmaloni The spectral properties of xanthophyll indicate a resemblance to a-carotene. NiLSSON and Karrer^^* converted perhydroxanthophyll into the dibromide and then removed the two bromine atoms by reductive fission. The perhydro- carotene C40H78 formed was optically active and the rotation had the same sign and was of the same magnitude as that of the perhydrocarotene obtained by the reduction of a-carotene. Hence xanthophyll is a dihydroxy-a-carotene. These investigations also proved that the hydroxyl group must occupy position 3 ' in the a-ionone ring, since a hydroxyl group on carbon atom 4' would have enolic character. Very recently Karrer, Koenig and Solmssen^^^ succeeded in isolating a-citraurin, an isomer of /5-citraurin discovered by Zechmeister and Tuzson^^^, by the careful potassium permanganate degradation of xanthophyll. The formula of xanthophyll was thus further confirmed. CHo CHo \V C CHo CHo CHo CHo /\ I I I I CHa C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO I I! HOCH C-CHs \^ / ^-Citraurin CH2 CHo CHo \/ C CH, CH, CH, CH, /\ I I I I CHj CH- CH = CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO HOCH C-CH3 \ /^ ' a-Citraurin CH 'Properties and Physical Constants Crystalline form: The pigment crystallises from methanol in violet prisms which have a metallic lustre and characteristic dovetail shape. The crystals contain one molecule of methanol of crystallisation. References p. 214-217. 8 XANTHOPHYLL 203 Melting point: 193° corr. Solubility: Xanthophyll is easily soluble in chloroform, benzene, acetone, ether and carbon disulphide, sparingly soluble in ethanol and methanol, and almost insoluble in petroleum ether, i g of xanthophyll dissolves in about 700 g of boiling methanol. Spectral properties (cf. Fig. 11, p. 352) Solvent: Absorption maxima}'^'' Carbon disulphide 508 475 445 m// Chloroform 487 456 428 m/f « Ethanol 476 446.5 420 vafi Petrol 477.5 447.5 420 van Methanol .473.5 4-44" 418 m/z Solutions of xanthophyll in carbon disulphide are red. Dilute solutions in benzene, ethano', ether and chloroform are golden yellow, while concentrated solutions in these solvents are orange in colour. Optical activity: Xanthophyll, like a-carotene exhibits strong optical activity. The specific rotation is [aYcd = +^45° (i^ ethyl acetate), [a]^^ = +160° (in chloroform) . Colour reactions*: Xanthophyll dissolves in concentrated sulphuric acid first with a green, then with a blue colouration. Concentrated formic acid gives a bright green, trichloracetic acid a dark blue, and antimony trichloride in chloroform solution an intense dark blue colouration. Partition test: On partition between petroleum ether and 90 % methanol, xanthophyll is found in the lower layer. Chromatographic behaviour: Xanthophyll is easily adsorbed on calcium carbonate and zinc carbonate from benzene solution. It is eluted with ether containing a few per cent of methanol. Detection and estimation: Xanthophyll is separated from other phyto- xanthins by adsorption on zinc carbonate (or calcium carbonate). It can be identified by a determination of the absorption maxima. According to Kuhn and Brockmann^^^, the colourimetric determination can be carried out using a standard solution of azobenzene in ethanol. Physiological properties: Xanthophyll possesses no vitamin A activity. According to voN Euler, Karrer and Zubrys, however, a vitamin A-active product is formed on treating the phytoxanthin with phosphorous tribromide^^^. Concerning the colour intensity of the antimony trichloride reaction, cf. H. v. Euler, P. Karrer and M. Rydbom, Ber. 62 (1929) 2445. References p. 214-21^. 204 CAROTENOIDS CONTAINING HYRDOXYL GROUPS XI Derivatives Perhydroxanthophyll C40H78O2 : This compound is obtained by the catalytic reduction of xanthophyll. It is a viscous, colourless oil which is weakly dextra- rotatory^2° [ajp = +28° in chloroform. Perhydroxanthophyll is much more soluble in organic solvents than xanthophyll, and can be esterified to give an oily diacetate^^^. Xanthophyll halogenides: On treating xanthophyll with bromine, an oxygen-free bromide C4oH4oBr22 is formed^22 -j-j-jg action of bromine dissolved in chloroform is milder and only 8 mols are absorbed^^^. Using iodine chloride, ten double bonds are saturated after 24 hours, but the eleventh double bond is only saturated after 7 daysi24. Xanthophyll diiodide C40H56O2I2 is a well-defined, beautifully crystalline compound, the analysis of which confirms the molecular weight of xantho- phylP^^. Xanthophyll is liberated unchanged from the iodide by means of sodium thiosulphate^^^. Synthetic esters of xanthophyll^'^'' : The two hydroxyl groups in xanthophyll can be esterified with acid anhydrides or acid chlorides in pyridine solution. The spectral properties of xanthophyll and its esters are largely identical, but in the partition test the esters show the opposite behaviour from xanthophyll and are found quantitatively in the upper layer. The melting points of the esters generally decrease with increasing length of the acid chain. The esters are easily soluble in benzene and petroleum ether, but very sparingly in alcohols. a) Xanthophyll diacetate C44H60O4: Crystallises from a mixture of benzene and methanol. M.p. 170°. b) Dipropionate C45H64O4: The dipropionate crystallises from a mixture of ben- zene and methanol in reddish-yellow plates, m.p. 138°. c) Dibutyrate C48H68O4: Reddish-yellow plates from methanol. M.p. 156°. d) Di-n-valerate C50H72O4 : The ester crystallises from a mixture of benzene and methanol in reddish-yellow plates, m.p. 128°. e. Di-ri-caproate €52^17504: Reddish-yellow plates from a mixture of benzene and methanol. M.p. 117°. f) Dioenanthate C54Hgo04: Reddish-yellow plates from a mixture of benzene and methanol. M.p. 111°. g) Dicaprylate C55H84O4: Reddish-yellow plates from a mixture of benzene and methanol. M.p. 108°. h) Dipalmitate, Helenien, C^^^^j^^O^: This compound was discovered by Kuhn and Winterstein^^s [^ ^j-jg blossom leaves of Helenimn autumnale. Helenien is widely distributed in the vegetable kingdom. A summary of plants containing helenien is given by Kuhn and Winter- STEIN^^*. References p. 214— 2iy. 8 XANTHOPYHLL 205 Helenien can be obtained synthetically from xanthophyll and palmitic acid chloridei29 j^ crystallises from ethanol in red needles, m.p. 92°. With regard to the colourimetric estimation, see Kuhn and Brockmann^^". i) Distearate C-j^-^^^i^i- ^^^ plates which appear yellow under the microscope. M.p. 87°i3i. k) Dibenzoate C54H64O4: Red plates from ethanol. M.p. about 165°i3i_ 1) Bis-/?-nitrobenzoate €54115208X2: This compound separates from benzene as a red micro-cr^'stalline powder, m.p. 210°^^^. Xanthophyll monomethyl ether C4iH5802^^^: The ether is obtained from the potassium derivative of xanthophyll (prepared by the interaction of xantho- phyll and potassium ^^r^.-amylate) and methyl iodide. It crystallises from methanol in needles, m.p. 150°. On partition between petroleum ether and 90 % methanol the pigment exhibits predominately epiphasic behaviour, but both layers are coloured. a-Citraurin CggHjoOg: CHo CHo C CHo CH.. CHo CHo /\ I I I I CH2 CH- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO HOCH C-CHj \ ^ a-Citraurin CH a-Citraurin was obtained by Karrer, Koenig and Solmssen^^^ by the careful potassium permanganate oxidation of xanthophyll. a-Citraurin is related to /S-citraurin^^* in the same way as a-apo-2-carotenal to /9-apo-2- carotenal. a-Citraurin crystallises from methanol in brilliant orange plates, m.p. 153°. Solvent Absorption maxima Carbon disulphide 514 480 449 m^ Petroleum ether 477 438 m^u Md = +372° (± 25°). (cf. Fig. 28, p. 359) Treatment of a-citraurin with hydroxylamine acetate affords the oxime, which crystallises from methanol in clustered needles, m.p. 148°. Solvent Absorption maxima Carbon disulphide 499 468 m^u Ethanol 470 440 ran References p. 214-21"/. 2o6 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI 9. XANTHOPHYLL EPOXIDE AND ITS TRANSFORMATION PRODUCTS Xanthophyll epoxide C4UH56O3: CH, CH, CH, CH, C CH3 CH3 CH, CH, C /\ I I I I /\ CHa C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CHg )0 ' ' HsC-C CHOH HOCH C \/ \ / \^ Xanthophyll epoxide CH CHg CHj Xanthophyll mono-epoxide was obtained by Karrer and Jucker^^^ by the oxidation of xanthophyll diacetate with monoperphthalic acid. It crystallises from a mixture of benzene and methanol in reddish-yellow crystals, m.p. 192° (uncorr., evacuated capillary). Recent investigations^^^^ have shown that this epoxide is widely distributed in blossoms and also occurs in large quantities (as much as 40% of the xanthophyll content) in leaves of all kinds. Since xanthophyll epoxide is readily isomerised to fiavoxanthin (see p. 207) by traces of acids, it is probable that the latter is often an artefact. TABLE 44 OCCURRENCE OF XANTHOPHYLL EPOXIDE Sources References Asters P. Karrer and E. Jucker, Helv. chim. Acta 26 (1943) 626. Elodea canadensis P. Karrer and J. Rutschmann, Helv. chim. Acta 28 (1945) 1526. — D. Hey, Biochem. J. 31 (1937) 532. Green or etiolated leaves P. Karrer, E. Krause-Voith and K. Steinlin, Helv. chim. Acta 31 (1948) 113. Kerria japonica DC P. Karrer and E. Jucker, Helv. chim. Acta 2g (1946) 1539. Laburnum anagyroides do. Ranunculus acer P. Karrer, E. Jucker, J. Rutschmann and K. Stein- lin, Helv. chim. Acta 28 (1945) 1146. Sarothamnus scoparius P. Karrer and E. Jucker, Helv. chim. Acta 2y (1944) 1585. Stinging nettles P. Karrer, E. Jucker, J. Rutschmann and K. Stein- lin, Helv. chim. Acta 28 (1945) 1146. Tragopogoyi pratensis do. Trollius eiiropaeus P. Karrer and E. Jucker, Helv. chim. Acta 2g (1946) 1539. The absorption spectrum of xanthophyll epoxide is very similar to that of violaxanthin (p. 195) and the two pigments are best distinguished by treat- ment with chloroform containing traces of hydrochloric acid when they are converted into fiavoxanthin (maxima 478, 449 m/^ in carbon disulphide) and auroxanthin (maxima 454, 423 m/^ in carbon disulphide), respectively. References p. 214— 2iy. TO FLAVOXANTHIN 207 Solvent Absorption ynaxima Carbon disulphide 501.5 472 m^ Benzene 482 453 m^ Petroleum ether ,47 1..- — ^442 m/z Ethanol 473 445 m// On treating xanthophyll epoxide with acetic anhydride in pyridine, xantho- phyll epoxide diacetate is obtained. This compound exhibits entirely epiphasic behaviour. M.p. 184-185° (uncorr., evacuated capillary) . Xanthophyll epoxide has approximately the same solubility in organic solvents as xanthophj-ll. It shows entirety hypophasic properties in the partition test. On treating an ethereal solution with concentrated aqueous hydrochloric acid, the latter assumes a blue colour. Xanthophyll epoxide is extremely unstable towards acids. Even traces of hydrochloric acid, such as occur, for instance, in chloroform which has been stored for some time, convert the pigment into the two isomeric furanoid oxides, flavoxanthin and chrysanthemaxanthin. These pigments are described below (p. 207, 211). Eloxanthin (Xanthophyll epoxide) During an investigation of the carotenoids from the leaves of elodea cana- densis, Hey^^^ discovered a previously unknown pigment for which he proposed the term eloxanthin. The properties of eloxanthin are so similar to those of xanthophyll epoxide, that the identity of the two pigments appeared very probable. Karrer and Rutschmann^^"^ were in fact able to show the presence of xanthophyll epoxide in elodea canadensis and as no second pigment showing the properties of eloxanthin was present, the identity of the two compounds is very probable. Natural xanthophyll epoxide (eloxanthin) is optically active, [p.Yc\ ^ +225° in benzene. The optical activity of the partially synthetic product has not yet been determined. 10. FLAVOXANTHIN C^oHggOg History 1932 KuHN and Brockmann^^^ isolate flavoxanthin from blossoms of Ranun- culus acer. 1942 Karrer and Rutschmann^^^ report the occurrence of this phytoxanthin in other plants and propose a provisional formula. 1945 Karrer and Jucker^*" carry out a partial synthesis of flavoxanthin which establishes the constitution of the pigment with a high degree of certainty. References p. 214— 21 j. 2o8 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Occurrence Flavoxanthin occurs fairly widely in plants, but only in small concentrations and not as the main pigment. TABLE 45 OCCURRENCE OF FLAVOXANTHIN Source References Dandelion P. Karrer and J. Rutschmann, Helv. chim. Acta 23 (1942) 1144. Ranunculus acer R. Kuhn and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. — P. Karrer, E. Jucker, J. Rutschmann and K. Steinlin, Helv. chim. Acta 28 (1945) 1146. . Sarothamnus scoparius P. Karrer and E. Jucker, Helv. chim. Acta 2y (1944) 1585. Senecio vernalis ( .'') R. Kuhn and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. Ulex europaeiis K. Schon, Biochem. J . 30 (1936) 1960. Viola tricolor P. Karrer and J. Rutschmann, Helv. chim. Acta 27 (1944) 1684. Preparation According to Kuhn and Brockmann^^*, the pigment is best extracted from crowfoot blossoms. The yield amounts to approximately 40 mg flavoxanthin from I kg of blossoms. Karrer and Rutschmann^^* extracted phytoxanthin from dandelion blossoms by means of petroleum ether and obtained 80 mg of pure flavoxanthin from 1.5 kg of dry blossoms. a) Flavoxanthin from Ranunculus acer: The dried and finely ground blossoms are extracted with methanol at room temperature and the solution is concentrated to a small volume in vacuum. The pigments are then extracted with ether and sapo- nified with alcoholic potassium hydroxide. After partitioning into an epiphasic and hypophasic fraction the phytoxanthins are chromatographed on calcium carbonate from a mixture of benzene and petrol, and the chromatogram is developed with petrol. The flavoxanthin is eluted with petrol containing 1% of methanol and crystallised repeatedly from methanol. b) Flavoxanthin from dandelion: The dried and finely ground dandeUon blossoms are continuously extracted with petroleum ether and the pigments are saponified with methanolic hydroxide. After a separation into hypophasic and epiphasic constituents, the former are subjected to a preliminary purification by adsorption on alumina. The crystalline mixture of phytoxanthins is adsorbed on zinc carbonate, and eluted with ether containing methanol. Pure flavoxanthin is obtained by crystallisation from methanol. References p. 214-217. FLA VOXANTHIN Chemical Constitution 209 The first attempts to elucidate the constitution of flavoxanthin were made by KuHN and Brockmann^^. These authors determined the empirical formula and the number of double bonds and reported the presence of three hydroxyl groups. According to more recent investigations^"* °, however, flavoxanthin contains only 2 hydroxyl groups, the third oxygen atom being present in the form of an ether grouping. The nature of this oxygen atom was only established by the investigation of Karrer and Jucker^^" in the course of which flavo- xanthin was partially synthesized. Flavoxanthin was obtained together with the isomeric chrysanthemaxanthin by the action of very dilute hydrochloric acid (in the form of aged chloroform) on xanthophyll epoxide. According to the discussion on p. 62 this transformation proceeds as follow. CHo CHo \/ C CH, /\ I CH, C-CH=CH-C=CHCH=CH-- I 1)0 HOCH C Epoxide residue ^ of xanthophyll- CH2 CH3 epoxide \V c /\ CH2 C=CH CH3 HOCH C CH-C=CHCH=CH- CH2I 0 Furanoid residue of CH, flavoxanthin Thus flavoxanthin and the isomeric chrysanthemaxanthin have the following constitution : CHo CHo \V 0 CHq CHo /\ \V CHs C=CH CH3 CH, CH, CH, C I I I I I I I /\ HOCH C CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH, I I H,C-C CHOH CH Flavoxanthin All the properties of the pigment are in complete agreement with this formulation. The partially synthetic product proved to be identical in all respects with natural flavoxanthin^^''-^^^. Properties and Physical Constants Crystalline form: Flavoxanthin crystallises from methanol on rapid cooling in narrow, clustered prisms, which are golden yellow in colour and possess a beautiful surface lustre. M.p. 184° (corr., evacuated capillary). References p. 214— 2IJ, Carotenoids 14 2IO CAROTENOTDS CONTAINING HYDROXYL GROUPS XI Solubility: The solubilities of flavoxanthin are similar to those of xantho- phyll. Flavoxanthin is easily soluble in chloroform, benzene, and acetone, more sparingly in methanol and ethanol, and almost insoluble in petroleum ethei . Spectral properties (cf. Fig. 12, p. 352) : Solvent Absorption maxima Carbon disulphide 479 449 m// Chloroform 459 430 m/i Petroleum ether 450 421 m/z Ethanol 448 421 m/i Optical activity: [a]c° = +190° (in benzene). Partition test: Flavoxanthin is entirely hypophasic on partition between 90% methanol and petroleum ether. Chromatographic behaviour: For the separation of flavoxanthin from other phytoxanthins, especially xanthophyll and chrysanthemaxanthin, zinc carbo- nate is a particularly suitable adsorbent^*". A good separation of the pigments can also be achieved by adsorption on calcium carbonate. Benzene is employed as solvent, and ether containing a little methanol is used as eluent. Flavo- xanthin is found below violaxanthin, but above chrysanthemaxanthin, on the chromatogram column. Xanthophyll is found in the lowest part of the column. Colour reactions: Concentrated sulphuric acid .... deep blue Trichloracetic acid blue Antimony trichloride in chloroform . blue Anhydrous formic acid green Picric acid in ether green Concentrated aqueous hydrochloric acid blue (not very stable, cf. violaxanthin) Identification and estimation: Flavoxanthin is separated from other phyto- xanthins by adsorption on zinc carbonate. Prolonged washing is required for the separation from chrysanthemaxanthin which may also be present. The pigment is identified by the determination of the absorption maxima and by the colour reaction with hydrochloric acid. Physiological behaviour: As would be expected from its structure, flavo- xanthin has no vitamin A activity. Derivative Flavoxanthin diacetate Z^^YI^qO^^'^'^: The diacetate crystallises from methanol in brilliant orange red leaflets, m.p. 157° (uncorr., evacuated capillary). References p. 214— 2iy. II CHRYS ANTHEM AX ANTHIN 211 II. CHRYSANTHEMAXANTHIN C4oH5g03 History 1943 Karrer and Jucker isolate chrysanthemaxanthin from red and yellow blossoms of asters^*^. 1944 The new phytoxanthin is found in blossoms of Sarothamnus scoparius and investigated in more detaiP**. 1945 Karrer and Jucker prepare chrysanthemaxanthin by a partial syn- thesis and thus establish its constitution^*^. Occurrence Up to the present time, chrysanthemaxanthin has not been frequently found in nature. It is always accompanied by xanthophyll and usually by fiavoxanthin. This fact allows certain conclusions to be drawn concerning its mode of formation in the plant (cf. the original communication by Karrer and JuckerI*^). TABLE 46 OCCURRENCE OF CHRYSANTHEMAXANTHIN IN BLOSSOMS Asters P. Karrer and E. Jucker, T/e/t'. c/u'm. ^cto 26 (1943) 626. Gorse P. Karrer and E. Jucker, Helv. chim. Acta 2y (1944) 1585. Ranunculus acer P. Karrer, E. Jucker, J. Rutschm.\nn and K. Stein- LiN, Helv. chim. Acta 28 (1945) 1146. Preparation The extraction of the pigment meets with some difficulty. The supply of large quantities of asters is costly, and the yield of chrysanthemaxanthin is small^*®. Larger quantities of this phytoxanthin can be obtained from gorse blossoms, but the picking of the blossoms must be done very carefully and their content of chrysanthemaxanthin depends on the season^*'. It is therefore preferable to prepare chrysanthemaxanthin synthetically from xanthophylP*^. For this purpose xanthophyll is converted into xanthophvU epoxide by the action of a dilute ethereal solution of monoperphthalic acid. By treatment with dilute hydrochloric acid, the epoxide is converted into a mixture of fiavoxanthin, chrysanthemaxanthin and a little xanthophyll. The separation and purification of the pigments can be achieved by adsorption on zinc carbonate from benzene solu- tion, followed by crystallisation of the phytoxanthins from a mixture of benzene and methanol. References p. 214-212. 2 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Chemical Constitution C CHj CHo CH2 C=^CH CH3 CH3 CH3 CH3 C I A I I I I I I HOCH C CH— C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CHj H3C-C CHOH Chrysanthemaxanthin ^ / CH Chrysanthemaxanthin has the same empirical formula as flavoxanthin^^^. It is identical with the latter in many respects, with the exception of the melting point, the reaction with concentrated aqueous hydrochloric acid and the strength of adsorption on zinc carbonate. On the basis of these facts and its mode of formation together with flavoxanthin by the action of dilute acids on xanthophyll epoxide, Karrer and Jucker^*^ assigned the above formula to chrysanthemaxanthin. The formula is in complete accord with all the properties of the pigment. The isomerism of flavoxanthin and chrysanthemaxanthin is presumably steric in origin. Structural isomerism is unlikely as the two pigments have identical absorption spectra. The isomerism may be due to the fact that the hydroxyl and ether groups in ring A are in czs-positions in one pigment and in ^raws-positions in the other. The question of this isomerism requires further investigation. Properties and Physical Constants Crystalline form: Chrysanthemaxanthin crystallises from a mixture of benzene and methanol in golden yellow leaflets. Melting point: 184-185° (uncorr., evacuated capillary). Solubility: Chrysanthemaxanthin exhibits the same solubilities as flavo- xanthin. It is readily soluble in chloroform, benzene, acetone and ether, a little more sparingly in ethanol and methanol, and almost insoluble in petroleum ether. Spectral properties: Solvent Absorption maxima Carbon disulphide 479 449 m// Chloroform 459 430 m^ Petroleum ether 450 421 m/i Ethanol 448 421 m/^ References p. 214-21'j. 12 LYCOPHYLL 213 Optical activity: Natural and partially synthetic chrysanthemaxanthin have the same optical activity^^". [a]l° = +180-190° (in benzene). Partition test: Chrysanthemaxanthin is entirely hypophasic in character. Chromatographic behaviour: Chrysanthemaxanthin is easily adsorbed on zinc carbonate (or calcium carbonate) from benzene solution. It is found below flavoxanthin but above xanthophyll epoxide on the chromatogram. Colour reactions: With concentrated sulphuric acid, chrysanthemaxanthin gives a dark blue colouration. In contrast to flavoxanthin, concentrated hydro- chloric acid produces no blue colouration. Detection and estimation: Chrysanthemaxanthin is separated from other phytoxanthins by adsorption on zinc carbonate and can be identified by the determination of the absorption maxima and by the lack of colour reaction with hydrochloric acid. Physiological behaviour: Chrysanthemaxanthin exhibits no vitamin A activity. 12. LYCOPHYLL C40H56O2 History and Occurrence 1936 Zechmeister and von Cholnoky isolate lycophyll from Solanum dulca- mara and also establish the occurrence of the new phytoxanthin in Solanum esculentum^^^. Preparation 17 Kg of fresh berries of Solanum dulcamara were dehydrated with ethanol and extracted at room temperature with peroxide-free ether. After removal of the solvent by distillation, the residue was dissolved in benzene and the mixture of pigments was adsorbed on calcium hydroxide. The adsorption was repeated several times and the pigment was finally crystallised from a mixture of benzene and methanol. The yield was 9 mg of lycophyll. Chemical Constitution CH, CH, CH, CH, C CH3 CH3 CH3 CH, C, y I I I I \ CH CHCH=CH-C=CHCH = CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH HOCH C-CHg HaC-C CHOH \ / Lycophyll \ / CH2 CHa References p. 214— 21 y. 214 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI Zechmeister and von Cholnoky assigned the above formula to lycophyll, but this formula could not be proved because of lack of material. The molecular formula C40H56O2 and the purely hypophasic behaviour of the pigment suggest that it is a dihydroxy-compound. The location of the absorption maxima, which are identical with those of lycopene, indicate the presence of 13 double bonds. For these reasons lycophyll is probably 4 :4'-dihydroxy lycopene. The presence of two hydroxyl groups is confirmed by the preparation of lycophyll dipalmi- tateis. Properties Lycophyll crystallises from a mixture of benzene and methanol in violet leaflets, or from benzene and petroleum ether in violet-red needles, m.p. 179° (corr.). It is readily soluble in carbon disulphide, less soluble in benzene and ethanol, and only very sparingly soluble in petroleum ether. On partition between methanol and petroleum ether, it is found quantitatively in the lower layer. Lycophyll is adsorbed somewhat more strongly than lycoxanthin on calcium hydroxide from benzene solution. Solvent AhsoyptioH ^naxinia Carbon disulphide 546 506 472 m/i Benzene 521 487 456 m// Petrol 504 473 444 m/i Ethanol 505 474 444 m/i Lycophyll dipalmitate: This compound crystallises from a mixture of benzene and methanol in violet-red needles ,m.p. 76° (corr.). It shows purely epiphasic behaviour and is easily soluble in carbon disulphide and benzene, less easily in petroleum ether and almost insoluble in ethanol. The light absorption pro- perties of the dipalmitate in the visible region are identical with those of lycophyll. REFERENCES 1. L. Zechmeister and L. v. Cholnoky, Ber. 6g (1936) 422. 2. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 268, 1084; 14 (1931) 614, 843. 3. R. KuHN and C. Grundmann, Ber. 67 (1934) 339, 1133. 4. R. KuHN and C. Grundmann, Ber. 6y (1934) 339, ii33- 5. R. Yamamoto and S. Tin, Sci. Pap. Inst, physic, chem. Res. 20 (i933) 411; Chem. Centr. 1933, I, 3090. 6. R. KuHN and C. Grundmann, Ber. 66 (1933) 1746. 7. P. Karrer and W. Schlientz, Helv. chim. Acta 17 (1934) 55- 8. R. KuHN and C. Grundmann, Ber. 66 (1933) 1746. 9. R. KuHN and C. Grundmann, Bey. 66 (1933) ^74^- 10. R. Yamamoto and Y. Kato, Chem. Ceyitr. 1934, II, 2993. 11. P. Karrer and W. Schlientz, Helv. chim. Acta 17 (1934) 55- 12. R. KuHN and C. Grundmann, Ber. 66 (1933) 1746. 13. P. Karrer and E. Jucker, Helv. chim. Acta 2g (1946) 229. 14. H. V. Euler, p. Karrer and E. Jucker, Helv. chim. Acta 30 (1947) 1159. 15. P. Karrer and E. Jucker, Helv. chim. Acta 29 (1946) 229. REFERENCES 215 16. H. V. EuLER, P. Karrer, E. Jucker, Helv. chim. Acta 30 (1947) ii59- 17. L. Zechmeister and R. M. Lemmon, /. Am. Chem. Soc. 66 (1944) 3i7- 18. P. Karrer, H. Salomon and H. Wehrli, Helv. chim. Acta 12 (1929) 790. — P. Karrer H. Wehrli and H. Helfenstein, Helv. chim. Acta 13 (1930) 268. 19. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 619; 15 (1932) 492; Arch. Sci. biol. 18 (1936) 36. 20. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 791; 13 (1930) 268. 21. R. KuHN and co-workers, Helv. chim. Acta 12 (1929) 499; Naturwissenschaften 18 (1930) 418; Ber. 63 (1930) 1489- 22. L. Zechmeister and co-workers, Z. physiol. Chem. igo (1930) 67; ig6 (1931) i99- 23. L. Zechmeister and L. v. Cholnoky, Ann. 481 (1930) 42. 24. P. Karrer and H. Wehrli, Helv. chim. Acta 13 (1930) 1104. 25. R. KuHN and A. Winterstein and W. Kaufmann, Ber. 63 (1930) 1489. 26. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 477. 27. P. Karrer and E. Jucker, Helv. chim. Acta 30 (1947) 266. 28. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 268; Helv. chim. Acta 14 (1931) 614; 15 (1932) 492. 29. P. Karrer, Arch. Scienze Biol. 18 (1933) 30. 30. R. KuHN and A. Winterstein, Ber. 65 (1932) 1873; Ber. 66 (1933) 429. 31. P. Karrer and co-workers, Helv. chim,. Acta 13 (1930) 1095. 32. R. KuHN and co-workers, Ber. 63 (1930) 1496. 33. P. Karrer and co-workers, Helv. chim. Acta 21 (1938) 448. 34. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 479. 35. P. Karrer and U. Solmssen, Helv. chim. Acta 21 (1938) 448. 36. R. KuHN and A. Smakula, Z. physiol. Chem. igy (1931) 161. 37. K. W. Hausser and A. Smakula, Z. angew. Chem. 47 (1934) 663; 48 (1935) 152. 38. K. W. Hausser, Z. tech. Phys. 15 (1934) i3- 39. L. Zechmeister and co-workers, Ber. 72 (1939) 1678. 40. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 43. 41. H. V. EuLER, P. Karrer and A. Zubrys, Helv. chim. Acta ly (1934) 24. 42. H. V. EuLER, P. Karrer, E. Klussmann and R. More, Helv. chim. Acta 15 (1930) 502. 43. R. Kuhn, A. Winterstein and W. Kaufmann, Ber. 63 (1930) 1496. 44. P. Karrer and co-workers, Helv. chim,. Acta 15 (1932) 490. 45. P. Karrer and T. Takahashi, Helv. chim. Acta 16 (1933) 1163. 46. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 479. 47. P. Karrer and A. Notthafft, Helv. chim. Acta 13 (1932) 1195. 48. R. Kuhn ,A. Winterstein and L. Kaufmann, Ber. 63 (1930) X497. 49. P. Karrer and W. Schlientz, Helc. chim. Acta ly (1934) 55- 50. R. Kuhn and W. Wiegand, Helv. chim. Acta 12 (1929) 499. 51. L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. i8g (1930) 159; Ann. 481 (1930) 42. 52. R. Kuhn and co-workers, Naturwissenschaften 18 (1930) 418; Ber. 63 (1930) 1489. 53. R. Kuhn, A. Winterstein and W. Kaufmann, Ber. 63 (1930) 1489. 54. R. Kuhn and W. Wiegand, Helv. chim. Acta 12 (1929) 499. 55. L. Zechmeister and L. v. Cholnoky, Ann. 481 (1930) 42. 56. R. Kuhn and H. Brockmann, Ber. 6y (1934) 59^. 57. K. W. Hausser and A. Smakula, Z. Angew. Chem. 4y (1934) 663; 48 (1935) 152. 58. R. Kuhn and K. Meyer, Z. physiol. Chem. 185 (1929) 193. — L. Zechmeister, Carotinoide, Julius Springer, Berlin 1934. 59. R. Kuhn and W. Wiegand, Helv. chim. Acta 12 (1929) 499. 60. P. Karrer, U. Solmssen and O. Walker, Helv. chim. Acta ly (1934) 417. 61. P. Karrer and W. Gugelmann, Helv. chim. Acta 20 (1937) 405- 62. L. Zechmeister and L. v. Cholnoky, Z. physiol. Chem. i8g (1930) 159; Ann. 481 (1930) 42. 63. R. Kuhn and co-workers, Ber. 63 (1930) 1489. 2i6 CAROTENOIDS CONTAINING HYDROXYL GROUPS XI 64. R. KuHN and H. Brockmann, Z. physiol. Chem. 206 (1932) 61. 65. L. Zechmeister and co-workers, Biochem. J. 32 (1938) 1305; Ber. y2 (1939) 1340, 1678; /. Am. Chem. Soc. 66 (1944) 317. 66. L. Zechmeister and R. M. Lemmon, /. Am. Chem. Soc. 66 (1944) 317. 67. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 68. P. Karrer and J. Rutschmann, Helv. chim.. Acta 2j (1944) 1684. 69. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 70. P. Karrer and J. Rutschmann, Helv. chim. Acta 2y (1944) 1684. — P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 71. P. Karrer and A. Oswald, Helv. chim. Acta 18 (1935) 1303. 72. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 72a. G. Tappi and P. Karrer, Helv. Chim. Acta 32 (1949) 50. 73. R. KuHN and A. Winterstein, Ber. 64 (1931) 326. 74. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 1044; 16 (1933) 977; 19 (1936) 1024; 27 (1944) 1684. 75. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 76. R. KuHN and A. Winterstein, Ber. 64 (1931) 326. 77. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 78. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 79. R. KuHN and A. Winterstein, Ber. 64 (1931) 326. 80. P. Karrer and R. More, Helv. chim. Acta 14 (1931) 1045. 81. P. Karrer and co-workers, Helv. chim. Acta ig (1936) 1024; Helv. chim. Acta2y (1944) 1684. 82. P. Karrer and J. Rutschmann, Helv. chim. Acta 2y (1944) 1684. 83. K. W. Hausser and A. Smakula, Z. angew. Chem. 4y (1934) 663; 48 (1935) 152; K. W. Hausser, Z. techn. Phys. 15 (1934) i3- 84. P. Karrer and J. Rutschmann, Helv. chim. Acta 2y (1944) 1684. — P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 85. P. Karrer and U. Solmssen, Helv. chim. Acta ig (1936) 1024. — R. Kuhn and A. Winterstein, Ber. 64 (1931) 332. 86. P. Karrer and J. Rutschmann, Helv. chim. Acta 2y (1944) 1684. 87. P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) 1624. 88. P. Karrer and J. Rutschmann, Helv. chim. Acta 2$ (1942) 1624. 89. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 90. P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) 1624; 27 (1944) S^o- 91. P. Karrer and J. Rutschmann, Helv. chim. Acta 2y (1944) 1684. 92. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 93. P. Karrer, E. Jucker and J. Rutschmann, Helv. chim. Acta 28 (1945) 1156. 94. J. J. Berzelius, Ann. 21 (1837) 261. 95. R. Willstatter and W. Mieg, Ann. 355 (1907) i. 96. R. Willstatter and H. H. Escher, Z. physiol. Chem. y6 (1912) 214. 97. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 268, 1094; 14 (1931) 614, 843; ■r<5 (1933) 977- 98. R. Willstatter and A. Stoll, Untersuchungen iiber Chlorophyll, Julius Springer, Berlin 1913; P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 614; R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 41; R. Kuhn and A. Winterstein, Naturwissenschaften 18 (1930) 754. 99. H. Junge, Z. physiol. Chem. 268 (1941) 179. 100. R. Kuhn, A. Winterstein and E. Lederer, Z. physiol. Chem. igy (1931) 153. — P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 625. loi. R. Kuhn, A. Winterstein and E. Lederer, Z. physiol. Chem. igy (1931) 153. 102. E. S. Miller, Chem. Centr. ig35, I, 3545. 103. L. Zechmeister and P. Tuzson, Ber. 63 (1930) 3203; Ber. 6y (1934) I7°- 104. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 268, 1084; 14 (1931) 614, 843; 16 (1933) 977. REFERENCES 217 105. R. WiLLSTATTER and W. MiEG, Ann. 355 (1907) i. 106. L. Zechmeister and P. Tuzson, Ber. 61 (1928) 2003. 107. R. PuMMERER and L. Rebmann, Ber. 61 (1928) 1099. 108. P. Karrer, a. Helfenstein and H. Wehrli, Helv. chim. Acta 13 (1930) 87. 109. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 709, 1099, 1102. no. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 1103. 111. P. Karrer, A. Zubrys and R. More, Helv. chim. Acta 16 (1933) 977. 112. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 631. 113. P. Karrer and co-workers, Helv. chim. Acta 13 (1930) 270, 1094. 114. R. NiLssoN, and P. Karrer, Helv. chim. Acta 14 (1931) 843. 115. P. Karrer, H. Koenig and U. Solmssen, Helv. chim. Acta 21 (1938) 445. 116. L. Zechmeister and P. Tuzson, Ber. 6g (1936) 1878. 117. For quantitative extinctions measurements see R. Kuhn and A. Smakula, Z. physiol. Chem. igj (1931) 163; K. W. Hausser and A. Smakula, Z. angew. Chem. 4j (1934) 663; 48 (1935) 152; K. W. Hausser, Z. Techn. Phys. 15 (1934) i3- For X-ray diffration pattern see G. Mackinney, /. Am. Chem. Soc. 56 (1934) 4^8. 118. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 43. 119. H. V. EuLER, P. Karrer and A. Zubrys, Helv. chim. Acta ly (1934) 24. 120. L. Zechmeister and P. Tuzson, Ber. 61 (1928) 2003; Ber. 62 (1929) 2226. 121. P. Karrer and S. Ishikawa, Helv. chim. Acta 13 (1930) 709, 1099. 122. R. WiLLSTATTER and H. H. Escher, Z. physiol. Chem. 64 (1910) 47. — H. Escher, Dissertation, 1909 Zurich. 123. L. Zechmeister and P. Tuzson, Ber. 62 (1929) 2226. 124. R. PuMMERER, L. Rebmann and W. Reindel, Ber. 62 (1929) 1411. 125. R. WiLLSTATTER and W. MiEG, Ann. 355 (1907) i. 126. P. Karrer and O. Walker, Helv. chim. Acta ly (1934) 43. 127. P. Karrer and S. Ishikawa, Helv. chim. Acta 13 (1930) 709, 1099. 128. R. Kuhn and A. Winterstein, Naturwissenschaften 18 (1930) 754. — R. Kuhn, E. Lederer, a. Winterstein, Z. physiol. Chem. igy (1931) 147, 150. 129. P. Karrer and S. Ishikawa, Helv. chim. Acta 13 (1930) 1099. 130. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 43. 131. P. Karrer and S. Ishikawa, Helv. chim. Acta 13 (1930) 713. 132. P. Karrer and B. Jirgensons, Helv. chim. Acta 13 (1930) 1103. 133. P. Karrer, H. Koenig and U. Solmssen, Helv. chim. Acta 21 (1938) 445. 134. L. Zechmeister and P. Tuzson, Ber. 6g (1936) 1878. — ■ P. Karrer and co-workers, Helv. chim. Acta 20 (1937) 682. 1020. 135. .P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 135a. P. Karrer, E. Krause-Voith and K. Steinlin, Helv. chim. Acta 31 (1948) 113. 136. D. Hey, Biochem. J. 31 (1937) 532- 137. P. Karrer and J. Rutschmann, Helv. chim. Acta 28 (1945) 1526. 138. R. Kuhn and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. 139. P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) 1144. 140. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 141. P. Karrer, E. Jucker and J. Rutschmann, Helv. chim. Acta 28 (1945) 1156. 142. P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) 1144. 143. P. Karrer and E. Jucker, Helv. chim. Acta 26 (1943) 626. 144. P. Karrer and E. Jucker, Helv. chim. Acta 27 (1944) 1585. 145. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. 146. P. Karrer and E. Jucker, Helv. chim. Acta 26 (1943) 626. 147. P. Karrer and E. Jucker, Helv. chim. Acta 27 (1944) 1585. 148. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 300. T49. P. Karrer and E. Jucker, Helv. chim. Acta 27 (1944) 1585. 150. P. Karrer, E. Jucker and J. Rutschmann, Helv. chim. Acta 28 (1945) 1156. 151. L. Zechmeister and L. v. Cholnoky, Ber. 69 (1936) 422. CHAPTER XII Carotenoids of known or largely known structure containing one or more carbonyl groups I. /3-CITRAURIN C30H40O2 History and Occurrence In the course of investigations of carotenoids from orange peel {Citrus aurantium), Zechmeister and Tuzson^ discovered a previously unknown pigment for which they proposed the term citraurin*. Citraurin occurs in oranges together with carotene, cryptoxanthin, zeaxanthin, xanthophyll and violaxanthin. It has not, so far, been isolated from any other source. Preparation^ Orange peels are dehydrated with ethanol and extracted with peroxide-free ether. After removal of the solvent in a partial vacuum, a red oily residue remains. In order to separate the pigments from the oily constituents, this residue is dissolved in petrol and chromatographed on calcium carbonate. The oily components, together with carotene and cryptoxanthin, are removed by prolonged washing. The violet-red zone which contains ^-citraurin is eluted with a mixture of ether and methanol, and the pigment esters are saponified with methanolic potassium hydroxide. After saponification is complete, the free phytoxanthins are dissolved in ether, the solution is evaporated to dryness and the residue is dissolved in warm carbon disulphide. On cooling this solution, much pigment material crystallises (violaxanthin etc.) while ^-citraurin remains in solution. It is adsorbed on a calcium carbonate column which is washed with carbon disulphide. ^-Citraurin forms a red zone in the chromatogram and is eluted with a mixture of ether and methanol. After removal of the solvent, the residue is dissolved in a little hot methanol, from which |8-citraurin crystallises in round, reddish aggregates on addition of a little water. 35 mg of pigment were obtained from 100 kg of oranges. P. Karrer and co-workers, Helv. chim. Acta 20 (1937) 682, 1020 proposed the term )5-citraurin for the pigment discovered by L. ZecHxMeister and P. TuzsoN^ in order to avoid confusion with the isomeric a-citraurin (cf. p. 205). References p. 2^3—25$. I ^-CITRAURIN 219 Chemical Constitution Cxlq Cliq \V /\ I I II CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CHO I II HOCH C-CHs \ / /9-Citraurin The composition of /S-citraurin (C30H40O2) , and the ease of oximation which suggests the presence of an aldehyde group, led Zechmeister and Tuzson^ to suggest that this pigment represents a degradation product of a C40 caro- tenoid. Karrer and Solmssen^ established the very close relationship between /S-citraurin and j3-apo-2-carotenaP. They proposed the formula shown above, according to which ^-citraurin is a 3-hydroxy-j3-apo-2-carotenal. The correct- ness of this formula was later established by Karrer and co-workers* and by Zechmeister and von Cholnoky^. The first-named authors obtained ^-citraurin by the permanganate degradation of zeaxanthin (cf. p. 184), while Zechmeister and von Cholnoky obtained the aldehyde by the hydrolysis of capsanthin (cf. p. 248). j3-Citraurin can also be prepared, though only in small amounts accompanied by a large proportion of a-citraurin, by the permanganate degra- dation of xanthophyll^. Finally, Karrer and Koenig' also succeeded in ob- taining the aldehyde by permanganate oxidation of capsanthin. Properties Crystalline form: /3-Citraurin crystallises from a mixture of benzene and petrol in very thin, yellow to orange plates which appear almost colourless under the microscope. Melting -point: 147° (corr., Berl-block, short thermometer). Solubility: j3-Citraurin dissolves easily in acetone, ethanol, ether, benzene and carbon disulphide. The solubility in petrol is very small, even at the boiling point. Spectral properties: Solvent Absorption maxima Carbon disulphide 525 490 457 m^u (diffuse) Benzene 497 467 m^u Petrol 488 459 m/i (sharp) Hexane 487 458 m/i Ethanol diffuse Solutions of the pigment in carbon disulphide have a beautiful red colour. Ethanol solutions are red, hexane and petrol solutions straw yellow, and benzene solutions yellowish-brown. References p. 253-255. 220 CAROTENOIDS CONTAINING CARBONYL GROUPS XII Colour reactions: On treating an ethereal solution of the aldehyde with concentrated aqueous hydrochloric acid, the latter assumes a blue colouration. Partition test: /3-Citraurin is hypophasic on partition between methanol and petroleum ether. Chromatographic Properties: On chromatography from carbon disulphide, j3-citraurin is adsorbed somewhat more strongly than cryptoxanthin and is found above the latter, but below zeaxanthin, on the column. Detection and estimation: After saponification, the pigment is found in the hypophasic fraction and is separated from other phytoxanthins by means of chromatographic adsorption analysis. It forms a zone with a characteristic red (not violet) colour, which is easily distinguishable from that of any other natural carotenoid. j3-Citraurin can be identified by the determination of the absorption maxima and, if necessary, by the prepartion of the oxime. Derivatives ^-Citraurin oxime C30H41O2N: This compound is obtained on treating j8-citraurin with free hydroxylamine^. The oxime crystallises from methanol in thin rods grouped into star-like formations. M.p. 188° (corr.). Solvent Absorption maxima [all sharp) Carbon disulphide 505 473 m/t Benzene 487 456 m/j. Petrol 474 444 m^a Hexane 473 443 m/i Ethanol 476 444 m/z The oxime is insoluble in cold petrol. The solubility in ethanol and acetone is somewhat greater. ^-Citraurin semicarbazone C31H43O2N3*: The semicarbazone crystallises from benzene in microscopic reddish-brown leaflets, which melt over a range near 190°. The semicarbazone is easily soluble in ethanol and acetone, but sparingly soluble in petrol. Solvent Absorption maxima Carbon disulphide 517 483 m/^ (diffuse) Benzene 498 463 m^ (diffuse) Hexane 485 454 vsifi (sharp) Ethanol 486 454 m^ (sharp) References p. 253-255. RHODOXANTHIN 2. RHODOXANTHIN C40H50O2 History 1893 MONTEVERDE^ observcs a new pigment in the reddish-brown leaves of Potamogeton nutans. The same pigment is later found by Tswett^" in a number of conifers, and described under the name "Thujorhodin". 1913 Monteverde and Lubimenko^^ isolate rhodoxanthin in the crystalline state. It is investigated in 1925 by Prat^^ q^^^ [^^ jg26 and 1927 by LlPPMAA^^. 1933 Kuhn and Brockmann^^ isolate rhodoxanthin from yew trees and propose a constitutional formula for the pigment. 1935 Karrer and Solmssen^^ convert dihydrorhodoxanthin into zeaxanthin and thus confirm the constitution of rhodoxanthin. Occurrences^ Rhodoxanthin is fairly widely distributed in nature in small quantities. It occurs in larger quantities in Taxus haccata L. (japonica). Source Aloe-species Bulbina annua Buxus Chamaecyparis Cryptomeria japonica Don. Cypressus Naitnocki Encephalartos Hilde- brandtii Equisetum-species Gasteria Gnetum- species Haworthia Juniperus virginiana L. Potamogeton naians Reseda odorata Retinospora pluniosa Scirpus References p. 2^3—235. TABLE 47 OCCURRENCE OF RHODOXANTHIN References T. LIPPMAA, Ber. hot. Ges. 44 (1926) 643, H. Kylin, Z. physiol. Chem. 163 (1927) 229. — H. Molisch, Ber. hot. Ges. 20 (1902) 442. T. Lippmaa, Ber. hot. Ges. 44 (1926) 643. T. Lippmaa, Ber. hot. Ges. 44 (1926) 643. T. Lippmaa, Ber. hot. Ges. 44 (1926) 643. M. TswETT, Compt. rend. 152 (1911) 788. do. M. W. LuBiMENKO, Rev. gen. Bot. 25 (1914) 475; Compt. rend. 158 (1914) 510. T. Lippmaa, Ber. bot. Ges. 44 (1926) 643. — S. Prat, Biochem. Z. 152 (1924) 495. T. Lippmaa, Ber. bot. Ges. 44 (1926) 643. N. A. Monteverde and V. N. Lubimenko, Bull. Acad. Sci. Petrograd [6] 7 (1913) 1105. T. Lippmaa, Ber. bot. Ges. 44 (1926) 643. M. Tswett, Compt. rend. 152 (1911) 788. N. A. Monteverde, Acta Horti Petropol. 13 (1893) 201. T. Lippmaa, Ber. bot. Ges. 44 (1926) 643. M. TswETT, Compt. rend. 152 (1911) 788. T. Lippmaa, Ber. bot. Ges. 44 (1926) 643. 