PRACTICAL
PLANT BIOCHEMISTRY

CAMBEIDGE UNIVEKSITY PEESS

C. F. CLAY, Manager

LONDON : FETTER LANE, E.G. 4

LONDON : H. K. LEWIS & CO., Ltd.

136, Gower Street, "W.C. 1
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2-4 Arthur St, New Oxford St, W.C. 2
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TOKYO: MARUZEN-KABUSHIKI-KAISHA

ALL EIGHTS EK8EEVED

PKACTICAL
PLANT BIOCHEMISTEY

BY

MUEIEL WHELDALE ONSLOW

FORMERLY FELLOW OF NEWNHAM COLLEGE, CAMBRIDGE, AND RESEARCH STUDENT

AT THE JOHN INNES HORTICULTURAL INSTITUTION, MERTON, SURREY.

AUTHOR OF THE ANTHOCYANIN PIGMENTS OF PLANTS.

^Ed'vtlOYl 1- ^

CAMBRIDGE

AT THE UNIVERSITY PRESS

1923

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1

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First Edition 1920
Second Edition 1923

(951

PRINTED IN GREAT BRITAIN

PHEFACE

THIS book is intended primarily for students of Botany. Such a
student's knowledge of plant products is usually obtained, on the
one hand, from Organic Chemistry, on the other hand, from Plant
Physiology ; between these two standpoints there is a gap, which, it is
hoped, the following pages may help to fill. It is essentially a text-book
for practicai work, an aspect of Plant Biochemistry which has received up
to the present time very little consideration in teaching. A number of
experiments have been devised and have been actually tested in practical
classes. These experiments should enable a student to extract from the
plant itself the chemical compounds of which it is constituted, and to learn
something of their properties. An elementary knowledge of Organic
Chemistry on the part of the student has been assumed, as it appeared
superfluous to incorporate the material which has already been so amply
presented in innumerable text-books.

My sincerest thanks are due to Dr F. F. Blackman, F.R.S., for criticism
and many suggestions throughout the writing of the book. I am further
indebted to Mr H. Raistrick, M.A., for help in various ways, especially in
reading the proof-sheets. I wish, in addition, to express my gratitude to
Professor F. G. Hopkins, F.R.S., for the great interest he has always shown
in the subject and for his kind and stimulating advice in connexion with
the scheme of teaching presented in the following pages.

M. W. O.

Cambridge,

February, 1920.

PREFACE TO THE SECOND EDITION

IN the present edition, some account, accompanied in most cases by
illustrative experiments, has been given of a number of substances, or
groups of substances, involved in plant metabolism, which were not in-
cluded in the first edition. These are notably the "vegetable acids,"
waxes, sterols, lecithins, inositol, phytin, the "essential oils" and nucleic
acid. Corrections have also been made in order to include more recent
additions to our knowledge on certain problems, as, for instance, those
connected with oxidizing enzymes.

Since it is advisable to keep the book as short as possible, a few of
the original experiments have been omitted to make space for others
considered to be of greater value to the student.

The chapter on the colloidal state is intended to give the student a
preliminary conception, only, of the importance of such phenomena.
Additional information, both as to theory and experiment, is to be found
in text-books which deal more exclusively with this subject.

Sufficient experience has not yet been gained to admit of the in-
clusion, in the present edition, of quantitative class-work in Plant Bio-
chemistry.

I am much indebted to Dr F. F. Blackman, F.R.S., for kindly assist-
ing with the proofs.

M. W. O.

Cambridge,
Deeemher, 1922.

CONTENTS

CHAP. PAGE

I. INTRODUCTION . 1

II. THE COLLOIDAL STATE 11

III. PLANT ENZYMES 18

IV. CHLOROPHYLL 27

V. CARBOHYDRATES . 42

VI. THE VEGETABLE ACIDS 81

VII. FATS AND ALLIED SUBSTANCES ... 89

VIII. AROMATIC COMPOUNDS 101

IX. PROTEINS AND AMINO-ACIDS . . . . 132

X. GLUCOSIDES 157

XL PLANT BASES 169

INDEX 183

Ml I

CHAPTEE I

INTRODUCTION

This chapter should be re-read after the remaining chapters have been studied.

All plants are made up of a complex organized mixture of chemical
substances, both organic and inorganic. As a preliminary to the study
of plant chemistry, the student should realize that the chemical com-
pounds which make up the living plant may be approximately grouped
into the six following classes. Thus, in later chapters, when reference
is made to any plant product, it will be understood, broadly speaking, to
which class it belongs, and what relationship it bears to other chemical
compounds.

The main classes may be enumerated as follows :

(1) Carbohydrates. The simplest members of this class are the sugars,
which are aldehydes and ketones of polyhydric alcohols of the methane
series of hydrocarbons. The more complex carbohydrates, such as starch,
cellulose, dextrins, gums and mucilages, are condensation products of the
simpler sugars. The sugars are found in solution in the cell-sap of living
cells throughout the plant. Cellulose, in the form of cell-walls, constitutes
an important part of the structure of the plant, and starch is one of the
most widely distributed solid "reserve materials."

(2) Vegetable acids. This term is usually applied to acids and hydroxy-
acids derived from the lower members of the methane, olefine and
acetylene series of hydrocarbons. Such acids as formic, acetic, valeric
and caproic are not readily detected in the plant. Nevertheless, it is
more than likely that they play an important part in metabolism, for
their amino derivatives, glycine, valine, etc. (see section 5) form con-
stituents of practically all proteins. The dibasic and hydroxy-acids, e.g.
oxalic, succinic, glutaric, malic, etc., are probably products of oxidation
of the sugars in respiration. Aspartic (amino-succinic) and glutaminic
(amino-glutaric) acids are also constituents of proteins.

(3) Fats. Chemically these are glycerides, that is glycerol esters, of
acids derived from the higher members of the methane and olefine series
of hydrocarbons, and they usually contain a large number of carbon atoms.
The fats occur as very fine globules deposited in the cells, especially in
the tissues of seeds where they form reserve materials, though they also
occur in other parts of plants.

2 INTRODUCTION [ch.

The lecithins, which are compounds of fats with phosphoric acid, are
probably present in all living cells and have an important metabolic
significance.

The above substances belong to the aliphatic series of organic com-
pounds, that is to the series in which the carbon atoms are united in
chains.

(4) Aromatic compounds. These are characterized by having the
carbon atoms united in a ring as in benzene. They may contain more
than one carbon ring, and, moreover, aliphatic groupings may be attached
to the carbon ring as side-chains. The number of aromatic substances is
very great, and every plant contains representatives of the class. Some
members are widely distributed ; others, as far as we know, are restricted
in their distribution, and may be peculiar to an order, a genus or even
a species. This class contains: (a) Phenols, i.e. hydroxy-derivatives of
T^enzene, such as phloroglucinol. (h) Aromatic alcohols, aldehydes and acids
derived from benzene ; various hydroxy-benzoic acids, such as gallic and
protocatechuic acids, are important, since, by condensation, they give
rise to tannins. Just as in the case of the carbohydrates, where simpler
compounds may become more complex by condensation, the soluble
crystalline acids condense to form the complex colloidal tannins. Of
other aromatic acids, the amino derivatives, such as phenylalanine and
tyrosine, form constituents of proteins, (c) Complex hydrocarbons, the
terpenes, accompanied by derivative alcohols, aldehydes, ketones and
esters. These form constituents of the "essential oils" obtained from
plants by steam distillation, and are responsible for most of the plant
scents, (d) Other members which contain more than one ring are the
water-soluble yellow, red, purple and blue pigments of plants, the yellow
being hydroxy -flavones and flavonols, the remainder, anthocyan pigments.

(5) Proteins. This large class contains substances which are in many
cases built up of groupings from both the aliphatic and aromatic series.
It includes not only the proteins but also their simpler derivatives, the
albumoses, peptones and polypeptides. In this case, as before, the simplest
derivatives, known as the amino-acids, are synthesized by condensation
to form the polypeptides, peptones, albumoses and proteins, in a series
of increasing complexity. The amino-acids are compounds, either of the
aliphatic, aromatic or heterocyclic (see 6) series, in which one or more
hydrogen atoms are replaced by the radicle NHg. They are soluble and
crystalline, but after condensing together, the final product, the protein,
only exists in either the solid or the colloidal state. Proteins, in the
latter condition, constitute the bulk of the complex material, protoplasm ;

i] INTRODUCTION 3

in the solid state, in the form of grains and granules, they occur as reserve
material in the cell.