222 CAROTENOIDS CONTAINING CARBONYL GROUPS XII Source References Selaginella N. A. Monteverde and V. N. Lubimenko, Bull. Acad. Sci. Petrograd [6J 7 (1913) 1105. — T. Lippmaa, Ber. hot. Ges. 44 (1916) 643. Taxus baccata R. Kuhn and H. Brockmann, Ber. 66 (1933) 828. M. TswETT, Compt. rend. 152 (1911) 788. Thuja orientalis L. M. Tswett, Compt. rend. 152 (1911) 788. Preparation" The ripe japonica fruit are mashed to a fine pulp and extracted with methanol in portions of 10 kg. The first two extracts are usually colourless to light red, and contain hardly any pigment, while the following methanol extracts are deep red in colour. The residue is then extracted with petroleum ether (b.p. 70-80°). The com- bined methanol extracts are diluted with water, and the pigments extracted with petrol. This petrol solution is combined with the petrol extract. Considerable purification of the pigment is achieved by repeated partitioning between methanol and petroleum ether. The last petrol solution is concentrated in vacuum almost to dryness. On cooling, rhodoxanthin crystallises. It is boiled first with a little metha- nol and then with petroleum ether. By this procedure about 70 mg of crude pigment are obtained Irom 10 kg of japonica fruit. Rhodoxanthin is purified by recrystallisation from a mixtur^of one part of benzene and four parts of methanol, or by slowly evaporating an ethanol solution. Chemical Constitution CH, CH, CH, CH., C CHo CH3 CH3 CH3 C /\ I I I I /\ CHa C=CHCH=C-CH = CHCH = C-CH=CHCH=CH-C=CHCH=CH-C=CHCH = C CHj 0=C C-CHg HgC-C C=0 \ /" Rhodoxanthin X / CH CH The constitution of rhodoxanthin was elucidated by Kuhn and Brock- MANN^'. Elementary analysis gave the molecular formula C^qYIc^qO^. On catalytic hydrogenation the pigment first rapidly absorbs 12 mols of hydrogen ; a further 2 mols are taken up on prolonged hydrogenation. This behaviour indicates the presence of 12 double bonds and two carbonyl groups. The presence of the latter was confirmed by the preparation of a dioxime, but whereas polyene aldehydes (e.g. lycopenal) react readily with hydroxylamine, rhodoxanthin only reacts with difficulty. For this reason, Kuhn and Brockmann concluded that two ketone groups are present in the pigment molecule, conjugated with the system of conjugated double bonds. This conclusion is in accord with the long-wavelength location of the absorption bands. By means of chromic acid oxidation, Kuhn and Brockmann^" established the presence of six side-chain methyl groups. References p. 253—255, 2 RHODOXANTHIN 223 Zerewitinoff determinations indicated the presence of one hydroxyl group, but this result must be due to partial enolisation of the ketone, since no acetyl derivative could be obtained by the action of acetic anhydride in pyridine. (This latter result could be due to the presence of a tertiary hydroxyl group, but this would not be in accord with the general properties of the pigment) . The constitution of rhodoxanthin has been confirmed by investigations of Karrer and Solmssen^^ in the course of which the pigment was converted into zeaxanthin via its dihydro-derivative, and the relationship between the two pigments was thus established. These investigations have already been described on p. 182. Properties Crystalline form: Rhodoxanthin crystallises from a mixture of benzene and methanol (1:4) in dark violet needles, combined in rosettes. From aqueous p^Tidine or ethanol it is obtained in thin, finely branched rods. If an ethanolic solution of rhodoxanthin is allowed to evaporate slowly, the pigment crystallises in well-formed leaflets. Melting point: 219° (corr., evacuated capillary). Solubility: The pigment is very easily soluble in pyridine, easily soluble in benzene and chloroform, very sparingly soluble in ethanol and methanol, and insoluble in petrol, hexane, and petroleum ether. spectral properties: Solvent Absorption maxima Carbon disulphide 564 525 491 m/i Chloroform 546 510 482 m/i Benzene 542 503,5 474 m/< Ethanol 538 496 (very difiuse) Petrol 524 489 458 m/i Petroleum ether 521 487 456 m^ Hexane 524 489 458 m/* (cf. Fig. 13 and 15, p. 353 and 354) Solutions of the pigment in petrol are yellow-red, solutions in methanol wine-red. The same difference was observed in the case of capsanthin. Zech- MEiSTER and PoLGAR^^ ascribe this phenomenon to the polar nature of the alcohol. Colour reactions: Rhodoxanthin dissolves in concentrated sulphuric acid with a deep blue colour. On treating a solution of the pigment in chloroform with antimony trichloride, an intense blue-violet colouration is observed. On shaking an ethereal solution of rhodoxanthin with 25% hydrochloric acid, References p. 253-255. 224 CAROTENOIDS CONTAINING CARBONYL GROUPS XII the latter is coloured a faint red- violet. With more concentrated acid the colouration is somewhat more intense. Partition test: On partitioning between petroleum ether and 90 % methanol, rhodoxanthin colours both layers. Chromatographic behaviour: In contrast to phytoxanthins, rhodoxanthin is not adsorbed on calcium carbonate. It is well adsorbed on alumina from a mixture of benzene and petrol. It forms a deep violet zone in the chromatogram and can be eluted with a mixture of petrol and methanol. Detection and estimation. After separation from other carotenoids by means of chromatographic analysis on alumina, rhodoxanthin can be identified by its absorption spectrum. Chemical behaviour: Rhodoxanthin is relatively stable towards atmospheric oxygen. Derivatives Rhodoxanthin dioxvme C40H52O2N2 : The dioxime is prepared by boiling a solution of rhodoxanthin in a little pyridine with a solution of 6 mols of hydroxylamine containing some sodium hydroxide". The dioxime crystallises from a mixture of pyridine and petrol in red squares, m.p. 227-228° (corr., evacuated capillary). Rhodoxanthin dioxime is less easily soluble in petrol and benzene, but more soluble in ethanol than rhodoxanthin. In contrast to the latter, it can be adsorbed on calcium carbonate from petrol. On alumina it is adsorbed so strongly that it cannot be eluted. Solvent Absorption maxima Carbon disulphide 516 483 453 m^ Chloroform 527 490 457 vcifi Benzene 527 490 457 m/i Ethanol 516 483 454 mix Petrol 516 483 453 m/^ Hexane 513 479 451 m/^ Petroleum ether 510 477 450 m/^ Dihydrorhodoxanthin C4QH52O2: CH, CH, CH, CH, C CH3 CH3 CII3 £'1x3 G /\ II I I /\ CH„ C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHg I II II I 0=C C-CHg H3C-C C=0 \ / Dih3'drorhodoxanthin \^ ^Z CHa CHa References p. 2^3—253. 3 MYXOXANTHIN 225 This compound was prepared by Kuhn and BrockmannI' by the reduction of a solution of rhodoxanthin in pyridine and glacial acetic acid with zinc. Dihydrorhodoxanthin crystallises from a mixture of benzene and methanol in golden yellow leaflets, m.p. 219° (corr., evacuated capillary). Its solubility is similar to that of rhodoxanthin. The chromophoric system of dihydrorhodo- xanthin is the same as that of j3-carotene and zeaxanthin, as shown by the similarity of the absorption maxima. Solvent Absorption maxima Dihydrorhodoxanthin Zeaxanthin Carbon disulphide ... 514 479 448 m/< 517 482 459 m^ Chloroform 492 460 431 m^t 495 462 429 m// Petrol 483 452 425 m/< 483.5 451.5 423 m^ Ethanol 480 450 422 mn 483 451 423 m/x Hexane(cf. Fig. 13. p. 353) 480 449 422 m/^ Dihydrorhodoxanthin is optically inactive. In solution (e.g. in piperidine or pyridine containing a small proportion of alcoholic potassium hydroxide) it is rapidly dehydrogenated by atmospheric oxygen to rhodoxanthin. On catalytic hydrogenation in decalin, the pigment absorbs 13 mols of hydrogen. By the reduction of dihydrorhodoxanthin Karrer and Solmssen (cf. p. 60, 183) ob- tained zeaxanthin. Dihydrorhodoxanthin dioxime C40H54O2N2: This derivative is obtained by a procedure analogous to that described for the preparation of rhodoxanthin dioxime (p. 224). The dioxime crystallises from ethanol in reddish-yellow needles, m.p. 226-227° (corr., in vacuum). Solvent Absorption maxima Carbon disulphide .... 514 479 448 m^ Petrol 483 451.5 424 m/z Hexane 480 449 422 m^ 3. MYXOXANTHIN C40H54O History ayid Occurrence Heilbron, Lythgoe and Phipers^^ found a previously unknown epiphasic carotenoid, which they termed myxoxanthin in the algae Rivularia nitida. This pigment was later observed in Oscillatoria ruhescens^^ and in Calothrix scopu- Preparation From Oscillatoria rubescens^'^ : The algae are dehydrated with methanol and then extracted first with methanol and then with ether. The combined extracts are References p. 253-253. • Carotenoids 15 226 CAROTENOI DS CONTAINING CARBONYL GROUPS XII concentrated under reduced pressure in nitrogen, and the pigments are saponified with aqueous potassium hydroxide and then separated into epiphasic and hypo- phasic fractions. Myxoxanthin is obtained from the epiphase and myxoxanthophyll can be extracted from the hypophase (p. 228). The epiphasic petroleum ether extract is washed free from alkaU, dried, and allowed to stand for a short time during which a considerable quantity of j3-caro- tene separates. The mother liquors are evaporated to dryness in vacuum, the residue is chromatographed on alumina, and the pigment of the central zone of the chro- matogram is chromatographed again from alumina. The myxoxanthin is then dissolved in a mixture of ether and methanol, the solution is concentrated, and colourless impurities are separated out by cooling. On further concentration and cooling, the mother liquors yield myxoxanthin. For further purification, the pigment is repeatedly crystallised from a mixture of pyridine and methanol. Chemical Constitution CH, CH, CH3 CH3 C CH3 CH3 CH3 CH3 CH /\ II I I \ CH, C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH r II i 11 CH, C-CH3 H3C-C CH \ / M3'xoxanthin( ?) \y' CHs, CO The constitution of myxoxanthin has not yet been fully elucidated. However, the investigations of Heilbron and Lythgoe^^ and Karrer and Rutschmann23 make the above formula very probable. Elementary analysis gave the empirical formula C40H54O. On microhydro- genation, myxoxanthin absorbed 12 mols of hydrogen, while myxoxanthin oxime requires 13 mols of hydrogen for saturation. This shows that the free pigment contains 12 double bonds and one carbonyl group. The latter can be reduced by means of aluminium isopropoxide and isopropylalcohol to give a secondary alcohol, myxoxanthol. This agrees in its spectral properties with y-carotene and rubixanthin and must therefore contain the same, or a very similar, chromophoric system. Since myxoxanthin exhibits vitamin A activity, it must further contain an unsubstituted j8-ionone ring. For reasons of analogy (e.g. astacene), it is assumed that the carbonyl group is cross-conjugated with the system of conjugated double bonds. (Carotenoid ketones which contain a ketone group attached terminally to the unsaturated system generally exhibit an absorption spectrum with 3 bands, whereas astacene and myxoxanthin exhibit only one band) . Properties Myxoxanthin crystallises from a mixture of pyridine and methanol in deep violet prisms, m.p. 168-169°. The pigment is easily soluble in a mixture of chloroform and ether, but only sparingly soluble in chloroform alone. On References p. 253—255* 4 MYXOXANTHOPHYLL 227 partition between methanol and petroleum ether it is entirely epiphasic. Myxoxanthin is adsorbed on calcium hydroxide and magnesium hydroxide, but not on calcium carbonate, from petroleum ether solution. Spectral -properties: Solvent Absorption maxima Carbon disulphide 488 m^ Chloroform 473 m/x Ethanol 470 m^ Petroleum ether 465 m^ Colour reactions: On adding concentrated sulphuric acid to a chloroform solution of myxoxanthin, the latter is coloured deep blue. Concentrated hydro- chloric acid produces no colour change in chloroform. Addition of hydrochloric acid to an ethereal solution produces a greenish-blue colouration. Myxoxanthin oxime C40H55ON: Brilliant, cinnabar-red plates, m.p. 195-196°. Solvent Absorption maxima Chloroform 463 m// Myxoxanthol C40H56O : Deep red crystals, m.p. 169-172°. Solvent Absorption maxima Carbon disulphide 529 494 464 m/n Chloroform 508 474 441 mjj, Petroleum ether 495 465 431 m/n \V \V /\ I I I I \ CHg C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CH2 C-CHa HsC-C CH \ y Myxoxanthol ( ?) ' \ y" CHj ' CH I OH 4. MYXOXANTHOPHYLL C40H56O7 In the course of investigations on Oscillatoria rubescens, Heilbron and Lythgoe^^ discovered a new hypophasic pigment for which they proposed the name myxoxanthophyll. The pigment occurs together with myxoxanthin, xanthophyll and /3-carotene. In recent times myxoxanthophyll has been in- vestigated by Karrer and Rutschmann^^. References p. 25J-255. 228 CAROTENOIDS CONTAINING CARBONYL GROUPS XII For the isolation of the pigment the hypophasic portion of oscillatoria-extracts (cf. p. 225) are used. The pigment is dissolved in ether, the solution is washed free from alkali, and dried over sodium sulphate, and the solvent is distilled off in vacuum. The deep red, resinuous residue is dissolved in chloroform, and chromatographed on calcium carbonate. After elution and removal of the solvent the myxoxanthophyll is dissolved in pyridine. Colourless impurities are removed by freezing and the mother liquors are then diluted with petroleum ether, when the pigment crystallises on cooling. The structure of myxoxanthophyll has been investigated mainly by Karrer and RuTSCHMANN^*. The molecular formula of the pigment is C^f^H^^O^^^- ^^. On microhydrogenation, lo mols of hydrogen are absorbed rapidly and an additional mol of hydrogen more slowly^*, indicating the presence of lo double bonds and one carbonyl group. Of the remaining six oxygen atoms, four are present as secondary hydroxyl groups, since myxoxanthophyll forms a tetraacetate. The latter still contains two free hydroxyl groups, as shown by Zerewitinoff determinations, but these cannot be esterified and are therefore probably tertiary in character^*. It should be remarked that the presence of a carbonyl group has not been definitely established, although the behaviour of the pigment and the long-wavelength absorption renders it very probable. Karrer and Rutschmann^^ tentatively proposed the following formula for myxoxanthophyll, which is in agreement with the properties of the pigment: CH, CH, CH3 CH3 OH C OH CH, CH, CH, CH3 CH OH \/\/ I I I I \/ CH C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH CH C-CHa HaC-C CH X\/'\ Myxoxanthophyll( ?) \/\ OH CHa OH CO OH It should be emphasised, however, that this constitution is by no means certain. Myxoxanthophyll crystallises from acetone in violet needles, m.p. 182° (uncorr., in vacuum)*. According to Heilbron and Lythgoe, the pigment is laevo-rotatory, [aj^d = -255°' (in ethanol). The pigment is readily soluble in pyridine and ethanol, more sparingly soluble in chloroform and acetone, and insoluble in petroleum ether, ether and benzene. Concentrated sulphuric acid produces a deep blue colouration in a chloroform solution of the pigment. No colouration is produced by concentrated hydrochloric acid. Solvent Absorption maxima Pyridine 526 489 458 m/i Chloroform 518 484 454 m/z Ethanol 503 471 445 m/^ * I. M. Heilbron and B. Lythgoe, /. Chem. Soc. 1936, 1376 record m.p. 169-170° C; but the determination was not carried out in vacuum and the sample was probably less pure. References p. 253-255. 5 ASTACENE AND ASTAXANTHIN 229 Myxoxanthophyll tetraacetate C^^^^iOi-i^: The ester is prepared by treating myxoxanthophyll with acetic anhydride in pyridine. It crystallises from methanol in lustrous violet leaflets, m.p. 131- 132°. Myxoxanthophyll tetraacetate is entirely hypophasic on partition between methanol and petroleum ether. Solvent Absorption maxima Carbon disulphide 544 508 479 mfx Karrer and Rutschmann^* also prepared myxoxanthophyll benzoate, but the compound was not obtained pure because of shortage of material. 5. ASTACENE C40H48O4 AND ASTAXANTHIN C40H52O4 Introduction The pigments of Crustacea have long aroused the interest of chemists and zoologists^^. It is only in recent years, however, that it has been possible to obtain some indication of the nature of these pigments (cf. p. 234), and that the investigations regarding the chemical constitution of these compounds has reached a certain degree of finality. The first pigment of this type was isolated in 1933 in the form of astacene from lobster shelP^. Astacene was later recognised as a j3-carotene tetraketone by Karrer and co-workers^^. Some years later, KuHN and Sorensen^^ found that the pigment from lobster eggs known as "ovoester" was not an ester of astacene but an entirely new pigment^^. It was termed astaxanthin, and it was shown that it is readily converted into astacene in alkaline solution under the influence of atmospheric oxygen. It therefore appeared possible that astacene is an artefact formed by the alkaline saponifi- cation of astaxanthin esters and that astaxanthin is the natural pigment^^. This supposition has been proved in a number of cases while in others the necessary experiments have not yet been carried out. It has been found that astaxanthin occurs fairly freciuently in the animal organism^^ and also in plants^'"*. The close relationship of the two carotenoids and the facile conversion of astaxanthin into astacene under the influence of atmospheric oxygen in alkaline solution are established without doubt. The two pigments will therefore be dealt with together in this chapter. History 1933 Kuhn and Lederer^^ isolate crystalline astacene from lobster shell. In the animal, the pigment is partly combined mth protein and partly esterified. References p. 253—255. 230 CAROTENOIDS CONTAINING CARBONYL GROUPS XII 1934-36 Karrer and co-workers^' carry out a detailed investigation of the new pigment and establish its constitution. 1938 KuHN and Sorensen^^ show that the eggs of the lobster do not contain esterified astacene ("ovoester"), but a new pigment, astaxanthin. They establish the constitution of astaxanthin and its close relationship to astacene. Occurrence It was originally assumed that astacene and astaxanthin are typical animal pigments. More recent investigations show, however, that these pigments are also present in plant organisms^^. Since the isolation of astacene from lobster (shell, hypodermis and eggs^^), numerous other sources of this carotenoid and of astaxanthin have been found. The pigment occurs either esterified or combined with protein. The protein adduct appears to be ionic in nature, with the protein as the positively charged component (cf. p. 235). TABLE 48 OCCURRENCE OF ASTAXANTHIN OR ASTACENE* ^^ Source References I. Green algae Haematococciis plnvialis+ R. Kuhn, J. Stene and N. A. Sorensen, Ber. y2 (1939) 1688. — J. TiscHER, Z. physiol. Chcm. 250 (1937) 147. II. Protozoa: Euglena heliorubescens'^ J. Tischer, Z. physiol. Chem. 23g (1936) 257; 267 (1941) 281. III. Spongiaria: Axinella crista-galli P. Karrer and U. Soimssen, Helv. chim. Acta 18 (1935) 915. IV. Crustacea : a) Malacostraca: 1) Schizopoda Euphausia J. C. Drummond and R. McWalter, /. exper. Biol. 12 (1934) 105. 2) Decapoda:' Astacus gamniarus'^ , Shell, hypodermis, eggs R. Kuhn and N. A. Sorensen, Ber. yi (1938) 1879. Cancer pagurus ■ R. Fabre and E. Lederer, Bttll. Sac. Ch-ini. Biol. 16 (1934) 105. Sources from which astaxanthin was isolated are marked"*". In other cases it is not known for certain whether astacene is the natural pigment or a transformation product of astaxanthin. References p. 253—255. ASTACENE AND ASTAXANTHIN 231 Source Eupagurns Prideauxii Leander serratus Maja sqninado, eggs Nephrops-species Palinurus vulgaris Portunus puber Potamobius astacus b) Copepoda: Calanus finniarchi anils'^ Heterocope saliens c) Phyllopoda: Holopedium gibberum d) Arthrostaca: Gammarus pulex'^ References E. Lederer, Bttll. Sac. Chim. Biol. 20 (1938) 554, 567, 611. R. Fabre and E. Lederer, Bull. Soc. Chim. Biol. 16 (1934) 105. R. KuHN, E. Lederer and A. Deutsch, Z. physiol. Chem. 220 (1933) 229. R. Fabre and E. Lederer, Bull. Soc. Chim. Biol. 16 (1934) 105. R. Fabre and E. Lederer, Bull. Soc. Chitn. Biol. 16 (1934) 105. R. Fabre and E. Lederer, Bull. Soc. Chim,. Biol. 16 (1934) 105. e.g. H. Willstaedt, Svensk Kem. Tidskr. 46 (1934) 205, 261. H. V. EuLER, H. Hellstr5m and E. Klussmann, Z. physiol. Chem. 228 (1934) 77. — E. Lederer, Bull. Soc. Chim. Biol. 20 (1938) 554, 567, 611. N. A. SoRENSEN, Kgl. Norske, Vid. Selsk. Skr. ig36 No. 1. do. do. V. MoUusca (Lamellibran- chiata): Lima txcavata N. A. SoRENSEN, Kgl. Norske Vid. Selsk. Skr. 1936 No. 1. VI. Echinoderma: Ophidiaster ophidianus Echinaster sepositus VII. Tunicata ( Ascidiacea). Dendrodoa grossularia'^ Halocynthia papulosa'^ P. Karrer and F. Benz, Helv. chim. Acta ly (1934) 412. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 915; E. Lederer, Bull. Soc. Chim. Biol. 20 (1938) 554, 567, 611. do. VIII. Fish: Beryx decadactylus , skin E. Lederer, Compt. rend. Soc. Biol. 118 (1935) 542. Carassius auratus, skin E. Lederer, Compt. rend. Soc. Biol. 118 (1935) 542. 2 32 CAROTENOIDS CONTAINING CARBONYL GROUPS XII Source Cyclopterus lurnpus, liver Lophius piscatorius, liver Perca fluviatilis , fins Regalecus glesne+ liver Salmo salar, muscle Salmo trutta'^, muscle Sebastes fnarinus, skin References N. A. SoRENSEN, Z. physiol. Chem. 235 (1935) 8. N. A. SoRENSEN, Tidskr. Kjemi Bergves 1935, 12. E. Lederer, Bull. Soc. Biol. 20 (1938) 554, 567, 611. N. A. SoRENSEN, Tidskr. Kjemi Bergves 1935, 12. — R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. N. A. Sorensen, Z. physiol. Chem. 235 (1935) 8. N. A. Sorensen and J. Stene, Kgl. Norske Vid. Selsk. Skr. 1938, No. 9. E. Lederer, Bull. Soc. Biol. 20 (1938) 554, 567, 611. IX. Reptiles: Clemmys insculpata, retina G. Wald and H. Zussman, /. biol. Chem. 122 (1938) 449. X. Birds: Chicken, retina+ Phasianus colchicus, 'Rosen'+ R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. — . G. Wald and H. Zussman, /. biol. Chem. 122 (1938) 449. R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. — H. Brockmann and O. Volker, Z. physiol. Chem. 224 (1934) 193. XI. Mammals: Balaenoptera niusculus, Fat S. Schmidt-Nielsen, N. A. Sorensen and B. Trumpy, Kgl. Norske Vid. Selsk. Skr. 5 (1932) 118. — G. N. Burkhardt, I. M. Heilbron, H. Jackson, E. G. Parry and I. A. Lovern, Biochem.J. 28 (1934) 1698. The occurrence of astaxanthin in the green algae Haematococcus pluvialis^^ is of especial interest as it disproves the view that astaxanthin is a purely animal carotenoid. As early as 1936, Tischer discovered a pigment in the flagellate Euglena heliorubescens^^ , which he termed euglenarhodon. Later he also found this piigment in the green algae Haemato coccus pluvialis^^. Subsequent investigations by KuHN, Stene and Sorensen^^ showed that euglenarhodon from Haemato- coccus pluvialis is identical with astacene. A reinvestigation of the pigment from Euglena heliorubescens by Tischer^^ led to the same result. KuHN and his collaborators have recently isolated astaxanthin, as well as j3-carotene, from the eggs of rainbow trout, Salmo irideus, and have shown that astaxanthin is the chemotactic principle responsible for attracting the trout sperms. References p. 253-255, ASTACENE AND ASTAXANTHIN 233 TABLE 49 MODE OF OCCURRENCE OF ASTAXANTHIN IN NATURE* Mode of Occurrence Epiphasic ester Free pigment phromoproteid blue-black green blue olive-brown or blue violet red olive-brown Source Astacus gammarus, hypodermis * Beryx decadactylus, skin * Carassius auratits, skin Chicken retina Euglena heliorubescens Haematococciis pluvialis * Perca fltiviatilis, fins Phasianus colchiciis 'Rosen' * Sebastes marinus, skin * Balaenoptera musculus, fat Calanus finmarchicus (partly esterified) Maja squinado, eggs Phasianus colchicus Regalecus glesne, liver Salmo irideus, eggs Salmo trutta Astacus gammarus, shell Astacus gammarus , eggs (ovoverdin) * Heferocope saliens Gammarus pulex Dendrodoa grossularia ■ * Lophius piscatorms * In the cases marked *, only astacene was isolated. It is not yet known for certain whether astacene is the natural pigment or whether it is formed from astaxanthin during the process of isolation. Preparation of Astacene front Lobster Shells^^ The shells of freshly killed animals are covered with 2N hydrochloric acid and left to stand until they have turned red. They are then washed with water and the hypodermis aie separated. The pigment is then extracted with acetone at room temperature, and transferred into petroleum ether by dilution of the extract with water. The petroleum ether solution is wa.shed with water and with 90% methanol, diluted with 2N sodium hydroxide and sufficient ethanol to produce a homogeneous solution, and left to stand in the dark at room temperature for 5 hours. After this period, sufi&cient wa^er is added to produce two layers. The ethanolic layer is separated, covered with a little petrol, and the astacene is precipitated by careful acidification %\dth acetic acid. The pigment is washed with hot water, dissolved in a small amount of highly purified pyridine and crystallised by addition of a little water. From 29 animals (12.8 kg Uac weight), the yield of thrice recny^stallised astacene was (i.265 g. Preparation of Astaxanthin from Lobster Eggs^'' 2.5 Kg of lobster eggs are crushed in a porcelain mortar, with the addition of acetone and solid carbon dioxide. The crushed mass is filtered and repeatedly References p. 2^3-255. 234 CAROTENOIDS CONTAINING CARBONYL GROUPS XII extracted on the filter with strongly cooled acetone. The deep red extract is covered with a fifth of its volume of petroleum ether, and diluted carefully with one volume of distilled water, when most of the pigment separates in beautifully glistening plates. After filtering, the petroleum ether solution is extracted with 90 % methanol, the methanol solution is covered with freshly distilled benzene, and a further quantity of astaxanthin is precipitated by careful addition of water. The two fractions are crystallised together from a mixture of pyridine and water^^. 750 mg of pure pigment are thus obtained. Two other methods of preparation of the two pigments are described by Karrer and co-workers^^. Chemical Constitution of Astacene C40H48O4: CH, CH3 CH, CH, C CH3 CH, CH, CH, C /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH, I i II I OC C-CHj HgC-C CO \^ / Astacene (Ketoform) \ / CO CO CH3 CH3 CH, CH,* \V \V C CH, CH, CH, CH, C /\ \ I I \ /\ CH C-CH=CH'C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH HOC C-CHg ■ HjC-C COH \ / Astacene (Enolform) \^ / CO CO The formula for astacene (3 13' 14 14' tetraketo-j3-carotene) was proposed by Karrer^^. Elementary analysis of the compound itself and of the dioxime gave the molecular formula C4QH48O4 which differs from that of j3-carotene only by the presence of 8 fewer hydrogen atoms and 4 additional oxygen atoms. From the molecular formula and the nature of the oxygen atoms, Karrer and co- workers concluded that the constitution of the pigment must be that of a tetraketo-j3-carotene. Astacene forms a dioxime which contains 4 active hydrogen atoms. Two of these are derived from the oxime residues, while the other two must be due to the enolisation of the other two carbonyl groups. The four keto groups therefore differ in nature, two being capable of forming an oxime, and the other two undergoing enolisation. Astacene gives a bis-phenazine derivative with o-phenylene diamine, which shows that each pair of carbonyl groups must be adjacent. By the oxidation of astacene with permanganate, Karrer and co-workers obtained dimethylmalonic acid. On oxidation of the bis-phenazine derivative, a:a-dimethylsuccinic acid was also isolated, thus establishing the presence of the 4 carbonyl groups in the 3:3':4:4'-positions. This is in accord with the finding of Willstaedt that astacene can be reduced References p. 253—253. 5 ASTACENE AND ASTAXANTHIN 235 by zinc dust and acetic acid in pyridine solution to give a light yellow deriv- ative*". Microhydrogenation of astacene showed the presence of 13 double bonds, two of which are formed by enolisation. Free astacene is only slightly enoHsed as shown by the slow etherification with diazomethane and by Zerewitinoff determinations. The weakly acidic nature of the pigment disappears on hydro- genation, as would be expected. Karrer, Loewe and Hubner*^ investigated the epiphasic astacene ester from lobster and described it as astacene dipalmitate. CH3 CH3 CH3 CH3 C CH, CH3 CH3 CH3 C /\ I I I I /\ CH C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH II II II II CH3(CH2)i4C00C C-CH3 HjC-C C00C-(CH2)i4-CH3 \ / Astacene dipalmitate \ / CO CO It is probable that the compound is actually the dipalmitic acid ester of astaxanthin. Chetnical Constitution of Astaxanthin C40H52O4: CH, CH, CH3 CH3 C CH3 CH3 CH3 CH3 C /X I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CHj HOCH C-CH3 HjC-C CHOH \ / Astaxanthin \ y^ CO CO The isolation of a crystalline "ovoester" from the eggs of lobster was described by Kuhn and Lederer^^. It was later shown by Kuhn and Soren- SEN that the "ovoester" is not an ester but a free phytoxanthin. It was named astaxanthin and has the molecular formula €40115204*2. By analogy with the formula of astacene established by Karrer and co-workers, Kuhn and Soren- SEN*2 assigned the structure of a 3:3'-dihydroxy-4:4'-diketo-|S-carotene to astaxanthin*. This formulation is based on the fact that, in the absence of air, astaxanthin forms a deep blue salt in potassium hydroxide solution, while under aerobic conditions the pigment absorbs exactly 2 mols of oxygen in alkaline solution and is converted into astacene. The conversion of astaxanthin into astacene represents the autoxidation of a di-a-ketol. In agreement with the The alternative structure, 4:4'-dihydroxy-3:3'-diketo-/3-carotene, is excluded by the spectral properties of astaxanthin (cf. p. 237). References p. 253-235. 236 CAROTENOIDS CONTAINING CARBONYL GROUPS XII formulation of the pigment, di-esters can be prepared (cf. p. 235). According to KuHN and Sorensen, the deep blue potassium salt of astaxanthin may be compared with the orange coloured potassium derivative of benzoin*, and the corresponding salts of dihydrocrocetin dimethyl ester and dihydrobixin- dimethyl ester*^^ ^nd may be formulated as follows: : CH3 CH3 CH3 CH3 C CH3 CH3 CH3 CH3 C CHa C-CH=CH-C = CHCH=CH-C=CHCH=CHCH=C-CH=CHCH = C-CH=CH-C CH, KOC C-CH, HjC-C COK V \^ c c j Potassium Salt of Astaxanthin (deep blue) I OK (^K Constitution of Ovoverdin^^ Ovoverdin is the name given by Kuhn and Leuerer^i to the blue-green chromoproteid which is the natural pigment of the lobster shell. According to Kuhn and Sorensen*^, ovoverdin is an enol salt somewhat analogous to the potassium derivatives of benzoin and astaxanthin. This formulation would explain the blue-green colour of the compound. CH3 CH3 CH3 CH3 \/ \V C CH3 CH3 CH3 CH3 c CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ C C-CH3 BX-G C -0 c c 0- \ ' I / 0- Ovoverdin (blue-green) 0- / Protein++ Prot"ein++ It is surprising, however, that ovoverdin is not autoxidisable, in contrast to the potassium derivative of astaxanthin. This fact is explained by Kuhn and Sorensen by assuming that the interaction with the protein component is not simply ionic in nature, but that additional binding forces are involved which result in a relatively stable combination of the pigment and the protein in the form of a molecular complex. The nature of the additional binding forces is not specified but a complex derived from one protein molecule with two separate links, rather than from two independent protein molecules, appears to be implied. * See note page 235. References p. 253-255. 5 ASTACENE AND ASTAXANTHIN 237 A number of investigations have been made regarding the nature of the protein component. Thus, Wyckoff^* determined a molecular weight of about 300.000 for ovoverdin. Kuhn and Sorensen*^ worked out a procedure for the purification of ovoverdin which involves the fractional adsorption of the chromoproteid on aluminium hydroxide and the fractional elution with disodium phosphate or ammonium sulphate. The molecular weight of ovoverdin purified in this manner was 144,000. According to Stern and Salomon*® the protein component of ovoverdin has albumin character. Properties and Derivatives of Astacene Crystalline form: The pigment crystallises from a mixture of pyridine and water in violet needles with a metallic lustre. Sometimes the needles are sickle- shaped. Melting point: 240-243° (corr., in vacuum, slow heating)^!; 241°*', 228°^. Solubility: Astacene is insoluble in water, very sparingly soluble in ether, petroleum ether and methanol, sparingly soluble in benzene, ethyl acetate and acetic acid, fairly soluble in carbon disulphide, and easily soluble in chloroform, pyridine and dioxan. Optical activity: Astacene is optically inactive. Partition test: On partition between petroleum ether and 90% methanol, astacene is found almost entirely in the lower layer. On addition of a little more water, the pigment is easily transferred to the petroleum ether layer, but remains in the lower layer in the presence of alkali*^. Chromatographic behaviour: Astacene is hardly adsorbed on calcium carbonate from a mixture of benzene and petrol. It is found in the top zone of the chroma- togram on adsorption on alumina from the same solvent mixture. It cannot be eluted from alumina either with a mixture of benzene and methanol or a mixture of pyridine and methanol. Spectral properties (cf. Fig. 16, p. 354) : Solvent Absorption ynaxitna Pyridine about 500 m/i (wide band) Carbon disulphide about 510 m/<*' Colours of solutions: Concentrated solutions of the pigment in pyridine are blood-red, dilute solutions orange-red. Colour reactions: Astacene dissolves in concentrated sulphuric acid with a deep blue colour. Treatment of the solution of the pigment in chloroform with antimony trichloride results in a blue-green colouration. References p. 253—255. /^ 238 CAROTENOIDS CONTAINING CARBONYL GROUPS XII Detection and estimation: Astacene differs from other carotenoids by its single-banded spectrum and its behaviour towards alkah. Chemical behaviour: Astacene is very stable towards atmospheric oxygen. Its general chemical behaviour is characterised by the capacity of two of the carbonyl groups to enolise and of the other two carbonyl groups to undergo ketonic reactions. Astacene dioxime CjnH=,nOjN,^": CH, CH, ^40^ ^50^-^4"^^ 2 CHo CHo w ■ \V /\ I I I I /\ CH C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH HOC C-CH, \/ C II NOH Astacene dioxime (Enol formula) HgC-C COH \/ C i NOH The dioxime separates from ethanol in black crystals. Bis-phenazine derivative CjjHjgN/^: CHo CHo C CH, CH, /\ I I CH2 C-CH=CH-C=CHCH=CH-C=CHCH= N=C C-CH, / \/ -C6H4^^N=C The bis-phenazine derivative was obtained by Karrer and Loewe^** by warming a solution of astacene with o-phenylene diamine in glacial acetic acid for one and a half hours on a water bath. The compound separates from benzene in deep violet crystals. As it cannot enolise, it gives rise to a:a-dimethyl- succinic acid on oxidation with permanganate (cf. p. 47), m.p. 224-225°. It shows an absorption maximum in carbon disulphide at about 515 m//^^. Astacene diacetate C^Jil^^O^: This derivative is obtained by allowing a solution of astacene and acetic anhydride in pyridine to stand for 16 hours^^. It crystallises from a mixture of pjnridine and water in black-violet crystals, m.p. 235° (uncorr., with decom- position). Astacene dipalmitate C^jHjogO/^ (Astacein): It was shown by Karrer and co-workers (cf. p. 235) that astacene (or astaxanthin) occur in the lobster esterified with palmitic acid. Kuhn and co- References p. 253-255. 5 ASTACENE AND ASTAXANTHIN 239 workers^^ prepared astacene dipalmitate from astacene and palmitic acid chloride. The ester crystalHses from petroleum ether in almost rectangular red leaflets, m.p. 121°. Astacin dipalmitate exhibits epiphasic properties. Properties and Derivatives of Astaxanthin Crystalline form: Astaxanthim crystallises from pyridine in lustrous plates. Melting point: 216° (with decomposition). Solubility: Only few data are recorded in the literature regarding the solubility of astaxanthin. The pigment is readily soluble in pyridine, from which it can be crystallised on addition of water. Spectral properties (cf. Fig. 15, p. 354) : In contrast to astacene, astaxanthin exhibits an absorption curve in which three definite maxima can be recognised^^. In pyridine, these maxima are located at 476, 493 and 513 m^^^. Optical activity: Astaxanthin is optically inactive^*. Partition test: Astaxanthin exhibits entirely hypophasic properties. Chromatographic behaviour: Kuhn and co-workers chromatographed asta- xanthin on cane sugar from a mixture of benzene and petroleum ether (1:4). Benzene was employed for elution. Colour reactions: The alkali salts of the pigment have a characteristic deep- blue colour which is only shown, however, if air is excluded. On admission of oxygen, there is an immediate colour change to red and dehydrogenation to astacene occurs. Detection and estimation: Astaxanthin can be readily identified by the colour reactions described above. Astaxanthin diacetate C44H5gOg^^ : CHo CHo CHo CHo C CH3 CH3 CHo CH3 C /\ I I I I /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C CH^ I II II L CH3COOCH C-CHj HjC-C CH-ooc-ce3 \^ / . Astaxanthin diacetate \^ / CO CO The diacetate is obtained by treating astaxanthin dissolved in pyridine with acetic anhydride. The ester crystallises from a mixture of pyridine and water in rugged, deep blue-black needles, m.p. 203-205° (Berl-Block, in vacuum, uncorr.). The ester is very little enolised in the cold. In the partition test, it is References p. 253—255. 240 CAROTENOIDS CONTAINING CARBONYL GROUPS XII found in the lower layer. The absorption maxima are located at slightly shorter wavelengths than those of the parent pigment. Astaxanthin dicaprylate CjgH-oOg^*: This ester is prepared by the treatment of astaxanthin in pyridine with caprylic acid chloride. It can be purified by chromatography on calcium car- bonate from petroleum ether solution. Ast^anthin dicaprylate crystallises from a mixture of petrol and ethanol in dark red crystals, m.p. 121-124° (not sharp, Berl-block, in vacuum). In the partition test with 90% methanol and petroleum ether, the ester is found almost quantitatively in the upper layer. The ester becomes hypophasic only on employing 97% methanol. AstaxantJiin dipalmitate C-^HugOg^® : This ester is prepared by a procedure analogous to that described for astaxanthin dicaprylate. It crystallises from a mixture of pyridine, methanol and water in flat, violet-red needles, m.p. 71.5-72.5°. Astaxanthin dipalmitate exhibits purely epiphasic behaviour. Astaxanthin monopalmitatc C5gH8205^' : This derivative 'is obtained by the esterification of astaxanthin with the calculated amount of palmitic acid chloride. The ester crystallises from a mixture of benzene and methanol in red spheres, m.p. 113.5-114.5° (corr.). Astaxanthin monopalmitatc is entirely epiphasic on partition between 90% methanol and petroleum ether. Astaxanthin esters from Haematococcus pluvialis. From Haematococcus pluvialis, Kuhn and Sorensen^^ isolated various esters of astaxanthin, the composition of which has not yet been definitely determined. Ovoverdin and Chromoproteids. The probable structure of ovoverdin has been described above (p. 236). It is very probable that the nature of thceprotein component is different in different organisms. A common characteristic of all these chromoproteids is their green or blue colour, water solubility, and great sensitivity towards heat, acids and organic solvents (with the exception of petroleum ether) . The protein of the chromoproteids is coagulated by these reagents, leaving the free pigment component, astaxanthin. 6. CAPSANTHIN C40H58O3 History 1817 Braconnot carries out the first investigations on the pigments of paprika^^. 1869 Thudichum recognises the close relationship of the paprika pigments to the carotenoids^", a relationship later confirmed by Pabst^^ and Kohl^^. References p. 253-255. 6 CAPS A NTH IN 241 1913 A number of investigators establish the spectroscopic similarity of lyco- pene to the paprika pigment^^. 1927 Zechmeister and von Cholnoky^* succeed in obtaining the pigment of Capsicum annuum (paprika) in a crystalline form. They propose the name capsanthin for the new carotenoid. 1927-35 Zechmeister and von Cholnoky^^, and Karrer and co-workers^^ elucidate the constitution of capsanthin. Occurrence Capsanthin is a rare carotenoid. Zechmeister and von Cholnoky®'' found the esterified pigment in the ripe pods of Capsicum annuum and Capsicum frutescens japonicum^^ and Karrer and Oswald^^ observed that the anthers of Lilium tigrinum contain capsanthin besides antheraxanthin (cf. p. 191). Preparation''^ The paprika pods are freed from their shells and seeds and dried at 35-40°. The finely ground material is then extracted at room temperature with petroleum ether (1 kg of pods requires % ^ of petroleum ether). The solution is diluted with a threefold volume of ether, 30 % methanolic potassium hydroxide is added, and the mixture is allowed to stand for 1-2 days at room temperature. (Concerning the control of the saponification, compare the original communication of Zechmeister and VON Cholxoky). At the end of this period the free phytoxanthins are dissolved in ether. The solution is washed until it shows a neutral reaction, dried over sodium sulphate, and most of the solvent is evaporated under reduced pressure. The ethereal residue is diluted with much petroleum ether and allowed to stand in the cold for 24 hours. In this way, 1.2-2 g of crude capsanthin is obtained from 1 kg of high quality paprika. After two recrystallisations from carbon disulphide, the yield amounts to 0.8-1.2 g of crystallised, but still inhomogeneous pigment. The sepa- ration from accompanying carotenoids (zeaxanthin, capsorubin) is effected by chromatography on calcium carbonate or zinc carbonate. Carbon disulphide is employed as solvent for developing the chromatogfam. A mixture of benzene and ether (1:1) is also suitable for this purpose''^. Cht>micul Constitution CM, CH, CH, CH, C CH, CH3 CH3 CH3 C /\ II II /\ CH- C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH3 r II I HOCH C-CHj HaC-CHa CHOH \ / Capsanthin \ y^ CHg CH2 The molecular formula of capsanthin CjoHggOa was determined by Zech- meister and VON Cholnoky'^'^. The same authors also established that the References p. 253—255. Carotenoids 16 242 CAROTENOIDS CONTAINING CARBONYL GROUPS XII pigment contains ten double bonds'^, which must be conjugated in view of the long- wavelengths location of the absorption maxima. Zerewitinoff deter- minations and esterification''* proved that only two of the three oxygen atoms are present as hydroxyl groups. (This result was confirmed by Zechmeister and VON Cholnoky'^^). The third oxygen atom belongs to a carbonyl group which is directly attached to the system of conjugated double bonds and cannot be oximated. The presence of a carbonyl group was indirectly proved by Zechmeis- ter and VON Cholnoky'^^ by showing that perhydrocapsanthin contains three hydroxyl groups which can be acetylated. This is in agreement with the results of microhydrogenation'^^ during which capsanthin takes up ii mols of hydrogen. The presence of a carbonyl group and its position in the conjugated system was confirmed by the investigations of Karrer and Hubner in the course of which capsanthin was converted into the corresponding alcohol, capsanthol, by reduction with aluminium isopropoxide and isopropyl alcohol. Capsanthol is a triol of the formula €401157(011)3, the longest wavelength absorption band of which is displaced by only 35 m/< towards shorter wavelengths as compared with capsanthin. This shows that the carbonyl group in capsanthin must be ter- minally conjugated rather than cross-conjugated, otherwise the displacement of the absorption maxima on reduction would be larger owing to the break in conjugation. Karrer and co-workers ^® subjected capsanthin to permanganate degra- dation and obtained a:a-dimethylmalonic acid and a:a-dimethylsuccinic acid. No a:a-dimethylglutaric acid was formed, so that the presence of an unsubsti- tuted j3-ionone ring is excluded. These findings are in agreement with the results of biological assay'^ which show that capsanthin has no vitamin A activity. A further conhrmation of the open-chain formula of capsanthin was provided by an investigation of Karrer and Jucker^", in the course of which a caro- tenoid containing the chromophoric system present in capsanthin was obtained b}' a rational partial synthesis (cf. p. 250). Properties Crystalline form: Capsanthin crystallises from carbon disulphide in deep carmine-red spheres. From petrol, the pigment is obtained in needles, and from methanol in prisms. Melting point: 176° (uncorr.)^^, 175-176° (corr.)^^^ Solubility: Capsanthin is readily soluble in acetone and chloroform, less soluble in methanol, ethanol, ether and benzene, only sparingly soluble in car- bon disulphide, and almost insoluble in petroleum ether^^. References p. 253-255. 6 CAPSANTHIN 243 spectral properties: Solvent Absorption maxima Carbon disulphide 542 503 m^ Petrol 505 475 m^ Benzene 520 486 m// (cf. Fig. 14, p. 353) Solutions of the pigment in ethanol are deep red. Solutions in petrol are lemon to orange-yellow. Colour reactions: On treating a solution of the pigment in chloroform with concentrated sulphuric acid, the latter assumes a deep blue colouration. On treating an ethereal solution of the pigment with concentrated aqueous hydro- chloric acid, no colour change is observed. Capsanthin gives a deep blue colouration with antimony trichloride in chloroform. Numerous other colour reactions have been described by Zechmeister^*. Optical activity: [aj^a = +36° (in chloroform). Partition test: On partition between petroleum ether and 90% methanol, capsanthin is found quantitatively in the lower layer. Chromatographic behaviour: Capsanthin is well adsorbed on calcium carbo- nate or zinc carbonate from carbon disulphide or from a mixture of benzene; and ether (1:1). It is found above violaxanthin on the chromatogram column. Elution is effected by means of methyl or ethyl alcohol or by means of an_ ether-methanol mixture (5:1)*. Detection and estimation: For a micro-method for the identification of capsanthin, compare Zechmeister^*. The simplest method of identifying the pigment is the determination of the absorption spectrum. According to Zech- MEiSTER^* the following reaction is characteristic of the pigment. A layer of 30% methanolic potassium hydroxide is formed under a solution of capsanthin in petroleum ether, and the mixture is allowed to stand undisturbed for one day. At the end of this period, deep red needles are formed. Physiological properties: According to B. v. Euler, H. v. Euler and Karrer^^ capsanthin has no vitamin A activity. Chemical behaviour: Capsanthin is not acidic in character but there are cer- tain indications that the pigment is at least partly enolised. Thus, when the pigment is chromatographed on calcium carbonate from benzene, two zones are regularly observed^®. The same behaviour was observed by Karrer and Concerning the observation of L. Zechmeister and L. v. Cholnoky that the pigment forms two zones in the chromatogram on calcium carbonate, cf. the section on cis-irans- isomerism, p. 248. References p. 253-25$. 