(6) Plant bases. This class contains (a) the amines or substitution
products of ammonia. Sometimes the hydrogen of ammonia is substi-
tuted by a group of some complexity which leads to the production of a
■compound of the heterocyclic type, i.e. with a ring containing both carbon
and nitrogen atoms. The pyrrole ring is an example which occurs in the
amino-acid, proline, in certain alkaloids (see below), and in the pigment
chlorophyll, (b) Purines. In connection with these substances we need
to consider two more heterocyclic rings, i.e. the pyrimidine and the
iminazole. The former may be regarded as the condensation product of
urea, which is possibly present in small quantities in plants, and an un-
saturated acid, e.g. acrylic acid. The pyrimidine ring is present in some
purines, the iminazole in the amino-acid, histidine. The remaining
purines contain a condensed pyrimidine and iminazole ring. Certain of
the purines become condensed together, in combination with phosphoric
acid and a pentose sugar, to form the nucleic acids. The latter, in com-
l)ination with proteins, as nucleoproteins, form a constituent, as their
name implies, of the nucleus, (c) The alkaloids are substances of con-
siderable complexity, containing various heterocyclic rings. Unlike the
simpler bases, they are restricted to a certain extent in their distribution.

It is not possible to include all classes of plant substances in the
above list and many others, such as the sulphur compounds, sterols,
phytin, etc., are referred to in the later chapters. It should be borne in
mind that the importance of a compound in plant metabolism is not
estimated by the amount of it occuring in the plant. Frequently, most
important substances occur in such small quantities that they are diffi-
cult to detect.

In order to appreciate the subject of plant chemistry, the plant,
which is familiar as a botanical entity, must be interpreted in chemical
terms. The principal classes of the more essential and widely distributed
compounds found in plants have already been indicated on the broadest
basis, so that they may now be referred to without additional comment.

From the botanical point of view, the plant may be regarded as a
structure composed of many living protoplasmic units enclosed in cell-
walls and combined together to form tissues. There are also certain
tissues, known as dead tissues, which assist in giving rigidity to the plant.
All these structural elements may, in time, be translated into terms of
chemical compounds.

1—2

4 INTKODUCTION [cr.

One of the chemical processes most frequently met with in the plant
is that of synthesis by condensation, with elimination of water, of large
complex molecules from smaller and simpler molecules. The formation
of cellulose, for instance, is a case in point. Cellulose has the composition
(CeHioOs)^ and, on hydrolysis with dilute acids, it yields glucose as a final
product. Hence it is concluded that the complex molecule of cellulose is
built up from the simpler carbohydrate by condensation. The synthesis
of proteins from amino-acids affords another example. These acids con-
tain either an aliphatic or aromatic nucleus (let it be R), and one or more
carboxyl and amino groups. Condensation takes place in the plant, with
elimination of water, according to the following scheme:

Ri Rii Riii Ra;

i , I I I

NHoCH— COiOH HInH-CH— COiOH HiNH • CH— COiOH HiNHCH— COOH

The products of such condensation, the proteins, vary among them-
selves according to the number and kind of amino-acids which take part
in the synthesis.

Two important results arise from this process. First, the substances
formed by condensation have molecules of a very large size ; secondly,
whereas the simple compounds, sugars and amino-acids, are soluble,
crystalline and diffusible, the condensation products are either insoluble,
e.g. cellulose, or exist in the colloidal state, as is the case of many proteins
and other plant constituents. As these very large molecules do not dialyze>
they remain where they are synthesized, and build up the solid structure
of the plant, as for instance, the cell-walls.

Matter in the colloidal state is of very great importance in the plant
and is probably responsible for many of the properties of ** living'^
material. Thus it will not be out of place, though it will be referred to
again in a later chapter, to make at this point a few remarks on the
colloidal state. It has been known for some time that certain metals,
e.g. gold and silver, and also certain metallic hydroxides and sulphides,
e.g. ferric hydroxide and arsenious sulphide, though insoluble in water
under ordinary conditions, can, by special methods, be obtained as solu-
tions which are clear to the unaided vision. Such solutions are termed
colloidal. Investigation has shown that the matter is not present in true
solution, but in a very finely divided state, i.e. as particles many times
larger than simple molecules, but smaller than the particles obtainable
by mechanical means of division. Such solutions are known as artificial
colloidal solutions, but there are a number of organic substances, with
very large molecules, such as proteins, starch, gums, agar, etc., which at

I] INTRODUCTION 5

once dissolve in water giving colloidal solutions. The main feature of
the colloidal state is that the system consists of two phases, or conditions
of matter. In the case of the artificial colloidal solutions first mentioned,
one state is solid, the gold particles; the other state is liquid, the water.
The solid is known as the dispersed phase, and the water as the continuous
phase, and such colloidal solutions are termed suspensoids. In the case
of proteins, starch, etc., both phases are liquid: the dispersed phase, a
concentrated solution of protein, etc.; the continuous phase, a dilute
solution of protein, etc. Such colloidal solutions are known as emulsoids.

An important point in connexion with the colloidal state is that the
molecules, or aggregates of molecules, forming the dispersed phase are
so large that they exhibit some of the phenomena of surface energy,
electrical charge, etc., associated with matter in mass. These properties
come to be of considerable importance, when we consider how large
a surface is presented by matter in this state in comparison with its mass.

A material in the plant upon which much interest naturally centres
is the protoplasm and the nucleus. It has been shown that the protoplasm
consists, chemically, largely of proteins in the colloidal state. It is itself
a liquid, and embedded in it are substances of various chemical constitu-
tion, in the form of granules of solid matter and also liquid globules.
Numerous chemical reactions are continually taking place in the proto-
plasm throughout the cell, and since many of these reactions can take
place both simultaneously and independently, the protoplasm must have
some form of organized structure. Though many phenomena of "life"
may be accounted for by the physical and chemical properties of such
substances as proteins, it is impossible to say, with our present knowledge,
how far all "living" phenomena may yet be explained in this way.

Some of the main lines of metabolic S3nitheses which take place in
the plant will next be considered. A fundamental fact which should be
borne in mind is that the green plant synthesizes all the complex
materials of which it is composed from the simple compounds, carbon
dioxide, water and certain inorganic salts. The most important factor,
perhaps, which figures in plant metabolism, is chlorophyll. The green
pigments of chlorophyll are esters of complex organic acids containing
the elements carbon, hydrogen, oxygen, nitrogen and magnesium. They
have the remarkable power of absorbing the radiant energy of the sun's
rays and of transforming it into chemical energy, by means of which
carbon dioxide and water are combined to form some organic compound,
possibly formaldehyde, from which a simple carbohydrate is readily
synthesized.

6 INTRODUCTION [ch.

If now the initial and final products of carbon assimilation be con-
sidered in detail, it will be seen that the process is one of reduction :
6C02 + 6H20 = C6Hi206 + 602.

This is confirmed by the fact that oxygen is evolved in the process-
Moreover, the plant accumulates a store of energy, since the final pro-
duct, the carbohydrate, has a higher potential energy than the system^
water and carbon dioxide. Hence carbon assimilation, in addition to-
providing a basis of organic material as a starting-point for all the main
metabolic functions, also provides a source of chemical energy by mean&
of which reactions in other directions are brought about.

The setting free of this accumulated energy constitutes the process
of respiration, which is, in reality, an oxidation of carbohydrate taking
place in tissues throughout the plant. It is the converse of carbon
assimilation, in that oxygen is absorbed and carbon dioxide and water
are formed. Thus these two processes, both so fundamental and essential
to the metabolism of the green plant, are constantly taking place side
by side in the same cell.

The first-formed carbohydrate, which is probably a hexose, is con-
densed in the plant, on the general lines we have previously indicated^
to form more complex disaccharides and polysaccharides, such as maltose,
cane-sugar, starch, cellulose, etc. Some of these products, such as the
disaccharides, form true solutions and may be present in the cell-sap ;
others, such as cellulose and starch, are present in the solid state, though
they contain considerable quantities of water. Others, again, such as
dextrin and gum, are present in the colloidal state. Thus, given an
initial carbohydrate and a source of energy, we may proceed to indicate
the other main lines of syntheses in the plant.