244 CAROTENOIDS CONTAINING CARBONYL GROUPS XII JucKER during the chromatographic adsorption of capsochrome^' (cf. p. 248). On standing in air, capsanthin is slowly oxidised. In an atmosphere of oxygen, oxidation occurs more rapidly. The uptake of oxygen is complete after about I month and amounts to about 29% by weight, corresponding to 10 mols of oxygen. The pigment wax of capsanthin is much more stable towards oxygen. According to Pummerer and Rebmann^, 6-8 mols of oxygen are absorbed on treating capsanthin with perbenzoic acid. On treatment with bromine in chloroform solution, 8 mols of bromine are absorbed. The thermal decomposition of the pigment gives rise to w-xylene. Derivatives Capsanthin diiodide: This compound is obtained on treating capsanthin with iodine in carbon disulphide solution. Capsanthin diiodide crystallises in flat needles which appear yellow-brown to black under the microscope^^. It is easily soluble in chloroform and acetone, a little less soluble in ethanol and ether and almost insoluble in petroleum ether. Perhydrocapsanthin: Capsanthin absorbs 10 mols of hydrogen on hydro- genation in acetic acid in the presence of platinum as catalyst; the carbonyl group remains unchanged. The perhydrocapsanthin thus obtained is a viscous, colourless oil which is much more soluble in organic solvents than capsanthin itself. On treating perhydrocapsanthin with sodium and alcohol, the carbonyl group is reduced and the completely hydrogenated triol C4oHg(,03 is obtained^". Capsanthol C40H50O3: CH, CH, . CH, CH3 C CH, CH3 CH, CH3 C /\ I I I I /\ CH2 C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- CHOHCH^ HOCH C-CH3 HgC-CHa CHOH \ / Capsanthol \^ CH2 CHj Karrer and Hubner^^ prepared capsanthol by the reduction of capsanthin with aluminium isopropoxide and isopropyl alcohol. The compound was purified by repeated adsorption on calcium hydroxide from benzene solution. Capsanthol crystallises from ethanol in reddish-brown leaflets, which appear yellow under the microscope. Melting point 175-176" (uncorr.). Capsanthol is sparingly soluble in boiling ethanol. Catalytic reduction shows the presence of 10 double bonds. References p. 253-255. 6 CAPSANTHIN 245 Solvent Absorption maxima Carbon disulpbide 508 477 vajx Pyridine 493 463 m/z Benzene 492 462 m^ Chloroform 486 456 m^ Ethanol 478 448 m/x Capsanthin diacetate C44H62O5: This compound was prepared by Zechmeister and von Cholnoky^^ by treating capsanthin with acetyl chloride in pyridine solution. The diacetate is purified by chromatography on calcium carbonate from petrol solution and by crystallisation from methanol. It separates in plates, m.p. 146.5° (corr.). The pigment is very soluble in chloroform, ether, carbon disulphide and benzene, and somewhat less soluble in methanol. On partition between methanol and petroleum ether, the ester is found quantitatively in the upper layer. Capsanthin dipropionate C^gHggOj: The dipropionate is prepared in the same way as the diacetate. It separates from a mixture of ethanol and carbon disulphide, or from ethanol alone, in crystals, m.p. 140°. Capsanthin dicaprate C60H94O5 : This compound is prepared in the. same way as capsanthin diacetate^^. M.p. 109° (corr.)*. The ester crystallises from a mixture of benzene and methanol in red plates with a violet tinge. It is readily soluble in petroleum ether, chloroform, ether, carbon disulphide and benzene. It is much less soluble in ethanol than the diacetate. [a\l%,^ == —61° in hexane. Capsanthin diniyristate CegHjioOg : The ester crystallises from a mixture of benzene and methanol in red needles, m.p. 88°. The solubihty is similar to that of the dicaprate, except that the dim^-Tistate is insoluble in alcohols. Capsanthin dipahnitate C^2^^iS^b '■ The dipalmitate is prepared and purified in the same way as the diacetate^*. It crystalhsed from a mixture of benzene and methanol in bordeaux-red plates, m.p. 95° (corr.)** ^^. For the absorption spectra in different solvents compare the original communication by Zech- meister and VON Cholnoky**. Capsanthin distearate C^qHI^^S^^: This compound is prepared by the same method as the other esters^^. M.p. 84°. The distearate shows great similarity to the dipalmitate both in appearance and solubility. Capsanthin dibenzoate Cg^HggOg: This ester crystallises from a mixture of benzene and methanol in red needles, and from a mixture of carbon disulphide * L. Zechmeister and L. v. Cholnoky, Ann. 487 (1931) 210, previously reported m.p. 102° (corr.). ** L. Zechmeister and L. v. Cholnoky, Ann. 509 (1934) 286, reported m.p. 92° (corr.). References p. 253—255. 246 CAROTENOIDS CONTAINING CARBONYL GROUPS XII and ethanol in leaflets, m.p. I2i-I22°(?). The dibenzoate is more soluble in alcohols than the fatty acid esters. Capsanthinone C40H58O5: CH3 CH3 OH3 CHj C CHo CH3 CH3 CH3 C /\ I I I I /\ CH2 OC-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj HOCH OC-CHa HsC-CHj CHOH \ / Capsanthinone \ / CH2 CH2 Capsanthinone is prepared by the oxidation of capsanthin diacetate with chromic acid^^. Capsanthinone diacetate crystallises from a mixture of benzene and petrol in glistening needles, m.p. 123-124° (corr.). The acetate is hypophasic. It is readily soluble in ethanol, ether, benzene and carbon disulphide, somewhat less soluble in acetone and practically insoluble in petrol. On treating an ethereal solution of the pigment with concentrated aqueous hydrochloric acid, the latter is coloured deep blue. Solvent Absorption maxima Carbon disulphide 541 503 468 m^ Benzene 524 487 454 m/t Hexane 503 472 440 m^ Anhydrocapsanih inane C43H5504 : CHo CH« CHo CHq G GH3 Gri3 GH3 GH3 C /\ I I I I /\ CHj C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj I II 1 HOCH — C-CO-CHs HgC-CHj CHOH Anhvdrocapsanthinone \ y' CH2 KuHN and Brockmann^'' obtained anhydro-j3-semicarotenone by splitting off water from j3-semicarotenone (cf. p. 140). Anhydrocapsanthinone is obtained by an analogous reaction from capsanthinone diacetate^®. The compound crystallises from methanol in small red needles which have no sharp melting point. On partition between methanol and petroleum ether, the pigment is found in the lower layer. In contrast to capsanthinone, the reaction with hydro- chloric acid is negative. The spectral properties are in agreement with the proposed structure, the chromophoric system of which differs only by an addi- tional conjugated double bond from that of capsanthinone. This results in the displacement of the maxima towards longer wavelengths by 16 m/,( compared with capsanthinone. References p. 233-235. 6 CAPSANTHIN 247 Solvent Absorption maxima Carbon disulphide 557 517 483 m/z Benzene 537 499 467 m// Cyc/ohexane 524 489 458 m/^ Hexane 518 483 453 m/f Capsanthylal CgoH^gOg^'-'* : CH3 CH3 CH3 CH3 C OHC-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj H,C-CH, CHOH Capsanthylal CH, Capsanthylal is formed by the oxidation of capsanthin diacetate with chromic acid^^. If an excess of oxidising agent is employed, smaller degradation products are also obtained. The aldehyde crystallises from 80 % methanol in needles which are grouped in star-like formations. M.p. 127° (corr.). Solvent Absorption ^naxiuia Carbon disulphide 518 483 452 m/i Hexane 483 452 m// Capsanthylal is readily soluble in benzene, ethanol and carbon disulphide, but only slightly soluble in petroleum ether. Capsanthylal monoxime C30H43O3N: Crystallises from methanol in needles, which are similar in colour to zeaxanthin. M.p. 184°. The oxime is readily soluble in benzene and carbon disulphide and somewhat less soluble in hexane and petrol. Solvent Absorption maxima Hexane 483 452 m/^ Capsylaldehyde C27H38O3 : CH, CH« I I I /\ OHC-CH=CH-C=CHCH=CHCH=C-CH = CHCH=C-CH = CH-CO CH^ H,C-CH, CHOH Capsylaldehvde CHj Capsylaldehyde has only been obtained crystalline in the form of its oxime. The formula shown above is assigned to this compound by Zechmeister and VON Cholnoky^^. The oxime crystallises in lemon-yellow needles, m.p. 172°. References p. 253-255. 248 CAROTENOIDS CONTAINING CARBONYL GROUPS XII It is hardly soluble in petrol, but somewhat more soluble in methanol, benzene and carbon disulphide. Solvent Absorption maxima Capsylaldehyde /3-Carotenone aldehyde Carbon disulphide . . 491 459 430 m/z 490 459 430 m/x Benzene 476 446 420 vajj, 478 448 421 m/x Hexane 458 431 m// 458 431 m/x 4-Hydroxy-p-caroteno7ie aldehyde C27H3e04®* : CHo CHo \V /\ I I I CH2 OC-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CH-CHO HOCH OC-CHa \ y/ 4-Hydroxy-)3-carotenone aldehyde CHa This aldehyde is formed besides capsylaldehyde and capsanthylal by the chromic acid oxidation of capsanthin diacetate. Only the oxime has so far been obtained in the crystalline state. It forms yellow cigar-shaped crystals, m.p. 189° (corr.). The oxime is readily soluble in benzene, methanol, hot petrol and hot hexane, but only sparingly in carbon disulphide. The absorption spectrum of the aldehyde coincides with that of the oxime, and is hardly different from that of capsylaldehyde. Solvent Absorption maxima Carbon disulphide 490.5 459.5 429 m/z Benzene 477 449 420 m/< Hexane 459 433 408 m/i Zechmeister and von Cholnoky^^ subjected capsanthin to a different kind of degradation, namely hydrolysis with aqueous alcoholic sodium hydro- xide in a sealed tube. The product obtained in this way was identified with citraurin (cf. p. 219), and the structures of both pigments were thus confirmed. Cis-Trans Isomers As early as 1937, Zechmeister and von Cholnoky^^ observed that pure capsanthin forms 2 zones in the chromatogram. This observation was hrst explained as due to the formation of the enol form of the pigments. These investigations were continued by the same two authors in 1940, and it was found that not only 2, but several zones appeared on the adsorption column^*"'. Following the investigations by Gillam and El Ridi^"^ and Zechmeister and co-workers^"2^ the phenomenon was then ascribed to cis-trans isomerisation. References p. 253-255. 6 CAPSANTHIN 249 By applying the usual methods (cf. p. 39), it is possible to isomerise capsanthin and capsanthin dipalmitate^^^ into various compounds which according to Zechmeister and von Cholnoky^"^ and PolgAr and Zechmeister^*'* are cis- trans isomers of the two pigments. Neo-capsanthin A could be obtained in a micro-crystalhne form, but no data are available regarding its melting point and elementary analysis. Solvent Absorption maxima Neo-A Neo-B Neo-C Carbon disulphide . . (532) (495) m^ Benzene (513) (481) m/f (513) (481) m,u (508) (479) m^ Hexane 496 465 m/^ The different isomers have the following optical rotations in benzene : Capsanthin [a\ = ± 0°(±5-10°) Neocapsanthin A [aJc = + 89° Neocapsanthin B [a]c = + 21° (± 5°) Neocapsanthin C [aj^ = + 27° ( ± 10°) Analogous experiments with capsanthin dipalmitate gave rise to two trans- formation products which exhibited the following absorption maxima : Carbon disulphide Benzene Hexane Capsanthin dipalmitate 541.5 502 m^ (519) (488) m/x 506 473 m/i Neocapsanthin dipalmitate I 535 499 m^< (512) (483) m/z 502 470 m/^ Neocapsanthin dipalmitate II 533 497 m/i (510) (482) m/i 496 465 m/z Optical rotations in petrol: Capsanthin dipalmitate [aj^ = —30° Neocapsanthin dipalmitate I [a],, = -22° Neocapsanthin dipalmitate II [a\ = -20° After iodine catalysis, the neo-capsanthins exhibit a well defined 'cis-peak' near 363 m^ in petrol. The possible configurations of the different isomers are discussed by Polgar and ZechmeisterI"*. Capsanthin mono-epoxide and Capsochrome Karrer and Jucker^"^ subjected capsanthin diacetate to oxidation with monoperphthalic acid and obtained a crystalline mono-epoxide C40H58O4: CH3 CHo CHo CHo C CHo CHo Clio CHo C /\ I I I r /\ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj )0 ' HaC-CH, CHOH HOCH C-CHs • \/ \^ / Capsanthin epoxide CH, CH2 References p. 25J-255. 250 CAROTENOIDS CONTAINING CARBONYL GROUPS XII The compound crystallises from a mixture of benzene and petroleum ether in leaflets and needles, m.p. 189° (uncorr., in vacuum). On shaking the ethereal solution of the pigment with concentrated aqueous hydrochloric acid, the latter assumes an unstable deep blue colouration. In the partition test the pigment is found quantitatively in the lower layer. On treating capsanthin epoxide with very dilute hydrochloric acid, it is isomerised into the stabler furanoid oxide, capsochrome. CHj CII3 CH CH, CH, CH, CH, C II I I I /\ CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH, I HaC-CHj CHOH Capsochrome \ / CHj Capsochrome crystallises well from a mixture of benzene and petroleum ether. Melting point 195° (uncorr., in vacuum). The pigment exhibits the same behaviour towards hydrochloric acid as capsanthin epoxide. It is hypophasic on partition between methanol and petroleum ether. Solvent Absorption maxima Capsanthin epoxide Capsochrome Carbon disulphide 534 499 515 482 m/i Chloroform 511 481 492 462 m/i (not sharp) Benzene 514 483 496 464 m^ Partially Synthetic Polyene Ketone Containing the Chromophoric System of Capsanthin By condensing j3-apo-2-carotenal^*'^ (I) with pinacolone (II), Karrer and JuCKER^*^' obtained the polyene ketone (III) which possesses the same chromo- phoric system as is present, according to Zechmeister and von Cholnoky^"^, in capsanthin. The absorption bands of the two pigments in the visible region are completely identical, which supports the formula proposed for capsanthin. \/ 0 L/Ho CHo CHo CHo /\ I I I I CHj C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO+CHsCO- C(CH3)j CHo C" CHo \/ I . II CHj References p. 253—253. 7 CAPSORUBIN 251 CH, CH, CH3 CH, \V \V C CH3 CH3 CH3 CH3 c CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH3 I II CH2 0*0x13 CHa Solvent Absorption maxima Polyene Ketone (III) Capsanthin Carbon disulphide 543 503 543 503 m^ Petroleum ether 503 473 503 475 m/z Most of the colour reactions exhibited by capsanthin on treatment with different acids and metalHc chlorides^* are also given by the polyene ketone. The two compounds, however, differ somewhat in their behaviour towards concentrated aqueous hydrochloric acid : the acid layer is coloured red on shaking with an ethereal solution of capsanthin but remains colourless in the case of the polyene ketone. 7. CAPSORUBIN C4oHgo04 History and Occurrence In 1934, Zechmeister and von Cholnoky^"^ isolated a new pigment, capsorubin, during the chromatographic purification of capsanthin. Capsorubin has so far only been found in Capsicum annuum. Preparation The paprika pods are pre-treated with ethanol, and then extracted with petroleum ether. The combined extracts are concentrated in vacuum and the pigment esters are chromatographed on calcium carbonate. After repeated chro- matographic adsorption, the capsorubin ester is saponified with methanolic pot- assium hydroxide, and the pigment is finally chromatographed repeatedly on calcium carbonate from carbon disulphide solution. For further purification, the pigment is crystallised from a mixture of benzene and petrol. The yield of analyti- cally pure pigment amounts to about 130 mg from 5 kg of pods. Chemical Constitution^'^^' ^^^ CHq CHo CHo CH.J 0 CH3 CH3 CH, CH3 C /\ I I I 1 /\ OHj 00-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CH2 HOCH CH,-CH, HoC-CH, CHOH CH2 OH2 References p. 253-255. 252 CAROTENOIDS CONTAINING CARBONYL GROUPS XII The formula for capsorubin proposed by Zechmeister and von Chol- NOKY^''^' ^"^ has not been completely established, but is in complete accord with all the properties of the pigment. The molecular formula of capsorubin is Qo-^6o04- It contains 9 conjugated double bonds and- 2 carbonyl groups. Acetylation shows the presence of two hydroxyl groups which, for reasons of analogy, are assigned to the same positions as in xanthophyll (p. 201) zea- xanthin (p. 183) and capsanthin (p. 242). The products of oxidation with chromic acid indicate the presence of 4 side-chain methyl groups. Properties^^^' io» Capsorubin crystallises from a mixture of benzene and petrol in violet- red needles. From carbon disulphide the pigment is obtained in rhombic plates. It is readily soluble in alcohol and acetone, more sparingly soluble in ether, benzene and carbon disulphide, and almost insoluble in petroleum ether. The chromatographic behaviour of capsorubin is similar to that of capsanthin. It is well adsorbed on calcium carbonate from carbon disulphide and is found above capsanthin on the column. Melting point 201° (corr.). On treating an ethereal solution of capsorubin with concentrated hydro- chloric acid, the latter immediately assumes a violet colouration, which later turns deep blue. With 25 % hydrochloric acid, no colour change is observed. The pigment is entirely hypophasic in the partition test. Solvent Absorption maxima Carbon disulphide 541.5 503 468 m^ Benzene 520 486 455 m/z Petrol 506 474 444 m/i Capsorubin diacetate C44Hg408: This compound is obtained by the acetylation of capsorubin in pyridine with acetyl chloride. The ester crystallises from methanol in square leaflets, m.p. 179° (corr.). It is readily soluble in petrol, benzene and methanol. Cis-Trans Isomers Zechmeister and von Cholnoky^^" examined the behaviour of capsorubin on treatment with iodine, on heating or on standing over long periods. They found that the pigment undergoes similar transformations as capsanthin (cf. p. 248). They were not able, however, to isolate any of the transformation products, which they regard as cis-trans isomers, in the crystalline state. The only data available are the absorption spectra and optical rotations. References p. 253-255. REFERENCES 253 Solvent Absorption maxima Carbon disulphide Benzene Hexane Capsorubin 541 502 467 524 489 455 502 470 471 mu Neocapsorubin A . . 533 495 460 517 483 451 498 466 (435) m/i Neocapsorubin B . . 535 497 462 518 484 453 500 467 (436) m^ Capsorubin dipalmitate 541.5 502.5 467 524 489 455 507 474 442 m/< Neocapsorubin dipalmitate I ... 536 496 463 521 486 452 502 470 439 m/i Neocapsorubin dipalmitate II . . . 533 495 460 518 484 450 499 467 437 m/i The optical rotations of the various transformation products in benzene are as follows : Capsorubin \_a\ = 0° Neocapsorubin A \xt\ — -134° Neocapsorubin B [a\ = - 69° Capsorubin dipalmitate [a]^. = 0° Neocapsorubin dipalmitate I [aj^ = -75° Neocapsorubin dipalmitate II [a]c = -15° REFERENCES 1. L. Zechmeister and P. Tuzson, Ber. 6g (1936) 1S78; yo (1937) 1966. 2. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) 682. 3. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) 682, 1020 (cf. p. 205). 4. P. Karrer and co-workers, Helv. chim. Acta 21 (1938) 448. 5. L. Zechmeister and L. v. Cholnoky, Ann. 530 (1937) 291. 6. P. Karrer, H. Koenig and U. Solmssen, Helv. chim. Acta 21 (1938) 445. 7. H. Koenig, Dissertation, Zurich 1940. 8. L. Zechmeister and L. v. Cholnoky, Ann. 530 (1937) 29^. g. N. A. Monteverde, Acta Horti Petropol. 13 (1893) 121. 10. M'. TswETT, Compt. rend. 152 (191 1) 788. 11. N. A. Monteverde and V. N. Lubimenko, Bull. Acad. Sci. Petrograd. 7 (1913) 1105. 12. S. PrAt, Biochem. Z. 152 (1924) 495. 13. T. Lippmaa, Compt. rend. 182 (1926), 867, 1040; Ber. bot. Ges. 44 (1926) 643. 14. R. Kuhn and H. Brockmann, Ber. 66 (1933) 828. 15. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 477. 16. Cf. T. Lippmaa, Das Rhodoxanthin, University of Tartu (1925). 17. R. Kuhn and H. Brockmann, Ber. 66 (1933) 828. 18. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 477- 19. A. Polgar and L. Zechmeister, /. Am. Chem. Soc. 66 (1944) 186. 20. I. M. Heilbron, B. Lythgoe and R. F. Phipers, Nature 136 (1935) 989. 21. I. M. Heilbron and B. Lythgoe, /. Chem. Soc. 1936, 1376. 22. J. Tischer, Z. physiol. Chem. 251 (1938) 109. 23. J. Rutschmann, Dissertation, Zurich 1946; Helv. chim. Acta 2j (1944) 1691. 24. P. Karrer and J. Rutschmann, Helv. chim. Acta 27 (1944) 1691. 25. G. Pouchet, /. Anat. Physiol. 12 (1876) 1-90, 113-165. — R. Maly, Sitzgsber. Akad. Wiss. Wien 83 (1881) 1126; Mh. Chem. 2 (1881) 351. — C. F. W. Kruckenberg, Vergleichend-physiologische Studien g2 (18S2). — W. Zopf, Beitr. Phys. Morph. nied. 254 CAROTENOIDS CONTAINING CARBONYL GROUPS XII Org. 3 (1893) 26. — M. Newbigin, /. Anat. u. Physiol. 21 (1897) 237. — G. Vegezzi, Dissertation, Fribourg, 1916. — J. Verne, Compt. rend. Soc. Biol. Paris 83 (1920) 963; g4 (1926) 1349; 97 (1927) 1290; Arch. Morph. 16 (1923) i. — E. Chatton, A. Lwoff and M. Parat, Compt. rend. Soc. Biol. Paris 94 (1926) 567. — E. Lonnberg and H. Hellstrom, Ark. Zool. A. 26 (1932) No. 7, etc. 26. R. KuHN and E. Lederer, Ber. 66 (1933) 4^8. 27. P. Karrer and co-workers, Helv. chim. Acta ij (1934) 4^2, 745; 18 (1935) 96; ig (1936) 479. 28. R. KuHN and N. A. Sorensen, Z. angew. Chem. 51 (1938) 465; Ber. 71 (1938) 1879. 29. R. KuHN J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. 30. J. TiscHER, Z. physiol. Chem. 267 (1941) 281. 31. R. KuHN and E. Lederer, Ber. 66 (1933) 488. 32. R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. — J. Tischer, Z. physiol. Chem. 267 (1941) 281. — Cf. p. 232. 33. R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. — J. Tischer, Z. physiol. Chem. 239 (1936) 257. 34. J. Tischer, Z. physiol. Chem. 250 (1937) 147; 252 (1938) 225. 35. R. KuHN, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. 36. J. Tischer, Z. physiol. Chem. 267 (1941) 281. 36a. M. Hartmann, F. Graf Meden, R. Kuhn and H. J. Bielig, Z. Naturforsch. 2b (1947) 330- 37. R. Kuhn and N. A. Sorensen, Ber. 71 (1938) 1879. 38. P. Karrer and co-workers, Helv. chim. Acta 17 (1934) 412, 745; ig (1936) 479. — W. Jaffe, Dissertation, Ziirich, 1939. 39. P. Karrer and co-workers, Helv. chim. Acta 17 (1934) 4^2; 17 (1934) 745; 18 (1935) 96, 19 (1936) 479- 40. H. Willstaedt, Svensk Kem. Tidskr. 46 (1934) 205. 41. P. Karrer, L. Loewe and H. Hubner, Helv. chim. Acta 18 (1935) 96. 42. R. Kuhn and N. A. Sorensen, Z. angew. Chem. 51 (1938) 465; Ber. 71 (1938) 1879. 43. Literature references are given by R. Kuhn and N. A. Sorensen, Ber. 71 (1938) 1882. 44. R. W. G. Wyckoff, Science 86 (1937) 311. 45. R. Kuhn and N. A. Sorensen, Z. angew. Chem. 51 (1938) 465. 46. K. G. Stern and K. Salomon, J. biol. Chem. 122 (1938) 461. 47. P. Karrer and F. Benz, Helv. Chim. Acta 17 (1934) 412. 48. R. Kuhn, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1688. In the recent literature, the value 228° is always given. 49. R. Kuhn, E. Lederer and A. Deutsch, Z. physiol. Chem. 220 (1933) 231. 50. P. Karrer and L. Loewe, Helv. chim. Acta 17 (1934) 745- 51. Cf. R. Kuhn and co-workers, Ber. 72 (1939) 1688. 52. R. Kuhn ,E. Lederer and A. Deutsch, Z. physiol. Chem. 220 (1933) 231. — Cf. P. Karrer and L. Loewe, Helv. chim. Acta 17 (1934) 745. 53. R. Kuhn and N. A. Sorensen, Ber. 71 (1938) 1879. 54. Cf. Ber. 71 (1938) 1886. 55. R. Kuhn and N. A. Sorensen, Ber. 71 (1938) 1887. 56. R. Kuhn and N. A. Sorensen, Ber. 71 (1938) 1888. 57. R. Kuhn, J. Stene and N. A. Sorensen, Ber. 72 (1939) 1701. 58. R. Kuhn and N. A. Sorensen, Ber. 72 (1939) 1699. 59. H. Braconnot, Ann. Chim. 6 (1817) 122, 133. 60. J. L. Thudichum, Proc. Roy. Soc. (London) 17 (1869) 253. 61. T. Pabst, Arch. Pharm. 230 (1892) 108. 62. F. G. Kohl, Untersuchungen tiber das Carotin mid seine physiologische Bedeutung in Pfianzen, Leipzig 1902. 63. A. Tschirch, Ber. bot. Ges. 22 (1904) 414. — B. M. Duggar, Washington, Univ. Stud, i (1913) 22. — N. A. Monteverde and C. N. Lubimenko, Bull. Acad. Sci Petrograd. Ser. 6, 7. II (1913) 1105- REFERENCES 255 64. L. Zechmeister and L. v. Cholnoky, Ann. 454 (1927) 54- 65. L. Zechmeister and L. v. Cholnoky. Ann. 455 (1927) 7°; 4^5 (1928) 288; Ber. 61 (1928) 1534; Ann. 487 (1931) 197; 489 (1931) i; 509 (1934) 269; 516 (1935) 30- 66. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 614; 19 (1936) 474- 67. L. Zechmeister and L. v. Cholnoky, Ann. 454 (1927) 57; -#'57 (1931) i97- 68. L. Zechmeister and L. v. Cholnoky, ^wm. 489 (1931) i- 69. P. Karrer and A. Oswald, Helv. chim. Acta 18 (1935) 1303- 70. L. Zechmeister and L. v. Cholnoky, Ann. 454 (1927) 54; 5^9 (i934) 269. 71. P. Karrer ans E. Jucker, Helv. chim. Acta 28 (1945) ii45- 72. L. Zechmeister and L. v. Cholnoky, ^mw. 509 (1934) 269. 73. L. Zechmeister and L. v. Cholnoky, y4wM. 516 (1935) 3°- 74. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 614. 75. L. Zechmeister and L. v. Cholnoky, Ann. 454 (1927) 54; 5og (1934) 269. 76. L. Zechmeister and L. v. Cholnoky, Ann. 516 (1934) 3°- 77. P. Karrer and H. Hubner, Helv. chim. Acta ig (1936) 474. 78. P. Karrer and co-workers, Helv. chim. Ada 14 (1931) 6i4- 79. B. V. EuLER, H. V. EuLER and P. Karrer, Helv. chim. Acta 12 (1929) 278. 80. P. Karrer and E. Jucker, Helv. chim. Acta 2y (1944) 1588. 81. P. Karrer and A. Oswald, Helv. chim. Acta 18 (1935) 1305. 82. L. Zechmeister and L. v. Cholnoky, Ann. 509 (1934) 287. 83. L. Zechmeister and L. v. Cholnoky, Ann. 509 (1934) 269; 454 (1927) 67. 84. L. Zechmeister, Carotinoide, Berlin 1934. 85. B. V. EuLER, H. V. EuLER and P. Karrer, Helv. chim. Acta 12 (1929) 278. 86. L. Zechmeister and L. v. Cholnoky, Ayin. 530 (1937) 291- 87. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 1143. 88. R. PuMMERER and L. Rebmann, Ber. 61 (1928) 1099. 89. L. Zechmeister and L. v. Cholnoky, Ann. 478 (1930) 103. 90. L. Zechmeister and L. v. Cholnoky, Ann. 516 (1935) 38. gi. P. Karrer and H. Hubner, Helv. chim. Acta 19 (1936) 476. 92. L. Zechmeister and I^ v. Cholnoky, Ann. 487 (1931) 210. 93. L. Zechmeister and L. v. Cholnoky, Ann. 509 (1934) 2S5. 94. L. Zechmeister and L. v. Cholnoky, Ann. 487 (1931) 209. 95. L. Zechmeister and I-. v. Cholnoky, Ann. 543 (1940) 248. 96. L. Zechmeister and L. v. Cholnoky, Ann. 523 (1936) loi. 97. R. KuHN and H. Brockmann, Ann. 516 (1935) 95. 98. R. KuHN and H. Brockmann, Ann. 516 (1935) 95. 99. L. Zechmeister and L. v. Cholnoky, Ann. 530 (1937) 291. 100. L'. Zechmeister and L. v. Cholnoky, Ann. 543 (1940) 248. loi. A. E. GiLLAM and M. S. El Ridi, Nature 136 (1935) 914; Biochem. J. 30 (1936) 1735 ; 31 (1937) 1605. 102. L. Zechmeister and co-workers. Nature 141 (1938) 249; Biochem. J. 32 (1938) 1305; Ber. 72 (1939) 1340; Ber. 72 (1939) 1678; 2039. 103. L. Zechmeister and L. v. Cholnoky, Ann. 543 (1940) 248. 104. A. PolgAr and L. Zechmeister, /. Am. Chem. Soc. 66 (1944) 186. 105. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 1143. 106. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) ^^2. 107. P. Karrer and E. Jucker, Helv. chim. Acta 27 (1944) 1588. 108. L. Zechmeister and L. v. Cholnoky, Ann. 516 (1935) 30. 109. L. Zechmeister and L. v. Cholnoky, Ann. 509 (1934) 269. IXC. L. Zechmeister and L. v. Cholnoky, Ann. 543 (1940) 248. CHAPTER XIII Carotenoid carboxylic acids I. BIXIN C25H30O4 History 1825 BoussiNGAULT^ is the first to describe bixin, the pigment of Orleans, which has since been investigated by numerous workers^. 1878 Etti^ succeeds in crystalhsing bixin. 1917 Heiduschka and Panzer^ carry out careful elementary analyses of bixin and are the first to assign the correct empirical formula to the pigment. 1928-33 KuHN and co-workers^ propose a structural formula for bixin which is confirmed by Karrer and co-workers by the total synthesis of per- hydronorbixin^. Occurrence Bixin has only been found in Bixa orellana. The fresh seeds of this plant are surrounded by an orange-red mass which contains most of the pigment. After drying, the seeds are surrounded by a brown-red crust. Bixin also occurs in other organs of the plant, e.g. the secretionary cells of the leaves, and appears in the form of numerous brown spots on the lower side of the leaf. Preparation"^ a) From commercial Orleans. The commercial preparation is finely ground and allowed to stand for several days covered with acetone. The material purified in this way is dried in air and the pigment is extracted with chloroform in a Soxhlet apparatus. It is crystallised from the same solvent, or from ethvl acetate or acetic acid. By this method good preparations of the labile bixin are obtained but the yield is decreased by the pre-extraction with acetone. b) From "pate de rocou". The red dough is stirred up with methanol and the bixin is converted into its ammonium salt by addition of ammonia. The salt is extracted with water and the solution is filtered. The filtrate is acidified with acetic acid, when bixin is precipitated as a red powder. It is filtered, washed with methanol and extracted with chloroform. The crude preparation is then recrystallised. In this way about 500 g of bixin are obtained from 25 kg of "pate de rocou". References p. 290-294. BIXIN 257 c) From bixa-seeds*. The seeds are covered with water and allowed to stand for several hours. They are then stirred mechanically and filtered through a sieve. The turbid solution is allowed to stand overnight in large percolators and the lower layer is separated and centrifuged. The residue is broken up and dried, first in air and then in vacuum over calcium chloride, until it is not brittle but can be pressed. After grinding in a mill, 100 kg of seeds yield about 5-6 kg of a material which contains 15-30% of bixin. This material, in portions of 200 g, is immediately covered with portions of two litres of ethanol, warmed to 60-65° on a waterbath, and ammonia is passed in until the colour change is complete and the solution contains free ammonia. The reaction mixture is allowed to stand for 20 minutes, filtered warm, and the residue is stirred up with 1 litre of ethanol. Into this mixture, ammonia is again passed at 60°. It is then allowed to stand for 1 hour and filtered. The combined filtrates precipitate ammonium bixate on cooling. In order to complete the precipitation, 1 ml of acetic acid is added to each litre of solution and the solution is vigorously stirred. After a short time, a dark red resin separates on the stirrer and on the walls of the vessel. The resin is separated from the mother liquor and treated with acetic acid, with vigorous mechanical stirring. After some hours, free bixin separates and can be filtered and dried in vacuum over sodium hydroxide and calcium chloride. It is then crystallised from acetic acid. (About 18 g of boiling glacial acetic acid are required to dissolve 1 g of crude bixin). About 120-160 g of pure bixin are obtained from 100 kg of seeds. Chemical Constitution CH3 CHj CH3 CHj II II HaCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- COOH Bixin The elucidation of the constitution of bixin extended over several years. Heiduschka and Panzer^ determined the correct molecular formula C25H30O4 An unsymmetrical structural formula was first proposed, but was abandoned when KuHN and Winterstein^ put forward the now accepted formula of bixin. This has been confirmed by the investigations of Karrer and co- workers^", in the course of which perhydronorbixin was synthesised. Herzig and Faltis^^ recognised that bixin was the monomethyl ester of an unsaturated dicarboxylic acid. Bixin absorbs 9 mols of hydrogen on cata- lytic hydrogenation and is thus converted into the half-ester of a saturated dicarboxylic acid. From the deep red colour of the pigment it can be concluded that the 9 double bonds are conjugated. By the ozonisation of methyl bixin RiNKES and van Hasselt^^ obtained the methyl ester of /3-acetylacrylic acid and methylglyoxal. Methylglyoxal must be derived from the grouping = CH.C(CH3) =, while the first product indicates the presence of the grouping: A detailed description is given by R. Kuhn and L. Ehmann, Helv. chim. Acta 12 (1929) 904, and E. ForcAt, Dissertation, Zurich, 1930. References p. 2^o-2g4. Carotenoids 17 258 CAROTENOID CARBOXYLIC ACIDS XIII H3C0-0C-CH=CH-C= CH3 As early as 1909, van Hasselt^^ observed the formation of w-xylene during the dry distillation of bixin. This finding was later confirmed by Herzig and Faltis^^ and indicates the presence of the grouping : = CH-C=CHCH=CH-C= CH3 CH3 KuHN and co-workers subjected bixin to oxidative degradation with per- manganate and, later, with chromic acid, and deduced the presence of 4 side chain methyl groups in both cases^^. When the structure of the ozonisation products I and II obtained by Rinkes^^ CHaOOC- CH=CH- C=CH- CHO OHC- CH=C- CHO I CH3 II CHj had been determined, Kuhn and Winterstein^' suggested the above structural formula for bixin, which is also based on the recognition of the symmetrical structure of carotene, lycopene and squalene (Karrer). The correctness of this formula was proved by the investigations of Karrer, in the course of which the position of the two terminal methyl groups was established by the oxidative degradation of partially hydrogenated bixin^^. By the degradation of perhydro- norbixin, Karrer and co-workers^^ obtained 3:7:12: i6-tetramethyloctadecan- i:i8-dialIII, OHC • CH2CHCH2CH»CH2CHCH2CH,CH2CH2CHCH2CH2CHi,CHCHjjCH0 II II CII3 CH3 CH3 CH3 III which was oxidised to the dicarboxylic acid and converted into perhydro- crocetin^^ (cf. p. 280). The elucidation of the constitution of bixin was completed by the synthesis of perhydronorbixin by Karrer and co-workers^^ and by the conversion of perhydrocrocetin into perhydronorbixin. The structure of both these pigments was thus confirmed^". The formation of m-toluic acid by the thermal decompo- sition of bixin^^, is also in accord with the above formula. The synthesis of perhydronorbixin was carried out as follows: CHo CHo I I H0CH,CHCH„CH,CH»CHCH20H References p. 2go-2g4. BIXIN 259 CHj CH3 BrCHaCHCHjCHaCHijCHCHjBr + 2 NaCH(COOR)a CH3 I CHs (ROOQaCHCHjCHCHaCHjCHijCHCHaCHCCOOR)^ CH, CH, HOOC- CHjCHaCHCHaCHjCHaCHCHaCHa- COOH CH, CH, ROOC-CHjCHaCHCHjCHaCHaCHCHjCHjCOOH Electrolysis OHo OHo CH, CH, ROOC- CHaCHjCHCH^CHjCHaCHCHaCHaCHaCHjCHCHaCHjiCHaCHCHjCHa- COOR CH, CH, CH, CH, HOOC- CHaCHaCHCHjCHaCHaCHCHjCHjCHaCHjCHCHaCHjCHjCHCHaCH,- COOH Perhydronorbixin With regard to the conversion of perhydrocrocetin into perhydronorbixin, see p.. 278. Stereochemistry of Bixin The first observation concerning a stereoisomer of bixin was made in 1913. In the course of the isolation of the pigment, Herzig and Faltis^^ accidentally obtained a new, higher melting form which they termed /3-bixin. It was later suggested by Karrer and collaborators^^, that the two forms may be cis-trans isomers. By the treatment of natural labile bixin with iodine^*, these workers obtained the stable form identical with the j3-bixin of Herzig and Faltis. The same transformation was also achieved with methyl bixin (p. 268) . It was thus shown that two series of compounds exist, one of which is derived from the labile natural bixin, and the other from the stable jS-bixin. A uniform nomen- clature for bixin derivatives was proposed by Karrer and Kuhn and is em- ployed in the sequel. References p. 2go-2g4. 26o CAROTENOID CARBOXYLIC ACIDS TABLE 50 NOMENCLATURE OF BIXIN DERIVATIVES^^ XIII M.p. Configu- ration New name Old name Formula Karrer Herzig and Faltis C2,H2e(COOH)2 254-255° cis Labile norbixin Norbixin Norbixin ICOOCH3 196° cis Labile bixin Bixin Bixin C22H2e(COOCH3)2 163-164° cis Labile methyl Bixin Bixin bixin methyl ester methyl ester C^^H^eiCOOH)^ >300° trans Stable norbixin Isonorbixin )3-Norbixin ICOOCH3 '-22^26 1 COOH 220° trans Stable bixin Isobixin ^-Bixin C,3H3«(COOCH3), 200-201° trans Stable methyl Isobixin- /3-Bixin bixin methyl ester methyl ester Karrer and Solmssen have attempted to solve the question as to which double bond of the bixin molecule possesses the czs-confignration^^. They subjected the bixins to permanganate degradation* and compared the products obtained from labile bixin and stable bixin. Each isomer yielded a different apo-l-norbixinal methyl ester I and, very probably, a different apo-2-norbixinal methyl ester II**, distinguished by their melting points and absorption spectra. Both bixins also give rise to an identical apo-3-norbixinal methyl ester III. These results show that the isomerism of the two bixins probably depends on the different configuration of the third double bond in the chain, counting from the unesterified carboxyl group. In view of the incompletely established diffe- rence of the apo-2-norbixinal methyl esters, it is possible that cis-trans isomerism of the second double bond may also be involved. CH, CH, CH, CH, HsCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO I CH, CH, CH, HsCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CHO II ^* Cf. p. 47. The identity of the two compounds could not be established with certainty because the apo-2-norbixinal methyl esters were only obtained crystalhne in the form of their oximes. References p. 2go—2g4. I BIXIN 261 CH3 CH3 CH3 I I I HjCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CHO III CH3 CH3 H H CH3 H,COOC-CH C CH C CH CH C=C C CH-COOH CH CH CH CH CH C CH CH Labile bixin CHs CHq CH^ I ! HjCOOC-CH C CH C CH CH CH CH CH CH CH CH CH CH C CH C CH-COOH Stable bixin CH3 CH3 The stereochemical configuration of the bixins has recently been investigated by Zechmeister and Escue^'. Instead of natural, labile bixin they chose the more easily chromatographed labile methylbixin for investigation, and sub- jected it to heat treatment, iodine catalysis and illumination with sunlight. These experiments yielded a number of transformation products, two of which, neomethylbixin A and neomethylbixin C, were obtained in a crystalline state. The other isomers could be distinguished by their optical properties. Neomethylbixin A crystallises from a mixture of benzene and methanol in long, narrow plates, m.p. 190-192° (corr.). It is more soluble and less stable than labile methyl bixin and exhibits maxima at 485 and 453 m^ in petroleum ether solution. Neomethylbixin C is obtained from a mixture of benzene and methanol in small, clustered needles, m.p. 150-151° (corr.). It exhibits absorption maxima at 479 and 448.5 m/z in petroleum ether solution. 1. Stable norbixin C24H28O4: CH3 CH3 CH3 CH3 HOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- COOH Norbixin The potassium salt of stable norbixin is obtained by boiling labile bixin with excess 10 % potassium hydroxide. The free acid separated on addition of hydrochloric acid to the aqueous solution^^. The dinitrile of norbixin can be obtained from bixin dialdehyde dioxime (cf. under lycopene, pp. 118 and 123) and yields norbixin on hydrolysis with methanolic potassium hydroxide. Stable norbixin crystallises from pyridine in glistening blue-red leaflets. It does not melt below 300°. It is fairly easily soluble in pyridine, very sparingly soluble in acetic acid and amyl alcohol, and almost insoluble in other organic solvents. References p. 2go-2g4. 262 CAROTENOID CARBOXYLIC ACIDS XIII Solvent Absorption maxima Carbon disulphide 527.5 492 457.5 m^ Chloroform 509 474,5 442 m/i On adding alkali to a suspension of stable norbixin in water, it is converted into a very sparingly soluble, crystalline yellow salt. The action of diazomethane on norbixin yields a stable methylbixin. Trans-norhixm. is stable in air. It dissolves in concentrated sulphuric acid with a greenish-blue colour. Stable bixin, the monom,ethyl ester of stable norbixin C25H30O4: Karrer and co-workers^* obtained stable bixin by allowing labile natural bixin to stand in chloroform solution in the presence of iodine. It crystallises from acetic acid or pyridine, or from acetone in flakes. M.p. 216-217° (uncorr., with decomposition). The solubility in organic solvents is considerably smaller than that of labile bixin. Solvent Absorption maxima Carbon disulphide 526.5 491 457 mfi Chloroform 509.5 475 443 m^ (of. Fig. 17, p. 355) On shaking a solution of stable bixin in acetic acid and pyridine with zinc dust for a short time, dihydrobixin is obtained. The same dihydrobixin is also formed from labile bixin^" under the same conditions. Stable methylbixin, the dimethyl ester of stable norbixin CjgHjjO^: Stable methylbixin can be prepared either by the isomerisation of labile methylbixin in the presence of iodine^"- ^^ or by the esterification of stable norbixin. The ester is also formed by shaking in air a solution of dihydro- methylbixin in piperidine, or in pyridine containing a little sodium hydroxide^^. CI13 CH3 CH3 CHj HjCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- COOCH, Stable methylbixin Stable methylbixin crystallises from a mixture of chloroform and ethanol in stout, blue- violet needles, m.p. 205-206° (corr.). On shaking a solution in pyridine containing a little acetic acid with zinc dust at 50°, the same dihydro- methylbixin is obtained as from labile methylbixin. Solvent Absorption maxima Carbon disulphide . . . 525.5 490 456.5 m/z Chloroform 509.5 475.5 444 m^ Hexane 484 450 425 405 m/z (cf. Fig. 19, p. 356) References p. 290-294. I BIXIN 263 Quantitative extinction measurements are reported by Hausser and Smakula^^. The fluorescence spectrum is described by Hausser, R. Kuhn and E. Kuhn^*. Stable apo-i-norbixinal methyl ester CjsHggOg : CH3 CH3 GI13 CHg HjCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CHO Apo-i-norbixinal methyl ester This ester is obtained by the controlled permanganate oxidation of stable bixin^^. The stable aldehyde is also formed by the isomerisation of labile apo- i-norbixinal methyl ester with iodine^^. It crystalHses in small prisms, m.p. 167°. Solvent Absorption maxima Carbon disulphide 509 478 m// Ethanol 487 456 m/f Petroleum ether 472.5 445 m^ Solvent Absorption maxima of oxime Carbon disulphide 509 478 m/i Ethanol 483 452 m// Petroleum ether 475 446 m// Stable apo-2-norbixinal methyl ester C20H24O3 : HaCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CHO Apo-2-norbixinal methyl ester This ester is obtained by the controlled oxidation of stable methylbixin with potassium permanganate^^. Only the oxime and semicarbazone, but not the compound itself, has so far been obtained in a crystalline state. Solvent Absorption maxima Carbon disulphide 483.5 453 vufi Petroleum ether 450 424 m/z Solvent Absorption maxima of oxime Carbon disulphide 481 451 m^ Ethanol 459 mfj. Absorption maxima of semicarbazone Carbon disulphide 493 462 m^ Ethanol 471 m/z Apo-3-norbixinal methyl ester C^gHjaOg^^: CHs CI13 CHo I I I HsCOOC- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CHO Apo-3-norbixinal methyl ester References p. 2go-2g4. 264 CAROTENOID CARBOXYLIC ACIDS XIII The same apo-3-norbixinal methyl ester, m.p. 147° is formed by the con- trolled permanganate oxidation of stable or labile methylbixin (cf. p. 260). It forms an oxime, m.p. 188° and a semicarbazone, m.p. 215°. On shaking an ethereal solution of the pigment with concentrated aqueous hydrochloric acid, no Mue colouration is observed. Solvent A hsorption maxima Carbon disulphide 455 427 m/i Petroleum ether 425 m/i Ethanol about 440 m/z (cf. Fig. 17, p. 355) Absorption maxima of semicarbazone Carbon disulphide 472 443 m/i Ethanol 449 m// Absorption maxima of the oxime Carbon disulphide 458 428 m/i Petroleum ether 428 408 m/z 2. Labile norbixin C24H28O4: Labile norbixin is obtained by the saponification of labile bixin^^ or of labile methylbixin^'^. It crystallises from acetic acid in stout red needles, m.p. 254-255°. Labile norbixin is readily soluble in pyridine, fairly readily soluble in acetic acid, ethanol and methanol, sparingly soluble in chloroform and methyl acetate, and almost insoluble in ether^^. It readily dissolves in aqueous alkalis. Labile norbixin is autoxidisable in air^^. By boiling a solution of the sodium salt with excess ammonia and 2.3 mols of titanium trichloride, Karrer and co-workers^® obtained dihydronorbixin. If a larger proportion of titanium trichloride is used, or on prolonged boiling, tetrahydro- or hexahydro -norbixin is formed. On prolonged heating with aqueous potassium hydroxide (less smoothly by employing ethanolic potassium hydroxide), labile norbixin is converted into stable norbixin^®. By boiling with 3 % methanolic hydrochloric acid, Karrer and Takahashi^'' obtained stable methylbixin, whereas labile methyl- bixin is formed by the methylation of labile bixin with diazomethane^^. On methylation of labile norbixin with dimethyl sulfate, labile bixin and labile methylbixin are obtained^^. For the action of chlorine and hydrogen chloride on the pigment, compare the communication of Heiduschka and Riffart*". Labile norbixin dissolves in concentrated sulphuric acid with a blue-green colour*^. The mono-potassium salt of labile norbixin is micro-crystalline. It is insoluble in water and sparingly soluble in ethanol. The di-potassium salt forms brown-red needles, readily soluble in water. When moist, it is readily oxidised in air. References p. 2go-2g4. I BIXIN 265 Solvent Absorption maxima Carbon disulphide 527 491 458 rtijU Chloroform 503 469.5 440 m/i Dihydronorbixin C24H30O4 : Karrer and co-workers prepared dihydronorbixin by boiling labile norbixin dissolved in 2.1 mols of dilute sodium hydroxide with excess ammonia and 2.3molsof titanium trichloride*^. Dihydronorbixin crystallises from ether in yellow clusters, which sinter at 197°. It readily dissolves in acetic acid, acetone and chloroform, but is only sparingly soluble in ether and almost insoluble in ligroin. It is readily oxidised in air. The large displacement (about 70 m/j,) of the absorption maxima on passing from norbixin to dihydronorbixin indicates that the two carboxyl groups are no longer conjugated with the system of conjugated double bonds and that the addition of hydrogen takes place at the i : 18 positions. I I I 1 HOOC-CH2CH=C-CH=CHCH=G-CH=CHCH=CH-C=CHCH=CH-C=CH-CH2-COOH Dihydronorbixin Solvent Absorption tnaxima Carbon disulphide 454 428 m// Chloroform 435 410 m/u. Perhydronorbixin C24H4g04 (3:7:12:16-tetramethyloctadecane-l :18-dicarboxy- lic acid) : This compound was obtained by Herzig and Faltis*^ by boiling perhydro- methylbixin with potassium hydroxide. The total synthesis was achieved by Karrer and Benz** by the method described above (p. 258). Perhydro- norbixin is a viscous, colourless oil. B.p. 25o°/o.3 mm., 245.5°/o.24 mm., 227°/o.03 mm.