The next most important line of syntheses is probably that which
gives rise to the nitrogen-containing constituents of the plant. Nitrogen
is absorbed by the green plant in the form of nitrates and ammonium
salts, but the processes which lead to the synthesis of some of the simplest
nitrogen-containing compounds, such as the amino-acids, are still very
obscure. Aliphatic and aromatic acids of various kinds are abundantly
present in the tissues, but the reactions by which the NHg groups are
introduced are by no means clear. There is little doubt, however, that
once the amino-acids are formed, condensation takes place as already
indicated, and more complex molecules, termed polypeptides, arise. Such
polypeptides have now been synthesized artificially by the condensation
of amino-acids. From the polypeptides, by further stages of condensation,
the albumoses, peptones, and finally proteins are produced.

I] INTRODUCTION 7

Another line of syntheses is that which leads to the production of
the fats and allied substances. The fats are mainly glycerides of acids
of the methane and olefine series, such as butyric, palmitic and oleic
acids. Like all other plant products the fats must either directly or
indirectly arise from the carbohydrates. There is evidence that the
origin is fairly direct, as, for instance, in fatty seeds when the fats take
the place of sugars in ripening. The sugars, as we know, are aldehydes
of the polyhydric alcohols of the methane series. It has been suggested,
though the actual stages have not been ascertained, that by various
oxidation and reduction processes, the sugars yield fatty acid residues
which then condense to form the fatty acids of high molecular weights
present in fats. By a converse process, the fats, especially when they
are stored as reserve materials in seeds, are broken up, and sugars are
again formed which pass to other parts of the germinating seedling, and
are there used in other synthetic processes.

A third main line of syntheses is that which gives rise to the aro-
matics of the plant. Since no ring compound is absorbed by the green
plant, it follows that by some process the aliphatic structure must be
transformed into the aromatic. Thus, for instance, the trihydric phenol,
phloroglucinol, might at some stage be formed from a hexose by conversion
of the aliphatic chain into a closed ring :

OH H OH OH
OHO— C— C— 0— C— CH2OH — 3H2O = CO— CH2— CO— CH2— CO— CH2
H OH H H I I

Glucose

H2 H

OC CO HOC COH

= 11= II I

H2C CH2 HC CH

\c/ \c/

O OH

Phloroglucinol

There is evidence that aromatic compounds, such as phloroglucinol,
tannins, flavones and anthocyanins are synthesized in the leaves, and that
sugar-feeding, by floating leaves in sugar solutions, leads to the increase
of aromatics in the tissues. When the ring structure has been once
synthesized, further changes can take place either by the addition of
side-chains to the ring or by the condensation of two or more rings. In
this way the great multitude of aromatic products present in the higher
plants may arise.

8 INTRODUCTION [ch.

Thus the cell can be pictured as a colloidal solution of proteins
endoAved with the properties of matter in mass and surrounded by a
permeable cell-wall of cellulose. The colloidal solution contains liquid
and solid particles of very varied chemical composition. In the proto-
plasm are spaces, vacuoles, filled with cell-sap also containing many
and various substances in solution. Throughout the protoplasm, which
probably has an organized structure, many kinds of chemical reactions
are continually in progress, some being the converse of others, as for
instance those of oxidation and reduction which can take place side by
side in the same cell.

Next will be considered the chemical reactions by which the various
metabolic changes in the plant are brought about. How are these pro-
cesses controlled and how do they take place ?

There is a large group of organic substances, termed enzymes, many
of which are present in every plant. They have a certain characteristic
in common, i.e. they bring about chemical reactions in the plant without
undergoing any permanent change : in other words they are organic
catalysts. Many of these reactions, which take place in the cell at
ordinary temperatures with considerable rapidity, need prolonged heating
at high temperatures when brought about by artificial means. Enzymes
can generally be extracted from the plant by water, especially if the
tissues are thoroughly disintegrated. Their chemical constitution is at
present unknown, and they are usually destroyed by temperatures greater
than 60° C. Moreover, many of the processes which they control in the
plant can be brought about by them in vitro under suitable conditions,
and it is by means of such experiments that information as to their role
in plant metabolism has been ascertained. The majority of known
enzymes control both hydrolysis and its converse, synthesis by conden-
sation with elimination of water, but under artificial conditions hydrolysis
most frequently occurs. The enzyme, diastase, for instance, found in all
starch -containing plants hydrolyzes in vitro starch to dextrin and maltose.
Similarly the enzyme, maltase, hydrolyzes maltose into glucose. Other
enzymes hydrolyze proteins into amino-acids, and others, again, hydro-
lyze fats into fatty acids and glycerol.