*5. D^° 0.953; n^ 1.468*5 i|- ^g insoluble in water. By esterification with diazomethane, or with a mixture of methanol and hydrogen chloride, Herzig and Faltis*^ obtained perhydromethyibixin. a:a'-Dihydroxyperhydronorbixin dimethyl ester CjeHj^Og (I) : CHo Gilo (I) HjCOOC- CH(OH)- CHa- CH- CHa- CHj- CHj- CH- CHaCH^ Karrer and co-workers*' converted perhydronorbixin into a : a'-dihydroxy- perhydronorbixin dimethyl ester (I) by the action of bromine and red phosphorus followed by potassium hydroxide and diazomethane. The ester is obtained as an almost colourless oil, b.p. 2i3-2i6°/o.i4 mm. References p. 2go-2g4. 266 CAROTENOID CARBOXYLIC ACIDS XIII 3:y:i2:i6-Tetramethyloctane-i:i8-dial C22H42O2 (II): The dialdehyde is obtained from a : a'-dihydrox5rperhydronorbixin dimethyl ester by the action of methyl magnesium iodide, followed by oxidation with lead tetraacetate*^. CH3 CH3 CH3 CH3 OHC- CHjCHCHaCHaCHjjCHCHjCHaCHaCHjCHCHjCHaCHaCHCHi,- CHO (11) The compound is a yellow oil with an intensive odour, reminiscent of ozone. B.p. 18570.3 mm. 2:6:11 :i5-Tetramethylhexadecane-i:i6-dicarboxylic acid C22H42O4 (III) : I I I I HOOC-CHaCHCHaCHaCHjCHCHjCHaCHaCHaCHCHaCHjCHjCHCHaCOOH (III) This dicarboxylic acid is obtained by the oxidation of 3 : 7 : 12 : i6-tetra- methyloctadecane-i : i8-dial with chromic oxide in acetic acid*^. B.p. 220°/ o.i.mm. The diamide, C22H44O2N2 is prepared by converting the acid (III) by means of thionyl chloride into the acid chloride and treating the latter with concentrated aqueous ammonia*®. It separates from ethyl acetate in crystals, m.p. 127°. i:i6-Dihydroxy-2:6:ii:i5-tetramethylhexadecane-i:i6-dicarboxylic acid dimethyl ester C^^B-.^O^ (IV) : This ester was obtained by Raudnitz and Peschel^o from 2:6:11:15- tetramethylhexadecane-i : i6-dicarboxylic acid by the action of bromine and red phosphorus, followed by potassium hydroxide and diazomethajie. CIi» CHo CHo CHo I I I I " H3COOCCH(OH)CHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2CH2CHCH(OH)COOCH3 (IV) The ester (IV) is a colourless oil which can be distilled in high vacuum. Concerning its conversion into perhydrocrocetin, see p. 280. 4:8:1 3:iy-Tetram,i thyleicosane-i :20-diol ( i:20-dihydroxybixane) C24H5QO2: CHo OHo GHo CHo I I I I HO" GH2CH2CH2CHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2CH2CHCH2CH2CH2' OH I : ao-dihydroxybixane This diol was prepared by Kuhn and Ehmann^^ by heating perhydro- methylbixin with sodium and amyl alcohol. It is a pale yellow oil which partly References p. 290-294. I BIXIN 267 solidifies in the cold. B.p. i98°/o.i2 mm. It is readily soluble in chloroform and benzene, but only dissolves in acetic acid, ethanol and petroleum ether on warming. 4:8:1 3:iy-Tetramethyleicosane (Bixane) C24H50: CH3 ^i^S V-'-tl3 v^ils II II H3CCH2CHJCHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2CH2CHCH2CH2CH3 Bixane Bixane can be prepared by heating i : 20-dihydroxybixane with 66 % aqueous hydrobromic acid for 15 hours in a sealed tube at 230° and then warming the dibromide formed with activated zinc and 60 % acetic acid at 100° for 14 hours. It is a mobile colourless liquid, which boils at i62°/o.5i mm (corr.) D^° 0.8054; np° 1.4502. Bixane is readily soluble in chloroform, carbon disulphide and petroleum ether, and more sparingly soluble in ethanol and acetic acid. Labile bixin, monomethyl oster of labile norbixin C25H30O4: Labile (natural) bixin crystallises from acetic acid in deep violet, dichroic prisms. From ethyl acetate the pigment is obtained in rhombs. On rapid heating it melts at 198°, on slow heating at 191.5°. 100 Ml of chloroform dissolve only 0.5 g of labile bixin at 18°. The pigment is even less soluble in ethanol, ether and cold acetic acid but readily soluble in boiling acetic acid, pyridine and nitrobenzene, i Litre of boiling ethyl acetate dissolves 4 g of labile bixin. Solvent Absorption maxima Carbon disulphide 523.5 489 457 m/z Chloroform 503 469.5 439 m// Bixin remains unchanged for long periods on keeping in air, but undergoes some decomposition on heating to 110°. By heating above the melting point, VAN Hasselt^^ obtained w-xylene (cf. p. 49). The isomerisation of labile bixin into the stable form has been described on p. 259^^. Pummerer, Rebmann and Reindel^* found that only about six of the double bonds in bixin are saturated by reaction with perbenzoic acid. With regard to a new method of oxidation of labile bixin with manganese acetate, see a communication by Viebock^^. By the reduction of labile bixin with sodium amalgam, Karrer and co- workers^^ obtained a light yellow oil which on oxidation with alkaline pot- assium permanganate gave succinic acid. With regard to the action of chlorine and bromine^', iodine in benzene^^ iodine chloride^*, hydrogen chloride^^ and thiocyanogen^* see the original com- munications. References p. 2go-2g4. 268 CAROTENOID CARBOXYLIC ACIDS XIII On treating labile bixin with methanolic potassium hydroxide, the pot- assium salt^** is first obtained, but this is converted by prolonged shaking, or by boiling for a short time, into labile norbixin. By boiling with aqueous ethanolic potassium hydroxide, stable norbixin can be obtained besides the labile form®^. Investigations by B. von Euler, H. von Euler and Karrer^^ j^^ve shown that labile bixin has no vitamin A activity. The pigment dissolves in concentrated sulphuric acid with a cornflower-blue colour. For further colour reactions see the communication by Kuhn and co-workers®'. The sodium salt of labile bixin crystallises from 70% ethanol in dark, copper-red crystals®*. The potassium salt forms deep violet needles which are readily soluble in ethanol and methanol, but insoluble in water. Dihydrobixin C25H32O4: Karrer and co-workers®^ prepared this compound by a method analogous to that employed for the preparation of dihydronorbixin (p. 265). Kuhn and Winterstein®® prepared dihydrobixin by brief shaking of labile or stable bixin in pyridine with zinc dust and acetic acid. M.p. 207-208° (uncorr.)®^ (For the constitution of dihydrobixin, see p. 265.) Solvent Absorption maxima Carbon disulphide 454 428 m^ Chloroform 435 410 m// (of. Fig. 20, p. 356) Perhydrobixin, perhydronorbixin mono-methyl ester CjsH^gOj: This compound is formed by the catalytic hydrogenation of labile bixin in acetic acid in the presence of a palladium-barium sulfate catalyst®'. It is a colourless oil, b.p. 2i3-2i7°/o.3 mm.®^ D^° 0.9368; n^" 1.4615. Labile methylbixin, dimethylester of labile norbixin C26H32O4: Labile methylbixin can be obtained by the esterification of labile bixin or labile norbixin with dimethyl sulphate®^. Labile methylbixin is also formed by the action of diazomethane on labile bixin dissolved in chloroform'", or on labile norbixin'^. It crystallises from ethyl acetate in red, pleochroic rhombs, m.p. 163° (uncorr.)'°. It is fairly readily soluble in chloroform, acetic acid and ethyl acetate, sparingly in ethanol and very sparingly in methanol. For quantitative extinction measurements, see the communication by Hausser and Smakula'^. Labile methylbixin can be converted into the stable form by the action of iodine in the same way as labile bixin''. Numerous investigations have been made regarding its oxidative degradation. For details, the original literature should be consulted'*. References p. 2go-2g4. I BIXIN 269 By brief shaking of a solution of labile methylbixin in pyridine with acetic acid and zinc dust, Kuhn and Winterstein'^ obtained dihydromethylbixin. Karrer and Takahashi^^ found that by the saponification of labile methyl bixin with ethanolic sodium hydroxide (i mol) at 65°, stable bixin is formed besides labile bixin. Labile methylbixin dissolves in concentrated sulphuric acid with an intense blue colour. Dihydromethylbixin CggHg^O^ : This compound is obtained by briefly shaking a solution of labile or stable methylbixin in pyridine and acetic acid with zinc dust". It crystallises in orange yellow leaflets, m.p. 180-182° (corr.). A piperidine solution of dihydromethyl bixin oxidises in air to give stable methylbixin''^. (For constitution, see p. 265). Solvent Absorption maxima Carbon disulphide 454 428 m/z Chloroform 435 410 m/n (of. Fig. 20, p. 356) Perhydronorbixin dimethyl ester, perhydromethylbixin CjgHjoO^: Perhydromethylbixin was prepared by Herzig and Faltis by the catalytic hydrogenation of labile methylbixin'^^. It is also obtained by the methylation of perhydronorbixin with diazomethane, or with methanol and hydrogen chloride''^. Karrer and co-workers^" methylated perhydrobixin to the dimethyl ester by treatment with dimethylsulphate and aqueous potassium hydroxide in acetone. B.p. 2ii°/o.3 mm. D^° 0.9234; n^° 1.456881. By heating with sodium and amyl alcohol, perhydromethylbixin is converted into 4:8:13: 17-tetra- methyleicosane-i : 20-dioF2. Perhydronorbixin diethyl ester C23H54O4: The total synthesis of this ester by Karrer and co-workers^^ has already been mentioned (p. 259). The compound is a colourless oil, b.p. 20770.3 mm. Perhydronorbixin diamide C24H48O2N2 : Perhydronorbixin is converted into the acid chloride by means of phos- phorous pentachloride or thionyl chloride and the acid chloride is treated with concentrated aqueous ammonia^*. The diamide separates from ethyl acetate or ether in colourless crystals, m.p. 111°. It is almost insoluble in ether but soluble in ethanol and chloroform. Perhydronorbixin bis-( 2:4:6-tribromoanilide) C33H5o02N2Br5 : The acid chloride of perhydronorbixin (see above) is reacted with 2:4:6- tribromoaniline^^ and the product is recr^'stallised from methyl acetate. The tribromanilide melts at 83°. References p. 2go-2g4. 270 CAROTENOID CARBOXYLIC ACIDS XIII Norbixin mono-ethyl ester, ethylnorbixin C26H3JO4 : Ethylnorbixin was prepared by van Hasselt by the saponification of labile bixin with ethanolic potassium hydroxide and treatment of the reaction product with diethyl sulphate. The diethyl norbixin which separated was then filtered and washed with dilute acid^^. The ester crystallises from ethyl acetate in red needles with a green lustre, m.p. 176°. It forms a potassium salt which crystallises in needles and is insoluble in water. Methylethylnorbixin C27H34O4 : a) M.p. 149°. This form is obtained by the methylation of ethylnorbixin*'. It crystallises in red rhombic crystals. b) M.p. 138° (ethylbixin) . This form is obtained by treating labile bixin in ethanol solution with one mol of potassium hydroxide and diethyl sulphate in the presence of ethyl acetate^. It separates from ethanol in red rhombic crystals. It is readily soluble in chloroform, ethyl acetate and acetone. Diethylnorbixin CggHjjO^ : The preparation of this ester is similar to that of ethylnorbixin. Diethyl- norbixin separates from acetone in blue crystals m.p. 121° ^*. Methyl-n-octylnorbixin (n-octyl bixin ester) C^^^f^O^: This ester was obtained by Karrer and Oswald^^ by the action of w-octyl iodide on potassium bixinate. Dark violet crystals from ethanol, m.p. 132°. Methyl-n-butylnorbixin (n-butyl bixin ester) C29H3g04: This ester is prepared in the same way as the previously described derivative. Dark crystals, m.p. 160°. Methyl-n-octadecyl norbixin (n-octadecyl bixin ester) C43H85O4 : Dark crystals, m.p. 118°*®. i:i:2o:20-Tetramethyldihydrobixinol C2gH4202 (I) : H3C CH3 CH3 CH3 CH3 CH3 HO- C- CH2CH=C- CH=CHCH=C- CH=CHCH=CH- C=CHCH=CH- C= CHCHjC- OH H3C (I) CH3 This compound was obtained by Karrer and Rubel^" by reacting di- hydrobixin methyl ester with methyl magnesium iodide. It crystallises from ethyl acetate in golden-yellow needles, m.p. 166-167° (uncorr.). It is entirely hypophasic in the partition test. References p. 2go-2g4. BIXIN Solvent Absorption maxima Carbon disulphide 455 429 m.u Chloroform 435 410 m/x 271 Labile apo-i-norbixinal methyl ester CjsHggOj: Karrer and Solmssen^^ subjected labile bixin to controlled permanganate degradation and obtained 3 different aldehydes which were identified as apo-i-, apo-2-, and apo-3-norbixinal methyl ester*. Apo-i-norbixinal methyl ester is obtained in the largest yield. M.p. 156°. Oxime, m.p. iS6°. Semicarbazone, m.p about 225°. Solvent Absorption maxima Carbon disulphide 505 475 ra/j. Petroleum ether 470 441 m/j, Ethanol about 484 m/z A bsorption maxima of the oxime Carbon disulphide 501 470 m// Ethanol 479 448 m/x Absorption maxima of the sem,icarbazone Carbon disulphide 515 487 m/z , Ethanol 487 460 m^ Labile apo-2-norbixinal methyl ester CjuHj^Og: This compound was only formed in very small amounts^^ and could not be obtained in a crystalline state. Solvent Absorption maxima Carbon disulphide 479.5 449 mjx Petroleum ether 446.5 421 rafj, Ethanol wide bands Absorption maxima of the oxime Carbon disulphide 478 449 m/n Ethanol 456 m/z Petroleum ether 447 m/x Absorption maxima of the semicarbazone Carbon disulphide 488.5 458 m/z Ethanol 465.5 mu The apo-3-norbixinal methyl ester was only obtained in one form. References p. 2go-2g4. 272 CAROTENOID CARBOXYLIC ACIDS 2. CROCETIN C20H24O4 XIII History 1818 AscHOFF^^ investigates the pigment of saffron and terms it crocin. 1852-1914 Different workers®* carry out investigations on crocin. The glyco- sidic nature of crocin is recognised. 1915 Decker®^ isolates the still inhomogeneous aglycon (crocetin) from crocin. 1927-33 Karrer and Salomon^® and Karrer and co-workers®^ elucidate the constitution of crocin and crocetin®^. Occurrence Crocin is the colouring principle of saffron which has been employed in different countries since early times. Crocin was shown by Karrer and Miki® to be the digentiobiose ester of crocetin. Besides crocin, Crocus sativus contains small quantities of crocetin* and also of ^-carotene, y-carotene, lycopene and zeaxanthin. A colourless glycoside, picrocrocin (saffron bitter) which is closely related to crocin, has also been isolated^"". TABLE 51 Source a) In blossoms: Crocus sativus L. Crocus albiflorus Kit., var. Neapolitanus hart. b) In fruit : Gardenia grandiflora Lour. c) In blossom leaves: Cedrela Toona Roxb. Crocus luteus Nyctanthes Arbor-tristis V erbascum phlomoidcs L. OCCURRENCE OF CROCETIN References B.Wmss, J . prakt. Chem. (1) 1 01 (1867) 65; Jb. Fortschr. d.Chem.j86y, 733. — of. B.Qu avr at, J . prakt. Chem. ^6 (1852) 68; Jb. Fortschr. d. Chem. 18 51, 532. R. KuHN, A. WiNTERSTEiN and W. WiEGAND, Helv. chim. Acta 11 (1928) 718. F. RocHLEDER and L. Mayer, /. prakt. Chem. (1) 72 (1857) 394; 7^ (1858) l; Jb. Fortschr. d. Chem. 1857, 490; 1858, 475. — R. Kuhn and co-workers, Helv. chim. Acta II (1928) 718. E. G. Hill and A. P. Sikkak, /. Chem. Sac. gi (1907) 1501. — A. G. Perkin, /. Chem. Soc. loi (1912) 1540. — R. Kuhn and A. Winterstein, Helv. chim. Acta 12 (1929) 496. R. Kuhn, A. Winterstein and W. Wiegand, Helv. chim. Acta 11 (1928) 718. E. G. Hill and A. P. Sikkar, /. Chem. Soc. gi (1907) 1501. L. ScHMiD and E. Kotter, Monatsh. 5g (1932) 340, 353. Concerning the stereochemistry of natural crocetin from Crocus sativus, see R. Kuhn and A. Winterstein, Ber. 66 (1933) 209; 67 (1934) 34^- References p. 2^0-294. CROCETIN 273 Stereochemistry of Crocetin The first observations on stereoisomers of crocetin are due to Kuhn and WiNTERSTEiN^"^ who obtained, besides the known crocetin dimethyl ester of m.p. 222°, an isomer of m.p. 141°. The lower melting isomer is converted to the higher-melting, stable ester on illumination. The isomerisation can also be brought about by other means, e.g. heat, iodine catalysis, and via the dihydro- derivative. Kuhn and co-workers regarded the isomerisation as a cis-trans change. From the ease of conversion of the lower into the high-melting form, Kuhn and WiNTERSTEiN concluded that the former is a cis and the latter a trans isomer of crocetin. The following nomenclature has been proposed^"^. TABLE 52 NOMENCLATURE OF CROCETINS New Nomenclature M.p. Configuration Old name Stable crocetin Stable crocetin monomethyl ester Stable crocetin dimethyl ester Labile crocetin dimethyl ester 285° 218° 222° 141° trans trans trans cis a-Crocetin /3-Crocetin y-Crocetin Pigment of Kuhn Preparatio/i a) Crocin: According to Karrer and Salomon^^^, saffron is dried at 90° and pre-extracted with ether. The material is then extracted with ethanol and oily components are precipitated with ether. After further addition of ether, crocin gradually separates out in a microcrystalline form. Appreciable amounts of oily material are also obtained; these consist largely of crocin, but their crystallisation is rather difficult because of the presence of resinous components and takes a long time. By repeated dissolution of this oil in hot ethanol, seeding with crocin and standing, several crops of crystals can be obtained which are combined and re- crystallised from the same solvent. b) Crocetin^''* : 500 g of dried saffron is pre-extracted with ether. The residue is dried in air and extracted with 70% ethanol. About half the solvent is evaporated and the residual solution is strongly diluted with water. It is saponified with a solution of 30 g of potassium hydroxide and 500 ml of water. After acidification with hydrochloric acid, a thick yellow precipitate separates which is dried on porous tile and subsequently saponified with 10% ethanolic potassium hydroxide. The potassium salt of crocetin thus obtained is filtered and treated with acetic acid. For further purification, crude crocetin is recrystallised from pyridine. With regard to the isolation of crocetin from blossom leaves of Crocus luteus, see the communication of Kuhn and co-workers^°^. The isolation of crocetin di- methyl ester from saffron is described by Karrer and Helfenstein^"^. References p. 2go-2g4. Carotenoids 18 274 CAROTENOID CARBOXYLIC ACIDS XIII Chemical Constitution of Crocetin The constitution of crocetin was elucidated by Karrer, Salomon and co- workers^^- ^''' ^^. As a result of this work (and the investigations on bixin) a real insight into the principles governing the structure of carotenoids was obtained for the first time. CH3 ^^3 ^113 '-'H3 HOOC-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-COOH Crocetin The main features of the constitution of crocetin were determined by Karrer and Salomon^"'. These authors recognised the polyene nature of the pigment and the presence of a system of conjugated double bonds with side- chain methyl groups, and identified perhydrocrocetin as an aliphatic, saturated, dicarboxylic acid. The molecular formula C20H24O4 was proposed by Kuhn and L'Orsa^"^ and confirmed by Karrer and co-workers. Karrer and Salomon^"^ established the presence of 7 double bonds in the crocetin molecule by means of catalytic hydrogenation and Kuhn and L'Orsa^"^ established the presence of 4 side-chain methyl groups by chromic acid degradation. The latter authors proposed an 'unsymmetrical' structure for crocetin, whereas Karrer and co- workers^^" put forward the formula shown above, the correctness of which was proved by the degradation of perhydrocrocetin to 6:ii-dimethylhexa- decane-2 : 15-dione^i^ and by the total synthesis of perhydrocrocetin^^^. Degradation of perhydrocrocetin to 6:ii-dimethylhexadecane-2:5-dione: CH3 CH3 CHj CH3 HOOCCHCHaCHaCHaCHCHjCHaCHjCHaCHCHaCHjCHgCH • COOH Perhydrocrocetin \ CHo CHo CH3 CH3 II II HOOCC- CHaCHjCHaCHCHaCHjCHjjCHaCHCHaCHaCHaC- COOH Bi Br CH3 CH3 CH3 CH3 H3COOC • CCHgCHaCHaCHCHaCHaCHaCHaCHCHaCHaCHaCOOCHs OH OH a:a'-Dihydroxyperhydrocrocetin dimethyl ester References p. 290-294. CROCETIN CH3 CH3 CH3 CH3 H3C\ II II /m, yC — CCH2CH2CH2CHCHgCH2CH2CH2CHCH2CH2CH2C — C\ H3C/ I I I I \CH3 HO OH HO OH CH, CH, CH, CH, OCCH2CH2CH2CHCH2CH2CH2OH2CHGH2CH2CH2OO 6 : 1 i-Dimethylhexadecane-2 : 1 5-dione Total synthesis of perhydrocrocetini^^ : CHo CH, I I HOCHoCHCH,CH,CH,CHCH,OH CH3 CH3 HOCH2CHCH2CH2CH2CHCH2OC2H 5 CH3 CH3 I I BrCH2CHCH2CH2CH2CHCH20C2H5 CH, CH, HOOCn HOO ^ CHCH2CHCH2CH2CH2CHCH2OC2H5 CH, CH, HOOC • CH2CH2CHCH2CH2CH2CHCH2OC2H6 Electrolysis CH3 CH3 CH3 CH3 II II H5C2OCH2CHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2CH2CHCH2OC2HS CH3 CH3 CH3 CH3 BrCH2CHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2CH2CHCH2Br References p. 290-2^4. 275 276 CAROTENOID CARBOXYLIC ACIDS XIII CHj CH, CH, CH3 HOCHjCHCHjCHjCHaCHCHaCHjCHaCHjCHCHjCHjCHjCHCHjOH CHg CH3 CH, CH5 HOOCCHCHaCHaCHjCHCHaCHjCHjCHjCHCHaCHaCHjCHCOOH Perhydrocrocetin Properties Stable Crocetin C20H24O4: The pigment is obtained from acetic anhydride in brick-red rhombs, m.p. 285°. It is very sparingly soluble in the common organic solvents, but fairly soluble in pyridine and in very dilute alkalis. Crocetin is fairly stable in air in the solid state; the surface of the crystals is decolourised, however, under the influence of light^^^. In alkaline solution on the other hand, the pigment ab- sorbs oxygen from the air even at 20° ; this oxidation is considerably accelerated by haemin^^*. With concentrated sulphuric acid, crocetin gives a deep blue colour which soon changes into violet and eventually into brown*. No colour- ation is observed with hydrochloric acid. For the separation of crocetin from other carotenoids, see the communi- cation by KuHN and Brockmann^^^. A microchemical method of identification is described by Tunmann^^^. Solvent Absorption maxima Carbon disulphide 482 453 426 mn Pyridine 464 436 411 m/z Chloroform 463 434,5 mfi Petrol 450.5 424.5 mn Hexane 445 420 400 m/i (cf. Fig. 22, p. 357) Disodium salt of crocetin C2oH2204Na2 : Orange-5^ellow needles^^'. Dipotassium salt of crocetin C20H22O4K2 '■ This compound is obtained from aqueous ethanol in yellow crystals^^'. Diammonium salt of crocetin C2oH2204(NH4)2: Red needles from ammoniacal aqueous ethanol"'. Dipyridine salt of crocetin C20H24O4.2C5H5N : Dark red plates from aqueous pyridine^i®. Crocin was first obtained in the crystalline state by Karrer and Salomon^^^. It was recognised as the digentiobiose ester of crocetin by Karrer and Miki^^o^ * Further colour reactions are described by P. Karrer and co-workers, Helv. chim. Acta II (1928) 1201. References p. 2^0-294. 2 CROCETIN 277 The sugar residues of crocin are very readily split off by alkalis. On working in aqueous media, crocetin is obtained, whereas in the presence of methanol, methyl esters of the pigment are formed^^^. Crocin C44H64O24 : CH3 CH3 CH3 CH3 -HCOO(>-C=CH-CH=CH-C=CH-CH=CH-CH=C^CH=CH-CH=C-COOCH 1 r 0 (CH0H)3 (CH0H)3 0 I CH CH 1 I I 0 1 1 ^ 1 i CHgO • CH(CH0H)3CH • CHjOH H0CH2CH(CH0H)3CH • OCHj Crocin dissolves readily in water to give an orange-red solution. M.p. 186° (with frothing). Crystals of crocin contain water of crystallisation which is only given up on prolonged drying in vacuum at 100°. Stable crocetin niononiethyl ester (/3-crocetin) C21H26O4: This mono-ester is obtained either by the replacement esteiification of crocin with 70 % methanol and potassium hydroxide, or by the esterification of cro- cetin with dimethyl sulphate^^^. The ester crystallises from chloroform in rectangular leaflets, m.p. 218°. Stable crocetin dimethyl ester (y-crocetin) C22H28O4: This ester is best prepared by the replacement esterification of crocin^^^, but it can also be obtained by the esterification of crocetin with diazomethane^^^. Hexagonal leaflets, m.p. 222.5° (corr.)^24_ (With regard to the isomerisation of labile crocetin dimethyl ester, see p. 273). Crocetin dimethyl ester can be distilled unchanged in vacuum. The dry-distillation is described by Kuhn and WlNTERSTEIN^^^. Solvent Absorption maxinia^^^ Petrol 450.5 424.5 m// Chloroform 463 434.5 m// (of. Fig. 18, p. 355) At 20°, one part of crocetin dimethyl ester dissolves in 100,000 parts of methanol. The solubility in ether is also very small. Labile crocetin dimethyl ester C22H28O4 : According to Kuhn and Winterstein^^', labile crocetin occurs esterified with gentiobiose in the blossoms of saffron {Crocus sativus). It has only been isolated in the form of its dimethyl ester. The labile dimethyl ester is formed together with the stable form by the action of dilute sodium hydroxide on a methanoHc extract of saffron. The References p. 2go-2g4. 278 CAROTENOID CARBOXYLIC ACIDS XIII separation of the two forms depends on the greater solubihty of the labile form in ether. The ester crystallises from methanol in elongated rectangular plates, m.p. 141°. Under the microscope the crystals appear yellow. One part of the labile ester dissolves in 5900 parts of methanol. Its behaviour towards light, iodine, or heat has already been described on p. 273. Reduction with zinc dust and acetic acid in pyridine yields a dihydroderivative which is identical with that obtained from the stable ester. By saponification with warm ethanolic potassium hydroxide, stable crocetin is formed. Solvent Absorption maxima Petrol 445 422 m/j. Chloroform 458 432.5 m/i Tricyclocrocetin C22H2404( ?) : This compound was obtained by Kuhn and Winterstein^^s jjy ^j^g (jj-y. distillation of stable crocetin dimethyl ester in vacuum, followed by chromato- graphy and saponification. It crystallises from methanol in colourless needles, m.p. 263-264°. On oxidation with chromic acid it yields 2.5 mols of acetic acid. Catalytic hydrogenation shows the presence of 4 double bonds. For the light absorption curve, the original communication should be consulted. Perkydrocrocetin dimethyl ester C22H42O4 : This perhydro-compound was obtained by Karrer and Salomon by the catalytic reduction of stable crocetin dimethyl ester^^g j|- ^g ^ viscous oil which partly sohdifies in the form of crystals, m.p. 27° ^^''. Perhydrocrocetin dimethyl ester boils at 198-210° at i mm and 180-185° at 0.05 mm. For the light absorption curve, see the communication by Karrer and Salomon^^i. 2:6:11 :i§-Tetramethylkexadecane-i:i6-diolC^QH.^202' This diol was obtained by Karrer and co-workers^^^ by the reduction of perhydroci ocetin dimethyl ester with sodium in alcohol according to Bouveault- Blanc. HO- CHjCHCHaCHjCHaCHCHaCHaCHaCHaCHCHaCHaCHaCHCHg- OH Colourless oil, b.p. i8o-i8i°/o.i mm (uncorr.). 2:6:ii:i5-Tetramethylhexadecane-i:i6-diol is converted by the action of hydrogen bromide into i: i6-dibromo-2:6: ii:i5-hexadecane, from which perhydronorbixin can be obtained by condensation with sodium malonic ester, saponification of the product and decarboxylation by heating to 200°^^'. References p. 2^0-2^4. CROCETIN 279 CH3 CH3 CH3 CH3 Br-CH2CHCH2CH2CH2CHCHaCH2CH2CHjjCHCH2CH2CH2CHCH2Br I CH3 CH3 CH3 CH3 HOOCx II II /COOH ^CH • CHjCHCHjCHijCHaCHCHaCHaCHaCHaCHCHaCHaCHaCHCHaCH^ HOOC/ \C00H CH3 CH3 CH3 CH3 HOOC- CHjCHaCHCHjCHaCHjCHCHjCHjCHaCHzCHCHaCHjCHaCHCHaCHa- COOH Perhydronorbixin (cf. p. 265) 2:6:ii:i§-Tetramethylhexadeca7te (Crocetane) C20H42: Karrer and Golde prepared crocetane from i:i6-dibromo-2:6: 11:15- tetramethylhexadecane by reduction with coppered zinc and dilute acetic acidi3*. CH3 CH3 CH3 CH3 H3C-CHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2CH2CHCH3 Crocetane Crocetane is a colourless oil, b.p. i35°/o.5 mm. It is readily soluble in petro- leum ether, chloroform and carbon disulphide and more sparingly soluble in ethanol and acetic acid. 6:ii-Dimethylhexadeca-3: 5:j:g:ii: i3-hexaeyi-2:iydicarboxylic acid. Dihydrucrocetin C20H26O4 (I)': HOOC- CHCH = CHCH = C-CH = CHCH=CH-C=CHCH=CH-CH- COOH I Dihydrocrocetin was prepared by Karrer, Helfenstein and Widmer^^^ by the reduction of crocetin with titanium ti ichloride in dilute sodium hydroxide and aqueous ammonia. It crystallises from ether in stout yellow needles, m.p. 192-193°. It is readily soluble in ethanol and acetic acid, more sparingly soluble in ether, and very sparingly soluble in water, ligroin and benzene. Dihydrocro- cetin is rapidly oxidised in air. With concentrated sulphuric acid, a wine-red colouration with a blue tinge is produced. Quantitative extinction measurements in ethanol solution are reported by Hausser and Smakula^^® (cf. Fig. 21, p. 356). References p. 290-2^4. 28o CAROTENOID CARBOXYLIC ACIDS XIII Dihydrocrocetin dimethyl ester C22H30O4 : This ester was obtained by Karrer and Helfenstein^^' by the action of diazomethane on dihydrocrocetin and by Kuhn and Winterstein by hydro- genation of stable^^^ or labile^^^ crocetin dimethyl ester in pyridine with zinc dust and acetic acid. This compound separates from ether in sulphur-yellow crystals, m.p. 96°. On shaking a solution in piperidine with air, stable crocetin dimethyl ester is rapidly formed^^^. If the pigment is dissolved in pyridine and little sodium hydroxide is added, a deep blue colomration is immediately produced. This rapidly changes to orange-red on contact with air, due to the oxidation of the dihydroderivative to the stable crocetin dimethyl ester^*". Dihydrocrocetin diethyl ester C2,;H3404 : This ester is prepared by treating dihydrocrocetin with diazoethane^*^. M.p. 62°. Dihydrocrocetin diethyl ester is very readily soluble in organic solvents. Hexahydrocrocetin C00H30O4 : Hexahydrocrocetin was prepared by Karrer and co-workers^*^ by the reduction of crocetin wdth excess titanium trichloride. It is a light yellow oil which is very stable towards atmospheric oxygen. On catalytic hydrogenation it is converted into perhydrocrocetin. On addition of concentrated sulphuric acid a brown-red colouration is produced. 6:ii-Dimethylhexadi>cane-2:i ^-dicavboxylic acid, perhydrocrocetin C20H38O4 : The total synthesis of this compound has already been described (p. 275). It is also formed by the catalytic hydrogenation of crocetin^*^. Perhydro- crocetin has also been prepared from i : i6-dihydroxy-2 : 6 : 11 : 15-tetramethyl- hexadecane-i : i6-dicarboxylic acid dimethyl ester (cf. p. 266)^**. The ester was treated with methyl magnesium iodide, the tetrahydroxyderivative formed was reacted with lead tetraacetate, and the resulting dialdehyde was oxidised with chromic acid and acetic acid. Perhydrocrocetin dianiide C20H40O2N2: Perhydrocrocetin is converted into the acid chloride by means of thionyl chloride and the acid chloride is reacted with concentrated aqueous am- moniai*^' i^^. M.p. 130°. a:a'-Dihydroxyperhydrocrocetin dimethyl ester C22H420g (I) : CH3 CHo CHo CHo II II HjCOOC- C-CH2CH2CH2CHCH2CH2CH2CH2CHCH2CH aCHaC- COOCH3 OH OH I References p. 2go-2g4. 3 AZAFRIN 28t This ester is prepared by the action of bromine and red phosphorous on perhydrocrocet in, conversion of the dibromide into the diol, and esterification with diazome thane. It is a viscous, colourless oil, b.p. 16570.04 mm. 6:ii-Dimethylhexadecane-2: 15-dione C18H34O2 : This compound is prepared from the diester (I)^*^ described above. The diketone is a liquid with a weakly aromatic smell. B.p. i32-i35°/o.05 mm. It forms a disemicarbazone, C20H40O4N6, which separates from ethanol in crystals, m.p. 168°. Crocelin Utrabromida C2oH2404Br4: This compoimd is obtained from crocetin by the action of bromine dissolved in chloroform^*''. It separates from a mixture of ethanol and ether in yellow crystals, m.p. 103-104° (with decomposition) . It is readily soluble in chloroform, ethanol, ether and acetic acid. 3. AZAFRIN C27H38O4 History 1885 Maisch^*^ is the first to describe the pigment of the roots of Escohedia scahrifolia. He names the pigment escobedine. 1911 LiEBERMANN^*^ succeeds in isolating escobedine in the crystalline form. He proposes a new name, azafrin, for the pigment. 1913-16 LiEBERMANN and SchillerI^" and Liebermann and Muhle^^^ attempt to elucidate the constitution of azafrin. 1931-33 KuHN and co-workers^^^ carry out investigations on azafrin in the course of which a structural formula is proposed and proved. Occurrence Up to the present time, azafrin, has only been found in two South American plants, Escobedia scabrifolia and Escohedia linearis^^^. Most of the pigment is contained in the roots, but some is also present in the stalks. Under the name of "azafran" or "azafranillo", azafrin is used in Paraguay for the colouring of fats. Preparation}^^ The finely ground material is extracted with benzene or chloroform in an extrac- tion apparatus, the solution is strongly concentrated and set aside in the cold. After about 24 hours the red pigment crystallises out. It is dissolved in 0.1 N alcoholic potassium hydroxide, the solution is filtered, and the pigment is preci- pitated with dilute acetic acid and recrystallised from toluene. From 3 kg of azafran, 7.5 g of pure pigment is obtained. References p. 2go-2g4. 282 CAROTENOID CARBOXYLIC ACIDS XIII According to a more recent procedure due to Kuhn and Winterstein^^^, the escobedia roots are first roughly ground, then finely ground in a spherical mill and exhaustively extracted with acetone in a Soxhlet apparatus. The dark brown solution is allowed to stand overnight, filtered to separate gelatinous material, and evaporated to a small volume in vacuum. The liquid residue soon solidifies to a crystalline mass which is filtered after addition of toluene. For further purification, the crude pigment is twice recrystallised from a mixture of acetone and toluene. Azafrin can also be purified by chromatographic adsorption on calcium carbonate from a petrol-benzene mixture. A less wasteful procedure consists of extracting the impurities with petrol from an alkaline solution of azafrin. Chemical Constitution \V C OH CH3 CH3 CHs CH2 C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- COOH I I CH2 C'CH3 \ / \ Azafrin^^* CHa OH The formula of azafrin was proposed by Kuhn and co-workers and is proved by the following facts.' Azafrin is a monocyclic carboxylic acid containing 7 double bonds which must be conjugated amongst themselves and with the carboxyl group^^^. The other two oxygen atoms are present in the form of 2 hydroxyl groups. These are tertiary in nature and must occupy neighbouring positions since on treatment with lead tetraacetate according to Criegee^", tetradecahydroazafrin yields a diketone (perhydroazafrinone)^^^: CHq CHo \V C Cxia CHo Clio /\ \ \ I CHj CO- CH2CH2CHCH2CH2CH2CHCH2CH2CH2CH2CHCH2CH2COOH CH, CO-CH, Perhydroazafrinone CH2 On careful oxidation with chromic acid, azafrin yields a diketone which was termed azafrinone by Kuhn and Deutsch^^^. Since this compound is optically inactive, it can be concluded that the rotatory power of azafrin is due to the two hydroxyl-bearing carbon atoms. Azafrinone absorbs at slightly longer wavelengths than azafrin. As the difference is of about the same magnitude as between ^-carotene and ^-semicarotenone (p. 139), it may be concluded that one of the carbonyl groups of azafrinone is in conjugation with the system of conjugated double bonds. References p. 2go-2g4. 3 AZAFRIN 283 CHo CH^ \V C CH3 CH3 CH3 /\ I I I CH2 CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- COOH CHis CO-CHa \ / Azafrinone CH2 On ozonisation azafrin yields a : a-dimethylglutaric acid and geronic acid. Chromic acid oxidation shows the presence of four side-chain methyl groups, the position of which follows from the nature of the three products obtained on thermal decomposition, namely w-xylene, toluene and w-toluic acid^^'. CHo CHo \/ C OH CH3 CH3 CH3 CH, C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- COOH CHj C-CHg Azafrin \/\ H3C CH2 OH 1 \ ^ Y CH— CH C CH y \ y V a :a-dimethyl- H3C •C CH CH C-COOH glutaric acid \ / \ y (p. 132) and geronic CH=C CH=CH acid (p. 132) \ m-toluic acid m-3i ylene CH3 The formula of azafrin was further confirmed by Kuhn and Brockmann by the conversion of azafrin into anhydroazafrinone amide which has also been obtained from /3-carotene (cf. p. 134), thus proving the relation between the two pigments^^". Chromic oxidation of azafrin yields azafrinone, which is converted via the acid chloride into azafrinone amide : CHq OHq \V C CH-i CHo CHo y\ 1 1 1 CHa CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CONHj CH-j C0-CH3 \ / Azafrinone amide CH2 By the action of potassium hydroxide, azafrinone amide is converted into anhydroazafrinone amide. CHo CHo * \/ /\ r I I CHj C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CONH,, CHj — C- CO- CHj Anhydroazafrinone amide References p. 2go-2g4. 284 CAROTENOID CARBOXYLIC ACIDS XIII Properties Crystalline form: Azafrin crystallises from benzene in microscopic orange- red needles combined into clusters. From toluene it crystallises in prisms. Melting point: 212-214° (corr.). Solubility: The pigment is insoluble in water, but dissolves in dilute alkali or alkali carbonate solutions. Azafrin is fairly soluble in chloroform, ethanol, acetic acid and benzene, but only very sparingly in ether. Solvent Absorption maxima Chloroform 458 428 m/z Pyridine 458 428 m/i Sodium hydroxide 447 422 m/z Optical activity: [a-Y^^^^^ = — 75° (in ethanol, c = 0.28). The optical rotation is not appreciably altered by the addition of boric acid^^^. Colour reactions*: Azafrin dissolves in concentrated sulphuric acid with an intense blue colour which changes to violet on addition of alcohol. On dissolving the pigment in acetic acid and adding concentrated hydrochloric acid, a violet colouration is produced after short boiling or on standing for several hours. On passing hydrogen chloride into a saturated chloroform solution, the latter assumes a cornflower-blue colour. Antimony trichloride in chloroform solution produces an emerald green colouration which changes into blue. Partition test: Azafrin exhibits entirely hypophasic properties. Chromatographic behaviour: Azafrin can be chromatographed on calcium carbonate from benzene-petrol solution. The chromatogram is developed with benzene. A mixture of benzene and methanol, or of pyridine and methanol is used for elution^^^. Derivatives Azafrinone €371^3304 (Formula p. 283): KuHN and Deutsch^^^ obtained azafrinone by the oxidation of azafrin in acetic acid and benzene with o.i N chromic acid solution. The diketone can also be prepared by the saponification of azafrinone methyl ester^®^. It separates from acetone in orange-red plates or needles, m.p. 191° (corr.). On catalytic hydrogenation, azafrinone absorbs 9 mols of hydrogen. Azafrinone is optically inactive. * R. KuHN, A. WiNTERSTEiN and H. Roth compare the colour reactions of azafrin, crocetin and norbixin, Ber. 64 (1931) 333. References p. 2go-2g4. 3 AZAFRIN 285 Solvent Absorption maxima Carbon disulphide 483 452 m^ Chloroform 472 440 m^ Petrol 454 429 m/z Azafrinone m.onoxim.e C27H37O4N: CHo CHi \/ C CH3 GS3 CHo /\ I I I CH2 CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- COOH CH, C=NOH \ / \ Azafrinone monoxime CH2 CH3 Like semi-j8-carotenone, azafrinone only forms a monoxime. It separates from acetone in crystals, m.p. 194° (corr.)^^^. A zafrinone methyl ester CgsHjgO^ : CHo CHo A r r r CH3 CO- CH=CH- C=CHCH=CH- C=CHCH=CHGH=C- CH=CH- COOH. I CH, CO-CH, Azafrinone methyl ester CHa This compound is obtained by the esterification of azafrinone suspended in ether with diazomethane^^^, or by the oxidation of azafrin methyl ester with chromic acid^®'*. The ester crystallises from petrol in red needles, m.p. 112° (corr.). It is sparingly soluble in hexane, somewhat more soluble in ethanol, ether and acetic acid, and easily soluble in chloroform, acetone, benzene, and pyridine. The chromatographic behaviour of azafrinone methyl ester is similar to that of azafrin methyl ester. Solvent Absorption maxima Carbon disulphide 483 452 m^ Chloroform 472 440 va.fi Petrol 454 429 m/i Azafrinone amide C27H37O3N: CH, CHo (of. Fig. 23, p. 357) C CHs CH3 CH3 CH2 CO- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- CO- NH^ CHa CO-CHs Azafrinone amide CH, References p. 2go-2g4. 286 CAROTENOID CARBOXYLIC ACIDS XIII KuHN and Brockmann^^^ converted azafrinone into the acid chloride by means of thionyl chloride and allowed the acid chloride to stand in benzene solution with ammonia. The reaction product was purified by chromatography on a calcium carbonate column from benzene solution. The amide crystallises from methanol in red needles, m.p. 177-178°. It is sparingly soluble in hexane, petroleum ether and petrol, but readily soluble in chloroform, carbon disulphide and hot benzene or methanol. Anhydroazafrinone C27H34O3: CHo CHo \V /\ I r I CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CH-COOH CH2 — C-CO-CHa Anhydroazafrinone Anhydroazafrinone is obtained by the action of potassium hydroxide on azafrinone^^^. It crystallises from dilute methanol in dark red prisms, m.p. 196°. The solubility in hexane, petroleum ether and petrol is very small; it is some- what better in chloroform, carbon disulphide, hot benzene or hot methanol. Solvent Absorption maxima Carbon disulphide 511 476 447 m^ Hexane 478 449 420 m/i Benzene 493 460 430 m// Petroleum ether 477 447 419 m/i Chloroform 493 459 433 m/z Ethanol (479) (449) m/< (diffuse) Anhydroazafrinone ni'thyl ester C28H3g03^'' : This ester is prepared by the esterification of anhydroazafrinone with diazo- methane. It separates from dilute methanol in crystals, m.p. 153°. Solvent Absorption maxima Petrol 479 448 420 m// Anhydroazafrinone oxime methyl ester C^^Yi^^O^'^^'' : CHo CHo \V C CHo CH3 CH« /\ I I I CH» C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- COOCH, r II CHa— C-C(CH3)=N0H Anhydroazafrinone oxime methyl ester is obtained by treating anhydro- azafrinone methyl ester with excess hydroxylamine and a little alkali. For References p. 2go-2g4. 3 AZAFRIN 287 purification, the reaction mixture is chromatographed on alumina from benzene solution. The derivative crystallises from dilute methanol in dark red leaflets with a violet lustre. M.p. 149-150°. Solvent Absorption maxima Petrol 476 447 420 m/^ Anhydroazafrinone amide C2-jii2b^2^-^^^ The preparation of anhydroazafrinone amide from /3-carotene or azafrinone has already been described (pp. 134 and 283). The amide crystallises from methanol in red-violet needles, m.p. 215°. It is sparingly soluble in hexane, petroleum ether and petrol, but somewhat more easily soluble in chloroform and hot benzene. Solvent Absorption maxima Carbon disulphide 508 474 444 vn.(x Hexane 475 444 419 m^ Petroleum ether 473 443 418 m/^ Benzene 477 447 420 vafi Chloroform 492 459 430 mn Ethanol (481) (450) mjx Perhydroazafri?i C27H52O4 : Perhydroazafrin can be obtained either by the catalytic hydrogenation of azafrin^^^ or by the saponification of perhydroazafrin methyl ester^^". It is a colourless, viscous oil which can be distilled in high vacuum. Optical rotation in ethanol.- [a]^ = —6.7° (c = 3.2)169. Apo-i-azafrinal CgjHjgOg: \/ C OH CH, CH, CH, /\/ I I I CHg C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CHO I I CHo C'CHj ^ Apo-i-azafrinal CHj OH This aldehyde was obtained by Karrer, Obst and Solmssen"^ by the careful permanganate oxidation of azafrin. It crystallises from benzene in orange-yellow needles, m.p. 17.1°. Solvent Absorption maxima Carbon disulphide 461 m/z Petroleum ether 431 m// Ethanol diffuse References p. 2go-2g4. 288 CAROTENOID CARBOXYLIC ACIDS XIII Apo-i-azafrinal oxime C25H3703N^'^: Melting point: 185°. Solvent Absorption maxima Carbon disulphide 445 416 m/x Petroleum ether 415 m/j, Ethanol 423 m/j, Azafrin methyl ester (methylazafrin) C2s^-i:)Oi^^'- CHo CH« \V C OH CH, CH, CH, /\/ I I I CH2 C- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CH- COOCH3 CHg C'CH» ^ Azafrin methyl ester CHj OH This ester is prepared by the esterification of azafrin with dimethylsulphate and potassium hydroxide. The ester crystalhses from methanol, or from a mixture of methanol and ether or acetic acid, in yellow-red leaflets or needles, m.p. 191°. It is very readily soluble in chloroform, and easily soluble in all organic solvents with the exception of petroleum ether and ligroin. Optical rotation in chloroform We^s-s = -32°. Solvent Absorption maxima Petrol 447 442.5 m/z Chloroform 458 428 m/i Carbon disulphide 476 445.5 419 m// (cf. Fig. 23, p. 357) With hydrogen chloride, hydrogen bromide, hydrogen iodide, perchloric acid, sulphuric acid or trichloracetic acid in acetic acid solution, azafrin methyl ester gives coloured addition products from which it cannot be regenerated^''^. With methyl magnesium iodide, 2 mols of methane are evolved^'^^. In biological tests methylazafrin exhibits no vitamin A activity^''^. j:8-Di7nethylnndecapentaene-ii-al-i-carboxylic acid methyl ester ("azafrinal I" methyl ester) C^^H-^gO^ (I) : 0CH-CH=CH-C=CHCH=CHCH=C-CH=CH-C00CH3 CH3 CH3 I This ester is formed besides other products by the chromic acid oxidation of methylazafrin"^. References p. 290-294. 3 AZAFRIN 289 It crystallises from dilute methanol in light yellow needles, m.p. 159-160°, It is readily soluble in chloroform, ethanol, carbon disulphide and benzene, and somewhat less readily soluble in petrol, petroleum ether and hexane. Solvent Absorption maxima Carbon disulphide 421 m// Chloroform 411 m/^ Benzene 410 m^ The oxime is obtained from the aldehyde on standing with hydroxylamine in ethanol. It crystallises from dilute methanol in yellow needles with a blue surface lustre. Melting point : 206-207°. Solvent Absorption maxitna Carbon disulphide 425 nifi Chloroform 413 m/i Benzene 412 m/x j:8-Dimethyldccapentaeve-i:io-dicarboxvlic acid CnH^^O^ (II) : The oxime described above is converted to the corresponding nitrile by boiling with acetic anhydride^'^. Hydrolysis of the nitrile yields the acid II. CH3 CHo I I HOOC-CH=CH-C=CHCH=CHCH=C-CH=CH-COOH II It forms yellow needles, m.p. 267-268°. It is insoluble in hexane, petroleum ether and petrol, sparingly soluble in chloroform, ethanol, carbon disulphide and benzene, and readily soluble in pyridine. Solvent Absorption maxima Carbon disulphide 419 m/z The potassium salt forms pale yellow leaflets. Dimethyl ester C^^Vi^rf)^: Formed by the esterification of the acid II with diazomethane^^^. Melting point: 175-176°. Solvent Absorption maxima Carbon disulphide 419 m/i Methyl ester nitrile C^jHj^^OjN : The preparation of this nitrile is described under 3 : 8-dimethyldecapentaene- I : lo-dicarboxylic acid. Golden yellow prisms from dilute methanol. Melting References p. 2go-2g4. Carotenoids 19 290 CAROTENOID CARBOXYLIC ACIDS XIII point 165° "'''. The nitrile is readily soluble in chloroform, benzene and hot methanol and sparingly soluble in cold methanol, petrol and petroleum ether. Solvent Absorption maxima Carbon disulphide 413 vn.^ Mono-amide C-^^^iriy^O^ : The amide is formed by boiling the methyl ester nitrile with potassium hydroxide"''. It crystallises from methanol in yellow prisms, m.p. 256-257°. 2:y-Dimethylnonatetraen-i-al-g-carboxylic acid methyl ester ("azafrinal-II" methyl est^r) QaHieOj (III) : CH3 ^113 OCH- C=CHCH=CHCH=C- CH=CH- COOCH3 III This compound is formed besides other products during the chromic acid oxidation of azafrin methyl ester"^. It is purified by repeated chromatography on alumina. It crystallises from 70 % methanol in light yellow prisms, m.p. 106°. It forms an oxime, CigHj^OgN, which crystallises from dilute methanol in light yellow prisms, m.p. 194° "^. Perhydroazafrin methyl 'ster C28H54O4 : KuHN, WiNTERSTEiN and RoTH^^ prepared this perhydroester by the catalytic hydrogenation of azafrin methyl ester. Colourless, viscous oil, b.p. i8o-200°/i mm. [a]^° = —9.0° (in ethanol). Azafrin ethyl ester C29H42O4 : Azafrin ethyl ester is obtained by esterification of azafrin with diethyl sulphate^^". It crystallises from ethanol in red prisms, m.p. 182° (corr., with decomposition) . REFERENCES 1. J. B. BoussiNGAULT, Ami. Chim. (2) 28 (1825) 440. 2. Cf. Wehmer, Pftanzenstoffe, 2nd edition, vol. 2, 796. — F. Czapek, Biochemie, 2nd ed. vol. 3, 576. — Karnot, Dissertation, Leipzig 1849; Jahresber. 1849, 457. — K. G. ZwicK, Ber. 30 (1897) 1972. — L. Marchlewski and L. Matejko, Anz. Akad. Wiss. Krakau, 1905, 745. — J. F. B. van Hasselt, Chem. Weekbl. 6 (1909) 480; Rec. 30 (191 1) 1,33 (1914) 192. — A. HEiDUSCHKAand H. Riffart, Arch. Pharm. 249 (igii) 4^. 3. C. Etti, Ber. 11 (1878) 864. 4. A. Heiduschka and A. Panzer, Ber. 50 (1917) 546 and 1525. 5. R. KuHN and co-workers, Helv. chim. Acta 11 (1928) 427; 12 (1929) 64; Ber. 64 (1931) 1732; Helv. chim. Acta 12 (1929) 904; Ber. 65 (1932) 646; 65 (1932) 1873. 6. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1218, 1399. 7. Cf. L. Zechmeister, Carotinoide, Berlin 1934. 8. A. Heiduschka and A. Panzer, Ber. 50 (1917) 546, 1525. REFERENCES 291 9. R. KuHN and A. Winterstein, Ber. 65 (1932) 646. 10. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1218, 1399. 11. J. Herzig and F. Faltis, Ann. 431 (1923) 40. 12. I. J. RiNKES, Chem. Weekbl. 12 (1915) 996 and I. J. Rinkes and J. F. B. van Hasselt, Chem. Weekbl. 13 (1916) 436, 1224, 14 (1917) 888. 13. J. F. B. VAN Hasselt, Chem. Weekbl. 6 (1909) 480. 14. J. Herzig and F. Faltis, Monatsh. 35 (1914) 997; Ber. 50 (1917) 927; Ann. 431 (1923) 40. 15. R. Kuhn, a. Winterstein and L. Karlovitz, Helv. chim. Acta 12 (1929) 64. — R. KuHN and F. L'Orsa, Ber. 64 (1931) i732- 16. I. J. Rinkes, Rec. 47 (1928) 934; 48 (1929) 603; 48 (1929) 1093- 17. R. Kuhn and A. Winterstein, Ber. 65 (1932) 646. — Cf. R. Kuhn and L. Ehmann, Helv. chim. Acta 12 (1929) 904. 18. P. Karrer, F. Benz, R. More, H. Raudnitz, M. Stoll and T. Takahashi, Helv. chim. Acta 15 (1932) 1218, 1399. 19. H. Raudnitz and J. Peschel, Ber. 66 (1933) 901. 20. P. Karrer and F. Benz, Helv. chim. Acta 16 (1933) 337. 21. R. Kuhn and A. Winterstein, Ber. 65 (1932) 1873. 22. J. Herzig and F. Faltis, Ann. 431 (1923) 40. 23. P. Karrer, A. Helfenstein, R. Widmer and T. B. van Itallie, Helv. chim. Acta 12 (1929) 741- 24. Cf. R. Kuhn and A. Winterstein, Ber. 65 (1932) 646; 66 (1933) 209. 25. Cf. O. Walker, Dissertation, Zurich 1935. 26. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) ^i9^- 27. L. Zechmeister and R. B. Escue, Science g6 (1942) 229; /. Am. Chem. Soc. 66 (1944) 322. 28. P. Karrer and co-workers, Helv. chim. Ada 12 (1929) 753. 29. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 754. Cf. Ref. 30. 30. R. Kuhn and A. Winterstein, Ber. 65 (1932) 650. 31. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 754. Cf. also R. Kuhn and A. Winterstein, Ber. 65 (1932) 650. 32. R. Kuhn and P. J. Drumm, Ber. 65 (1932) 1459. — R. Kuhn and co-workers, 65 (1932) 1785. 33. K. W. Hausser and A. Smakula, Z. angew. Chem. 47 (1934) 663; A. Smakula, 48 (1935) 152. 34. K. W. Hausser, R. Kuhn and E. Kuhn, Z. physik. Chem. B 2g (1935) 452. 35. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) 1396. 36. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 752. — Cf. J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (191 1) 6. 37. P. Karrer and T. Takahashi, Helv. chim. Acta 16 (1933) 287. 38. P. Herzig and F. Faltis, Ann. 431 (1923) 60. . 39. J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (1911) n. 40. A. Heiduschka and H. Riffart, Arch. Pharm. 24^ (1911) 47. 41. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 752. — Cf. J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (191 1) 6. 42. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 746, 754. — Cf. P. Karrer and F. Rubel, Helv. chim. Acta ly (1934) 773- 43. J. Herzig and F. Faltis, Ann. 431 (1923) 51. 44. P. Karrer and F. Benz, Helv. chim. Acta 16 (1933) 337- 45. R. Kuhn and L. Ehmann, Helv. chim. Acta 12 (1929) 905. 46. J. Herzig and F. Faltis, Ann. 431 {1923) 51. 47. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1409. 48. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1410. 49. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1411. 50. H. Raudnitz and J. Peschel, Ber. 66 (1933) 901. 292 CAROTENOID CARBOXYLIC ACIDS XIII 51. R. KuHN and L. Ehmann, Helv. chim. Acta 12 (1929) 904. 52. J. F. B. VAN Hasselt, Rec. Trav. chim. Pays-Bas 30 (191 1) 31. 53. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 754. — Cf. R. Kuhn and A. WiNTERSTEiN, Ber. 65 (1932) 650. 54. R. PuMMERER, L. Rebmann and W. Reindel, Ber. 62 (1929) 1417. 55. F. Viebock, Ber. 6y (1934) 377- 56. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1417. 57. A. Heiduschka and H. Riffart, Arch. Pharm. 249 (191 1) 43. — J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (191 1) 26. 58'. C. Liebermann and G. Muhle, Ber. 48 (1915) 1657. 59. A. Heiduschka and H. Riffart, Arch. Pharm. 24g (1911) 43. 60. J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (191 1) 17. 61. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 750. 62. B. V. EuLER, H. v. EuLER and P. Karrer, Helv. chim. Acta 12 (1929) 278. 63. R. Kuhn and co-workers, Helv. chim. Acta 11 (1928) 723. 64. L. MARCHLEWSKiandL. Matejko.^w^. Akad.Wiss.Krakau.igo^, 749 (C. 1906,11, 1265). 65. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 748, 755. 66. R. Kuhn and A. Winterstein, Ber. 65 (1932) 650. 67. J. Herzig and F. Faltis, Ann. 431 (1923) 49. — Cf. R. Kuhn and co-workers, Helv. chim. Acta 11 (1928) 723; 12 (1929) 910. 68. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 749. 69. J. F. B. VAN Hasselt, Rec. Trav. chim. Pays-Bas 30 (191 1) 8. 70. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 751. 71. J. Herzig and F. Faltis, Ann. 431 (1923) 61. 72. K. W. Hausser and A. Smakula, Z. angew. Chem. 4j (1934) 662; A. Smakula, Z. angew. Chem. 48 (1935) I52- 73. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 753. — R. Kuhn and A. Winterstein, Ber. 65 (1932) 650. 74. I. J. Rinkes, Chem. Centr. 1916, I, 336. — I. J. Rinkes and J. F. B. van Hasselt, Chem. Centr. igiy, I, 208, II, 680. — R. Kuhn, A. Winterstein and L. Karlovitz, Helv. chim. Acta 12 (1929) 66. — I. J. Rinkes and J. F. B. van Hasselt, Chem. Centr. 1916, II, 390; Chem. Centr. igiy, I, 208, II, 680. — I. J. Rinkes, Rec. Trav. chim. Pays-Bas 48 (1929) 1093. 75. R. Kuhn and A. Winterstein, Ber. 65 (1932) 650. 76. P. Karrer and T. Takahashi, Helv. chim. Acta 16 (1933) 288. 77. R. Kuhn and A. Winterstein, Ber. 63 (1932) 650. 78. R. Kuhn and co-workers, Ber. 65 (1932) 1459, 1785. 79. J. Herzig and F. Faltis, Ann. 431 (1923) 48. 80. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 751. 81. R. Kuhn and L. Ehmann, Helv. chim. Acta 12 (1929) 905. 82. R. Kuhn and L. Ehmann, Helv. chim. Acta 12 (1929) 905. — P. Karrer and co- workers, Helv. chim. Acta 15 (1932) 1406. 83. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1404. 84. F. Faltis and F. Viebock, Ber. 62 (1929) 706. — P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 750; 75 (1932) 1416. 85. P. Karrer and co-workers, Helv. chim. Acta 75 (1932) 141 7. 86. J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (1911) 13. 87. J. F. B. van Hasselt, Chem. Weekbl. 6 (1909) 482; Rec. Trav. chim. Pays-Bas 30 (1911) 14. 88. J. F. B. van Hasselt, Rec. Trav. chim. Pays-Bas 30 (1911) 13. 89. A. Oswald, Dissertation, Ziirich 1939. 90. P. Karrer and F. Rubel, Helv. chim. Acta ly (1934) 773. 91. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) 1396. 92. P. Karrer and U. Solmssen, Helv. chim. Acta 20 (1937) i396- 93. Aschoff, Ber. Jb. 51 (1818) 142. REFERENCES 293 94. B. Quadrat, /. prakt. Chem. 36 (1852) 68. — F. Rochleder, /. praki. Chem. 74 (1858) I. — B. Weiss, /. prakt. Chem. loi (1867) 65. — R. Kayser, Ber. ly (1884) 2228. — E. Fischer, Ber. 21 (1888) 988. — E. Schunck and L. Marchlewski, Ann. 2y8 (1894) 349. — Pfyhl and Scherz, Z. Unters. d. Genussmittel 16 (1906) 237. — F. Decker, Arch. Pharm. 252 (1914) I39- 95. F. Decker ,Arch. Pharm. 252 (1914) 139. 96. P. Karrer and H. Salomon, Helv. chim. Acta 10 (1927) 397; ^i (1928) 513; 11 (1928) 711; 16 (1933) 643- 97. P. Karrer and co-workers, Helv. chim. Acta 12 (1929) 985; I3 (1930) 392; 15 (1932) 1218; 1399; 16 (1933) 297- 98. Cf. R. KuHN and co-workers, Helv. chim. Acta 11 (1928) 716; 12 (1929) 64; Ber. 64 (1931) 1732. 99. P. Karrer and K. Miki, Helv. chim. Acta 12 (1929) 985. 100. E. Winterstein and J. Teleczky, Helv. chim. Acta 5 (1922) 376. — R. Kuhn and A. Winterstein, Naturwissenschaften, 21 (1933) 527; •Be>'. 67 (1934) 344. loi. R. Kuhn and A. Winterstein, Ber. 66 (1933) 209; 67 (1934) 344- — Cf. also Ber. 65 (1932) 1785. 102. Cf. O. Walker, Dissertation Ziirich igj^. — R. Kuhn and A. Winterstein, Ben 66 {1933) 209. 103. P. Karrer and H. Salomon, Helv. chim. Acta 11 (1928) 513. — Cf. R. Kuhn and A. Winterstein, Ber. 67 (1934) 344. 104. P. Karrer and co-workers, //e/f. cAwz. y^c^a jo (1927) 397; jj (1928) 513; Jj (1930) 392. 105. R. Kuhn and co-workers, Helv. chim. Acta 11 (1928) 716. 106. P. Karrer and A. Helfenstein, Helv. chim. Acta 13 (1930) 392. 107. P. Karrer and H. Salomon, Helv. chim. Acta 11 (1928) 513. 108. R. KuKN and F. L'Orsa, Ber. 64 (1931) 1732; Z. angew. Chem. 44 (1931) 847. 109. R. Kuhn and F. L'Orsa, Ber. 64 (1931) 1732; Z. angew. Chem. 44 (1931) 847. no. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1399. 111. P. Karrer and co-workers, Helv. chim. Acta 16 (1933) 297. 112. P. Karrer, F. Benz and M. Stoll, Helv. chim. Acta 16 (1933) 297. 113. A. G. Perkin, /. Chem. Soc. loi (1912) 1541. 114. R. Kuhn and K. Meyer, Z. physiol. Chem. 185 (1929) 193. 115. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 42. 116. O. Tunmann, Chem. Centr. igi6, II, 279; Apoth.-Ztg. 31 (1916) 237. 117. F. Decker, Arch. Pharm. 252 (1914) 147. — R. Kuhn and co-workers, Helv. chim. Acta II (1928) 722. 118. P. Karrer and H. Salomon, Helv. chim. Acta 10 (1927) 402. 119. P. Karrer and H. Salomon, Helv. chim. Acta 11 (1928) 513. 120. P. Karrer and K. Miki, Helv. chim. Acta 12 (1929) 985. 121. P. Karrer and A. Helfenstein, Helv. chim. Acta 13 (1930) 392. 122. P. Karrer and A. Helfenstein, Helv. chim. Acta 13 (1930) 396. — P. Karrer and H. Salomon, Helv. chim. Acta 10 (1927) 397. 123. P. Karrer and co-workers, Helv. chim. Acta 15 (1932) 1418. 124. R. Kuhn and F. L'Orsa, Ber. 64 (1931) 1732. 125. R. Kuhn and A. Winterstein, Ber. 65 (1932) 1876; 66 (1933) i733- 126. Quantitative extinction measurements: K. W. Hausser and A. Smakula, Z. angew. Chem. 47 (1934) 663; 48 (1935) 152. — K. W. Hausser, Z. techn. phys. 15 (1934) 13. — P. Karrer and H. Salomon, Helv. chim. Acta 11 (1928) 516. 127. R. Kuhn and A. Winterstein, Ber. 66 (1933) 209; 67 (1934) 348. 128. R. Kuhn and A. Winterstein, Ber. 66 (1933) ^737- 129. P. Karrer and H. Salomon, Helv. chim. Acta 11 (1928) 515, 524. 130. R. Kuhn and F. L'Orsa, Ber. 64 (1931) 1735. 131. P. Karrer and H. Salomon, Helv. chim. Acta 11 (1928) 515, 524. 132. P. Karrer and T. Golde. Helv. chim. Acta 13 (1930) 707; cf. P. Karrer and co- workers, Helv. chim. Acta 15 (1932) 1406. 294 CAROTENOID CARBOXYLIC ACIDS XIII 133. P. Karrer and F. Benz, Helv. chim. Acta 16 (1933) 337. 134. P. Karrer and T. Golde, Helv. chim. Acta 13 (1930) 707; cf. P. Karrer and co- workers, Helv. chim. Acta 15 (1932) 1406. 135. P. Karrer , A. Helfenstein and R. Widmer, Helv. chim. Acta 11 (1928) 1207. 136. K. W. Hausser and A. Smakula, Z. angew. Chem. 47 (1934) 663; 48 (1935) 152. 137. P. Karrer and A. Helfenstein, Helv. chim. Acta 13 (1930) 392. 138. R. Kuhn and P. J. Drumm, Ber. 65 (1932) 1459, ref. 6. 139. R. Kuhn and A. Winterstein, Ber. 66 (1933) 209. 140. R. Kuhn and co-workers, Ber. 63 (1932) 1785. 141. P. Karrer and A. Helfenstein, Helv. chim. Acta 13 (1930) 397. 142. P. Karrer and co-workers, Helv. chim Acta 11 (1928) 1207. 143. R. Kuhn and F. L'Orsa, Ber. 64 (1931) 1735. 144. H. Raudnitz and J. Peschel, Ber. 66 (1933) 901. 145. P. Karrer, F. Benz and M. Stoll, Helv. chim. Acta 16 (1933) 297. 146. P. Karrer and co-workers, Helv. chim,. Acta 15 (1932) 1408. 147. F. Decker, Arch. Pharm. 252 (1914) 155. 148. Dragendorf, Heilpflanzen 608, 1885. 149. C. Liebermann, Ber. 44 (1911) 850. 150. C. Liebermann and W. Schiller, Ber. 46 (1913) 1973. 151. C. Liebermann and G. Muhle, Ber. 48 (1915) 1653. 152. R. Kuhn and A. Deutsch, Ber. 66 (1933) 883. — . R. Kuhn and H. Brockmann, Ber. 67 (1934) 885. Ann. 516 (1935) 104. 153. Cf. Y. Takeda and T. Ohta, Z. physiol. Chem. 258 (1939) 6. 154. R. Kuhn and co-workers, Ber. 64 (1931) 333; 65 (1932) 1873. 155. R. Kuhn and A. Deutsch, Ber. 66 (1933) 883. 156. R. Kuhn, A. Winterstein and H. Roth, Ber. 64 (1931) 333. 157. R. Criegee, Ber. 64 (1931) 260. 158. R. Kuhn and A. Deutsch, Ber. 66 (1933) 883. 159. R. Kuhn and A. Winterstein, Ber. 65 (1932) 1873. 160. R. Kuhn and H. Brockmann, Ann. 516 (1935) 95. 161. R. Kuhn and A. Deutsch, Ber. 66 (1933) 883. 162. R. Kuhn and A. Deutsch, Ber. 66 (1933) 883. 163. R. Kuhn and A. Deutsch, Ber. 66 (1933) 891. — Cf. R. Kuhn and H. Brockmann, Ann. 516 (1935) 131- 164. R. Kuhn and A. Deutsch, Ber. 66 (1933) 891. — Cf. R. Kuhn and H. Brockmann, Ann. 516 (1935) 131- 165. R. Kuhn and H. Brockmann, Ber. 6y (1934) 885; Ann. 516 (1935) 132. 166. R. Kuhn and H. Brockmann, Ann. 516 (1935) i3i- 167. R. Kuhn and H. Brockmann, Ann. 516 (1935) 132. 168. R. Kuhn and H. Brockmann, Ann. 516 (1935) 133. 169. R. Kuhn, A. Winterstein and H. Roth, Ber. 64 (1931) 340. — C. Liebermann and G. Muhle, Ber. 48 (1915) 1653. 170. R. Kuhn and A. Deutsch, Ber. 66 (1933) 890. 171. P. Karrer, H. Obst and U. Solmssen, Helv. chim. Acta 21 (1938) 451. 172. C. Liebermann and W. Schiller, Ber. 46 (1913) 1977. — R. Kuhn and co-workers, Ber. 64 (1931) 338. 173. R. Kuhn, A. Winterstein and H. Roth, Ber. 64 (1931) 333. 174. R. Kuhn and H. Brockmann, Z. physiol. Chem. 221 (1933) 133. 175. R. Kuhn and H. Brockmann, Ann. 516 (1935) 104, 134. 176. R. Kuhn and H. Brockmann, Ann. 516 (1935) 136-137. 177. R. Kuhn and H. Brockmann, Ann. 516 (1935) 136-137. 178. R. Kuhn and H. Brockmann, Ann. 516 (1935) 139. 179. R. Kuhn ,A. Winterstein and H. Roth, Ber. 64 (1931) 340. — Cf. C. Liebermann and G. Muhle, Ber. 48 (1916) 1653. 180. R. Kuhn, A. Winterstein and H. Roth, Ber. 64 (193 1) 339. CHAPTER XIV Carotenoids of partly or completely unknown structure I. RHODOVIOLASCIN C42HgQ02 History 1873 Lankester^ is the first to investigate the pigments of purple bacteria, subsequent^ studied by other workers^. 1905 Archichovskji^ succeeds in separating a green pigment not identical with chlorophyll from the red pigments of purple bacteria. 1907 MoLiscH* carries out a detailed investigation of the red pigments. 1935-40 Karrer and co-workers^ investigate the pigment mixture from rhodovibrio-bacteria and thiocystis-bacteria. They isolate a number of different polyene pigments and partly elucidate the constitution of rhodoviolascin. Occurrence Rhodoviolascin has hitherto been found only in rhodovibrio-bacteria^ and in thiocystis-bacteria^. (According to Zechmeister and co-workers', spirillo- xanthin from rhodospirillum rubrum^ is identical with rhodoviolascin) . Preparation^- ^° For the method of growing rhodovibrio-bacteria, compare the communication by Karrer and Solmssen*. The bacteria are dehydrated with ethanol and ex- haustively extracted with carbon disulphide. After removal of the solvent by distillation, an almost black residue remains which still contains much elementary sulphur. (The latter is formed by reduction processes during the growth of the bacteria in the magnesium sulphate-containing nutrient solution). The black residue is dissolved in a ligroin-methanol mixture, the solution is decanted from sulphur and diluted with a little water. Rhodoviolascin is precipitated as a dark- red crystalline powder at the boundary between the ligroin and methanol. It is filtered off and sealed in evacuated ampoules. The ligroin solution, which contains the other carotenoids and also some more rhodoviolascin, is extracted several times with methanol to remove the bacterio-chlorophyll, washed with water, dried over sodium sulphate and concentrated by distillation. The residue is dissolved in a little References p. 341-343. 296 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV benzene and chromatographed on calcium hydroxide. The chromatogram is first developed with a mixture of benzene and petroleum ether and then with petroleum ether alone. The salmon-red zone of rhodoviolascin is eluted with a mixture of methanol and benzene, the solvent is removed by distillation and the remaining pigment is combined with the crystalline rhodoviolascin (see above). The following pigments were obtained from the other chromatogram zones : rhodovibrin, rhodopin, rhodopurpurin, /3-carotene( ?) and flavorhodin. For further purification, the crude rhodoviolascin is chromatographed several times on calcium hydroxide and finally recrystallised from benzene. In order to obtain 0.9 g of pigment, Karrer and Koenig worked up 19,320 1 of ripe bacteria nutrient solution in the course of two years. Chemical Constitution CHo CHo CHq CHa CH CHo CHq CHi CHo CH / I I I I V CHa CH-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH Cfi^ I II II I HaCO-C C-CHs HjC-C C-OCHs X/ Rhodoviolascin(?) (I) \/' CH CH Karrer and Solmssen^^ proposed a tentative structural formula for rhodoviolascin which was subsequently modified by Karrer and Koenig^^ (Formula I). However, even the modified structure cannot yet be regarded as established with certainty. The molecular formula of rhodoviolascin is C42Hgo02. Methoxy 1 determinations show the presence of two methoxyl groups. On catalytic hydrogenation, 13 moles of hydrogen are taken up. The long- wavelength location of the absorption maxima (573.5, 534, 496 m// in carbon disulphide) shows that all the double bonds must be in conjugation. The pigment does not react with hydroxylamine and yields no dihydro-derivative on treatment with pyridine, acetic acid and zinc. In contrast to carotenoid diketones, it exhibits the same absorption spectrum in petrol and methanol. Karrer and Koenig subjected rhodoviolascin to stepwise degradation with permanganate^^ and obtained at least 6 different oxidation products. From these, bixindialdehyde could be isolated and identified, thus establishing the structure of the central part of the molecule from carbon atom 6 to carbon atom 27. Besides bixindialdehyde, another dialdehyde was obtained in very small yield which was found above bixindialdehyde on the chromatogram and had absorption maxima located at 40 m/* towards longer wavelengths. It must therefore contain 2 conjugated double bonds more than bixindialdehyde. Methoxyl determination gave a negative result and the compound is probably 2 : 6 : 10 : 15 : ig : 23-hexamethyltetracosaundecaene-i : 24-dial (II) : References p. 341-343. 2 RHODOPIN 297 CH- CH=CH- C=CHCH=CH- C=CHCH=CHCH=C- CH=CHCH=C- CH=CH- CH ill I I II 0 C-CH, CH, CH3 CH3 CH3 HaC-C 0 CH CH The formation of this compound from rhodoviolascin is in agreement with formula I. Properties Crystalline form: Rhodoviolascin crystallises from benzene in beautifully- glistening, deep red, spindle-shaped crystals. Melting point: 218°. Solubility: The pigment is almost insoluble in petroleum, ether, ligroin and methanol. It is somewhat more soluble in hot benzene. Partition test: Rhodoviolascin is entirely epiphasic in character. Optical activity: At the high dilution of the pigment solution which is necessary in view of the high colour intensity, no optical rotation was observed. Spectral properties: Solvent Absorption maxima Carbon disulphide 573.5 534 496 m/z Chloroform 544 507 476 m/< Benzene 548 511 482 n\fx Ethanol 526 491 (465)m/< (of. Fig. 25, p. 358) Colour reactions: With antimony trichloride in chloroform solution, a blue colouration is produced with an absorption maximum at 642 m/^. Chromatographic behaviour: Rhodoviolascin can be easily chromatographed on calcium hydroxide from benzene solution. It is adsorbed below rhodopin, but above rhodopurpurin. 2. RHODOPIN C40H58O* Rhodopin was first isolated by Karrer and Solmssen^^ from rhodovibrio- bacteria. Later investigations have shown that this pigment is also present in thiocystis-bacteria^*. For the isolation of rhodopin one proceeds first as in the preparation of rhodo- violascin (p. 295). After the crv^stallisation of the rhodoviolascin, the petroleum ether mother liquors are chromatographed on calcium hydroxide. Rhodopin is eluted with a benzene-methanol mixture from the upper part of the chromatogram. The formula C4oH5gO is also in agreement with the analytical data. References p. 341-343. 298 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV The solvent is removed by distillation and the residue is repeatedly adsorbed on calcium hydroxide. Only in this way is it possible to separate rhodopin from rhodo- violascin and rhodovibrin. Finally the pigment is re-crystallised from a mixture of carbon disulphide and petroleum ether. The structure of rhodopin is not yet definitely known. It has the molecular formula C^oHggO or C4QH56O. Zerewitinoff determinations showed the presence of one active hydrogen atom, but no acetyl derivative of rhodopin could be prepared. Microhydrogenation indicated the presence of 12 double bonds, which are probably all conjugated. No carbonyl group was detected and on reduction of the pigment with zinc dust and acetic acid no change in the spec- trum was observed. Methoxyl determinations gave a negative result. Karrer and KoENiG^^ attempted to obtain some information regarding the structure of rhodopin by oxidative degradation with permanganate. The amount of material available was, however, too small for the identification of the degrada- ation products. Rhodopin crystallises from a mixture of carbon disulphide and petroleum ether in deep red crystals which appear as small clusters of needles and prisms under the microscope. Melting point: 171° (after previous sintering). Rhodopin exhibits epiphasic behaviour on partition between petroleum ether and 90 % methanol. Solvent Absorption maxima Carbon disulphide 547 508 478 m/f Chloroform 521 486 453 mfi Petroleum ether 501 470 440 m// Ethanol (absolute) 505 474 (445)m/i (cf. Fig. 26, p. 358) 3. RHODOVIBRIN During the chromatographic purification of rhodopin on calcium hydroxide, Karrer and Solmssen* obtained another hitherto unknown carotenoid for which they proposed the name rhodovibrin. Rhodovibrin is adsorbed somewhat more strongly than rhodopin on the chromatographic column and can thus be separated from the latter. Repeated chromatographic adsorption is necessary, however, as the two pigments are only separated with difficulty. Rhodovibrin crystallises form a mixture of carbon disulphide and petroleum ether in small deep-red crystal clusters which are almost indistinguishable in appearance from those of rhodopin and melt at 168°. The absorption maxima are located at 556 and 517 m// in carbon disulphide solution. The pigment could not be obtained in a completely pure state as it is only present in very small quantities in The literature is summarised under rhodopin, p. 297; cf. H. Koenig, Dissertation, Zurich, 1940, p. 29. References p. 341-343. 5 FLA VORHODIN 299 rhodovibrio-bacteria, but it appears to have the molecular formula C4oH5g02 or C4(,H5g02. It is improbable that both oxygen atoms are present as hydroxyl groups since rhodovibrin, like rhodopin, is epiphasic in the partition test. Methoxyl determinations also gave a negative result. 4. RHODOPURPURIN Rhodopurpurin occurs in rhodovibrio-bacteria in very small amounts and was isolated from the latter by Karrer and Solmssen* by the procedure described for the preparation of rho^pviolascin and rhodopin (pp. 295 and 297). It is adsorbed below rhodopin in the chromatogram on calcium hydroxide and is isolated by elution, dissolution in petroleum ether and concentration of the solution. The pigment crystallises from petroleum ether in fine microscopic needles which are partly combined in clusters and melt at 161-162°. Ultimate analysis shows that the compound is probably a hydrocarbon of the formula ^40^56 ^^ ^40^58- On partition between methanol and petroleum ether, rhodopurpurin is entirely epiphasic. In its spectral properties it shows great similarity to lycopene ; its identity with the latter is still uncertain, however. Solvent Absorption ynaxima Carbon disulphide 550 511 479 m/< Chloroform 527 487 (458)m// Petroleum ether 502 472 m/t Benzene 527 490 m/i 5. FLAVORHODIN This pigment was also obtained by Karrer and Solmssen* from rhodo- vibrio-bacteria. It is obtained from the lowest zones of the calcium hydroxide- chromatogram in the preparation of rhodopin**. The constitution of flavorhodin is unknown. The pigment is easily soluble in petroleum ether, benzene, chloroform acetone and ether, but only sparingly soluble in ethanol and methanol. It is entirely epiphasic in the partition test. Solvent Absorption maxima Carbon disulphide 503 472 441 m/n Chloroform 482 453 m/z Petroleum ether 470 442 m/x Ethanol 471 443 m/u (cf. Fig. 27, p. 359) In its optical properties, flavorhodin is reminiscent of sarcinin (p. 319), but the question of the identity of these two pigments is still unsettled. For a summary of the literature, see under rhodopin, p. 297. ** Cf. p. 297. References p. 341-J4J. 300 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV 6. APHANIN C40H54O History and Occurrence Although the pigments of blue algae have been repeatedly investigated within recent years, our present knowledge concerning these compounds is still very incomplete. In 1927, Kylin^^ investigated extracts of the blue algae Calotrix scopulorum by capillary-analytical methods, and found, besides carotene, three new pigments. These were not, however, isolated in a pure state and analysed. In 1936, Heilbron and Lythgoe investigated the carotenoids from Oscillatoria ruhescens^^, and reported the presence of j3-carotene and xantho- phyll, as well as of two new pigments, myxoxanthin and myxoxanthophyll (cf. p. 225). Very recently, Karrer and Rutschmann" reinvestigated the polyene pigments from Oscillatoria rubescens and found a previously unknown acidic pigment, oscillaxanthin, besides /3-carotene, zeaxanthin, myxoxanthin and myxoxanthophyll, but no xanthophyll (cf. p. 335). TisCHER^^ investigated the carotenoids of the blue algae Aphanizomenon flos-aquae and was able to isolate four new polyene pigments besides j8-carotene. He proposed the term aphanin, aphanicin, flavacin and aphanizophyll for these pigments. They have so far only been observed in Aphanizomenon flos- aquae. Preparation^^ The algae Aphanizomenon flos-aquae are collected Irom their aqueous suspen- sions by means of nets, freed from impurities and dehydrated with ethanol. The residue, still alcohol-moist, is mixed with sand and ammonium sulphate and ex- tracted at room temperature with ether which has been freshly distilled from sodium. The ether is removed by distillation and the aqueous ethanolic residue is extracted with petroleum ether (Extract A). The petroleum ether-insoluble pigments from the alcoholic layer r re obtained in the form of a red flocculent precipitate b}^ salting out with ammonium sulphate. The alcoholic extracts which remain after the dehydration of the algae also yield a small amount of red pigment, which is combined with that obtained by salting-out and dissolved in pyridine (Extract B). The material remaining after extraction with ether is once more extracted with ethanol at 40° (Extract C). The petroleum ether extract A is adsoibed on alumina and the chromatogram is developed with petrol. The carotenoids are adsorbed on the column in the following downward sequence: 1. aphanicin, 2. aphanin, 3. flavacin and 4. /3-caro- tene*. a) Aphanicin: The pigment is again chromatographed on alumina, saponified with methanolic potassium hydroxide after elution and once more chromatographed on alumina. Aphanicin is eluted with petrol containing a little ethanol and the solu- For a detailed description, see J. Tischer, Z. physiol. Chem. 251 (1938) 109. References p. 341-342. APHANIN 301 tion is washed with water. The solvent is evaporated and the residue is recrystalHsed from petrol. From 50 kg of fresh moist algae, which yield 2 ^4 kg of dry material, a total of about 50 mg of pure aphanicin was obtained. b) Aphanin: The aphanin zone in the chromatogram is washed through into the filtrate. After removal of the solvent by distillation, the pigment can readily be obtained in a crystalline state. For further purification, the pigment is adsorbed on alumina and crystallised from a mixture of benzene and methanol (1 : 10). The yield of aphanin from 50 kg of algae amounted to about 110 mg. c) Flavacin: The pigments from the third zone of the first chromatogram are repeatedly chromatographed on alumina. The colourless impurities are frozen out from the petroleum ether solution, and the mother liquors are saponified with inethanolic potassium hydroxide, and then extracted with petroleum ether. After evaporation of the solvent a small amount of flavacin crystallises from the residue. It can be purified by repeated re-crystallisation from a mixture of benzene and methanol. The fourth zone of the chromatogram yields analytically pure /S-carotene. From the pyridine solution B and the ethanol extract C, aphanizophyll is obtained. The pyridine solution B is evaporated to dryness in vacuum and the residue is saponified with ethanolic potassium hydroxide. The reaction mixture is acidified with acetic acid and the pigment extracted with ether. This solution is adsorbed on sodium sulphate and the aphanizophyll is eluted with methanol and crystallised from acetone. For further purification, the pigment is chromato- graphed on calcium carbonate and repeatedly recrystallised from acetone, and finally from chloroform. From 50 kg of fresh algae, about 10 mg aphanizophyll were obtained in this wsiy. Working up of the e.xtract C yielded a further very small quantity of the pigment^'. Chemical Constitution^^ CH, CH, CH, CH, /\ 1 r I I /■•\ CH2 C-CH=CH-C=CHCH = CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-C6 2 CHg CH2 .C-CHs HjC-CS' 3'C=0 \ / Aphanin ( ?) \^'/ CHg CH2 The main features of the constitution of aphanin were established by TiscHER^ who proposed the above formula. Elementary analysis indicated the molecular formula C4oH5^0. On microhydrogenation the pigment absorbed 11 mols of hydrogen rapidly and an additional mol of hydrogen slowly. This behaviour suggested the presence of a carbonyl group which was confirmed by the preparation of a well-crystallised oxime. It seems almost certain that the carbonyl group is not conjugated with the system of conjugated double bonds as the absorption maxima of the oxime and of the parent compound have the same wavelength location. This is also in agreement with the fact that the absorption maxima in different solvents such as petrol and ethanol are the same, whereas carotenoids containing carbonyl groups conjugated with the References p. 341-343. 302 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV ethylenic double bonds exhibit a different behaviour (cf. p. 56). The test for the presence of an isopropylidene grouping gave a negative result, thus excluding a y-carotene type structure. On permanganate degradation, almost 5 (4.77) mols of acetic acid were obtained. This result is not in contradiction with the proposed formula for aphanin since cryptoxanthin, which contains six side- chain methyl groups, also gives rise to only 4.85 mols of acetic acid^^. The reduction of aphanin with aluminium isopropoxide and isopropylalcohol yielded an alcohol, aphanol, with an absorption maximum displaced by about 10 vafx towards shorter wavelengths as compared with aphanin (in petrol). TiscHER suggested that the structure of aphanol is identical with that of cryptoxanthin. If this suggestion is correct, the carbonyl group must be in the 3'-position, but since aphanol was not obtained crystalline and was not analysed, this question must still remain open. In animal feeding tests, aphanin exhibits half the vitamin A activity of ^-carotene, so that it must contain one unsubstituted /3-ionone ring according to present views. For that reason the constitution of one half of the molecule must be assumed to correspond to that of j3-carotene. The fact that the pigment is optically inactive is also in agreement with the proposed formula. Properties Crystalline form: Aphanin crystallises from a mixture of benzene and methanol in large, spear-like, blue-black leaflets which are often combined in rosettes and exhibit a pronounced graphitic lustre. From a mixture of benzene and petrol, aphanin is obtained in stout prismatic crystals. Melting point: 176° (corr., crystallised from benzene-methanoP^). 180° (corr., crystallised from benzene-petrol^"). Solubility: Aphanin is very readily soluble in carbon disulphide, chloroform and benzene, less readily soluble in pyridine, ether and petrol, and very sparingly soluble in methanol. Spectral properties: Solvent Absorption range Absorption maxima Carbon disulphide . 475-555 533.5 494 m/t Chloroform .... 455-520 504 474 m^ Benzene 455-520 505 472 m/i Petrol, b. p. 70-80° . 445-510 494 460 m/i Pyridine 460-525 507.5 477 m/i Methanol 445-505 491.5 457 m// TiscHER^^ describes the absorption spectrum of aphanin as follows: "The absorption spectrum exhibits two maxima separated by a more weakly ab- References p. 341-343. APHANIN 303 sorbing zone, giving the appearance of a single wide absorption band with 2 maxima. The wide absorption range has no sharp hmits and although the two maxima are clearly recognised their centres can only by approximately determined." Solutions of the pigment in petrol and methanol are yellow, solutions in benzene, chloroform and pyridine are orange, and solutions in carbon disulphide are red-orange. Optical activity: Aphanin shows no optical rotation. Partition test: On partition between petroleum ether and 90% methanol, the pigment is found entirely in the upper layer; if 95% methanol is used, however, the lower layer is also weakly coloured. Chromatographic behaviour: Aphanin is adsorbed somewhat more strongly than j3-carotene, but more weakly than aphanicin, on alumina from petrol solution. If the chromatogram is developed with ether, the colour changes to brown-red; on washing with benzene or chloroform, it becomes dark violet. Colour reactions: Concentrated aqueous hydrochloric acid gives no blue colouration with an ethereal solution of aphanin. A chloroform solution of the pigment assumes a deep blue colour which gradually changes to blue-green on addition of concentrated sulphuric acid. With antimony trichloride in chloro- form, aphanin exhibits a brownish-violet colour which turns blue-violet on fading*. Physiological properties: Aphanin exhibits vitamin A activity which is about half as great as that of j3-carotene^^. Detection and estimation: The separation of aphanin from the other carote- noids of Aphanizomenon flos-aquae is carried out by means of chromatographic adsorption on alumina. By this means, the pigment can be separated from the more strongly adsorbed aphanicin and can be identified by its absorption spectrum. Derivatives Aphanin oxime C40H55ON: This compound is obtained on treating a solution of aphanin in pyridine with free hydroxy famine. For purification, the crystallised oxime is chromato- graphed on alumina and recrystallised from a mixture of benzene and methanol. M.p. 208° (corr.). The oxime is rather sparingly soluble in benzene. For further colour reactions, cf. the original communication, Z. physiol. Chem. 251 (1938) 109. References p. J41-343. 304 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV Solvent Absorption range Absorption maxima Carbon disulphide 475-545 530 492 m^ Chloroform . . . 455-520 504 472 van Benzene .... 455-520 505 472 m/z Petrol (70-80°) . 445-520 494 459 m/z Pyridine .... 460-525 509 477 mfi Methanol .... 445-505 491 457 mil The spectrum of aphanin oxime exhibits two bands. The Hmits of the wide absorption band are less clear than in the case of aphanin and cannot be deter- mined exactly^®. For various colour reactions of the oxime, the original commu- nication^^ should be consulted. Aphanol C^^Yi^^O: During the reduction of aphanin with aluminium isopropoxide and iso- propylalcohol a compound which shows the absorption spectrum of crypto- xanthin is obtained amongst other products. According to Tischer^^ this compound is identical in structure with cryptoxanthin. 7. APHANICIN This pigment was first isolated by Tischer^^ from Aphanizomenon flos- aquae*. Chemical Constitution Very little is yet known concerning the constitution of aphanicin. Various considerations lead Tischer to regard aphanicin as a "di-carotenoid" of the empirical formula CgoH^QgOg, consisting of 2 molecules of aphanin combined by an oxygen bridge. As compounds of this type have not otherwise been encountered in nature, nor prepared synthetically, this suggestion can only be accepted with reserve. Experimentally the following results were obtained. The elementary analysis of aphanicin gave the values C, 86.14%; H, 9.35% (Calculated for C4(,H540: C, 87.20: H, 9.89%). The pigment formed an oxime, found: N, 3.97%. (C40H55ON requires N, 2.48%. Calculated for C40H55ON. yaNHgOH, 3.61 %**). The aphanicin molecule thus contains at least i carbonyl group. On microhydrogenation, aphanicin absorbs 12 mols of hydrogen. As the absorption spectrum of aphanicin is very similar to that of aphanin, it may be assumed that the two compounds contain very similar or identical chromo- phoric systems. Aphanicin has only about half the vitamin A activity of For the isolation of the pigment, see under aphanin, p. 300. ** Addition products of free hydroxylamine and carotenoids have been observed, J. Tischer, Z. physiol. Chem. 26y (1941) 281. References p. 341-343. 7 APHANICIN 305 aphanin*. Reduction of the pigment with aluminium isopropoxide and iso- propyl alcohol yields a product, aphanicol, the absorption maxima of which are displaced by about 10 m/^ towards shorter wavelength as compared with aphanicin (in petrol). From this fact and from the identical location of the absorption maxima in petrol and methanol it may be concluded that the carbo- nyl group is not in conjugation with the system of conjugated double bonds. Properties Crystalline form: Aphanicin crystallises from a mixture of benzene and methanol with a little more difficulty than aphanin and forms red-violet prismatic needles with a strong metallic lustre. Melting point: 190° (corr., crystallised from benzene-methanol) . 195° (corr., crystallised from benzene-petrol). Solubility: The pigment is even less soluble in methanol than aphanin. In other solvents the solubility of the two pigments is about equal. Spectral properties: Solvent Absorption range Absorption maxima Carbon disulphide . 475-555 533 494 m^ Chloroform .... 455-520 504 474 ra/x Benzene 455-520 505 474 m/f Petrol, b.p. 70-80° . 445-510 494 462 m^ Pyridine 460-525 507.5 478 m^ Methanol 445-505 491.5 457 m^i The colours of solutions of aphanicin in organic solvents are the same as those of aphanin, see p. 303. Optical activity: No data have been recorded. Partition test: On partition of aphanicin between petroleum ether and 95 % methanol, a somewhat higher proportion of the pigment is found in the alcoholic layer than in the case of aphanin. Even with 90 % methanol the lower layer is slightly coloured. Chromatographic behaviour: Aphanicin is more strongly adsorbed than aphanin on alumina from a mixture of benzene and petrol (1:1) and can thus be readily separated from the lattei. The two zones are also distinguished by the depth of their colours, the lower zone being a darker bordeaux-red than the upper zone. It has recently been shown that steric differences have a great influence on vitamin A activity, cf. L. Zechmeister and co-workers. Arch. Biochem. 5 (1944) 107; 7 (1945) 247; 7 (1945) 157- References p. 341-343. Carotenoids 20 3o6 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV Colour reactions: Aphanicin shows almost the same colour reactions as aphanin. Table 53 shows the reactions which give different results. TABLE 53 COLOUR REACTIONS DISTINGUISHING BETWEEN APHANIN AND. APHANICIN Reagent Colouration Aphanin Aphanicin Trichloracetic acid in chloro- form Arsenic trichloride in chloro- form faint brown -violet brown, changing to sepia red-violet, stable brown, changing to violet Antimony trichloride in chloroform Antimony trichloride in ether brown-violet, fading yellow-brown brown-violet reddish-brown Physiological behaviour^^: Aphanicin exhibits about half the vitamin A activity of aphanin. Detection and Estimation: Aphanicin can be separated from the other carotenoids of Aphanizomenon flos-aquae by chromatographic adsorption and is identified by means of its spectral properties and its colour reactions. Derivatives Aphanicin oxime: Aphanicin oxime is prepared in the same way as aphanin oxime. M.p. 241° Solvent Absorption range Absorption maxima Carbon disulphide . 460-545 529 492 m^l Chloroform .... 450-520 504 474 m// Benzene 455-520 505 472 m/< Petrol b.p. 70-80° . 445-505 493 461 m/z Pyridine 460-525 508 478 m/z Methanol 445-505 491 456 mfi Aphanicol: This compound is prepared in the same way as aphanol^*. Aphanicol could not be obtained crystalline. Solvent Absorption maxima Carbon disulphide 516 482 m/i Petrol 484 454 m/i References p. 341—343. FLA VACIN 8. FLAVACIN 307 ^ TisCHER^^ discovered flavacin together with aphanin, aphanicin and aphanizophyll in the blue algae Aphanizomenon flos-aquae. The isolation of the pigment is described in connection with the preparation of aphanin on p. 301. TABLE 54 ■. COMPARISON OF SOME PROPERTIES OF MUTATOCHROME AND FLAVACIN Properties Flavacin Mutatochrome Melting point 155° (corr.)* 163-164° (uncorr.) in vacuum Absorption maximum in carbon disulphide 490 457 (424) m/< 489.5 459 m// • Absorption maximum in petrol 458 428 mfjL 456 427 m^ Action of concentrated hydrochloric acid no blue colouration very faint blue coloura- tion which only appears gradually Partition test epiphasic epiphasic Position in the chromatogram above /S-carotene above j8-carotene This melting point was not yet constant. J. TiscHER employed 30% hydrochloric acid for this reaction, whereas P. Karrer and E. Jucker employed 37% hydrochloric acid but still only observed a very weak blue colouration. Chemical Constitution Very little is known concerning the structure of flavacin. The pigment is found below aphanin but above /3-carotene in the chromatogram. It has purely epiphasic character which suggests that it may be a hydrocarbon. The ab- sorption maxima in carbon disulphide are located at 490 and 457 m/i, so that the chromophoric system of flavacin must contain fewer conjugated double bonds than, foi instance, that of jS-caiotene. Since, however, the latter is more weakly adsorbed on the chromatographic column, flavacin must contain an additional functional group which increases its strength of adsorption. One is thus tempted to compare flavacin with mutatochrome^^. The two compounds have, in fact, largely the same pioperties. The question of the identity of the two pigments must still be left open, however, as a direct comparison has not yet been made. References p. J41—J4J. 3o8 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV 9. APHANIZOPHYLL* The chemical constitution of aphanizophyll is still unknown. The pigment con- tains a hydroxy! group which can be esterified and a carbonyl group which reacts with hydroxylamine. Elementary analysis, however, gave very low values for the carbon content (C, 70.15 %; H, 9.42 %), so that no conclusions regarding the structure of the pigment can be drawn on the basis of the analytical results. TiscHER^^ points out the similarity between aphanizophyll and myxoxantho- phyll^^, but does not regard the two pigments as identical. Properties Crystalline form: The phytoxanthin crystallises from methanol in prismatic crystals combined in rosettes. From pyridine it is obtained in circular, finely tufted crystals. Melting point: 172-173° corr. Solubility: Aphanizophyll dissolves readily in pyridine and ethanol and somewhat less readily in acetone, ether and acetic acid. It is completely insoluble in benzene, petrol and carbon disulphide. Optical rotation: No data have been recorded. Spectral properties: Solvent Absorption maxima Pyridine . . , ..... . . 