Until fairly recently the fact escaped notice that such reactions are
reversible, and that these enzymes in situ in the plant may, according
to the conditions, control not only the hydrolytic but also the corre-
sponding synthetic process. The latter may also be brought about, though
not readily, in vitro. This, and other evidence, leads us to believe that
enzymes in the plant control the reactions in both directions.

I] INTRODUCTION 9

Hydrolysis, and synthesis with elimination of water are not however
the only processes catalyzed by enzymes. There is another type of these
•catalysts, the oxidizing enzymes, which bring about oxidation of sub-
stances in the plant, notably of aromatics. In addition, there is the
•enzyme, zymase, which decomposes sugar with the production of alcohol
and carbon dioxide.

The question which now arises is — How many reactions in the plant
are catalyzed by enzymes ? It is conceivable that a greater number of
enzymes may exist than are at present known, but that they are unable
to be extracted by our present methods of isolation. A certain number
of reactions probably take place in the cell-sap between the substances
in solution ; others are catalyzed by enzymes which are supposed to be
intimately connected with the protoplasm, but there are an enormous
number to which there is at present no clue as to how they are brought
about, such, for instance, as the synthesis of carbohydrates from carbon
•dioxide and water, and the formation of the benzene ring from the open
carbon chain. Such processes are usually said to be controlled by the
" living protoplasm," but what exactly is the significance of this expres-
sion is at present beyond our knowledge.

Finally, also, little is known of the question as to how the various
lines of metabolic syntheses in different parts of plants are regulated
and correlated with each other. Some of the phenomena involved are
shortly outlined as follows. There is undoubtedly, under suitable con-
ditions, a constant synthesis of sugars in the leaves. In all probability
aromatic substances are also synthesized in the same organs, for there
is evidence that there is an increase of these compounds in the leaf if
translocation through the petiole is prevented. It is possible that amino-
acids also are formed in the leaf The above products are constantly
translocated to the growing organs as material for growth. They may,
nevertheless, be temporarily stored in the tissues where they have been
synthesized, and of this there is evidence in at least one case, e.g. starch
in the leaf. But, apart from the immediate use for growth, there is
in practically every plant, some tissue where, owing to some unknown
stimulus (causing probably changes in permeability of the cell-mem-
branes), accumulation of compounds occurs. This accumulation is
characteristic of organs from which growth will take place when it is
impossible for the plant to obtain fresh supplies by carbon assimilation,
as, for example, of bulbs, rhizomes, tubers, buds, seeds, fruits and woody
tissues. In these cases, in due time, the products stored supply the
growing shoots.

10 INTRODUCTION [ch. i

During storage, simple sugars, amino-acids, etc. have been condensed
to form insoluble, colloidal, or large molecules of starch, fats, aleurone,
cane-sugar, etc. These will remain until they are hydrolyzed by enzymes
when they can supply the growing shoots. Such stores are termed
" reserve materials." The actual stimuli involved in bringing about and
regulating this storage are unknown, but they are probably connected
with the life cycle of the particular plant under consideration and its
adaptation to external conditions.

REFERENCES

1. Abderhalden, E. Handbuch der biochemischen Arbeitsmethoden. Berlin,
1910.

2. Abderhalden, B. Biochemisches Handlexikon. Berlin, 1911.

3. Allen's Commercial Organic Analysis. London, 1909-1917.

4. BertrandjG., and Thomas, P. Practical Biological Chemistry. Translated
by H. A. Colwell. London, 1920.

5. Cole, S. W. Practical Physiological Chemistry. Cambridge, 1920. 6th ed.

6. Czapek, P. Biochemie der Pflanzen. Jena, Bd. 1, 1913, Bd. 2, 1920, Bd. 3,
1921.

7. Haas, P., and Hill, T. G. The Chemistry of Plant Products. London, 192L
3rd ed.

8. Palladin, V. I. Plant Physiology. Edited by B. E Livingston. Philadelphia,
1918.

9. Plimmer, R. H. A. Practical Organic and Biochemistry. London, 1918.
3rd ed.

10. "Wehmer, 0. Die Pflanzenstoffe. Jena, 1911.