531 494 462 m/j. Chloroform 523 487.5 457 m/x Methanol 507 475 444 m/n Solutions of the pigment in methanol are yellow, solutions in chloroform deep red, and solutions in pyridine blood-red. Partition test: Aphanizophyll is entirely hypophasic on partition between petroleum and methanol. Chromatographic behaviour: Aphanizophyll is adsorbed so strongly on alumina and calcium hydroxide that it is almost impossible to elute it. It is also readily adsorbed on calcium carbonate or sodium sulphate from chloroform or ether solution but can be completely eluted with methanol. Colour reactions: A solution of aphanizophyll in chloroform shows the follow- ing colour reactions. Concentrated sulphuric acid produces a blue colour. Concentrated nitric acid gives a blue colour which changes to green and eventually fades. Antimony trichloride in chloroform gives a blue colour which * For the isolation of this pigment, see under aphanin, p. 301. References p. 241-343- lo FUCOXANTHIN 309 changes to violet. An ethereal solution of the pigment gives a slowly fading bluish-green colour with 30 % hydrochloric acid. Detection and estimation: Aphanizophyll can be separated from accompa- nying carotenoids by means of chromatographic analysis and can be identified by determination of the absorption maxima, by partition between methanol and petroleum ether, and by the blue colouration produced with concentrated hydrochloric acid. Derivatives Aphanizophyll can be esterified with palmitic acid chloride. In contrast to the parent pigment, the ester is readily soluble in benzene, petrol and caibon disulphide. It crystallises from methanol in needles, which melt even on warming by hand. Solvent Absorption maxima Carbon disulphide 547 506 474 m/z Benzene 524 489 457 m/i The ester exhibits the same absorption maxima in pyridine, chloroform and methanol as the parent compound. It is more weakly adsorbed than aphanizo- phyll. On alumina, the ester is adsorbed with an orange-red colour and can be readily eluted, but it is not sufficiently adsorbed on calcium carbonate. On partition between petroleum ether and 90 % methanol, it exhibits pronounced epiphasic behaviour. With 95 % methanol, the lower layer is also definitely coloured. It is possible that aphanizophyll occurs as the palmitic ester in algae, as considerable amounts of palmitic acid could be isolated from the hypophasic fraction^®. Apart from one or more hydroxyl groups, aphanizophyll also contains a carbonyl group since an oxime can be prepared. The oxime is soluble in benzene and is well adsorbed on calcium carbonate. It exhibits the same absorption spectrum as aphanizophyll in different solvents. The absorption maxima in benzene are located at 524, 489 and 457 m/^. In view of the spectroscopic properties of the oxime it may be concluded that the carbonyl group is not in conjugation with the system of conjugated double bonds. Neither the oxime nor the ester could be obtained in the crystalline state and analysed. 10. FUCOXANTHIN C4oH5e06 History 1867 RoSANOFF^'' suggests that brown algae contain a yellow pigment besides chlorophyll. The pigment was also observed shortly afterwards by Kraus and Millardet^^ and termed phycoxanthin. References p. 341-343. 3IO CAROTENOIDS OF UNCERTAIN STRUCTURE XIV 1837 SoRBY^^ shows that brown algae contains not only one but, in his view, three yellow pigments which he terms xanthophyll, lichnoxanthin and fucoxanthin. 1906 TswETT^^succeeds in separating the pigments of brown algae into carotene, fucoxanthophyll and fucoxanthin by chromatographic adsorption. 1914 WiLLSTATTER and Page^^ isolate fucoxanthin in the crystalline state for the first time and investigate this pigment in detail. 1931-35 Karrer and co-workers^^, Kuhn and Winterstein^^ and Heilbron and Phipers^* carry out investigations on the constitution of fucoxanthin. Occurrence Fucoxanthin has been found mainly in Phaeophyceae where it occurs together with chlorophyll (mainly chlorophyll a) and other carotenoids such as carotene and xanthophyll. The following species of brown algae contain the pigment." Fucus virsoides, Dictyota, Cystosira and Laminaria^^. According to Heilbron and Phipers^*, dried brown algae {Fucus vesiculosus) contain j3-carotene and zeaxanthin, whereas fresh algae contain /S-carotene and fucoxanthin. More recent investigations^'*^ show that j8-carotene is the main carotenoid pigment of the male gametes, while fucoxanthin is present mainly in the female gametes. Fucoxanthin also occurs in Zygnema pectinatum, Polysiphonia nigrescens^^ and in diatoms^^'^'. Preparation^^ Air-dried brown algae are minced in a mincing machine and exhaustively extracted at room temperature with 90 % methanol. After dilution with water, chlorophyll is extracted from this solution with petroleum ether and the mother liquors are diluted with more water, covered with petroleum ether and allowed to stand for 24 hours. At the end of this period most of the fucoxanthin has separated as a brown precipitate at the boundary between the two solvents, and can be filtered off. The mother liquors which contain very little additional pigment, are discarded. The pigment is purified by crystallisation from methanol and a little water, and a second time from methanol alone. The yield of pure pigment from about 15 kg of air dried algae amounts to about 2 g. If, on the other hand, the algae are several weeks old, the pigment can no longer be isolated and only a veiy small amount of impure pigment is obtained^®. Using the procedure of Karrer and co-workens'^ just described, only fuco- xanthin is isolated from brown algae. WiLLSxAtter and Page^* have described a considerably more complicated method of preparation in which xanthophyll is also isolated and subsequently separated from fucoxanthin. Chemical Constitution Karrer and coworkers^^ obtained the molecular formula C4oHgg06 for fucoxanthin whereas I. M. Heilbron and R. F. Phipers*" obtained the formula References p. 341-343. lo FUCOXANTHIN 311 QoHeoOe- The nature of the oxygen atoms in fucoxanthin has not yet been clari- fied. The results obtained by Karrer and co-workers*^ indicated 4-5 hydro xyl groups, whereas the results of Kuhn and Winterstein^^ indicated 6. On catalytic hydrogenation 10 mols of hydrogen are absorbed*^ ; but the analysis of the perhydrocompound gave values in agreement with the formula C^oHygOg*". On vigorous oxidation with permanganate, four mols of acetic acid are obtained. From these results the number of side-chain methyl groups can be deduced. From the oxidation products of the pigment with alkaline per- manganate, a : a-dimethylmalonic acid can be isolated (Karrer and co- workers^i). The fact that no a : a-dimethylsuccinic acid or a:a-dimethylglutaric acid are formed indicates that the two ends of the fucoxanthin molecule are highly substituted by hydroxyl groups (cf. p. 201). These results do not, however, lead to any definite constitution for the pigment. Heilbron and Phipers*" proposed the following formula for fucoxanthin. CH3 CH3 CH3 CH3 C CH3 CH3 CH3 CH3 C /\ \ \ \ \ /\ CHa CO-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CO CHj HOCH CH-OH HO-CH HCOH \y\ Fucoxanthin(?) /\/ CHg CH3 CH3 CHj, The proposed constitution is not, however, in accord with all the properties of fucoxanthin. The chromophoric system of the proposed structure corresponds to that of j3-carotenone (cf . p. 141) and the absorption maxima would be expected to be located at considerably longer wavelengths than those observed. Further- more, a diketone of this type should be reduced by zinc and acetic acid to a dihydro-derivative, as in the caseof j8-carotenone. Fucoxanthin does not undergo such a reaction. The proposed formulation of the pigment is thus unacceptable. Properties Crystalline form: Fucoxanthin crystallises from methanol in brown-red prisms with a blue lustre. The crystals contain 3 molecules of methanol of cry- stallisation. From dilute ethanol or acetone, the pigment is obtained in deep red hexagonal plates containing 2 molecules of water. From a mixture of ether and petroleum ether, the pigment crystallises in needles without solvent of crystallisation. Melting point: 159.5-160.5° (corr., Wilstatter and Page^^), 166-168° (uncoir.)*". References p. 341-343. 312 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV Solubility: Fucoxanthin is readily soluble in ethanol, somewhat less soluble in carbon disulphide, sparingly soluble in ether, and quite insoluble in petroleum ether. lOO g of hot methanol will dissolve 1.66 g of the pigment. Spectral properties: Fucoxanthin exhibits an absorption spectrum similar to that of xanthophyll, but the bands are less sharp. In carbon disulphide 510 477 445 m/n (diffuse) In chloroform . . . 492 457 m/t (cell thickness 2 mm)*^ (cf. Fig. 9, p. 351) A solution of fucoxanthin in ether is orange-yellow, a solution in ethanol is reddish, and a solution in carbon disulphide is red. Colour reactions: On shaking an ethereal solution of fucoxanthin with 25 % aqueous hydrochloric acid, the latter is coloured deep-blue. Optical rotation: According to Karrer and co-workeis*^ fucoxanthin has a specific rotation of [a]p = + 72.5° ± 9° (in chloroform). Heilbron and Phipers*", on the other hand, find that the pigment does not rotate the plane of polarised hght. Chromatographic behaviour: Hardly any data are available regarding the chromatographic adsorption of fucoxanthin*^. In order to estabhsh whether the pigments investigated by different workers were identical or mixtures of different substances, a renewed detailed chromatographic investigation of fucoxanthin would be desirable. Detection and estimation: As far as is known at the present time, the pigment occurs mainly in brown algae. It is accompanied by j8-carotene, zeaxanthin and xanthophyll from which it can be easily distinguished by means of its absorption maxima and its colour reaction with hydrochloric acid. Chemical properties: Although fucoxanthin has no acidic properties, it is not indifferent towards alcoholic alkah since methanolic potassium hydroxide dissolves the pigment much more quickly than methanol alone. On acidification, no fucoxanthin can be regenerated from this solution, but new compounds are obtained which Heilbron and Phipers*" termed isofucoxanthins. Nothing definite is yet known concerning the nature of these compounds. Heilbron suggests that isofucoxanthins are formed by an aldol condensation but this suggestion has not been proved. Isofucoxanthins are much more strongly basic than fucoxanthin. Even 0.001 % hydrochloric acid removes the blue colour of an ethereal solution of these new pigments. The absorption maxima of isofucoxanthin are displaced towaids shorter wavelengths with respect to fucoxanthin. References p. 341-343. II GAZANIAXANTHIN 313 The behaviour of fucoxanthin towards hydrochloric acid has akeady been described on p, 312. Willstatter and Page^^ showed that a definite amount (4 mols) of hydrochloric acid is consumed in this reaction. The compound formed has the formula 640115403.4 HCl. Crystalline fucoxanthin is fairly stable in air, no uptake of oxygen being observed during a period of several weeks. However, the pigment absorbs water from the air and forms various hydrates. If an ethereal solution of the pigment is allowed to stand in the presence of iodine, a tetraiodide of fucoxanthin soon crystallises. This derivative forms short, pointed, violet-black prisms with a coppery lustre. M.p. 134-135° (corr., after slight sintering). Little is known concerning the pigments** designated as "fucoxanthin a, b, c" and their homogeneity is uncertain; the same applies to "neofuco- xanthin A" and "neofucoxanthin B"*^. II. GAZANIAXANTHIN History and Occurrence 1938 ScHON isolates a new phytoxanthin, gazaniaxanthin, from the blossoms of Gazania rigens^^. 1943 Zechmeister and Schroeder*' propose a tentative formula for gazania- xanthin. Preparation^'' 1 kg of blossom leaves of Gazania rigens are dried at 40-50° and extracted with petroleum ether at room temperature. After saponification with methanolic pot- assium hydroxide, the pigment mixture is chromatographed on calcium hydroxide from petroleum ether solution. Depending on the age of the blossoms, the yield of gazaniaxanthin varies between 380 and 620 mg. Chemical Constitution CH, CH, CH3 CH, C CH3 CH, CH, CH, CH /\ I I I I \ CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH CH^ HOCH C-CHs HjC-C CH, \ y^ Gazaniaxanthin ( ?) \ / CH2 CHa According to Schon*^, gazaniaxanthin has the molecular formula C4oHg40 or C4oH5gO. The oxygen atom is present in the form of a hydroxyl group, as shown by Zerewitinoff determinations and the preparation of an acetate. On catalytic hydrogenation gazaniaxanthin absorbs 11 mols of hydrogen*'. According to Zechmeister and Schroeder*' the molecular formula is C40H58O References p. 341-243. 314 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV and ozonisation yields 0.85 mols of acetone. On the basis of these results Zechmeister and Schroeder proposed the above formula for gazaniaxanthin. Apart from the uncertainty regarding the position of the hydroxyl group, this formula partly contradicts the experimental results. According to all previous experience in the carotenoid series, acetone is only obtained on ozonisation in the presence of an isopropylidene grouping (CH3)2C = C ... It is not easy to see why the grouping (CH3)2CH2... should also give rise to nearly i mol (0.85) of acetone on ozonisation. (Zechmeister and Schroeder^' cite the analogous case of thymol which on ozonisation yields 0.3 mols of acetone). Further experimental study is clearly required for a complete elucidation of the consti- tution of gazaniaxanthin. Properties Gazaniaxanthin crystallises from a mixture of benzene and methanol in glistening red plates, m.p. 133-134°. It is found below rubixanthin but above cryptoxanthin in the chromatogram on calcium hydroxide. It shows the same behaviour in the partition test as other phytoxanthins containing a hydroxyl group. Solvent Absorption maxima Carbon disulphide 531 494.5 461 m/n Benzene 509 476 447.5 m/i Petroleum ether 494.5 462.5 434.5 m/j, Ethanol 494.5 462 434.5 m/i According to the experiments of Zechmeister and Schroeder*', gazania- xanthin exhibits no vitamin A activity. It may be concluded from this result that the hydroxyl group is substituted in the ^-ionone ring. Ga "an iaxanth in monoacetate: This compound is formed by treating gazaniaxanthin with acetic anhydride in pyridine. It crystallises from benzene or methanol in stout needles and from a mixture of petroleum ether and methanol in curved needles, m.p. 83-85°. The spectral properties correspond to those of gazaniaxanthin. Cis-trans Isomers Zechmeister and Schroeder*' converted gazaniaxanthin into a complex mixture of isomers by the usual methods (p. 39). So far it has not been possible to obtain these compounds in a crystalline state and to investigate them in detail. References p. 341-343. 12 CELAXANTHIN 315 12. CELAXANTHIN C4oH5gO* History and Occurrence In the course of investigations on the red berries of Celastrus scandens, A.L. Le Rosen and Zechmeister^ found, besides /3-carotene, cryptoxanthin, zea- xanthin, rubixanthin(?) and two unknown pigments present in very small amounts, a new phytoxanthin, for which they proposed the name celaxanthin. In its spectral properties the new pigment shows close similarity to rhodovio- lascin (p. 297) and torulin (p. 329) so that these pigments are probably closely related. Preparation^ The air-dried, bright red flesh of Celastrus berries (230 g) was extracted for a short time with a mixture of petroleum ether and methanol (8:1.5) in a shaker. It was then dried at 40°, finely ground, and again exhaustively extracted with the same solvent mixture. The pigments obtained from the combined extracts were dissolved in petroleum ether, the solution was washed free from methanol, dried over sodiuin sulphate, and chromatographed on calcium hydroxide. The chromato- gram was developed either with benzene or a mixture of benzene and acetone. -The celaxanthin esters were found in the second zone from the top of the column, beneath the zone of a phytoxanthin with absorption maxima at very long wave- lengths (587, 547.5 m/x in carbon disulphide). The celaxanthin zone was arbitrarily divided into two zones and the pigments were separately subjected to renewed chromatography. They were then eluted with a mixture of benzene and methanol (3:1), the solvent was evaporated and the pigments were precipitated with methanol from a petroleum ether solution. The celaxanthin ester obtained in this manner was saponified at room temperature with methanolic potassium hydroxide. The reaction mixture was worked up in the usual way and the pigment obtained was purified by adsorption on calcium hydroxide from a little benzene, and the column was washed with a mixture of petroleum ether and acetone (10:1). After elution and removal of the solvent by distillation, the celaxanthin was crystallised from a mixture of ethanol'and carbon disulphide or benzene. Chemical Constitution CH3 CH3 c OH CH-CH = 1 II = CH CH3 •C=CHCH= CH3 = CH-C=CHCH=CHCH = CH3 = C-CH= =CHCH= CH3 =C-CH: = CH CH3 CH3 V /\ •C CH2 1 II CH C-CHg CH Celaxanthin( ? ) H3C •C CHOH CH, The results of elementary analysis suggests a molecular formula C40H54O or CjoHggO. The occurrence of a natural celaxanthin ester shows that the oxygen is present in the form of a hydroxyl group. As the pigment does not show the * Or C^oHj.O. References p. 341-343. 3i6 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV properties of an enol, the grouping =C.(OH) can be excluded. On ozonisation, Le Rosen and Zechmeister obtained 0.55 mols of acetone per mol of pigment (after substr action of the blank) thus indicating the presence of an isopropylidene grouping. Finally, the long-wavelength location of the ab- sorption maxima indicates the presence of 13 conjugated bonds. The position of the hydroxyl group is not certain ; for reason of analogy it is assumed to be at carbon atom 3'. The experimental results are in agreement with the proposed formula for celaxanthin, but do not exclude the possibility of other structures. Properties Crystalline form: Celaxanthin crystallises from petroleum ether and ethanol in long needles which are combined in rosettes or clusters. In bulk, the pigment has the appearence of a dark red crystalline powder, somewhat reminiscent of lycopene. Melting point: 209-210° (corr., Berl-Block, capillary filled with carbon dioxide). Another sample of the pigment had m.p. 204-205°. Solubility: Celaxanthin is sparingly soluble in carbon disulphide and benzene at room temperature. It is slightly soluble in petroleum ether and almost insoluble in methanol and ethanol. Partition test: On partition between petroleum ether and 85 % methanol, the pigment is entirely epiphasic. With 95 % methanol the lower layer is also coloured. Optical activity: The optical activity could not be determined because of the high colour intensity. Spectral properties: Solvent Absorption maxima Carbon disulphide . . 562 521 487 455 xxxfi Ethanol 520.5 488 455 m/z Petroleum ether . . . 520 486.5 456 (429)m^i Chromatographic behaviour: Celaxanthin is well adsorbed on calcium hydroxide from petroleum ether or from a mixture of petroleum ether and acetone. Benzene, or a mixture of benzene and acetone can also be used as solvents for this adsorbent. The pigment can be eluted with the same solvents, but containing a little methanol. Cis-trans Isomers Le Rosen and Zechmeister found that celaxanthin isreversibly isomerised on warming in the same way as torulin (cf. p. 329). Le Rosen and Zechmeister References p. 341-343. 13 PETALOXANTHIN 317 ascribe this change to cis-trans isomerism and distinguish three neo-celaxan- thins* : Solvent Absorption maxima in petroleum ether Neocelaxanthin A . . 534 497 464 (433.5) m// Neocelaxanthin B . . 530 493.5 460 (431.5) m/< Neocelaxanthin C . . 536 496.5 461 (432) m/^ The neotonilins, also obtained by le Rosen and Zechmeister, exhibit spectral properties similar to the neocelaxanthins, but can be easily separated from the latter by chromatography on calcium hydroxide. (The two natural pigments can be separated in the same way). 13. PETALOXANTHIN C4oH5g03** History and Occurrence The blossoms of Cucurhita Pepo were first examined for carotenoids in 1914 by MiCHAUD and Tristan*^ In 1936, Zechmeister, Beres and Ujhelyi^" isolated from this source a new phytoxanthin, for which they proposed the name petaloxanthin. (The obvious name cucurbitaxanthin could not be used as this had already been employed by Suginome and Ueno to designate lutein obtained from Cucurhita maxima Duch^^). Petaloxanthin has so far only been found in pumpkin blossoms. Preparation^'^ 1 kg of material (from male pumpkin blossoms) is exhaustively extracted with ether in the cold. The combined extracts are concentrated and the residue is saponified with methanolic potassium hydroxide at room temperature. The reaction mixture is covered with petroleum ether and the pigments are divided into an epiphasic and hypophasic fraction by addition of a little water. The pigments of the hypophasic fraction are extracted with ether and the solution is washed and dried, and the solvent evaporated. The residue is dissolved in a little carbon di- sulphide, a small portion remaining undissolved. This less soluble fraction is dissolved in more carbon disulphide. The solution is slightly cooled to precipitate colourless impurities and the mother liquors are twice chromatographed on calcium carbonate. The petaloxanthin is eluted with methanol-containing ether and crystallised from a mixture of carbon disulphide and petroleum ether. From 1 kg of ground blossoms, 20 mg of pigment are obtained in this way. Chemical Constitution Very httle is at present known regarding the structure of petaloxanthin. The molecular formula C40H56O3 or C4oH5g03, the absorption spectrum (maxima * None of these isomerisation products were obtained in a crystalline state. ** The formula C40H58O3 is also a possible one for petaloxanthin. References p. 241—343 ■ 3i8 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV at 514.5 and 481 m/^ in carbon disulphide) , the chromatographic behaviour, the blue colouration with concentrated hydrochloric acid, and the melting point all indicate a close relationship to, or even identity with, antheraxanthin (cf. p. 191). The two pigments can, however, be chromatographically separated, antheraxanthin being found in the upper zone. This behaviour is reminiscent of the pair of isomers, flavoxanthin and chrysanthemaxanthin. These two pigments also have closely similar properties but can be separated on a zinc carbonate chromatogram, and also differ in their reaction with hydrochloric acid, only flavoxanthin giving a blue colouration (cf. p. 210). The blue colour- ation produced on shaking an ethereal solution of petaloxanthin with concen- trated aqueous hydrochloric acid is weaker than the colouration produced with antheraxanthin. The nature of the three oxygen atoms present in petaloxanthin is also unknown. The strong adsorption of the pigment on calcium carbonate suggests that it contains two hydroxyl groups. The presence of a third hydroxyl group is unlikely, since a compound containing three hydroxyl groups should be moie strongly adsorbed than antheraxanthin which contains 2 hydroxyl and one epoxide group. A re-investigation of petaloxanthin thus appears very desirable. Properties Crystalline form: The pigment crystallises from a mixture of carbon di- sulphide and petroleum ether in long, spear-like crystals. From ethanol it is obtained in silky, lustrous, light-yellow leaflets which appear under the micro- scope as long, thin, straw-yellow squares. (A microphotograph will be found in the communication by Zechmeister and co-workers^^). Solubility: Petaloxanthin is easily soluble in benzene, less easily soluble in carbon disulphide, sparingly soluble in cold ethanol and almost insoluble in petroleum ether and petrol. Melting point: 211-212° (corr., oil-bath), 202° (Berl-Block, short thermo- meter). Spectral properties: Solvent Absorption maxima Carbon disulphide 514.5 481 m/z Chloroform 492 460.5 m/t Benzene 494 460.5 m/z Ethanol 483 451.5 m/i Optical activity: No data are available. Partition test: Petaloxanthin is entirely hypophasic. Chromatographic behaviour: The pigment is well adsorbed on calcium carbonate from carbon disulphide solution, or on calcium hydroxide from References p. 341-343. 13 SARCININ AND S A RCI N AX ANTHI N 319 benzene solution, and is found below antheraxanthin but above zeaxanthin in the chromatogram. Colour reactions: On shaking an ethereal solution of petaloxanthin with 37 % aqueous hydrochloric acid, a pale blue colouration is observed which eventually changes to violet -blue. Detection and estimation: The separation of petaloxanthin from other phytoxanthins is effected by adsorption on calcium carbonate or calcium hydroxide. The pigment can be identified by determining the absorption maxima and by the hydrochloric acid reaction. TABLE 55 COMPARISON OF SOME PROPERTIES OF PETALOXANTHIN AND ANTHERAXANTHIN Petaloxanthin Antheraxanthin Empirical formula C40H56O3 for C40H5SO3) C40H56O3 Number of hydroxyl groups probably 2 2 Number of double bonds ? 10, all conjugated Melting point 202=^ (uncorr.) (Berl- block) 205° (uncorr.) Mixed melting point 198° (uncorr.) (Berl- block) Position in chromatogram lower higher Colour reaction with concen- blue, soon changes to blue, soon fades trated hydrochloric acid violet-blue Absorption maxima in carbon 514.5 481 m^ 510 475 m// disulphide Absorption maxima in ethanol 483 451.5 m/z 479 449 m/i 14. SARCININ AND S ARCIN AX ANTHIN The pigments of Sarcina lutea were investigated by Chargaff and DiERYCK^^ and by Chargaff^* who isolated a new carotenoid termed sarcinin. The constitution of this pigment is till entirely unknown. It may be a hydro- carbon, but the amount isolated was so small that no conclusive data could be obtained. In petroleum ether solution, sarcinin exhibits absorption maxima at 469, 440 and 415 m//. Besides sarcinin, Sarcina lutea contains another new phytoxanthin which has the same optical properties. In 1936, Nakamura^^ isolated from Sarcina lutea a yellow carotenoid which he regarded as a phytoxanthin ester. This pigment exhibits absorption maxima at 490, 460 and 433 m/x in carbon disulphide solution. This carotenoid is References p. 341-343. 320 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV evidently not the hypophasic pigment of Chargaff, since the latter exhibits absorption maxima at longer wavelengths. Very recently the pigments of Sarcina lutea have been reinvestigated by YosHiHARU Takeda and Tatuo Ohta^^ who described the isolation of a new carotenoid which they termed sarcinaxanthin. From 385 g of dried bacteria, 3.4 mg of sarcinaxanthin were obtained. The pigment crystalhsed from a mixture of benzene and petrol in tufted red spheroids, m.p. 149-150° (Kofler- HiLBCK micro-melting point apparatus). On partition between petroleum ether and methanol, sarcinaxanthin behaves like a phytoxanthin containing one hydTOxyl group. It is fairly strongly adsorbed on alumina fiom petrol solution and can only be eluted with difficulty by means of petrol containing some ethanol. Solvent Absorption maxima Carbon disulphide 499 466.5 436 m/< Chloroform 480 451 423 m/< Petrol 469 440 (415) m// Benzene 481 451 424 m/z Ethanol 469.5 441 (415) m/x It appears that sarcinaxanthin may be identical with the hypophasic carotenoid reported — though not isolated — by Chargaff^^- ^*. 15. TARAXANTHIN C4oH5g04 History and Occurrence During an investigation of the carotenoids from the blossoms Taraxacum officinale (dandelion), Kuhn and Lederer" discovered a new pigment which they termed taraxanthin. Later studies by Karrer and Rutschmann^s showed that the occurrence of taraxanthin evidently depends on geographical and chmatic factors since the material investigated by the last-named authors did not contain the pigment. Numerous references to the occurrence of taraxanthin are given in the Hterature, but the pigment never occurs in large concentrations and its isolation is relatively tedious. References p. 241-343. 15 TARAXANTHIN 321 Source a) In blossoms." Helianthus annuus Impatiens noli tangere Leontodon autumnalis Ranunculus acer Taraxacum officinale T us silage Far far a Ulex europaeus b) In hips : Rosa canina Rosa damascena Mill. Rosa rubiginosa c) In algae : Cladophora Sauteri Nitella opaca Oedogonium Rhodymenia palmata d) In liver: (Lophius piscatorius) TABLE 56 OCCURRENCE OF TARAXANTHIN* Literature references L. Zechmeister and P. Tuzson, Ber. by (1934) 170. R. KuHN and E. Lederer, Z. physiol. Chem. 213 (1932) 188. do. R. KuHN and H. Brockmann, Z. physiol. Chem. 213 (1932) 192. — P. Karrer, E. Jucker, J. Rutsch- MANN and K. Steinlin, Helv. chim. Acta 28 (1945) 1146. R. Kuhn and E. Lederer, Z. physiol. Chem. 200 (1931) 108. P. Karrer and R. More, Helv. chim. Acta 15 (1932) 863. K. ScHON, Biochem. J. 30 (1936) 1960. R. Kuhn and C. Grundmann, Ber. 67 (1934) 339, 1133. do. do. I. M. Heilbron, E: G. Parry and R. F. Phipers, Biochem. J. 29 (1935) 1376. do. do. do. N. A. Sorensen, Chem. Centr. 1934, II, 682. — I. M. Heilbron and coworkers, Biochem. J. 29 (1935) 1379. Preparatiow''^ The dried and finely ground dandelion blossoms are extracted with a mixture of acetone and petroleum ether (1:1) at room temperature. The pigment is trans- ferred to the petroleum ether layer by addition of water, and saponified with ethanolic potassium hydroxide. The free phytoxanthins are dissolved in methanol, the solution is covered with a layer of petroleum ether, and the pigments are precipitated by very careful addition of water. The pigments are crystallised from methanol in order to remove colourless impurities. In this way about 400 mg of fairly pure phytoxanthin are obtained from 15,000 dandelion blossoms (without cups). The phytoxanthins are separated bv chromatographic adsorption on calcium carbonate from a mixture of benzene and petrol. Taraxanthin is ad- According to E. Lederer, Bull. Soc. Chim. biol. 20 (1938) 554 the skins of various species of fish contain taraxanthin. These findings require further confirmation. References p. 341-343. Carotenoids 21 322 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV sorbed on the uppermost part of the column and can be eluted with a mixture of ether and methanol. It is purified by precipitation with water from methanol solution covered with petroleum ether and subsequent crystallisation from metha- nol. About 40 mg of taraxanthin are obtained from 200 mg of phytoxanthin mixture. Taraxanthin can be prepared more simply from spring cabbage^®. The blossoms are dried, finely ground, and extracted with petroleum ether at room temperature. The phytoxanthin esters are saponified with ethanolic potassium hydroxide and the products are separated into an epiphasic and an hypophasic fraction. The free phytoxanthins are precipitated from the latter by the addition of water under a layer of petroleum ether. The pigment thus obtained is again dissolved in methanol, precipitated with water and finally recrystallised from methanol. In this way 4 mg of pure taraxanthin are obtained from about 500 blossoms. Chemical Constitution The constitution of taraxanthin is still unknown. The molecular formula C4oH5g04 shows that the pigment is isomeric with violaxanthin (cf. p. 193). The similar spectral properties of the two pigments indicate that they contain similar chromophoric systems. KuHN and Lederer^' obtained values corresponding to 3-4 active hydrogen atoms in Zerewitinoff determinations. On catalytic hydrogenation, tara- xanthin absorbs 11 mols of hydrogen and the composition of the perhydro- derivatives shows that the pigment contains two isocyclic rings. Properties Crystalline form: Taraxanthin crystallises from methanol in fine lustrous prisms or plates. Melting point: 185-186° (uncorr.)6o, 184-185° (corr.)«i. Solubility: The pigment is readily soluble in benzene, ether, ethanol and methanol, but insoluble in petroleum ether. Spectral properties: Solvent: Absorption maxima:* Carbon disulphide 501 469 441 m// Petrol 472 443 m// (cf. Fig. 24, p. 358) Optical activity: [a]g^ § = +200° in ethyl acetate solution. Partition test: Taraxanthin exhibits entirely hypophasic properties. Chromatographic behaviour: The pigment is well adsorbed on zinc carbonate from benzene solution (cf. Kuhn and Brockmann^^). Quantitative extinction measurements are reported by K. W. Hausser and A. Smakula, Z. angew. Chem. ^7 (1934) 663; 48 (1935) 152. — K. W. Hausser, Z. techn. Pkys. 15 (1934) 13- References p. 341-243. i6 ' ESCHSCHOLTZXANTHIN 323 Colour reactions: In contrast to violaxanthin, taraxanthin in ethereal solution does not give a blue colouration on shaking with concentrated aqueous hydrochloric acid. Detection and estimation: Taraxanthin is separated from other phyto- xanthins by adsorption on zinc carbonate from benzene solution. It can be identified by the determination of the absorption maxima and by the negative hydrochloric acid reaction. For the colourimetric determination, cf. Kuhn and Brockmann^^. Cis-trans Isomers of Taraxanthin Zechmeister and Tuzson^^ studied the behaviour of taraxanthin in the presence of iodine and found that the chromatogram of the reaction products contained several bands which were ascribed to cis-trans isomers of natural taraxanthin. Three neo-taraxanthins could be distinguished; they were charac- terised by their absorption spectra in carbon disulphide. None of these pigments has yet been obtained in the crystalline state. Solvent: Absorption maxima: Neo-taraxanthin A 494.5 464 434 m^ Neo-taraxanthin B 497 470.5 443 m^ Neo-taraxanthin C 480 449 m// Taraxanthin (natural) . . . 501 469 440 m/z 16. ESCHSCHOLTZXANTHIN In the course of investigations on the pigments from the blossoms of Eschscholtzia calif ornica, Strain^^ discovered a previously unknown carotenoid for which he proposed the term eschscholtzxanthin. For the isolation of the pigment, the blossoms are dried at 45-47°, finely ground and extracted with petroleum ether. The extract is concentrated and saponified with methanolic potassium hydroxide. The pigments are separated into epiphasic and hypophasic fractions, and the latter are extracted with ether. The ethereal solution is concentrated in vacuum and on cooUng most of the eschscholtzxanthin crystallises out. About 4 g of crude product are obtained from 1.15 kg of dried blossoms. The pigment is purified by repeated re-crystallisation from acetone or chromatographic absorption. (Products of equal purity are obtained by the two methods). Only a beginning has been made in the elucidation of the constitution of eschscholtzxanthin. The molecular formula is C40H54O2 (± Hg)*. The two oxygen atoms are present as hydroxyl groups. On microhydrogenation the pigment takes up 12 mols of hydrogen. The long-wavelength location of the The analytical figures are in better agreement with the formula C^QHggOg than with the formula C4QHg402, but the differences are too small to reach a definite conclusion. References p. 341—343. 324 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV absorption maxima indicates that the 12 double bonds are in conjugation. Eschscholtzxanthin is extremely unstable in air. The rate of oxygen uptake is different in different organic solvents and is not increased by haemin, in contrast to the behaviour of lycopene^*. If solutions of eschscholtzxanthin are warmed, the absorption bands are displaced. Melting point: 185-186° (corr., Berl-block). [ajg^^g = +225° ± 12° (in chloroform). Solvent: Absorption maxima: Carbon disulphide 536 502 475 mfi Benzene 516 485 458 m/j, Chloroform 513 484 456 m/^ Ethanol 503 472 446 m/j, Pyridine 521 489 463 m/z On adding concentrated sulphuric acid to a solution of the pigment in chloroform, the latter assumes a blue colouration. No colouration is obtained with concentrated hydrochloric acid. Eschscholtzxanthin can be chromatographed on magnesium carbonate or calcium carbonate from carbon disulphide or benzene solution. It is found above zeaxanthin but below capsanthin in the chromatogram. The results so far obtained in these preliminary investigations suggest that eschscholtzxanthin may be a dihydroxy-y-carotene; all its properties are in accord with such a structure. Esters of Eschscholtzxanthin 1. Diacetate: Melts over the range 200-240° with decomposition. [a]6678 : + 132° (in chloroform). 2. Dipalmitate: M.p. 100-110°. 3. Dibenzoate: M.p. 133°. [ajg^^g = — 142°. 4. Di-p-nitrobenzoate: M.p. 260° [aj^g^g = — 234°. 5. Dioleate: Could not be crystallised. 17. ECHINENONE During investigations on the pigments from sea urchin {Strongylocentrotus lividiis*), Lederer^^ isolated a new carotenoid, echinenone. For the preparation of echinenone, the sexual glands of sea urchins are ground with sand and extracted at room temperature with acetone. These extracts are combined with those obtained from the back shells of the animals and concentrated * In his first communication dealing with echinenone, E. Lederer [Compt. rend. 201 (1935) 300] erroneously described the sea urchins as Echinus esculentus. The error was corrected in the second communication®^. References p. 341-343. 17 ECHINENONE 325 in vacuum. The residual acetone solution is then diluted with water and the pigments are first extracted with petroleum ether and then with benzene. From the petroleum ether solution mainly polyene hydrocarbons are obtained, while the benzene extract contains mainly phytoxanthins and is worked up for pentaxanthin (p. 327). The petroleum ether solution is shaken with 90% methanol and the phytoxanthins thus extracted are worked up together with those obtained from the benzene solution. For the isolation of echinenone, the petroleum ether extract is washed with water and then saponified with methanohc potassium hydroxide. After working up in the usual way and freezing out accompanying steroids, the pigments are adsorbed on a column of calcium carbonate from petroleum ether solution. The echinenone fraction is separated and chromatographed on alumina. a-Carotene and /3-carotene are adsorbed on the lower part of the column. The echinenone fraction found in the upper part of the column is still not homogeneous, so that the pigment must be chromatographed repeatedly on alumina and on calcium carbonate. It is finally crystallised from a mixture of benzene and methanol. From 400 sea urchins about 4 mg of echinenone can be obtained in this manner. A certain amount of information is available regarding the chemical con- stitution of this pigment, but the structure* has not yet been fully clarified. The elementary analysis of echinenone gives the molecular formula C40H58O ( ± H2) . It produces strong vitamin A activity in rats and therefore probably contains an unsubstituted ^-ionone ring. The single-banded (?) absorption spectrum** suggests the presence of a carbonyl group and the epiphasic behaviour of the pigment excludes the presence of a hydroxyl group. In its general properties echinenone is reminiscent of /3-semicarotenone (p. 139). Lederer suggested the possibility that echinenone may be identical with myxoxanthin (p. 225). Some of the properties of the two compounds are in fact in good agreement, but Heilbron considers it unlikely that the two pigments are identical. (Private communication to Lederer). Echinenone crystallises from petroleum ether or from a mixture of benzene and methanol in violet needles with a metallic lustre, m.p. 178-179°. (On a single occasion Lederer obtained crystals melting at 193°, but the m.p. 178-179° is more probably correct). The absorption maxima of the pigment in carbon disulphide solution are located at 520, 488 and 450 mfi**. On partition between methanol and petroleum ether, echinenone exhibits purely epiphasic behaviour. By treatment with iodine the pigment can be precipitated from petroleum ether solution as the iodide from which it can be A possible structure for echinenone is proposed by E. Lederer and T. Moore, Nature, London, 737 (1936) 996. ** E. Lederer observed a single band at 488 m/n on examination in a visual spectro- meter. Photoelectric determinations by R. Kuhn showed the presence of two additional weak bands at 520 and 450 m^. E. Lederer considers it possible that these weak bands belong not to echinenone itself but to an accompanying carotenoid. This question requires further investigation. References p. 341-34.3. 326 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV regenerated by treatment with thiosulphate. Echinenone gives no blue colouration with concentrated aqueous hydrochloric acid in ethereal solution. l8. PECTENOXANTHIN Lederer^® investigated the pigments which occur during the develop- ment of the sexual organs of St. Jacques shell {Pecten maximus) and discovered a new carotenoid for which he proposed the term pectenoxanthin*. For the isolation of pectenoxanthin, the finely cut sexual organs of the shells are treated with acetone. Water is added and the pigments are extracted from the solution with petroleum ether. They are transferred to 90 % methanol and the solution is strongly concentrated in vacuum. On diluting the methanolic residue with a little petroleum ether, pectenoxanthin crystallises out. For further purification the pigment is adsorbed on a column of calcium carbonate and then crystallised from a mixture of pyridine and water. From 500 sex organs about 50 mg of pure pectenoxanthin were obtained in this way. No conclusive evidence is at present available regarding the chemical constitution of pectenoxanthin. The molecular formula appears to be C40H54O3 (ih Hg). Microhydrogenation showed the presence of 11 double bonds. Zere- WITINOFF determination yielded a quantity of methane corresponding to 2 atoms of active hydrogen. Thus it appears that 2 oxygen atoms are present in the form of hydroxyl groups. The nature of the third oxygen atom is as yet un- known. The pectenoxanthin molecule does not appear to contain an unsub- stituted j3-ionone ring as the compound exhibits no vitamin A activity. Solvent: Absorption maxima: Carbon disulphide 518 488 454 m^ Benzene 496 464 434 mn Petroleum ether 488 458 vafi The pigment crystallises from aqueous pyridine in long yellow-brown prisms, m.p. 182". Pectenoxanthin is adsorbed above zeaxanthin on calcium carbonate. The pigment is much more readily soluble in 90 % methanol than in petroleum ether. It is readily soluble in benzene, carbon disulphide and pyridine. With concentrated sulphuric acid it produces a deep blue colouration. No colouration is observed with aqueous hydrochloric acid. 19. pentaxanthin Besides echinenone, Lederer isolated a further previously unknown pigment, pentaxanthin, from sea urchin {Sirongylocentrotus lividus)^''. * P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 915 discovered a caro- tenoid which exhibits similar absorption maxima as pectenoxanthin in pecten jacobaeus. The two pigments are possibly identical. References p. 341-343. 20 SULCATOXANTHIN 327 For the isolation of the pigment, the phytoxanthin solutions obtained during the preparation of echinenone (p. 325) are washed with water, dried and adsorbed on alumina. After elution, pentaxanthin, which is accompanied by other phyto- xanthins, is again chromatographed on calcium carbonate. This operation is repeated once again and the pigment is then recrystallised from benzene. From 400 sea urchins about 40 mg of pure pentaxanthin are obtained. The chemical constitution of pentaxanthin is largely unknown. The molecular formula is C40H56O5 (± Hg). Only thiee of the five oxygen atoms appear to be present in the form of hydroxyl groups. Microhydrogenation indicates the presence of 12 double bonds in the molecule. Lederer assumes that II double bonds are in conjugation, while the remainder of the hydrogen is used to saturate a carbonyl group or an isolated ethylenic double bond. It should be remarked, however, that the longest wavelength location of. the absorption maximum in carbon disulphide solution produced by 11 conjugated double bonds would be expected to be near 520 m/^, (e.g. jS-carotene) , whereas the absorption maxima of pentaxanthin are located at 506, 474 and 444 m^. The pigment crystallises from benzene in red needles, m.p. 209-210°. Pentaxanthin is adsorbed more strongly than xanthophyll on calcium carbonate. Solvent: Absorption niaxitna: Carbon disulphide 506 474 444 m^a Benzene 487 456 424 m/i Methanol (445) 475 (505) m/i The pigment is very sparingly soluble in ether and petroleum ether, some- what more soluble in benzene and readily soluble in carbon disulphide and chloroform. 20. SULCATOXANTHIN Heilbron and co-workers^ isolated a previously unknown carotenoid, sulcatoxanthin, from Anemonia sulcata. 500 finely cut plants were extracted with a mixture of acetone and ether (1:1), the combined extracts were concentrated in vacuum, and the pigments were extracted with petroleum ether. The solution was then repeatedly shaken with 65 % methanol. Only a small quantity of pigment exhibiting absorption maxima at 478 and 450 m/i in carbon disulphide remained in the ether layer. The sulcato- xanthin was extracted from the methanolic solution with petroleum ether and chromatographed on alumina. It was then adsorbed on calcium carbonate from carbon disulphide solution and finally recrystallised from a mixture of ether and petroleum ether. The yield amounted to about 50 mg. Very little is known about the chemical constitution of sulcatoxanthin. The molecular formula appears to be C40H52O8. The high oxygen content is References p. 341-343. 328 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV reflected in the solubility in 65 % methanol. The pigment is insoluble in petroleum ether, sparingly soluble in carbon disulphide and readily soluble in benzene and ethanol. Sulcatoxanthin crystallises from a mixture of ether and petroleum ether in deep scarlet-red needles which show no sharp melting point but sinter at 110°, soften at 125° and have completely melted at 130°. The pigment is decomposed by the action of alkali. Concentrated sulphuric acid produces a blue colouration. Solvent: Absorption maxima: Carbon disulphide 516 482 (450) m/j. 21. GLYCYMERIN This carotenoid was isolated by Lederer^^ and by Fabre and Lederer''*' from the sexual organs of Pedunculus glycymeris shells. On working up a larger quantity of shells, glycymerin could no longer by obtained'^, a mixture of several phytoxanthins being found instead. As the isolation of the pigment could not be repeated and as Lederer himself obtained a very small quantity which he did not consider as pure, the existence of this pigment must be regarded as doubtful. Glycymerin crystallises in irregular brown-violet crystal aggregates, m.p. 148-153°. It is almost insoluble in petroleum ether, but fairly readily soluble in methanol. In carbon disulphide solution the pigment exhibits a single absorption band at 495 m/ii. It exhibited no acidic properties, thus differing from astacene. It is believed that the pigment occurs partly esterified in the shell. Glycymerin has also been observed in the coat of Pedunculus glycymeris and in the liver of Mytilus edulis^^' '". 22. CYNTHIAXANTHIN In the course of his investigations of the pigments of different species of ascidiae, Lederer found besides astacene, a new pigment, for which he proposed the name cynthiaxanthin, in Halocynthia pipillosa''^. The same source was later examined by Karrer and Solmssen'^^, but no cynthiaxanthin was obtained. In some respects cynthiaxanthin possesses properties very similar to those of zeaxanthin. Chromatography of a mixture of the two pigments, however, gives rise to two zones, the lower of which contains cynthiaxanthin. It is therefore improbable that the two pigments are identical. Cynthiaxanthin also differs in chromatographic properties from pectenoxanthin, since pecteno- xanthin is adsorbed above zeaxanthin on calcium carbonate. For the isolation of cynthiaxanthin, the animals (15 specimens weighing 120 g) are dissected and extracted with acetone. The pigments are transferred to petroleum References p. 341-343. 23 TORULIN 329 ether by dilution with water and the petroleum ether solution is extracted with 90 % methanol. a-Carotene, /5-carotene and esterified astacene (astaxanthin ?) remain in the petroleum ether layer. The hypophasic pigments are taken up in benzene and chrbmatographed on calcium carbonate. Two main zones are formed, the upper yielding astacene after saponification, while the lower contains the new pigment, cynthiaxanthin. By elution with aqueous methanol and crystallisation from the same solvent under petroleum ether, the pigment was obtained in small, yellow- orange needles. After recrystallisation from aqueous ethanol the m.p. was 188-190°. The yield was 1 mg. The pigment does not give a blue colouration with concentrated aqueous hydrochloric acid. Solvent: Absorption maxima: Carbon disulphide 517 483 451 rrifi Petroleum ether 482 452 m/i 23. TORULIN During investigations of the carotenoids of Torula rubra, Lederer'^- '^ found besides ^-carotene and two unknown pigments present in very small amounts, another new carotenoid for which he proposed the term torulin. Later Karrer and Rutschmann'^ also isolated torulin besides torularhodin (p. 330) from the same source. Torulin appears to be fairly widely distributed in nature. Besides Torula rubra, it has been found in Sporobolomyces roseus, Sporobolomyces salmonicolor , Lycogala epidendron^^ and Rhodotorula Sanniei'^'^. For the isolation of torulin, red yeast is extracted with acetone, the mixture of pigments is extracted with petroleum ether after addition of water, and the petro- leum ether solution is extracted with ethanoland saponified with ethanolic potassium hydroxide. After working up in the usual way the epiphasic pigments are dissolved in a little petroleum ether and the solution is allowed to stand in the cold to allow considerable quantities of steroids to crystallise out. The mother liquors are then chromatographed on alumina, the ^-carotene in the lower portion of the chromato- gram is separated, andthetorulin, which is adsorbed above ^-carotene, is crystallised from petroleum ether in the cold. The pigment is further purified by crystallisation from benzene and methanol. Torulin melts at 185°. Solvent: Absorption maxima:''^ Carbon disulphide ..... 565 525 491 m/i* Pyridine 545 508 475 m^M Benzene 541 503 470 m/i Ethanol 520 486 456 m^ Chloroform 539 501 469 m/x Nothing definite is known at the present time concerning the chemical constitution of the pigment. Lederer'^^ found a certain similarity between E. Lederer records absorption maxima at 563, 520 and 488 m^ in carbon di- sulphide. References p. 341-343. 330 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV torulin and rhodoviolascin (p. 295) and suggested that the former may be formed from rhodoviolascin by cyclisation of one half of the molecule, which would explain the shorter wavelength location of the absorption maxima. This suggestion, however, lacks experimental foundation. 24. TORULARHODIN C37H48O2 History and Occurrence In 1933, Lederer''^ investigated the carotenoids of Torula rubra. Besides ^-carotene, he found the previously unknown pigment torulin, as well as two other polyene pigments, one of which appeared to be a hydrocarbon while the other exhibited acidic properties. Fink and Zenger^" later carried out similar investigations and also observed the two last-mentioned pigments. The investigation of Torula rubra pigments was continued by Karrer and Rutschmann^^. These authors were able to elucidate the main features of the constitution of the acidic pigment which they termed torularhodin. Up to the present time torularhodin has only been observed in the red yea.st-Torula rubra. Preparation^^' ^^ After pre-extraction with ethanol in an atmosphere of carbon dioxide, the yeast cells are ground with quartz sand in a large porcelain mortar and then extracted with acetone in vacuum. The acetone solution is diluted with water and the pigments are extracted with a small quantity of petroleum ether. For the separation of the pigments this solution is adsorbed on alumina and the chromatogram is developed with a mixture of ether and methanol. Torularhodin forms an upper light-red zone in the chromatogram and is eluted with a mixture of ether and acetic acid (10:1). This solution is washed free from acid, the ether is evaporated and the oily residue is dissolved in a little methanol. The methanol solution is allowed to stand for 2-3 weeks at room temperature in an evacuated tube. During this period, torularhodin crystallises out, together with a considerable quantity of aliphatic acids, which are removed by washing with petroleum ether. The torularhodin remains undissolved and is obtained in a fairly pure condition after boiling with petroleum ether and methanol. The yield from 1 kg of yeast amounted to about 3-5 mg. For analysis, the pigment is reprecipitated several times from a mixture of benzene and methanol, but nevertheless still contains varying amounts of ash. Chemical Constitution''^ Torularhodin probably has the molecular formula C37H48O2 and contains 12 conjugated double bonds. It is a monocarboxylic acid, as shown by its behaviour towards alkalis and the preparation of a well-crystalhsed mono- methyl ester. The latter exhibits vitamin A activity which is considerably References p. 34.1-343. 24 TORULARHODIN 331 weaker than that of j3-carotene. Since growth-promoting properties are generally- associated with the presence of an unsubstituted jS-ionone ring, it appears probable that such a ring is present in torularhodin. On the basis of these facts, the following possible structural formula, which is in agreement with the analytical data, the number of double bonds and the absorption spectrum, can be deduced. C CH3 • CI13 CH3 CH3 /\ I I II CH2 C-CH=CH-C=CHCH=CH-C=CHCH=CHCH=C-CH=CHCH=C-CH=CH-CH COOH CH2 C"CH3 H3C'C CH \/ Torularhodin( ?) \ Z' CH2 CH In accordance with its acidic nature, torularhodin forms salts with alkalis. The salts exhibit the same optical properties as the free pigments, showing that salt formation does not result in any structural changes. The pigment is entirely indifferent to hydro xylamine in boiling ethanol and towards boiling alcoholic potassium hydroxide. Torularhodin is reduced by zinc dust and acetic acid in pyridine. This reaction is of considerable interest, as it has previously only been observed with carotenoids containing systems of conjugated double bonds terminated by two carbonyl or carboxyl groups (e.g. bixin, crocetin, rhodo- xanthin, j3-carotenone etc.). Torularhodin is rapidly reduced under the same conditions to give a light yellow product exhibiting the following absorption maxima. Solvent: Absorption maxima: Carbon disulphide 482 453 m^ Petroleum ether 453 m/j, This compound shows an acidic reaction and is more hypophasic than torularhodin on partition between methanol and petroleum ether. The displace- ment of the absorption bands towards the violet relative to the parent pigment is extraordinarily large for a dihydroderivative. Properties Crystalline form: Torularhodin crystallises in fine red needles on slowly evaporating a methanol-ether solution. From a mixture of benzene and methanol, the compound is obtained as a violet-black crystalline powder. Melting point: 201-203° (with decomposition, uncorr., in vacuum) References p. 341-343. 332 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV Solubility: Tonilarhodin is easily soluble in carbon disulphide, chloroform and pyridine, less easily soluble in ether, benzene and hot ethanol, very sparingly soluble in methanol and almost insoluble in petroleum ether. Solvent: Absorption maxima: Carbon disulphide 582 541 502 m/x Benzene 557 519 485 m/i Petrol 537 501 (467) m^ Pyridine 558 518 485 mpi Chloroform 554 515 (483) m/z Methanol 529 493 (460) m/i Ethanol 532 495 463 van The large difference in the location of the absorption maxima in carbon disulphide and in petrol or ethanol, is remarkable. The differences in the positions of the longest wavelength bands amount to 45-50 m//, whereas in other carotenoids the differences are usually of the order of 30-40 m/i. Optical activity: No data have been recorded. Partition test: On partition between petroleum ether and 95 % methanol, the two zones are about equally coloured. Chromatographic behaviour: Tonilarhodin is very strongly adsorbed on zinc carbonate from benzene solution, and forms a deep-violet zone in the uppermost part of the column. On alumina, the pigment is adsorbed with a light red colour. The chromatogram on zinc carbonate is developed with benzene, while the chromatogram on alumina is developed with a mixture of ether and methanol. The pigment is eluted with a mixture of ether and acetic acid (10:1). Colour reactions: Torularhodin exhibits a different behaviour towards antimony trichloride and towards strong acids than any other known carotenoid. With antimony trichloride, a permanganate-red colouration is at first produced which immediately fades. After some time the solution assumes a faint blue colour. Anhydrous formic acid and concentrated sulphuric acid also decolourise the yellow-red torularhodin solution ; on standing, however, a faint blue colour appears. On adding trichloracetic acid to a solution of the pigment in chloro- form, a faint green colouration is observed after initial bleaching. Detection and estimation: Torularhodin is identified by its acidic character and by means of its long- wavelength spectrum. Torularhodin methyl ester C3gH47C02CH3 The methyl ester is formed by the action of diazomethane on a solution of torularhodin in benzene. It crystallises from a mixture of benzene and methanol in dark red needles, m.p. 172-173°, uncorr. The absorption spectrum of the ester differs shghtly from that of the free acid. References p. 341-243. 26 VIOLERYTHRIN 333' Solvent: Absorption tnaxima: Carbon disulphide 581 541 502 m/j, Benzene 554 517 484 m/x Petrol 533 498 468 m/x Pyridine 560 519 485 m/x Ethanol 533 496 464 m/x 25. ACTINIOERYTHRIN This pigment was first obtained by Lederer®^ from sea anemones Actinia equina and was shortly afterwards isolated from the same source by Heilbron and co-workers^. For the isolation of the pigment^^- '", the finely cut anemones are extracted with a mixture of ether and acetone (1:1) and the solution is concentrated under reduced pressure. The pigments are transferred to petroleum ether and the solvent is again evaporated. By dilution of the residue with acetone, part of the accompanying phosphatides and steroids can be precipitated. The remainder is frozen out. The pigments remaining in the mother liquors are taken up in petroleum ether and chromatographed on alumina. Actinioerythrin forms a violet-black zone in the upper part of the chromatogram. After elution, the pigment is again adsorbed on calcium carbonate and finally crystallised from absolute ethanol. 30 mg of actin- ioerythrin were obtained from 500 anemones. Very little is yet known about the constitution of actinioerythrin. It is not even certain whether it is a carotenoid. According to Lederer^^, actinioerythrin is the ester of a coloured acid. This is supported by the low melting point and by the good solubility in petroleum ether. On alkaline Hy- drolysis, however, Heilbron and co-workers^ obtained a compound, violery- thrin, which showed no acidic properties towards dilute alkalis (cf. below). It was therefore assumed that violerythrin contains one or more enol groups which are isomerised to the stable ketoform. Actinioerythrin crystallises from ethanol in brown-violet rhombs, m.p. 85°. It is sparingly soluble in ethanol, but readily soluble in petroleum ether, carbon disulphide, chloroform and pyridine. Solvent: Absorption maxima: Carbon disulphide 574 533 495 m/x Petroleum ether 534 497 470 m/x Ethanol 577-518 m/< (broadband) 26. VIOLERYTHRIN Violerythrin was obtained by Heilbron, Jackson and Jones^ by the alkaline hydrolysis of actinioerythrin (of above). The fact that hydrolysis References p. J41—34J. 334 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV occurs on shaking actinioerythrin with alkaHs was previously observed by Fabre and Lederer'^* who were unable, however, to isolate a homogeneous derivative and therefore assumed that decomposition of the pigment had taken place. Violerythrin can only be obtained in relatively good yields by carrying out the hydrolysis under very carefully controlled conditions. According to Heilbron and co-workers, a solution of actinioerythrin in petroleum ether is shaken at room temperature with 2.5% methanolic sodium hydroxide until the pigment has been entirely transferred to the lower layer (about 1 hour). The reaction mixture is then diluted with water, acidified with acetic acid and extracted with ether. The ethereal solution is washed and dried, the solvent is evaporated and the residue is crystallised from aqueous pyridine. 1 mg of viol- erythrin was obtained in this way from 5 mg of actinioerythrin. The structure of this pigment is still unknown. It is not certain whether it belongs to the carotenoid series. Violerythrin crystallises from aqueous pyridine in dark-violet micro- crystals, m.p. 191-192°. It dissolves in carbon disulphide with a purple-red colour, in alcohol and ether with a violet-red colour, in acetone and pyridine with a blue colour, and in benzene with a deep-blue colour. The absorption maxima in carbon disulphide are located at 625, 576 and 540 vafi. 27. 8-CAROTENE WiNTERSTEiN®^ examined the shells of Gonocaryum pyriforme for carote- noids and found besides lycopene, a-carotene, ^-carotene and y-carotene, a new pigment for which he proposed the term 8-carotene. In the chromatogram, S-carotene is found between y-carotene and j8-carotene. It could not be obtained in a crystalline state so that its existence cannot be regarded as proven. Solvent: Absorption maxima: Carbon disulphide 526 490 457 mfi Chloroform 503 470 440 m/i Hexane 490 458 428 m/< WiNTERSTEiN^^ regards 6-carotene as the hitherto unknown carotene- isomer derived from one half-molecule of a-carotene and one half-molecule of y-carotene. 28. FENICOTTERIN During an investigation of the pigments responsible for the red colour of the fat of flamingo, C. Manunta^* discovered a carotenoid which is similar to astacene but differs from the latter by a shorter-wavelength absorption maximum (487 m/^ in carbon disulphide). As this compound was not obtained in a pure state or analysed, its existence cannot be regarded as certain. References p. 341-343. 30 TROLLIXANTHIN 335 29. OSCILLAXANTHIN In the course of an investigation of the carotenoids of Oscillatoria rubescens, Karrer and Rutschmann^^ found, besides myxoxanthin, myxoxanthophyll, zeaxanthin* and )3-carotene, a hitherto unknown pigment for which they proposed the term oscillaxanthin. For the isolation of this pigment, the algae are dehydrated with ethanol, dried and extracted with warm methanol. This extract is combined with that obtained from the dehydration and the solutions are strongly concentrated. They are then saponified with aqueous potassium hydroxide and extracted with a large volume of ether, and the aqueous phase is almost neutralised with dilute sulphuric acid. From this solution the methanol is removed by distillation and the remaining aqueous solution is slightly acidified with dilute sulphuric acid (to pH4-5). The solution is then again extracted with ether whereby most of the saponification products of chlorophyll are removed. From the mother liquors, oscillaxanthin can then be extracted with ethyl acetate. After evaporation of the solvent the pigment is taken up in acetone and chromatographed on zinc carbonate. After elution, the oscilla- xanthin is further purified by precipitation from ethyl acetate by means of ether. The pigment could not be obtained in a pure state because of the small amount of material available. Oscillaxanthin exhibits acidic properties. It is readily soluble in alcohol, pyridine and acetone, but almost insoluble in ether, benzene, carbon disulphide and petroleum ether. Oscillaxanthin esters are readily soluble in ether, benzene, pyridine and chloroform, but almost insoluble in ethanol. The constitution of oscillaxanthin is still entirely unknown. Solvent: Absorption maxima: Carbon disulphide 568 528 494 m^** Methanol 531 496 464 m/t Pyridine 552 514 483 m/< Antimony trichloride produces a blue-green, concentrated sulphuric acid a blue, and concentrated hydrochloric acid an unstable blue colouration. 30. TROLLIXANTHIN AND TROLLICHROME C4oH5g04 During investigations of the carotenoids of Trollius europaeus, Karrer and JucKER^^ discovered a new pigment which had previously escaped notice and for which they proposed the name trollixanthin^'^. It posseses the properties of an epoxide. In addition to trollixanthin, the flowers also contained jS-carotene, I. M. Heilbron and B. Lythgoe, /. Chem. Soc. 1936, 1376 found myxoxanthin, myxoxanthophyll, ;3-carotene and xanthophyll, but no zeaxanthin in Oscillatoria rubescens. Absorption maxima in carbon disulphide refer to solutions prepared by diluting one drop of an alcoholic solution of the pigment with a large volume of carbon disulphide. References p. 341-343. 336 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV xanthophyll, xanthophyll epoxide and another new carotenoid, also of epoxide character. The latter was not obtained in sufficient quantity for more detailed investigation. For the preparation of trollixanthin, the blossoms are dried at about 40° and are exhaustively extracted with petroleum ether at room temperature. The combined extracts are concentrated to a small volume in vacuum and the residue is saponified with methanolic potassium hydroxide at room temperature. The pigments are then separated into an epiphasic and a hypophasic fraction. The latter is repeatedly boiled with ligroin to remove colourless products and the undissolved portion is chromatographed on zinc carbonate. Trollixanthin is adsorbed on the upper part of the column and can be separated from the less strongly adsorbed xanthophyll epoxide. It is eluted with methanol-containing ether and is recrystallised from benzene. The constitution of the pigment is still unknown. The molecular formula of trollixanthin is C4{,H5g04^. Trollixanthin is a monoepoxide ; by the action of dilute mineral acid it is converted into a furanoid derivative, trollichrome, the absorption maxima of which are displaced by 22 m^i towards shorter wavelengths (in carbon disulphide solution) . The remaining oxygen atoms are present in the form of hydroxyl groups; the latter are responsible for the stronger adsorption of trollixanthin as compared with xanthophyll epoxide on zinc carbonate. In view of the close similarity in the spectral properties of trollixanthin and xanthophyll epoxide, and of trollichrome, flavoxanthin and chrysanthema- xanthin, it is probable that similar chromophoric systems are present. Trolli- xanthin crystallises from benzene in thin, clustered light-yellow leaflets, m.p. 199° (uncorr., in vacuum). On shaking an ethereal solution of the pigment with concentrated aqueous hydrochloric acid, the latter assumes a weak blue co- louration, as in the case of most carotenoid monoepoxides. Solvent: Absorption maxima: Carbon disulphide 501 473 m^ Ethanol 474 447 m/x Benzene 483 457 m/z Chloroform . . . . 482 455 m/j. On partition between methanol and petroleum ether, trollixanthin is entirely hypophasic. Trollichrome separates from benzene in light yellow crystals, m.p. 206-208° (uncorr., in vacuum). The hydrochloric acid reaction and partition test are the same as with trollixanthin, Solvent: Absorption maxima: Carbon disulphide 479 450 m/j. Ethanol 451 424 m/t Benzene 459 432 m/i Chloroform 458 430 m/j, References p. J41-J4J. 32 CANARYXANTHOPHYLL 337 31. HAEMATOXANTHIN During investigations of the spores of Haematococcus pluvialis, Tischer^^ found, besides jS-carotene, a-carotene*, xanthophyll, zeaxanthin andastacene**, a previously unknown carotenoid for which he proposed the name haemato- xanthin. About 6 g of the red spores of Haematococcus pluvialis were available for the isolation of the carotenoid. The spores were ground with quartz sand under acetone and exhaustively extracted with this solvent at room temperature. The extract was diluted with water and the pigments were extracted with petroleum ether. After removing the solvent by distillation, a red resinous residue remained which was dissolved in a little petrol and saponified with methanolic potassium hydroxide. The pigments were then divided in the usual way into epiphasic and hypophasic fractions and the epiphasic fraction was adsorbed on a column of calcium hydroxide. A very small quantity of haematoxanthin was eluted from the upper part of the chromatogram with alcohol-containing petrol and recrystallised from petrol. The pigment could not be obtained entirely pure because of lack of material. It is not yet known whether haematoxanthin occurs in algae as an ester^". The constitution of the pigment is unknown. Haematoxanthin is entirely epiphasic on partition between petroleum ether and 90 % methanol. If 95 % methanol is employed, the lower layer is also weakly coloured. The crude material melts at 205°. The pigment crystallises from petrol in brown-violet leaflets. Solvent: Absorption range Absorption maxima Carbon disulphide 463-563 513 m/x Petrol (b.p. 70-80") .... 450-515 478 m/z Ether 445-515 480 m/i 32. CANARYXANTHOPHYLL AND PICOFULVIN In the course of their investigations of the yellow pigments of different birds, Brockmann and Volker^^ found that the carotenoids present were derived mostly from xanthophyll or, occasionally from zeaxanthin***. By means of feeding tests, these authors were able to prove that the feathers of canaries only attain a yellow colour if the diet contains xant hophyll or zea- xanthinf. In the digestive tract, the xanthophyll is converted into canary- J. TiscHER, Z. physiol. Chem. 2^2 (1938) 225. ** In his paper, J. Tischer reports the isolation of "Euglenarhodon". This carotenoid later proved to be identical with astacene and thus does not require a special name. The yellow pigment of the budgerigar (Melopsitfacus undulatus) is not a carotenoid. f Polyene pigments such as jS-carotene, lycopene, violaxanthin and taraxanthin are not assimilated by birds. In canaries which have turned white by being kept on a suitable diet, the yeUow colour is only restored on feeding xanthophyll or zeaxanthin, but not on feeding a carotenoid hydrocarbon, or violaxanthin or taraxanthin. References p. 341-343. Carotenoids 22 338 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV xanthophyll and probably also into picofulvin, previously observed by Krukenberg. Neither canaryxanthophyll nor picofulvin could be obtained in the crystal- line state or investigated in detail, so that their constitutions are still unknown. If the diet contains zeaxanthin, the feathers also assume a yellow colour, but the pigments formed are not identical with those formed from a xanthophyll- containing diet. In this case the pigments appear to be decomposition products of zeaxanthin which do not exhibit sharp absorption maxima. The following is a brief summary of the pigments of the feathers of some birds*. Bird Pigment Bullfinch Red decomposition products Canary Canaryxanthophyll Green finch Canaryxanthophyll, xanthophyll Grey wagtail Xanthophyll Oriole Xanthophyll, canaryxanthophyll Weaver Xanthophyll, canaryxanthophyll, decomposition products Woodpecker Picofulvin, xanthophyll Canaryxanthophyll exhibits absorption maxima at 472, 443 and 418 m/i in ethanol. Aqueous 25 % hydrochloric acid produces no displacement of the absorption maxima towards shorter wavelengths in an ethereal solution of the pigment. Picofulvin exhibits absorption maxima at 450 and 424 m/^ in ethanol. This pigment differs from fiavoxanthin in its negative hydrochloric acid reac- tion. Canaryxanthophyll is adsorbed more strongly than xanthophyll on calcium carbonate. 33. LEPROTIN C40H54 Leprotin was isolated by Grundmann and Takeda^^ from a pure strain of acid-resisting bacteria of a leper. The same pigment was later found by Takeda and Ohta^^ in Mycobacterium phlei. For the isolation of leprotin, the bacteria are dried and extracted with acetone. The extracts are saponified and the pigments of the epiphasic fraction are chromatographed on alumina. After elution and evaporation of the solvent, leprotin is crystalHsed from a mixture of benzene and methanol. The pigment is very similar to ^-carotene but differs from the latter in melting point, in the stronger adsorption on alumina, and in the absence of vitamin A activity^*. The molecular formula of leprotin appears to be C^^Yi^^^. On microhydro- genation 12 mols of hydrogen are absorbed^^. Leprotin crystallises from a mixture of benzene and methanol in fine, Further information will be found on pp. 92 and 93—95. References p. 341-343. 36 MYTILOXANTHIN 339 felted, copper-red needles which melt at 198-200°. (Kofler block). On treating a chloroform solution of the pigment with antimony trichloride, a stable blue colouration is produced. Solvent: Absorption maxima: Carbon disulphide 517 479 447 m/i Chloroform 495 460 428 m^ Petrol 484 452 425 m// 34. SALMON ACID The pigments responsible for the red colouration of the flesh of salmon [Salmo salar) have been the subject of a number of investigations with partly contradictory results. In 1885, Krukenberg and Wagner^* estabhshed the presence of three carotenoids, namely xanthophyll, carotene and an unknown pigment. In 1933, the latter was obtained in a crystalline state by von Euler, Hellstrom and Malmberg^® and termed salmon acid. Salmon acid was later studied by Emmerie, van Eekelen, Josephi and Wolff^^. The chemical nature of salmon acid is still unknown. According to von Euler and co-workers^^, salmon acid is readily soluble in acetic acid and can be precipitated from the solution with alkali. It forms blue-black crystals and shows a single broad absorption maximum at 485 m/i in pyridine solution. A very weak subsidiary maximum at 525 m^ is also observed. Emmerie and co-workers, on the other hand, report that the pigment exhibits an absorption band near 500 m/< in pyridine solution. In the partition test salmon acid is found entirely in the 90 % methanol layer, 35. asterin acid During an investigation of the carotenoids of the back skin of Asterias rubens and of the eggs of Coregonus albula^^, von Euler and co-workers^^ discovered a previously unknown polyene pigment which they termed asterin acid. The compound possesses properties similar to those of astacene (or astaxanthin) . In a later investigation of Asterias rubens, Karrer and Rubel^''*' found that astacene was present. Asterin acid is therefore probably identical with astacene. 36. MYTILOXANTHIN In the course of extensive investigations concerning the part played by carotenoids in the metabolism of Mytilus californianus shells, Scheer^"^ established the presence of zeaxanthin and of another new pigment, mytilo- xanthin. The latter occurs as such in sea shells and appears to play a part in their metabolism. The structure of mytiloxanthin is still unknown. It appears to References p. 341-343. 340 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV be an acid or an enol, as its colour is reversibly changed on addition of alkalis. This behaviour and the single-banded absorption spectrum (maximum at 500 vcifx in carbon disulphide) are reminiscent of astacene, but mytiloxanthin has a much lower melting point (140-144°). (The melting point of astacene is 228°). 37. CAROTENOID FROM THE BLOSSOMS OF SATIN OAK [Grevillea robusta) Zechmeister and Polgar^^^ found, besides j3-carotene, cryptoxanthin and xanthophyll, a previously unknown hypophasic carotenoid in the blossoms of satin oak {Grevillea robusta). The pigment is only present in very small amounts, so that neither its physical properties nor its constitution could be determined. It crystallises in long plates. Solvent: Absorption maxima: Carbon disulphide 490.5 457 m/i Benzene 479.5 440.5 ran Petrol 457.5 430 raft 38. CAROTENOIDS FROM DIATOMS, BROWN ALGAE AND DINOFLAGELLATES According to Strain, Manning and Hardin^"^, certain species of diatoms and brown algae contain, besides fucoxanthin, a number of other carotenoids which were named diatoxanthin, diadinoxanthin, neofucoxanthin A and neo- fucoxanthin B. The chemical nature of these carotenoids is still entirely unknown. According to the same authors, dinofiagellates Peridinium cinctum contain pigments which were named dinoxanthin, neodinoxanthin, diadinoxanthin, neodiadinoxanthin and peridinin. The chemical nature of these compounds is also unknown. 39. carotenoid from neurospora A new carotenoid named neurosporen has been isolated by Haxo^*^* from the fungus Neurospora crassa. Neurosporen occurs together with lycopene, y-carotene and rhodoviolascin (spirilloxanthin) and smaller quantities of j8-carotene, and of four other pigments resembling 8-carotene, rhodopurpurin, lycoxanthin and rhodopin. Neurosporen was obtained crystalline, m.p. 124°. It is a hydrocarbon and has the molecular formula C^oHgg (± 2H). Solvent: Absorption maxima: Carbon disulphide 502.5 470.5 439.5 mn Petroleum ether 470 441.5 myt* References p. 341-343. REFERENCES 341 REFERENCES 1. E. R. Lankester, Quart. J. Mikroscop. Science 13 (1873) 408; 16 (1876) 27. 2. E. Warming, cited by H. Molisch, Die Purpurbakterien, Jena 1907, p. 2. — T. W. Engelmann, Boi. Ztg. 46 (1888) 661, 667, 693, 709. — S. Winogradsky, Bot. Ztg. 45 (1887) 489, 513, 529, 545, 569, 585, 606. — O. BuTSCHLi, tjber den Bau der Bakterien und verwandter Organismen, Leipzig 1890. — G. A. Nadson, Bull. Jard.bot. St. Peters- bourg 3 (1903) 109. 3. V. Archichovskji, Bot. Zentr. gg (1905) 25. 4. H. Molisch, Die Purpurbakterien, Jena 1907. 5. Cf. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 1306- 6. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 25; i^ (i935) 1306; ig (1936) 3; ig (1936) 1019; P. Karrer, U. Solmssen and H. Koenig, Helv. chim. Acta 21 (1938) 454; P. Karrer and H. Koenig, Helv. chim. Acta 23 (1940) 460. 7. A. PolgAr, C. B. van Niel and L. Zechmeister, Arch. Biochem. 5 (1944) 243. 8. C. B. VAN Niel and J. H. C. Smith, Arch. Microbiol. 6 (i935) 219- 9. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 25. 1306; ^9 (1936) 3- 1019: P. Karrer, U. Solmssen and H. Koenig, Helv. chim. Acta 21 (1938) 454; P. Karrer and H. Koenig, Helv. chim. Acta 23 (1940) 460. ID. Cf. H. Koenig, Dissertation, Zurich, 1940, p. 54. 11. P. Karrer and U. Solmssen, Helv. chim. Acta ig (1936) 1019. 12. P. Karrer and H. Koenig, Helv. chim. Acta 23 (1940) 460. 13. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 25, 1306; ig (1936) 3. 1019; P. Karrer, U. Solmssen and H. Koenig, Helv. chim. Acta 21 (1938) 454. 14. P. Karrer and U. Solmssen, Helv. chim. Acta ig (1936) 1019. — H. Koenig, Dissertation, Univ. Ziirich 1940. 15. H. Kylin, Z. physiol. Chem. 166 (1927) 50. — Cf. Kungl. Fysiogr. Sallskid Lund Fohr. 7 No. 12 (1937) I- 16. I. M. Heilbron and B. Lithgoe, /. Chem. Soc. ig36, 1376. 17. P. Karrer and J. Rutschmann, Helv. chim. Acta 27 (1944) 1691. — J. Rutsch- mann. Dissertation, Ziirich, 1946. 18. J. Tischer, Z. physiol. Chem. 251 (1938) 109; 260 (i939) 257. 19. J. Tischer, Z. physiol. Chem. 251 (1938) 127. 20. J. Tischer, Z. physiol. Chem. 260 (1939) 257. 21. R. Kuhn and C. Grundmann, Ber. 66 (1933) 1746. 22. J. Tischer, Z. physiol. Chem. 231 (1938) 109. 23. A. Scheunert and K. H. Wagner, Z. physiol. Chem. 260 (i939) 272. 24. J. Tischer, Z. physiol. Chem. 260 (1939) 269, 270. 25. P. Karrer and E. Jucker, Helv. chim. Acta 28 (1945) 427. — Cf. p. 147. 26. Z. physiol. Chem. 260 (1939) 267. 27. S. RosANOFF, Mem. Soc. Sci. nat. Cherbourg 13 (1867) 195. 28. G. Kraus and A. Millardet, Compt. rend. 66 (1868) 505; Compt. rend. 68 (1869) 462. 29. H. C. SoRBY, Proc. Roy. Soc. London 21 (1873) 474. 30. M. TswETT, Ber. dtsch. bot. Ges. 24 (1906) 234. 31. R. WiLLSTATTER and H. J. Page, Ann. 404 (1914) 237. 32. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 623; Z. angew. Chem. 12 (1929) 918. 33. R. KuHN and A. Winterstein, Ber. 64 (193 1) 326. 34. I. M. Heilbron and R. F. Phipers, Biochem. J. 2g (1935) i373- 34a. P. W. Carter, L. C. Cross, I. M. Heilbron and E. R. H. Jones, Biochem. J. 43 (1948) 349. 35. I. M. Heilbron and co-workers, Proc. Roy. Soc. (London), B 128 (1939) 82. 36. H. H. Strain and W. M. Manning, /. Am. Chem. Soc. 64 {1942) 1235. — H. H. Strain, W. M. Manning and G. Hardin, /. biol. Chem. 148 (1943) 655. Biochem. Bull. 86 (1944) 169. 342 CAROTENOIDS OF UNCERTAIN STRUCTURE XIV 37. R. ^ETovsKY, Collection of Czechoslovak, chem. Communications 13 (1948) 631. 38. P. Karrer and co-workers, Helv. chim. Acta 14 (1931) 628. 39. R. WiLLSTATTER and H. J. Page, Ann. 404 (1914) 253. 40. I. M. Heilbron and R. F. Phipers, Biochem.J. 29 (1935) 1369- 41. P. Karrer and co-workers, Helv. chim. Acta 14 {1931) 623. 42. H. V. EuLER, P. Karrer, E. Klussmann and R. More, Helv. chim. Acta 15 (1932) 502. 43. M. TswETT, Ber. dtsch. hot. Ges. 24 (1906) 234. — I. M. Heilbron and R. F. Phipers, Biochem. J . 2g (1935) i373- 44. H. H. Strain, W. M. Manning, /. Am. Chem. Sac. 64 (1942) 1235. 45. H. H. Strain, W. M. Manning, G. Hardin, Biochem. Bull. 86 (1944)- — /• biol. Chem. 148 (1943) 655. 46. K. ScHON, Biochem. J. 32 (1938) 1566. 47. L. Zechmeister and W. A. Schroeder, /. Am. Chem. Soc. 65 (1943) i535- 48. A. L. Le Rosen and L. Zechmeister, Arch. Biochem. i (1943) 17. 49. G. MiCHAUD and J. F. Tristan, Arch. Sci. phys. nat. Geneve 37 (1914) 47- 50. L. Zechmeister, T. B^res and E. Ujhelyi, Ber. 69 (1936) 573- — Cf. also Ber. 68 (1935) 1321. 51. H. Suginome and K. Ueno, Bull. Soc. chem. Jap. 6 (1931) 221. 52. L. Zechmeister and co-workers, Ber. 69 (1936) 573- 53. E. Chargaff and J. Dieryck, Naturwissenschaften 20 (1932) 872. 54. E. Chargaff, Compt. rend. 197 (i933) 94^- 55. T. Nakamura, Bull. chem. Soc. Japan 11 (1936) 176- 56. Yoshiharu Takeda and Tatuo Ohta, Z. physiol. Chem. 268 (1941) I-H- 57. R. Kuhn and E. Lederer, Z. physiol. Chem. 200 (1931) 108. 58. P. Karrer and J. Rutschmann, Helv. chim. Acta 25 (1942) ii44- 59. R. Kuhn and E. Lederer, Z. physiol. Chem. 213 (1932) 188. 60. K. Schon, Biochem. J. 30 (1936) i960. 61. R. Kuhn and H. Brockmann, Z. physiol. Chem. 206 (1932) 41- 62. L. Zechmeister and P. Tuzson, Ber. 72 (1939) 1340- 63. H. H. Strain, /. hiol. Chem. 123 (1938) 425. 64. Cf. W. Franke, Z. physiol. Chem. 212 (1932) 234. 65. E. Lederer, Recherches sur les Carotenoides des Animaux, These, Paris, 1938, p. 40. 66. R. Lederer, Compt. rend. Soc. Biol. 116 (1934) 15°: ^^7 (i934) 4ii- 67. E. Lederer, Compt. rend. 201 (1934) 3°°: These, Paris 1938. 68. I. M. Heilbron, H. Jackson and R. N. Jones, Biochem. J. 29 {1935) 1384- 69. E. Lederer, Compt. rend. Soc. Biol. 113 (i933) i39i- 70. R. Fabre and E. Lederer, Bull. Soc. Chim. Biol. 16 (1934) io5- 71. E. Lederer, These, Paris 1938, p. 27. 72. E. Lederer, Compt. rend. Soc. Biol. 116 (1934) ^5°'> ^^7 (i934) 1086. 73. P. Karrer and U. Solmssen, Helv. chim. Acta 18 (1935) 9i5- 74. E. Lederer, Compt. rend. 197 (1933) 1694; Compt. rend. Soc. Biol. 117, 1083; These, Paris 1938, p. 66; Bull. Soc. Chim. Biol. 20 (1938) 554- 75. Cf. H. Fink and E. Zenger, Wschr. Brauerei 51 (1934) 89- 76. P. Karrer and J. Rutschmann, Helv. chim. Acta 29 (1946) 355. 77. C. Fromageot, Jou6 Leon Tchang, Arch. Mikrobiol. 2 (1938) 424. 78. E. Lederer, These, Paris 1938, p. 68. 79. E. Lederer, Compt. rend. 197 (1933) 1694. 80. H. Fink and E. Zenger, Wschr. Brauerei, 51 (1934) 89. 81. P. Karrer and J. Rutschmann, Helv. chim. Acta 26 (1943) 2109; 28 {i945) 795; 29 (1946) 355. 82. J. Rutschmann, Dissertation, Ziirich, 1946. 83. A. Winterstein, Z. physiol. Chem. 219 (1933) 249. 84. Carmela Manunta, Helv. chim. Acta 22 (1939) 1153. 85. P. Karrer and J. Rutschmann, Helv. chim. Acta 27 (1944) 1691. 86. P. Karrer and E. Jucker, Helv. chim. Acta 29 (1946) 1539- REFERENCES 343 87. P. Karrer and A. Notthafft. Helv. chini. Acta 15 (1932) 1195. 88. P. Karrer and E. Krause-Voith, Helv. chim. Acta 30 (1947) 1772. 89. J. Tischer, Z. physiol. Chem. 250 (1937) 147. 90. J. Tischer, Z. physiol. Chem. 252 (1937) 225. yi. H. Brockmann and O. Volker, Z. physiol. Chem. 224 (1934) I93- 92. C. Grundmann and Y. Takeda, Naturwissenschaften 23 (1937) 27. 93. Y. Takeda and T. Ohia, Z. physiol. Chem. 258 (1939) 6. 94. Y. Takeda and T. Ohta, Z. physiol. Chem. 26y (1941) 171. 95. C. F. W. Krukenberg and H. Wagner, Z. Biol. 21 (1885) 25. — Cf. Newbigin, Colour in nature, a study in biology, London 1898 p. 1-344. 96. H. V. EuLER, H. Hellstrom and M. Malmberg, Svensk Kem. Tidskr. 43 (1933) 151. 97. A. Emmerie, M. van Eekelen, B. Josephi and L. K. Wolff, Acta. Brev. neerl. Physiol. Pharm. Microbiol. 4 (1934) 139. 98. H. V. EuLER and co-workers, Z. physiol. Chem. 22S (1934) 77- 99. H. V. EuLER and co-workers, Z. physiol. Chem. 22 j (1934) 89. 100. F. RuBEL, Dissertation, Zurich 1936. loi. B. T. ScHEER, /. biol. Chem. 136 (1940) 275. 102. L. Zechmeister and A. Polgar, /. biol. Chem. 140 (1941) i. 103. H. H. Strain, W. M. Manning, G. Hardin, Biochem. Bull. 86 (1944) 169. 104. F. Haxo, Arch. Biochem. 20 (1949) 400. Plate I. Crystalline forms of some carotenoids /^-Carotene from petrol a-Carotene from petrol Lycopene from petrol Xanthophyll from methanol-ether Zeaxanthin from methanol-ether Violaxanthin from methanol-ether Plate II. Crystalline forms of some carotenoids Taraxanthin from methanol-ether Fucoxanthin from methanol-etlier Capsanthin from carbon disulphide- petrol Crocetin dimethyl ester from chloroform-ethanol (top) Crocetin from pyridine (bottom) '"/'V X "^^ / Bixin from ethvl acetate Azafrin metliyl ester from toluene (top) Azafrin from toluene (bottom) Light absorption curves of some carotenoids Fig. 4. Lycopene in hexane* Z. angew. Chem. 4j (1934) 664 logt. ' 5.0 4.5 i.O 3.5 300 350 400 450 500 ■ X in rrtL Fig. 5. y-Carotene in hexane (- Ber. 66 (1933) 408 and carbon disulphide (- * b' is the molar extinction coefficient given by e' = 2.3/c/ x log (lo/I), where lo = intensity of incident light, 1= intensity of transmitted light, c~ concentration in gm.-mols/l, and ^= cell length in cm. 350 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS fi't 10' A« / \ J^ / \ c N^^X w>roo?°-r . k Fig. 6. )3-Carotene in hexane Z. angew. Chem. 47 (1934) 664 300 200 100 fnl /^ s r\ ^ f c v^^-x w>-ooOC-<^ ...A V- Fig. 7. a-Carotene in hexane Z. angew. Chem. 47 (1934) 664 500 -^X inmfi e'\ 10^ ~7^ p / \ ^'^^w^ >.o--x»o— ""^"^ V Fig. 8. Zeaxanthin in ethanol Z. angew. Chem. 4y (1934) ^^4 500 -»- A in mu. LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 351 logEut 3.0 2.5 2.0 1.6 ■ / • ^' \ ^ \ \ \ I rK / / / / / / V 1 \ \ \ / " / V\ 270 300 350 400 450 500 — -A in ma Fig. 9. Violaxanthin in ethanol; Fucoxanthin in hexane* Helv. chim. Acta 26 (1943) 117 3.0 2.5 , \ A • \ / \ ■ M • / \^ \ 1 \ \ /^ < y \ 250 350 400 ■ X in mil Fig. 10. Auroxanthin in ethanol Helv. chim Acta 25 (1942) 1624 * E is the extinction, one per cent, one centimetre, given by E '° ^ ijcl x log (lo/I). where I^ and I are the intensities of the incident and transmitted light, respectively, c=^ concentration in gms/ioo ml, and /==cell length in cms. 352 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 3.6 3.1 2.6 \ i<°\ I \ : \ h ^ .... \, ,, 250 300 350 ^00 «50 500 Fig. II. Xanthophyll in hexane Helv. chim. Acta 26 (1943) 117 -A "1 Till. Fig. 12. Flavoxanthin in benzene Z. physiol. Chem. 213 (1932) 194 300-10- 200 ■ / / / / / / / 1 1 / / / 1 1 / 1 y .J f \ \ \ \ \ \ 1 / • / f • / \ \ > \ \ \ \ \ \ )l . .1. 1 — 1 — X. itbO 500 550 -*-X in mu. Fig. 13. Rhodoxanthin — ) and dihydrorhodoxanthin (- Ber. 66 (1933) 828 in hexane LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 353 500 -m-Xin mu Fig. 14. Capsanthin in hexane Helv. chim. Acta 26 (1943) n? Fig. 15. Rhodoxanthin -) and astaxanthin ( ) in hexane Helv. chim. Acta 26 (1943) nS 354 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 80 X /0» 70 60 50 40 30 20 /^ / / / ^ / \ / \ y / \ -^ \ 10 \ J30 350 UOO 450 500 Fig. 1 6. Astacene in pyridine Ber. 66 (1933) 492 550 600 —X in ma Fig. 17. Bixin : Bixindialdehyde ; Apo-3-norbixinal methyl ester from stable bixin, all in ethanol Helv. chim. Acta 26 (1943) 120 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 355 3.6 3.1 2.6 ?.1 A ■ A \ ,.^ : ^ rw \ 230 300 350 WO 450 Fig. 18. Crocetin dimethyl ester in ethanol Helv. chim. Acta 26 (1943) 120 500 300-10^ e't 200 100 A^ .^..^.^^ __^ r \ K 500 Fig. 19. Methylbixin in hexane Z. angew. Chem. 47 (1934) ^62 356 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS Fig. 20. - Dihvdrobixin ; Dihydromethylbixin in ethanol Helv. chim. Acta 26 (1943) 120 1 .J ,„■! - ^200 ID flfl 1 pjy > 1 / 5 . . , . r^ y. . . .K a 1 1 1 1 200 300 220 250 i,QO 500 »-A/n ma Fig. 21. Dihydrocrocetin in hexane Z. angew. Cheni. 47 (1934) 662 300 350 UQO Fig. 22. Crocetin in ethanol Z. angew. Chem. 4y (1934) 662 ^50 490 X in mp. LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 357 J T r\ 150 .10^ / X / / \ ^ , ,-N 1 1 / ' \ \ / \ \ " / \ \ f^ \ \ f 1 / / / / / / 1 1 1 \ \ \ \ \ \ \ \ \ \ \ \ \ / / \ \ / / \ i\ / • / / \\ / / / / y \\ v> X Fig. 23. 350 450 500 Methylazafrin ; — Methylazafrinone, in ethanol Ber. 66 (i933) 890 ^ 200 100 aa y / 1 < v.