11. Wester, D. H. Anleitung zur Darstellung phytochemischer Uebungs-
praparate. Berlin, 1913.

CHAPTER II

THE COLLOIDAL STATE

Many of the substances of which the plant is built up exist in the living
cell in the colloidal state, and it is therefore important that some account
should be given of this condition of matter.

There are many organic products found in the plant (and also in the
animal), such as starch, various proteins, gums, etc., that apparently dis-
solve in water, giving a solution which, as a rule, only differs from an
ordinary solution by being opalescent. In addition, it has been known
for a long time that various inorganic substances, such as sulphides of
arsenic and antimony, hydroxide of iron, and also certain metals (gold,
silver), can, by special methods, be obtained in " solution," though in
ordinary circumstances they are quite insoluble. The above examples
are representative of colloidal solutions.

A property which all the above solutions possess is that the substance
dissolved will not pass through a parchment membrane, i.e. will not
dialyze, whereas if a solution of sodium chloride in water is separated
from pure water by a parchment membrane, the salt will pass through
the membrane until the concentration of the sodium chloride is equal
on either side of it.

The conclusion drawn from investigations of various kinds is that in
the colloidal solutions the substances dissolved exist in the state, either
of aggregates of molecules, or of very large molecules, and hence are
unable to pass through the pores of the parchment.

Moreover, certain distinctions can be drawn between colloidal solu-
tions : some, like those of gold, silver, metallic sulphides, hydroxides and
in fact most inorganic substances, are very sensitive to the presence of
small amounts of inorganic salts, i.e. electrolytes, and are precipitated
by them, but will not as a rule go into solution again. Also such col-
loidal solutions are very little more viscous than pure water. The organic
substances, on the other hand, are only precipitated from colloidal solu-
tions by comparatively large quantities of electrolytes. The viscosity,
moreover, of these solutions is greater than that of water, and is, in fact,
considerable, even if the percentage of dissolved matter is small.

12 THE COLLOIDAL STATE [ch.

Hence two terms have been employed for the above-mentioned types
of colloidal solutions : those of gold, silver, etc., are termed suspensoids
(suspensoid sols): those of starch, proteins, etc., emulsoids (emulsoid
sols).

The essential feature of both forms is that they are systems consisting
of two phases, or conditions of matter, known respectively as the " dis-
persed " phase and the " continuous " phase.

A suspensoid may be defined as having a dispersed phase composed
of ultramicroscopic particles or aggregates of molecules suspended in a
continuous phase composed of a liquid.

An emulsoid may be defined as having a dispersed phase composed
of ultramicroscopic drops of a highly concentrated solution of the sub-
stance suspended in a continuous phase composed of a dilute solution of
the same substance.

As a rule, therefore, the difference between a suspensoid and an
emulsoid is that, whereas in the former the liquid is restricted to the
continuous phase, and the solid to the dispersed phase, in an emulsoid
both phases are liquid, though containing different proportions of the
dissolved substance.

The terms suspensoid and emulsoid are used on account of the re-
semblance of these states of matter respectively to suspensions and
emulsions. If microscopic particles of a solid are shaken up in water,
what is known as a suspension is obtained ; in time, however, the solid
particles, if heavy enough, will settle and separate from the water, and
the whole process can be repeated. Thus a suspension differs from a
suspensoid solution in that the latter is stable, though, if precipitated,
the reaction is usually not reversible.

V If two liquids which are insoluble in each other, such as oil and
water, are shaken up together, finely divided drops of oil in water are
obtained. This is known as an emulsion. In time, however, the oil
separates from the water, because the tension on the films of water
separating the oil drops, when in contact, is too great, and they break,
with the result that the oil drops coalesce. But if, instead of water, a
solution of soap, saponins, or certain other substances is used, the surface
tension of the water is so lowered that the films of soap solution separating
the oil drops are permanent, and a system is obtained consisting of minute
drops of oil separated by soap solution. This system resembles an organic
colloidal solution, as, for instance, that of protein in which we suppose
a concentrated solution of protein exists in drops separated by a dilute
solution of protein. Milk and latex constitute natural emulsions.

II] THE COLLOIDAL STATE 13

Expt. 1. Formatio7i of a suspension. Precipitate a solution of barium chloride
with some sulphuric acid and shake up well the fine precipitate of barium sulphate.
Note the gradual settling of the precipitate.