>-A^ ,-c^W:--^ .,A 200 wo Fig. 24. Taraxanthin in ethanol Z. angew. Chem. 47 (1934) 664 -X in m; f Carotenoids 23 358 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 250 300 350 400 450 Fig. 25. Rhodoviolascin in hexane Helv. chim. Acta 26 (1943) 118 500 550 ■ ^Xin m/i logs 3.4 2.9 2.4 1.9 250 300 350 400 450 Fig. 26. Rhodopin in ethanol Helv. chim. Acta 26 (1943) 118 500 550 ^•Xinmfi LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 359 250 300 550 \inmji Fig. 27. Flavorhodin in hexane Helv. chim. Acta 26 (1943) 119 Fig. 28. a-Citraurin ; a-Apo-2-Carotenal, in hexane Helv. chim. Acta 26 (1943) 119 36o LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS log E urn 3.6 3.1 2.6 2.1 \ \\ A /\ ' \ J 1.- 1 ■ < . \ 1 250 300 350 400 450 500 ^Xmma Fig. 29. ^-Apo-2-Carotenal; - - - - Carotenone, in hexane Helv. chim. Acta 26 (1943) 119 >°S^Um 500 Fig. 30. Dihydro-;S-carotene in hexane Helv. chim. Acta 23 (1940) 958 LIGHT ABSORPTION CURVES OF SOME CAROTENOIDS 361 t Iflr 3.7- 3.6 3.5 14 3.3 3.2 3.1 3.0 29 2.6- 2.7 2.6 2.5 2.U 2.3 2.2. Fig /cm • Isocarotene - Dihydro-fi-Carofene - Lycopene ■fi- Carotene •a-Carotene A ft A 500 -^X in mil absorption of a-Carotene, /^-Carotene, Lycopene, Dihydro-^-carotene, and Isocarotene in hexane Helv. chim. Acta 23 (1940) 956 Index of vegetable and animal sources of carotenoids Abramis brama 96 Abutilon Darwini 72 - megapotamicum 72 - nervosum 72 Acacia acuminata (oil) 98, 128 Acacia decurrens var. mollis 71, 127, 19 - discolor 71, 127, 198 - linifolia 71, 127, 198 - longifolia 71, 127, 198 Acanthis flammea 93 Accra bullata 85 Achnantidium lanceolata 79 Acmaea virginea 85 Actinia equina 88, 333 Actinophloeus angustifolia 74, 115 - Macarthurii 74, 113 Adonis vemalis 70 Aecidio- and Basidio-spores 82 Aeolis papillosa 85 Aesculus calif ornica Nuttall 151 Afzelia cunazensis 75 Aglaonema commutatum 74 - nitidum 74 115 - oblongifolium 74, 115 - oblongifolium var. Curtisii 74, 115 - simplex 74, 115 Agonus cataphractus 96 Ahnfeltia plicata 81 Alcyonidium gelatinosum 88 Alcyonium digitatum 88 Aleuria aurantiaca 82, 161 Alfalfa 98 Algae, blue 301 - brown 128, 309, 340 - green 230 Allium siculum 70 AUomyces 82, 161 Aloe species 221 - vera 70 Alyssum saxatile 71 Amaryllidaceae 70, 74 Ammodytes lanceolatus 96 Ampelis garrula 93 Ampelisca tenuicornis 83 Ampelopsis hederacea 75 Amphibia 98 Amphineures 84 Amphiporus pulcher 87 Amphitrite afiinis 87 Amphiura chiajei 86 - filiformis 86 Amsinckia douglasiana De Candolle 151 Anabaena flos-aquae 78 Anacardiaceae 75 Ananas sativus 74, 199 Anapagurus chiroacanthus 83 Anas penelope 93 - platyrhyncha 93 - platyrhyncha domestica 94 Anemonia sulcata 88, 327 Angiospermae 73 Anguilla anguilla 96 Anomia ephippium 84 Anonaceae 74 Anser domesticus 93, 94 Antedon petasus 87 Antherina presbyter 96 Aphanizomenon flos aquae 78, 128, 300, 304 Aphiga minuta 96 Aphrodite aculeata 87 Apistonema Carteri 78 Aplysia rosea 85 Aplysina aerophoba 88 Apocynaceae 76 Aporrhais pes pelecani 85 Apricot-peach 74 Aprosmictus melanurus 94 Araceae 74 Arbutus Unedo 76, 115, 126, 151, 175, 194, 199 Archontophoenix Alexandrae 74, 115 Areca Alicae 74, 115 Arenicola marina 87 - piscatorum 87 Aricia norvegica 87 Armeria vulgaris 72 3^4 INDEX OF VEGETABLE AND ANIMAL SOURCES Arnica montana 72 Amoglossus megastoma 96 Arthropods 83 Arthrostaca 231 Arum italicum 74, 115 - maculatum 74, 115 - orientale 74, 115 Ascidia virginea 89 Asclepiadaceae 72 Asclepias curassavica 72 Ascobolus- species 82 Ascomycetes 82 Ascophyllum nodosum 80 Asparagus officinalis 74 Asphodelus cerasiferus 70 Astacus fluviatilis 83 - gammarus 83, 230, 234 Astarte banksi 84 - sulcata 84 Asteracanthion glacialis 86 Asterias glacialis 86 - miilleri 86 - rubens 86, 339 Asterina gibbosa 86 Asteroidea 86 Aster species 72, 199, 206 Astropecten aurantiacus 86 - irregularis 86 Astur gentilis 94 Atropa Belladonna 72 Attalea gomphococca 74 Avena sativa 73 Axinea rugosa 88 Axinella crista-galli SS, 230 Axinus flexuosus 84 Bacillariophyta 79 Bacillus Grasberger 77, 114, 128, 161 - Lombardo Pellegrini 77, 114, 128, 161 Bacteria 77 Bacterium chrysogloea 77 - egregium 77 - halobium 77 - Sarcina aurantica 114 Balaenoptera musculus 232 Balanus balanus 83 - crenatus 93 Balsaminaceae 72 Bangia species 81 Barbus fluviatilis 96 Barley germ 73 Basidiomycetes 82 Basidiospores 82 Batrachier species 98 Batrachospermum moniliforme 81 Bee's wax 98 Belone belone 96 - rostrata 96 Berberidaceae 71, 74 Berberis vulgaris 74 Beryx decadactylus 96, 231 Beta vulgaris 67 Birds 91, 232 Bixaceae 75 Bixa orellana 75, 256 Blood of pregnant women 90 Blood of umbilical cord 91 Blood serum 90, 93, 129, 175 Bombinator igneus 98 Bombyx mori 83 Bone marrow 90 Boraginaceae 72 Bothus maximus 96 - rhombus 96 Botrydium granulatum 79 - species 79 Botryllus schlosseri pallus 89 Brachiopoda 88 Brassica campestris 67, 74 - nigra 74 - Rapa 67 Brissopsis lyrifera 87 Bromeliaceae 70, 74 Bryonia dioica 76, 113 Bryozoa 88 Buccinum undatum 85 Bufo calamita 98 - viridis 98 - vulgaris 98 Bugula neritina 88 Bulbine annua 221 Bulbine semibarbata 70 Bulbochaete setigera 80 - species 80 Bullfinch 338 Buphthalmum salicifolium 72 Butia capitata 125, 161, 164 - eriospatha Becc. 125 Butter 116, 175 Buxus 221 Cacalia coccinea 72 Cacatua roseicapilla 94 Calanus finmarchianus 83, 231 Calceolaria rugosa 72 - scabiosaefolia 72 - species 72 INDEX OF VEGETABLE AND ANIMAL SOURCES 365 Calendula arvensis 72 - officinalis 72, 114, 127, 193, 198 CallionjTnus lyra 96 Calliostoma miliare 85 Callithamnion hiemale 81 Calocaris macandreae 83 Calocera cornea 82 - palmata 82 - viscosa 82 Calothrix-scopulorum 78, 225, 300 - species 78 Caltha palustris 70, 127, 198 Calurus auriceps 94 Calyptrocalyx spicatus 74, 115 Camellia species 151 Campanulaceae 72 Campethera nubica 94 Canary 338 Cancer pagurus 83, 230 Cannabis sativa 74 Cantharellus species 151 - cibarius 82, 116 - infundibuliformis 82, 116 - lutescens 82, 116 Caprifoliaceae 76 Capsicum annuum 175, 181, 199, 241, 251 - frutescens jap. 76, 126, 181, 241 - japonicum 126 Capulus hungaricus 85 Carassius auratus 96, 231 Carausius morosus 83 Caranx trachurus 96 Carcinus maenas 83 Cardinalis virginianus 94 Cardium echinatum 84 - norvegicum 84 - tuberculatum 84 Carduelis carduelis 94 - spinus (feathers) 94 Caricaceae 75 Carica Papaya 75, 175, 194 Carinella annulata 87 Carrot leaves 98 Carrots 130, 161 Carj'ophyllia smith i 88 Caterpillar of the cabbage butterfly 83 • Cedrela Toona 71, 272 Celastraceae 75 Celastrus scandens 67, 75, 175, 181, Centrolabrus exoletus 96 Centropristes stiratus 17 Cephaleurus albidus 80 Cephaleurus laevis 80 - minimus 80 - parasiticus 80 - solutus 80 Ceramium diaphanum 81 - rubrum 81 Ceratium furco 79 - fusus 79 - tripos 79 Cerebratulus fuscus 87 - marginatus 87 Certhiola mexicana 94 Chaetoderma nitidulum 84 Chaetopterus variopedatus 87 Chamaecyparis 221 Chamaeleon vulgaris 98 Chantransia species 81 Chara ceratophylla Wallr. 80, 114, 161 - fragilis 80 Charoph^i;a 80 Cheiranthus Cheiri 71 - Senoneri 71 Chelidonium majus 71 Chicken 232 Chlamydomonas eugametos f. simplex II Chlorella protothecoides 79 - variegata 79 - vulgaris 151 Chloris chloris 94 Chloronerpes aurulentus 94 - kirki 94 - jTicatensis 94 Chlorophanes atricapilla 94 Chlorophyceae 79 Chondrosia reniformis 88 Chondrus crispus 81 Chorda Filum 80 Chordata 89 ChromuUna Rosanoffii 78 Chr^-santhemum 72 - frutescens 72 - segetum 72 Chrysemis scripta elegans 98, 161 Chrj'somonadales 78 Chrysomonadina 78 Chrysoptilus punctigula 94 Chytridium species 82 Ciona intestinalis 89 Cirratulus cirratus 87 - tentaculatus 87 Citrullus vulgaris 76, 114, 127, 151, 162 Citrus aurantium 75, 115, 127, 181, 194, 199, 218 - decumana L. 114 366 INDEX OF VEGETABLE AND ANIMAL SOURCES Citrus grandis 75 - grandis Osbeck 75, 114 - Limonum 75 - madurensis 75, 127, 199 - maxima 151 - poonensis hort. 75, 127, 175, 194 Cladophorales 80 Cladophora glomerata 80 - rupestris 80 - Sauteri 80, 128, 151, 200, 321 Cladostephus spongiosus 80 Clavellina lepadiformis 89 Clemmys insculpata 98, 232 Clivia miniata 70 - species 74 Clupea harengus 96 Clythra quadripunctata 83 Coccinella septempunctata 83, 116 Coccospongia species 88 Cochleodesma praetenue 84 Coelenteraten 88 Colaptes auratus 94 - olivaceus 94 Coleoptera coccinella 83 Coleosporium Pulsatilla 82 - euphrasie 82 - senecionis 82, 151 Colostrum 91 Colutea media 71 Compositae 72, 76 Conjugatae 79 Convallaria majalis 74, 114, 127, 151, 199 Copepoda 231 Coprosma baueri Endlicher 151 Corallina officinalis 81 Corbula gibba 84 Coregonus albula 96, 339 Corella parallelogramma 89 Corpora rubra 91, 129 Corpus luteum 91, 129 Corydalis lutea 71 Corynebacterium 77 - carotenum 77 Cotinga coerulea 94 Cotoneaster occidentalis 74 - species 74 Cottus bubalis 96 - scorpius 96 Cow dung 90, 129 Cranberries 115, 199 Crangon allmani 83 Crania anomala 88 Crataegus Crus galli 74 Crenilabrus melops 96 Crenilabrus suillus 96 Crepis aurea 72 - species 72 Cribella aculata 86 Crinoidea 87 Crocus albiflorus kit; var. Neapolitanus hort. 272 - luteus 70, 272 - neapolitanus 70 - reticulatus 70 - sativus 70, 128, 162, 181, 272 - variegatus 70 Crossaster papposus 86 Cruciferae 71, 74 Crustaceae 83, 230 Cryptogames 76, 77 Cryptomeria jap. Don. 221 Cucumaria elongata 87 - lactea 87 Cucumis Melo 76 Cucurbitaceae 72, 76 Cucurbita foetidissima 72 - maxima 76, 127, 151, 194, 199, 317 - melanosperma 72 - Pepo 72, 76, 175, 181, 198, 317 Cultellus pellucidus 84 Cuscuta salina 115, 151, 162, 173 - subinclusa 115, 151, 162, 173 Cutleria multifida 80 Cyanophyceae 78 Cyclopterus lumpus 96, 151, 231 Cymatopleura solea 79 Cymbirhynchus macrorhynchus 94 Cynthia papillosa 89 Cypressus Naitnocki 221 Cyprina islandica 84 Cyprinus auratus 96 - carpio 96 Cypripedium Argus 70 - Boxallii 70 - insigne 70 Cystoclonium purpurascens 81 Cystosira 310 - abrontanifolia 80 Cytisus laburnum 197 - sagittalis 71 D Dacryomyces stillatus 82 Dahlias (anther) 73 Dandelion 208 Daucus Carota 67, 126, 151 Decapoda 230 Delesseria sanguinea 81 Dendrobium thyrsiflorum 70 INDEX OF VEGETABLE AND ANIMAL SOURCES 367 Dendrodoa grossularia 89, 151, 231 Dendronotus frondosus 85 Dendropicos cardinalis 94 Dentalium entale 85 Desmarestia aculeata 80 Diaptomus bacillifer 8^ Diatomeae 79, 128, 340 Dicotyledoneae 70, 74 Dictyota 310 - dichotoma 80 - polypodioides 81 Dilsea edulis 81 Dimorphotheca aurantiaca 73, 115 Dinoflagellatae 79, 340 Dinophysis acuta 79 - laevis 79 Dioscoraceae 74 Diospyros costata 76, 114, 127, 151,175, 181, 194 - Kaki 76, 114, 181 Diphyllodes magnifica 94 Ditiola radicata 82 ' Doris repanda 85 Doronicum Columnae 73 - excelsum 73 - Pardalianches 73 - plantagineum 73 Dosina exoleta 85 Doto coronata 85 Dracaena draco L. 151 Dryobates major 94 Dryocopus auratus 94 Dumontia filiformis 81 Dung 90, 129 Dysidea fragilis 88 Ebalia tumefacta 83 Ebenaceae 76 Echinaster sepositus 87, 231 Echinodemis 86, 231 Echinoidea 87 Echinus esculentus 87 Eclectus polychlorus 94 Ectocarpaceae species 81 Ectocarpus siliculosus 81 - tomentosus 81 Egg yolk 93, 98, 175, 181, 200 Elachistea species 81 Elaeagnaceae 75 Elaeis guineensis 74, 115 - melanococca 74, 115 Eleginus navaga 96 Elodea canadensis 98, 206, 207 Emarginula crassa 85 Emarginula fissura 85 Emberiza citrinella 94 - icterica 94 Embiotocidae 96 Encephalartos Hildebrandtii 221 Enteromorpha compressa 80 - intestinalis 80 Enteropneusta 89 Epiactis prolifera 88 Epimedium macranthum 71 Equisetum species 221 Eranthis hiemalis 70 Ericaceae 75 Eriobotyza japonica 74 Erysimum Perofskianum 71 Erythroxylaceae 75 Erythroxylon coca 75, 115 - novogranatense 75, 114 Eschscholtzia californica 71, 323 Escobedia linearis 281 - scabrifolia 67, 281 Esox anguilla 96 - lucius 96 - reticulatus 17 Esperia foliata 88 Eugenia uniflora 75 Euglena heliorubescens 79, 230 Euglenales 79 Euglena sanguinea 79 - viridis 79 Eumenia crassa 87 Eumycetes 82 Eunotia pectinalis 79 Eupagurus prideauxii 83, 230 Euphone nigricollis 94 Eurynome aspera 83 Evonymus europaeus 75, 181 - fortunei L. 125, 164 - japonicus 75, 116 - latifolius 75 Fabiana indica 72 Faeces 90, 129 Fagop3n-um esculentum 74 Fat tissues 90, 93, 129 Feathers 92, 200 Ferula species 72 Ficus carica L. 151 Finch, green 338 Fish 95, 96, 231 - oil 151, 152 Flagellatae 78 Flamingo (fat) 334 Flustra foliacea 88 368 INDEX OF VEGETABLE AND ANIMAL SOURCES Flustra securifrons 88 Formosa tea leaves 151 Forsythia Fortune! 72 - viridissima 72 Fragilaria species 79 Fringilla canaria 94 Fritillaria imperialis 70 Frogs (retina) 98 Fruit 73 Fucus ceranoides 81 - nodosus 81 - serratus 81 - species 81 - versoides 81, 310 - vesiculosus 81, 128, 181, 310 Fundulus parvipinnis 96 Fungi 82 Furcellaria festigiata 81 Gadus aeglefinus 96 - callarias 96, 97 - esmarkii 97 - merlangus 97 - minutus 97 - poUachius 97 - virens 97 Gaidropsarus cimbrius 97 - mustela 97 Gaillardia splendens 73 Galathea intermedia 83 Gallstones 91, 129 Gallus bankiva domesticus 94 Gammarus pulex 231 Gardenia grandiflora 76, 272 - jasminoides 76 - lucida 76 Gasteria 221 Gasterosteus aculeatus 97 - spinachia 97 Gastropoda 85 Gazania rigens 73, 115, 127, 162, 173, 198, 313 Gazania splendens 73 Gelidium corneum 81 Genista racemosa 71 - tinctoria 71 - tridentata 71, 127, 151, 198 Gephyrea 88 Geum coccineum 71 - montanum 71 Gibbula cineraria 85 - tumida 85 Gigartina stellata 81 Glands 91 Glaucium luteum 71 Glenochrysis maritima 78 Glenodinium species 79 Glycera goesii 87 Gnetum species 221 Gobius niger 97 Gomphonema species 79 Gongora galeata 70 Goniaster equestris 86 Gonocaryum obovatum 75, 115, 151 Gonocaryum pyriforme 75, 115, 127, 151, 162, 334 Gorse 151, 211 Gossypium 75 - hirsutum 75 Gramineae 70, 73 Green finch 338 Grey wagtail 338 Grevillea robusta 70, 175, 340 Gymnodinium helix 79 Gymnospermae 73 Gymnosporangium juniperinum 82 - juniperi-virginianae 82 H Haematococcus pluvialis (Sphaerella pluvialis) 79, 128, 151, 200, 230, 240, 337. 348 Halcampa duodecirrhata 89 Halichondria albescens 89 - caruncula 89 - incrustans 89 - panicea 89 - rosea 89 - seriata 89 Halydrys siliquosa 81 Halma Bucklandi 89 Halocynthia papillosa 231, 328 Halyseris polypodioides 81, 181 Haploops tubicula 83 Harmothoe sarsii 87 Harrimania kupferi 89 Haworthia 221 Heart tissue 91 Hedera helix L. 151 Helenium autumnale 73, 198 - autumnale var. grandicephalum 73 Helianthus annuus 73, 76, 175, 198, 321 Heliopsis scabrae cinniaeflorae 73 - scabra major 73 Hemerocallis Middendorffii 70 Hemichordata 89 Henricia sanguinolenta 86 Heterocontae 79 Heterocope saliens 231 INDEX OF VEGETABLE AND ANIMAL SOURCES 369 Hieracium aurantiacum 73 - murorum 73 - Pilosella 73 Hippasteria phrygiana 86 Hippoglossus hippoglossus 97 - platessoides 97 Hippophae rhamnoides 75, 181 Hircinia spinosula 89 Holopedium gibberum 231 Holothuria brunnea 87 - nigra 87 - poli 87 - tubulosa 87 Holothurioidea 87 Hordeum sativum 73 Human fat tissues 90, 181, 200 Human liver 91, 115, 181, 194, 200 Human serum 90, 116 Hungarian wheat blossoms 70 Hyas araneus 83 Hydrodictyon utriculatum 79 Hydrurus penicillatus 78 Hyla arborea 98 Hymeniacidon sanguineum 89, 151, 162 Hypophyses of cattle 91 Hypoxanthus rivolii 94 I Icacinaceae 75 Idothea baltica 83 - emarginata 83 - neglecta 83 Idya furcata 84 Impatiens noli tangere 72, 321 Inner organs 91 Insects 83 Integuments 131 Intestine and subcutaneous fat of horses and cattle 90, 93, 129 Inula Helenium 73 Ipomoea Batatas 67, 151 Iridaceae 70 Iris (chicks) 176 Iris Pseudacorus 70, 194 Isatis tinctoria 71 Ithaginis cruentatus 94 Ixia crocata 70 Jasminum Sambac 72 Jaundiced potatoes 67 Juniperus virginiana L. 221 K Kerria japonica 71, 127, 198, 206 Kidneys 91, 129 Kleinia Galpinii 73 Kniphofia aloides 70 Labiatae 72 Labrus bergsnyltrus 97 - melops 97 - ossifagus 97 Laburnum anagyroides 71, 127, 193, 206 Lacerta agilis 98 - muralis 98 Lacuna divaricata 85 Ladanum hybridum 72 Laetmonice filicomis 87 Lamellibranchiata 84, 231 Laminaria 310 - digitata 81 - saccharina 81 Laurencia pinnatifida 81 Leander serratus 84, 231 Leathesia marina 81 Leaves 206 Leda parvula 85 Leguminosae 71, 75 Lemania fiuviatilis 81 - mamillosa 81 Leontodon autumnalis 73, 198, 321 Leotia lubrica 82 Lepidonotus squamatus 88 Lepidopleurus cancellatus 84 Lepralia foliacea 88 Leuciscus rutilus (liver) 97 Leuconia fossei 89 Libocedrus decurrens Torrey 151 Liliaceae 70, 74 Lilium bulbiferum 70 - bulbiferum ssp. croceum 70 - candidum 70, 192 - tigrinum 70, igi, 241 Lima excavata 231 - hians 85 - loscombei 85 Linaceae 75 Linum usitatissimum 75 Liriodendron tulipifera 71 Littorina littorea 85 Liver 91, 115, 129, 181, 194, 200 Loasaceae 72 Loasa (Cajophora) lateritia 72 Locusta viridissima 83 Locusts 83 Lonicera tatarica 76 - xylosteum 76 Lophius piscatorius (liver) 97, 231, 321 370 INDEX OF VEGETABLE AND ANIMAL SOURCES Lota vulgaris (roe) 97 Lotus comiculatus 71 Loxia curvirostra 94 Lucernaria quadricornis 89 Lucina borealis 85 Luffa species 76, 199 Luidia sarsii 86 Lumbrinereis fragilis 88 Lycaste aromatica 70 Lycium barbarum 76, 181 — carolinianum 76 — halimifolium 76, 181 — ovatum 76 Lycogala epidendron 78, 128, 329 — flavofuscum 78 Lycopersicum ceraciforme 76 — esculentum 76, 125, 151 Lyonsia norvegica 85 Lyrurus tetrix 94 M Magnoliaceae 71 Magnolia grandiflora L. Phoenix 151 Maize, yellow 128, 152 Maja squinado 84, 231 Malacobdella grossa 87 Malaeostraca 230 Malvaceae 72, 75 Malva parviflora L. 151 Mammals 90, 232 Manettia bicolor 72 Mangifera indica 75, 127, 152, 199 Marine soil 99, 152 Marrow (human) 90 Marsh soil 200 Masdevallia Veitchiana 70 Meconopsis cambrica 71 Megaloprepia magnifica 94 Melampsora aecidioides 82 — salicis capreae 82 Meliaceae 71 Melilotus officinalis 71 Melosira species 79 Mesothuria intestinalis 87 Metridium dianthus 89 — senile 89 Micrococcus erythromyxa 77 — rhodochrous 77 Microcosmus sulcatus 89 Microcystis flos-aquae 78 Milk, human 129 Milk fat (all mammals) 91, 129 Milvus 94 Mimuluslongiflorus72, 115, 125, 162, 164 — moschatus 72 Modiolaria marmorata 85 Molgula occulta 89 Molluscs 84, 231 Molva molva 97 Momordica Balsamina 72, 72, 116, 199 - Charantia 76, 116 Monocotyledoneae 70, 73 Moraceae 74 Morone americana 17 Moss species 151 Motacilla cinerea 94 Mucor flavus 82 Mullus barbatus 97 Munida banffia 84 Muraena helena 97 Musaceae 70, 74 Musa paradisiaca 74, 200 Muxilla mammillaris 89 Mya truncata 85 Mycobacterium lacticola 77 - leprae 77 - phlei 77, 162, 176, 200, 338 Myristicaceae 74 Myristica fragrans 74 Myrtaceae 75 Mysis flexuosa 84 Mytilus californianus 85, 339 - edulis S^, 328 Myxomycetes 78 N Nacella pellucida 85 Narcissus poeticus 70 - Pseudonarcissus 70 - Tazetta 70 Nassa incrassata 86 - reticulata 86 Nasturtium species 71 Natica nitida 86 Navicula species 79 - torquatum 79 Nectria cinnabarina 82 Nemalion multiiidum 81 Nemertini 87 Nenga Polycephalus 74 Neoamphitrite figulus 88 Neohela monstrosa 84 Nephrops norvegicus 84 - species 231 Nephthys caeca 88 - ciliata 88 Neptunea antiqua 86 Nereis pelagica 88 - virens 88 Nerophis aequoreus 97 - ophidion 97 INDEX OF VEGETABLE AND ANIMAL SOURCES 371 Nertera depressa 76, 116 Nerves 91 Neurospora crassa 82 Nitella opaca 80, 128, 200, 321 - spores 80 - syncarpa 80, 162 Nitzschia closterium 79, 176 - Palea 79 - sigmoidea 79 - species 79 Nodularia species 78 Nonnea lutea 72 Nostoc species 78 Nucula sulcata 85 Nuphar luteum 70 Nyctanthes Arbor-tristis 72, 272 Nymphaceae 70 O Odonthalia dentata 81 Odontoglossum species 70 Oedipoda coerulescens 83 Oedipoda miniata 83 Oedogoniales 80 Oedogonium 128, 152, 200, 321 - species 80 Oenothera biennis 72 Oenotheraceae 72 Oleaceae 72 Oncidium species 70 Ophidiaster ophidianus 86, 231 Ophiocoma nigra 86 Ophiopholis aculeata 86 Ophiothrix fragilis 86 Ophiura affinis 86 - texturata 86 Ophiuroidea 86 Opisthobranchia 85 Orange peel 176 Orchidaceae 70 Oriole 338 Oriolus galbula 95 - oriolus 95 - xanthonotus 95 Orthagoriscus mola 97, 151 Oryza sativa 73 Oscillatoria 78 - Froelichii 78 - lapotricha 78 - limosa 78 - rubescens 78, 225, 227, 300, 335 Osmerus eperlanus 97 P Padina Pavonia 81 Pagurus bernhardus 84 Pagurus rubescens 84 Palaemon fabricii 84 - serratus 84 Palinurus vulgaris 84, 231 Palmae 74 Palmellococcus miniatus 79 Palmfruit 74 Palm oil 74, 114, 116, 128, 152, 162 Pandalus borealis 84 - brevirostris 84 - montagui 84 Pandanaceae 73 Pandanus polycephalus 73, 116 Papaveraceae 71 PapiUina suberea 89 Paprika 130, 152 Paradisea papuana 95 - rubra 95 Paroaria cucullata 95 Parthenocissus quinquefolia 151 Parus coeruleus 95 - major 95 Passifloraceae 75 Passiflora coerulea 75, 114 Pastinaca sativa 67 Patella vulgaris 86 Peat 200 Pecten jacobaeus 85 - maximus 8^, 326 - opercularis 85 - septemradiatus 85 - striatus 85 - tigrinus 85 Pectinaria belgica 88 Pectunculus glycimeris 85, 328 Pedaliaceae 76 Perca flavescens 17 - fluviatilis 97, 231 Peridinium cinctum 340 - divergens 79 Perillus bioculatus 83 Peripheral nerve 91 Permatula phosphorea 89 Petroselinum hortense 151 Peziza aurantia 82 - (Lachnum) bicolor 82 - (Lachnea) scutellata 82 Phaeophyceae 80 Phanerogams 67 Phascolosoma elongatum 88 Phasianus colchicus 95, 233 - colchicus X torquatus 95 Philline aperta 85 Phlogoena cruenta 95 Phoenicopteris antiquorum 95 372 INDEX OF VEGETABLE AND ANIMAL SOURCES Phoenix 151 Pholis gunellus 97 Phormidium vulgare 78 Phragmidium violaceum 82 Phycomyces 82 Phycomycetes 82 Phycopeltis amboinemis 80 - aurea 80 - epiphyton 80 - maritima 80 - Treubii 80 Phyllobium domorphum 79 - incertum 79 - Naegelii 79 Phyllophora Brodiaei 81 - membranifolia 81 Phyllophorus lucidus 87 Phyllopoda 231 Phylloscopus sibilatrix 95 Physalis Alkekengi 76, 176, 181 - Franchetii 76, 176, 181 Picidae species 95 Picus canus 95 - major 95 - viridis 95 Pieris brassicae 83 Pilayella littoralis 81 Pilobolus crystallinus 82 - Kleinii 82 - Oedipus 82 Pinicola species 95 Pinus radiata Don 151 Pirus aucuparia 127 Placenta 91, 129 Plankton 99 Pleotrachelus fulgens 82 Pleurobranchus species 85 Pleurococcus pluvialis 79 Pleuronectes flesus 97 - kitt 97 - limanda 97 - microcephalus 97 - platessa 97 Plocamium coccineum 81 Ploceus cucullatus 95 Plumbaginaceae 72 Polyalthia species 74 Polychaeta 87 Polygonaceae 74 Poly ides rotundus 81 Polymnia nebulosa 88 Polynoe spinifera 88 Polysiphonia fastigiata 81 - nigrescens 81, 310 - species 81 Polystigma ochraceum = Polystigma fulvum 82 - rubruTO 82 Pontophilus spinosus 84 Porania pulvillus 86 Porcellana longicomis 84 Porphyra hiemalis 81 - laciniata 81 - umbilicalis 81 - vulgaris 81 Portunus depurator 84 - longicomis 84 - puber 84, 231 - pusillus 84 Potamobius astacus 84, 231 Potamogeton natans 221 Potentilla erecta (Tormentilla) Potatoes, jaundiced 67 - sweet 67 Potentilla erecta (Tormentilla) 71 Prasiola species 79 Priapulus caudatus 88 Primuiaceae 72 Primula acaulis 72 - officinalis 72 Prionotus carolinus 17 Prorocentrum micans 79 Prosobranchia 85 Protanthea simplex 89 Proteaceae 70 Protococcales 79 Protococcus pluvialis (Pleurococcus pluvialis) 79 - vulgaris 79 Protozoa 230 Prunus armeniaca 74, 114, 127, 162 - persica 75, 181, 200 Psammechinus miliaris 87 Psammobia ferroensis 85 Psolus phantapus 87 Ptychandra elegans 74 - glauca 74 Ptychosperma elegans 114 Puccinia coronata 82 - coronifera 82 Purple bacteria 77, 295 Purpurea lapillus 86 Pyracantha angustifolia Schneid. 125, 164 - coccinia 99, 162 Pyrocephalus rubineus 95 Pyromelana franciscana 95 Pyrrhocoris apterus 83 Pyrrhula pyrrhula 95 - vulgaris 95 INDEX OF VEGETABLE AND ANIMAL SOURCES 373 Quercus agrifolia 151 R Radiella spinolaria 89 Raja batis 97 - clavata 97 Rana bufo 98 - esculenta 98, 181, 200 - temporaria 98 Raniceps raninus 97 Ranunculaceae 70 Ranunculus acer (R. Steveni?) 70, 159, 193. 198, 206, 208, 211, 321 - arvensis 71, 198 - auricomus 71 - califomicus Bentham 151 - Ficaria 71 - gramineus 71 - repens 71 - Steveni 71, 198 Raphanus Raphanistrum 71 Regalecus glesne 97, 152, 231 Regulus regulus 95 Reniera aquaeductus 89 Reptiles 98, 232 Reseda odorata 221 Retina 91 Retinospora plumosa 221 Rhodobacillus palustris 77 Rhodomela subfusca 81 - virgata 81 Rhodophyceae 81 Rhodotorula Sanniei 162, 329 Rhodovibrio-bacteria 78, 297, 299 Rhodymenia palmata 81, 128, 152, 200, Rhynchota 83 [321 Ribes aureum 71 Rissoa species 86 Rivularia atra 78 - nitida 78, 225 - species 78 Roe 129 Roots 67 Rosa canina 75, 114, 127, 173, 181, 321 Rosaceae 71, 74 Rosa damascena 75, 127, 173, 181, 321 - rubiginosa 75, 114, 127, 162, 173, 181 , 200, 321 - rugosa Thumb. 75, 114, 162, 173 - yellow species 71 Rubiaceae 72, 76 RubusChamaemorus 75,1 14,162,173,181 Rudbeckia Neumannii 73, 181, 199 Rutaceae 75 Rye germ oil 73, 91, 152 Sabal (serenaea) serrulatum 74 Sabella penicillus 88 Saccharomyces species 82 Saffron (Crocus sativus) 70, 115 Sagartia undata 89 - viduata 89 Salamandra maculosa 98 Salmo irideus 97 - salar 97, 232, 339 - trutta 97, 232 Salmon (flesh) 129 Sambucus nigra 76 Sarcina aurantiaca 78, 182 Sarcina lutea 78, 319 Sardine oil 99 Sarothamnus scoparius 71, 127, 199, 206, 208, 211 Saxicava rugosa 85 Saxifragaceae 71 Scalaria elatior 86 Scalpellum scalpellum 84 Scaphopoda 85 Schizoneura lanigera 83 Schizophyta 77 Schizopoda Euphausia 230 Scirpus 221 Scomber scombrus 97 Scophthalmus norvegicus 97 Scorpaena scrofa 97 Scotinosphaera paradoxa 79 Scrophulariaceae 72 Sea and marine soil 99, 152 Seaweed 99 Sebastes marinus 97, 232 Secretions 91 Seeds 73 Sedum acre L. 151 Selaginella 221 Seleucides alba 95 Senecio Doronicum 73, 182 - vernalis 73, 208 Sequoia sempervirens Engelmann 151 Serinus canaria 95, 181 - canaria serinus 95 Serum 90, 116, 200 Sesamum indicum 76 Shark 97 Sheep dung 90, 129 Silphium perfoliatum 73 Sinapis officinalis 71, 193 Siphocampylus bicolor 72 Carotenoids 24 374 INDEX OF VEGETABLE AND ANIMAL SOURCES Siphonales 80 Siphonostoma diplochaitos 88 Siphostoma typhle 97 Sisymbrium Sophia 71 Sittace macao 95 Skin 91 - yellow 91 Solanaceae 72, 76 Solanum Balbisii 76, 114 - corymbosum 76 - decasepalum 76, 116 - Dulcamara 76, 114, 171 - esculentum 171 - Hendersonii 76, 182 - Lycopersicum 76, 114, 127, 182 - Pseudocapsicum 76 - tuberosum 151 Solaster papposa 86 Solea solea 97 - variegata 97 - vulgaris 97 Solen ensis 85 Somateria moUissima 95 Sorbus Aria 75 - aucuparia 75, 152 - aucuparia dulcis 75 - suecica 75 Soya beans 75, 152 Spaeroplea species 80 Spartium junceum 71 Spatangus purpureus 87 Spathularia flavida 82 Spermothamnion roseolum 81 Sphacellaria cirrhosa 81 Sphaerella pluvialis see Haematococcus pluvialis Sphaerostilbe coccaphili 82 Sphaerotilus roseus 78 Sphinx ligustri 83 Spinachia spinachia 97 Spirillum rubrum Esmarch 78 Spirogyra crassa 79 - maxima 79 Spirontocaris lilljeborgii 84 Spisula solida 85 - subtruncata 85 Spleen 91 Sponges 88 Spongiaria 230 Sporobolomyces roseus 82, 329 - salmonicolor 82, 329 Staphylococcus aureus 182 - pyrogenes aureus 78 Stemonitis ferruginea 78 - fusca 78 Stenogorgia rosea 89 Stenorhynchus species 84 Stenotomus chrysops 17 Stichastrella endeca 86 - rosea 86 Stinging nettles 193, 206 Strelitzia Reginae 70 Strepotothrix corallinus 78 Strongylocentrotus drobachiensis 87 - lividus 87, 324, 326 Styela rustica 89 Stylarioides plumosus 88 Stylifer stylifera 86 Stypocaulon scoparium 81 Suberites domuncula 89 - ficus 89 - flavus 89 - massa 89 Sunflower oil 76 Swamp 99 Sweet potatoes 67 Synaspadix petrichiana 74, 116 Syndosmia alba 85 - nitida 85 Syngnatus acus 97 Synoicum pulmonaria 89 Tabernaemontana pentasticta 76, 116 Tagetes aurea 73 - erecta 73, 199 - grandiflora 73, 193, 199 - nana 73, 199 - patula 73, 199 Tamus communis 74, 113, 114, 117, 171 Tangerines 176 Tapes puUastra 85 Taraxacum officinale 73, 193, 199, 320, Taxaceae 73 [321 Taxus baccata 73, 116, 127, 222 Tealina felina 89 Tedania muggiana 89 Telekia speciosissima 73 Tellina crassa 85 Tentorium semisuberites 89 Terebella species 88 - stroemii 88 TerebratuUna caput serpentis 88 Testes 91 Tethya lyncurium 89 Tetrao tetrix 95 Thallochrysis litoralis 78 Thea species 151 Thelepus cincinnatus 88 Thermopsis lanceolata 71 INDEX OF VEGETABLE AND ANIMAL SOURCES 375 Thiocystis-bacteria 78, 115, 297 Thracia convexa 85 Thuja orientalis L. 221 Thyone fusus 87 Tiga tridactyla 95 Tillandsia splendens 70 Timotheegrass bacteria 84 Tolj^othrix species 78 Tonicella marmorea 84 Tormentilla 71 Tortoise 98 Torula rubra 78, 82, 128, 329, 330 Trachinus draco 97 Tragopogon pratensis 73, 127, 159, 193, 199, 206 Tremella mesenterica 82 Trentepohlia aurea 80, 128 - aureum-tomentosum 80 - bisporangiata 80 - crassiaepta 80 - Cyania 80 - jolithus 80 - moniliformis 80 - umbrina 80 Trichosanthes species 76, 116 Trigla gurnardus 97 - hirundo 97 Triphragmium ulmariae 82 Triticum vulgare 73 Tritogenaphis rudbeckiae 83 Tritoma hombergi 85 Triton cristatus 98 Tritonia aurea (Ixia crocata) 70 Trivia europaea 86 Trochus zizyphinus 86 Trogon massena 95 Trollius asiaticus 71 - eiiropaeus 71, 128, 199, 206, 335 Tropaeolaceae 71 Tropaeolum majus 71 Tubularia indivisa 89 - Tubularia larynx 89 Tulipa Gesneriana 70 - hortensis 70 Tulips, yellow variety 70, 193 Tunicata 89, 231 Turdus merula (beak skin) 95 Turritella communis 86 Tussilago Farfara 73, 193, 321 Ulvales 80 Umbelliferae 72 Umbilical cord 91 Uredo (Coleosporium) euphrasie 82 Uromyces alchemille 82 Urtica urens L. 151 Urticina felina 89 Uvularia grandiflora 70 V Vaccinium Vitis idaea 75, 116, 182 Vaucheria hamata 80 ^ species 80 Velutina velutina 86 Venus fasciata 85 - gallina 85 - ovata 85 Verbascum phlomoides L. 272 - species 72 - Thapsus 72 Vertebrates 90 Viburnum Lantana 76 - Opulus 76 Vicia, violet blue varieties 71, 115 Vigna sinensis 75 Viola biflora 72 - cornuta var. Daldowie 72 - lutea 72 - odorata 72 - tricolor 72, 182, 194, 196, 199, 208 Violaceae 72 Vitaceae 75 Vulsella barbata 85 - modiolus 85 Volvox species 79 W Wagtail, grey 338 Waldsteinia geoides 91 Washingtonia lilifera Wendland 151 Weaver 338 Wheat germ 70, 73, 200 Winter asters 199, 206, 211 Woodpecker 338 Worms 87 Y Yellow maize 128, 152 Yellow skin of diabetic subjects 91 - through special diet 91 U Ulex europaeus 71, 128, 194, 208, 321 - Gallii 71, 128, 152 Ulothrichales 80 Ulva lactuca 80 Zea-Mays 73, 176, 182 Zeus faber 97 Zoarces viviparus 97 Zygnema cruciatum 79 Zygnema pectinatum 79, 310 Carotenoids 24* Subject Index Absorption curve 8 Absorption maximum 53 Absorption spectrum 53, 58 Acetic acid 43 Acetone 27, 117 Actinioerythrin 333 Adsorption 24, 25, 26 Alcohol 27 Alumina 26 Aluminium oxide 26 Ammonium sulphate 237 Anhydrocapsanthinone 246 Anhydrosemi-/?-carotenone 140 Anhydroazafrinone 286 Anhydroazafrinone amide 137, 283, 287 Anhydroazafrinone methyl ester 286 Anhydroazafrinone oxime methyl ester 286 Antheraxanthin 33, 191 Antimony trichloride 7 Aphanicin - Constitution 304 - Derivatives 306 - Preparation 300 - Properties 305 Aphanicin oxime 306 Aphanicol 306 Aphanin 32, 300 - Constitution 301 - Derivatives 303 - History 300 - Preparation 300 - Properties 302 Aphanin oxime 303 Aphanizophyll - Derivatives 309 - Preparation - Properties 308 Aphanizophyll oxime 309 Aphanizophyll palmitate 309 Aphanol 304 Apo-i-azafrinal 287 Apo-i-azafrinal oxime 288 Apo-i-bixin dialdehyde 123 a-Apo-2-carotenal 156, 359 yff-Apo-2-carotenal 144 y0-Apo-4-carotenal 145 a-Apo-2-carotenal oxime 156 yS-Apo-2-carotenal oxime 144 /ff-Apo-4-carotenal oxime 145 y5-Apo-2-carotenal semicarbazone 144 a-Apo-2-carotenol 157 y5-Apo-2-carotenol 360 y8-Apo-4-carotenol 145 Apo-2-lycopenal 122 Apo-3-lycopenal 123 Apo-2 : i2-lycopenedial 123 Apo-3 : i2-lycopenedial 123 Apo-i-norbixinal methyl ester 260 Apo-2-norbixinal methyl ester 260 Apo-3-norbixinal methyl ester 260, 355 Apo-i-norbixinal methyl ester (labile) 260, 271 Apo-2-norbixinal methyl ester (labile) 260, 271 Apo-i-norbixinal methyl ester (stable) 260, 263 Apo-2-norbixinal methyl ester (stable) 260, 263 Apo-3-norbixinal methyl ester (stable) 263 Arsenic trichloride 7 Assimilation 10 Astacene 35 ,229 - Constitution 234 - Derivatives 238 - History 229 - Preparation 233 - Properties 237, 354 Astacene diacetate 238 Astacene dioxime 238 Astacene dipalmitate 235, 238 Astaxanthin 35, 229 - Constitution 235 - Derivatives 239 - History 229 378 SUBJECT INDEX Astaxanthin, Preparation 233 - Properties 239, 354 Astaxanthin diacetate 239 Astaxanthin dicaprylate 240 Astaxanthin dipalmitate 240 Astaxanthin monopalmitate 240 Asterin acid 339 Aurochrome 147 Auroxanthin 34, 191, 195 - Constitution 196 - Derivatives - History 196 - Preparation 196 - Properties 197, 351 Azafrin 35, 281 - Constitution 282 - Derivatives 284 - History 281 - Preparation 281 - Properties 284 Azafrin ethyl ester 290 Azafrin methyl ester 288, 299 Azafrinal-I-methyl ester 288 Azafrinal-II-methyl ester 290 Azafrinone 283, 284 Azafrinone amide 283, 285 Azafrinone methyl ester 285, 296 Azafrinone monoxime 285 Azobenzene 8 B Benzene 27 Bisanhydro-y5-carotenone 141 Bixane 267 Bixin 8, 36, 256, 267 - Constitution 257 - History 256 - Occurrence 256 - Preparation 256 - Properties 355 - Stereoisomerism 259 Bixin (labile) 267 Bixin (stable) 262 Bixindialdehyde 118, 123, 355 Bixindialdehyde dioxime 119, 261 Bixin w-butyl ester 270 Bromine addition 44 Calcium carbonate 26 Calcium hydroxide 26 Calcium oxide 26 Calcium phosphate 26 Calcium sulphate 26 Canary xanthophy 11 337 Capsanthin 34, 240 - Constitution 241 - Derivatives 244, 249 - History 240 - Occurrence 241 - Preparation 241 - Properties 242, 353 - Stereoisomerism 248 Capsanthin diacetate 245 Capsanthin dibenzoate 245 Capsanthin dicaprat'e 245 Capsanthin diiodide 244 Capsanthin dimyristate 245 Capsanthin dipalmitate 245 Capsanthin dipropionate 245 Capsanthin distearate 245 Capsanthin epoxide 249 Capsathinone 246 Capsanthinone diacetate 246 Capsanthol 244 Capsanthylal 247 Capsanthylal monoxime 247 Capsochrome 250 Capsorubin 34, 251 - Constitution 251 - History 251 - Occurrence 251 - Preparation 251 - Properties 252 - Stereoisomerism 252 Capsorubin diacetate 252 Capsylaldehyde 247 Carbon tetrachloride 27 Carbonyl group, detection and estima- tion of 46 Carbonyl group, influence on light ab- sorption of 56 Carboxyl group, determination of 46 Carotenase 12 a-Carotene 30, 150 - Constitution 153 - History 150 - Occurrence 150 - Preparation 152 - Properties 153, 350, 361 /ff-Carotene 8, 30, 126 - Constitution 131 - Degradation reactions 133 - Derivatives 136 - History 126 - Occurrence 126 - Preparation 130 - Properties 135, 350, 361 - Stereoisomerism 149 SUBJECT INDEX 379 y-Carotene 30, 161 - Constitution 163 - History 161 - Occurrence 161 - Preparation 162 - Properties 163, 349 - Stereoisomerism 164 (5-Carotene 334 e-Carotene 79 /^-Carotene di-epoxide 146 a-Carotene iodide 157 a-Carotene mono-epoxide 31, 158 y5-Carotene mono-epoxide 146 /?-Carotene oxide 137 /?-Carotene oxides 145 - Chemical properties 148 - Physiological properties 148 Carotenoid from Satin oak 340 Carotenoid epoxides 15, 63 /?-Carotenone 140, 360 yS-Carotenone aldehyde 143 a-Carotone 156 Carr-Price reagent 7 Celaxanthin 315 - Constitution 315 - History 315 - Preparation 315 - Properties 316 - Stereoisomers 316 Chloroform 27 Chromatography 23 Chromatophores 5 Chromic acid oxidation 48 Chromoproteids 240 Chrysanthemaxanthin 33, 211 Cis-peak 40 a-Citraurin 205, 359 /?-Citraurin 35, 184, 220, 248 - Constitution 219 - Derivatives 220 - History 218 - Preparation 218 - Properties 219 yS-Citraurin oxime 220 - Citraurin semicarbazone 220 Citroxanthin 31, 211 - Constitution 212 - History 211 - Occurrence 211 - Preparation 211 - Properties 212 Colorimetry 8 Colour and constitution 50, ^t, Colour reactions 7 Crocetane 279 Crocetin 36, 272 - Constitution 274 - Derivatives 276 - History 272 - Nomenclature 273 - Occurrence 272 - Preparation 273 - Properties 276, 357 - Stereoisomers 273 Crocetin digentiobiose ester 276 Crocetin dimethyl ester (labile) 277, 355 Crocetin dimethyl ester (stable) 277 Crocetin monomethyl ester (stable) 277 Crocetin tetrabromide 281 Cryptochrome 179 Cryptofiavin 178 Cryptoxanthin 32, 175 - Constitution 176 - Derivatives 178 - History 175 - Occurrence 175 - Preparation 176 - Properties 177 - Stereoisomers 180 Cryptoxanthin di-epoxide 179 Cryptoxanthin mono-acetate 178 Cryptoxanthin mono-epoxide 178 Cyclohexane 43 Cynthiaxanthin 238 D Decalin 43 Dehydro-/?-carotene (Isocarotene) 137 Dehydrolycopene 60, 121 Diazomethane 262, 264 I : i6-Dibromo-2 : 6 : 11 : 15-tetra- methylhexadecane 279 Diethyl norbixin 270 Dihydrobixin 262, 268, 356 Dihydro-a-carotene 155, 159, 160 Dihydro-y6-carotene 136, 159, 160, 360, 361 Dihydro-yS-carotenone 142 Dihydrocrocetin 279, 356 Dihydrocrocetin diethyl ester 280 Dihydrocrocetin dimethyl ester 280 Dihydromethylbixin 262, 269, 356 Dihydronorbixin 265 Dihydrophytol 118 Dihydrorhodoxanthin 224, 353 Dihydrorhodoxanthin dioxime 225 a : a-Dihydroxyperhydrocrocetin di- methyl ester 280 38o SUBJECT INDEX a : a-Dihydroxyperhydronorbixin di- methyl ester 265 Dihydroxy-semi-/3-carotenone 139 1 : i6-Dihydroxy-2 : 6 : 11 : 15-tetra- methylhexadecane-i : i6-dicar- boxylic acid dimethyl ester 266 a : a-Dimethyl-(5-acetyl valeric acid 47 y : y-Dimethyl-(5-acetyl valeric acid 47 3 : 8-Dimethyldecapentaene-i : lo-di- carboxylic acid 289 3 : 8-Dimethyldecapentaene-i : lo-di- carboxylic acid dimethyl ester 289 3 : 8-Dimethyldecapentaene-i : lo-di- carboxylic acid monoamide 290 a : a-Dimethylglutaric acid 47, 131 6 : ii-Dimethylhexadecane-2 : 15-di- carboxylic acid 280 6 : ii-Dimethylhexadeca-3 : 5 : 7 : 9 : : II : i3-hexene-2 : 15-dicarboxylic acid 279 6 : ii-Dimethylhexadecane-2 : 15-dione 275, 281 a : a-Dimethylmalonic acid 47, 131 2 : 6-Dimethylnaphthalene 49, 132 2 : 7-Dimethylnonatetraene-i-al-9- carboxylic acid methyl ester 290 a : a-Dimethylsuccinic acid 47, 131 Dimethyl sulphate 264 3 : 8-Dimethylundecapentaene-ii-al- i-carboxylic acid methyl ester 288 Diphenylpolyenes 60 Double bond, detection of 43 Double bond, influence on light absorp- tion of 54 Echinenone 324 Eloxanthin 207 Elution 27 Epiphasic carotenoids 21, 22 Epoxides 61 Epoxide group, influence on light ab- sorption of 54 Eschholtzxanthin 323 Eschholtzxanthin diacetate 324 Eschholtzxanthin dibenzoate 324 Eschholtzxanthin di-p-nitrobenzoate 324 Eschholtzxanthin dioleate 324 Eschholtzxanthin dipalmitate 324 Ethanol 27 Ether 27 Ethyl acetate 27, 43 Ethylnorbixin 270 Fennicotterin 334 Fluorescence spectrum 8 Flavacin 307 - Constitution 307 - Preparation 309 Flavochrome 158 Flavorhodin 299, 359 Flavoxanthin 33, 207 - Constitution 209 - Derivatives 210 - History 207 - Occurrence 208 - Preparation 208 - Properties 209, 352 Flavoxanthin diacetate 210 Fucoxanthin 309 - Constitution 310 - History 309 - Occurrence 310 - Preparation 310 - Properties 311, 351 Fuller's earth 26 Furanoid oxides 62, 63 Gazaniaxanthin 313 - Constitution 313 - History 313 - Preparation 313 - Properties 314 - Stereoisomers 314 Geronic acid 47, 131 Glycymerin 328 H Haematoxanthin 337 Helenien 204 Hemeralopia 16 Hexahydrocrocetin 280 Hexamethyltetracosaundecaenedial 296 Hexane 43 Hydrochloric acid 7 Hydrogenation 43 Hydroxy-a-carotene Hydroxy-/?-carotene Hydroxyl group, determination of 45 Hydroxy 1 group, influence on light ab- sorption of 55 Hydroxy semi-/?-carotenone Hypophasic carotenoids 21 Insect pigments 6 Inulin 26 SUBJECT INDEX 381 a-Ionone ring 47 /?-Ionone ring 47, 49 /?-Ionone ring, in relation to vitamin A activity 15 Isocarotene 137, 361 Isogeronic acid 153 Isolation of carotenoids 20 Isomerism 38 Isoprene 29 IsopropyUdene group, determination of 45 K Kaolin 26 Kieselguhr 26 L Leprotin 338 Levulinic aldehyde 119 Levulinic acid 119 Liver 12 Luteochrome 148 Lycopersene 124 Lycopene 30, 113 - Constitution 117 - Derivatives 121 - Formation 119 - History 113 - Isolation 116 - Occurrence 113 - Properties 120, 349, 361 Lycopenal 118, 122 Lycophyll 31, 213 - Constitution 213 - History 213 - Preparation 213 - Properties 214 Lycophyll dipalmitate 214 Lycoxanthin 31, 171 - Constitution 171 - Historj^ 171 - Preparation 171 - Properties 172 Lycoxanthin monoacetate 172 M Magnesium oxide 26 Manganese acetate 267 Methanol 27 Methylazafrin 357 Methylazafrinone 357 Methoxyl group, determination of 45 Methyl bixin (labile) 259, 261, 264, 356 Methyl bixin (stable) 262, 264 Methyl w-butyl norbixin 270 Methylene chloride 27 Methyl ethyl norbixin 270 Methyl glyoxal 257 Methyl heptenone 118 Methyl->z-octadecyl norbixin 270 Methyl-M-octyl norbixin 270 Microhydrogenation 44 Molecular weight, determination of 51 Mutatochrome 31, 138, 147 Mutatoxanthin 100, 104 Mytiloxanthin 339 Myxoxanthin 32, 225 - Constitution 226 - History 225 - Preparation 225 - Properties 226 Myxoxanthin oxime 227 Myxoxanthol 227 Myxoxanthophyll 227 Myxoxanthophyll tetra-acetate 229 N Neocapsanthin A 249 Neocapsanthin B 249 Neocapsanthin C 249 Neocapsanthin dipalmitate I 249 Neocapsanthin dipalmitate II 249 Neocapsorubin A 253 Neocapsorubin B 253 Neocapsorubin dipalmitate I 253 Neocapsorubin dipalmitate II 253 Neo-a-carotene 157 Neo-/?-carotene 150 Neo-y-carotene 164 Neocelaxanthin 317 Neocryptoxanthin 180 Neohydroxy-/?-carotene Neolycopene 124 Neomethylbixin A 261 - C 261 Neosemi-/?-carotenone 140 Neotaraxanthin 323 Neozeaxanthin 188, 189 Neurospora 340 Nitric acid 7 NorbLxin 119 Norbixin (labile) 264 Norbixin (stable) 261 Norbixin monoethyl ester 270 Norite A 26 w-Octadecyl bixin ester 270 w-Octyl bixin ester 270 Optical rotation 49 Oscillaxanthin 335 382 Ovoester 235 Ovoverdin 6, 236, 240 Oximation 46 Oxidation 47 Ozonisation 45 Palladium oxide 43 Palmitic acid chloride 205 Paprika pigment 240, 241 Pectenoxanthin 326 Pentaxanthin 326 Perchloric acid 7 Permanganate oxidation 47 Perhydroazafrin 287 Perhydroazafrin methyl ester 290 Perhydroazafrinone 282 Perhydrobixin 268 Perhydrocapsanthin 244 Perhydrocarotene 136 Perhydrocrocetin 60, 274, 275, 280 Perhydrocrocetin diamide 280 Perhydrocrocetin dimethyl ester 278 Perhydrolycopene 60, 117, 121 Perhydromethyl bixin 265, 269 Perhydronorbixin 60, 258, 265, 279 Perhydronorbixin diamide 269 Perhydronorbixin diethyl ester 269 Perhydronorbixin dimethyl ester 269 Perhydronorbixin mono-methyl ester 268 PerhydrophysaUen 188 Perhydroviolaxanthin 195 Perhydroxanthophyll 204 Perhydrozeaxanthin 185 Permonophthahc acid Petaloxanthin 317 - Constitution 317 - History and Occurrence 317 - Preparation 317 - Properties 318 Petroleum ether 27 o-Phenylene diamine 238 Physahen 186 - Constitution 186 - Derivatives 188 - Occurrence 186 - Preparation 187 - Properties 187 Physalien iodide 188 Physalienone 188 Phytoxanthin ester 6 Picofulvin 337 Plastids 5 Platinum oxide 43 SUBJECT INDEX Porphyropsin 16, 17 Pro-y-carotene 164 Prolycopene 125 M-Propylalcohol 27 Provitamins A 11, 13, 14 Pseudo-a-carotene 150 Pyridine 34 R Reduction with aluminium isopropoxide 242 Retina 16 RetinenCj 17 RetinenCg 17 Rhodopin 297, 358 Rhodopsin 16, 17 Rhodopurpurin 299 Rhodovibrin 298 Rhodoviolascin 31, 295 - Constitution 296 - History 298 - Occurrence 295 - Preparation 295 - Properties 297, 358 Rhodoxanthin 34, 221 - Constitution 222 - Derivatives 224 - History 221 - Occurrence 221 - Preparation 222 - Properties 223, 353, 354 Rhodoxanthin dioxime 224 Rubichrome 32 Rubixanthin 32, 172 - Constitution 173 - History 172 - Occurrence 172 - Preparation 173 - Properties 174 Salmon acid 339 Sarcinaxanthin 319 Sarcinin 319 Semi-a-carotenone 155 Semi-y5-carotenone 139 Semi-a-carotenone monoxime 155 Spectral analysis 38 Spectroscopy 8 Spirilloxanthin Squalene 258 Sucrose 26 Sulcatoxanthin 327 Sulphuric acid 6, 7 Synthesis of carotenoids 60 SUBJECT INDEX 383 Talcum 26 Taraxanthin 320 - Constitution 322 - History and occurrence 320 - Preparation 321 - Properties 322, 358 - Stereoisomers 323 Tetraketo-y6-carotene (astacene) 234 1 : I : 20 : 20-Tetramethyldihydrobixi- nol 270 4 : 8 : 13 : 17-Tetramethyleicosane 267 4 : 8 : 13 : 17-Tetramethyleicosane- 1-20 -diol (i : 20-dihydroxybixane) 266 2:6:11: 15-Tetramethylhexadecane 279 2:6:11 : 15-Tetramethylhexadecane- I :i6-dicarboxylic acid diamide 266 2 : 6 : II : 15-Tetramethylhexadecanedi- I : i6-diol 278 3 : 7 : 12 : i6-Tetramethyloctadeca- i:i8-diol 266 3 : 7 : 12 : i6-Tetramethyloctane-i :i8- diol 166 Thermal degradation 48 Titanium trichloride 264 Toluene 118 w-Toluic acid 258 Tomato pigment 120 Tomato preserves 116 Torularhodin 35, 330 - Constitution 330 - History and occurrence 330 - Preparation 330 - Properties 331 Torularhodin methyl ester 332 Torulin 329 Trichloroacetic acid 7 Trichloroethylene 27 Tricyclocrocetin 278 TroUichrome 335 Trollixanthin 335 U Ultraviolet light absorption 53 Violaxanthin 34, 190, 193 - Constitution 194 - Derivatives 195 - History 193 - Occurrence 193 - Preparation 194 - Properties 351 Violaxanthin dibenzoate 196 Violaxanthin di-^j-nitrobenzoate 196 Violerythrin 333 Visual process 16 Visual purple 16, 17 Vitamin A 12 Vitamin Aj 17 Vitamin Ag 17 Vitamin A acid 13 Vitamin A activity 13, 49 Vitamin Aj-aldehyde 17 Vitamin Ag-aldehyde 17 Vitamin A methyl ester 13 W Water 27 Water solubility of carotenoids 5 Xanthophyll 33, 197 - Constitution 201 - Derivatives 204 - History 197 - Occurrence 198 - Preparation 200 - Properties 202, 352 Xanthophyll diacetate 204 Xanthophyll dibenzoate 205 Xanthophyll dibutyrate 204 Xanthophyll di-«-caprate 204 Xanthophyll dicaprylate 204 Xanthophyll diiodide 204 Xanthophyll dioenanthate 204 Xanthophyll di-p-nitrobenzoate Xanthophyll dipalmitate 205 Xanthophyll dipropionate 204 Xanthophyll distearate 205 Xanthophyll di-w-valerate 204 Xanthophyll epoxide 33, 206 Xanthophyll halogenides 204 Xanthophyll monomethyl ether 205 m- Xylene 49, 118 Zeaxanthin 33, 180 - Constitution 183 - Derivatives 185 - Formation 182 - History 180 - Occurrence 180 - Preparation 182 - Properties 184, 350 - Stereoisomers 188 Zeaxanthin diacetate 185 Zeaxanthin dibutyrate 186 Zeaxanthin dicaprate 186 384 SUBJECT INDEX Zeaxanthin dicaprylate 186 Zeaxanthin halogenides 185 Zeaxanthin di-epoxide (Violaxanthin) Zeaxanthin mono-epoxide (Antheraxan- 100 thin) Zeaxanthin dilaureate 186 Zeaxanthin monomethyl ether 185 Zeaxanthin dimethyl ether 185 Zeaxanthin monopalmitate 186 Zeaxanthin dipropionate 186 Zeisel determination 45 Zeaxanthin distearate 186 Zerewitinoff determination 45 Zeaxanthin di-M-valerate 186 Zinc carbonate 26 fii,|i':'''P:iifi;i.^.4.;:'':;ci;;UTi|ii:p^^^^^ 1