Expt. 2. Formation of an eynulsi'on. Take a drop of olive oil in a test-tube and
half fill the tube with alcohol. Shake well and pour into a beaker of water. A fine
white emulsion of oil in water will be formed from which the oil will not separate.
By this method the oil is obtained in such small drops that stability is ensured.

Take about equal quantities of olive oil in two test-tubes and add an equal
quantity of water to each. To one tube add a drop or two of 10 % caustic alkali
solution. Shake both test-tubes well. An emulsion is formed in both, but in the
tube without alkali the oil will separate out on standing. In the other tube the
emulsion is permanent. This is due to the fact that the olive oil (unless specially
purified) contains some free fatty acid. The latter forms soap with the alkali (see
p. 93) and renders the emulsion permanent.

Expt. 3. Preparation of suspensoid sols, (a) Gold. Take two 100 c.c. measuring
cylinders and thoroughly clean them with nitric acid, and afterwards wash well with
freshly distilled water. In one make a 0*5 % solution of tannic acid (using the purest
sample obtainable) in water. In the other, 2 c.c. of commercial 1 % gold chloride are
made up to 100 c.c. with water. Use freshly distilled water in both cases. Mix equal
portions of the two solutions in a clean beaker. A purple colloidal solution of gold
will be formed. If three parts of the chloride solution are mixed with one part of the
tannin solution, and both solutions heated before mixing, a red colloidal solution is
obtained. (6) Silver. Take 5 c.c. of a 1 % solution of silver nitrate and add dilute am-
monia solution until the precipitate first formed just disappears, and then dilute with
100 c.c. of water. Mix equal volumes of this solution and the tannic acid prepared
for (a). A colloidal solution of silver will be formed which is clear brown by trans-
mitted light, but has a green fluorescence by reflected light, (c) Ferric hydroxide.
Take 5 c.c. of a filtered 33% solution of ferric chloride and pour into 500 c.c. of
boiling distilled water in a beaker. A colloidal ferric hydroxide sol is formed and the
colour changes to a deep brown-red. The yellow solution of ferric chloride is de-
composed by excess of water with the production of a soluble colloidal form of ferric
hydroxide, and hydrochloric acid is set free, (d) Arsenic tristdphide. Take 2 gms.
of arsenious acid and boil with 150 c.c. of distilled water, filter and cool. Then pass
sulphuretted hydrogen through the solution. A colloidal solution of the sulphide is
formed which is orange, with a greenish surface.

The above sols should be kept for further experiment [see Expt. 8].

Expt. 4. Preparation ofemulsoid sols, (a) Starch. Weigh out 2 gms. of dry starch,
and mix well with a little cold distilled water. Boil 100 c.c. of distilled water in a
flask, and, when boiling, pour in the starch paste and boil for a few minutes longer,
stirring well all the time. A colloidal solution of starch is obtained which is faintly
opalescent. It is not afifected by heating and does not change its state on cooling,
(6) Gum arabic. Make a 5 7o solution of gum arabic by boiling 5 gms. with 100 c.c.
of distilled water. Note that a sticky or viscous solution is formed which froths on
shaking, (c) Protein. Weigh out 10 gms. of white flour and add 100 c.c. of distilled
water. Let the mixture stand for 2 or 3 hours and then filter. The extract contains

14 THE COLLOIDAL STATE [ch.

protein. Note that the solution froths on shaking, {d) Soap. Make a 5-10 % solution
of soap in distilled water. It is opalescent and froths strongly.

The above sols should be kept for further experiment [see Expt. 9].

Expt. 5. Dialysis of starch and salt solution. Make a 2 % solution of starch in
water, as in Expt. 4 (a), and mix it with an equal volume of a 2 ^f^ solution of sodium
chloride in water. Pour the mixture into a parchment dialyzer and dialyze in a
beaker of distilled water. (The dialyzer should first be carefully tested to ascertain
of there be a leak.) Test the liquid in the beaker with solutions of both iodine and
silver nitrate. Some precipitate of chloride will be given, but no blue colour with
iodine. After 24 hours, test the liquid again. There will be an increased amount of
silver chloride formed, but a negative result with iodine. On addition of iodine to
the liquid in the dialyzer, a blue colour is obtained. Hence we may assume that the
colloidal starch does not pass through the membrane.

Some substances, such as gelatine (animal) and agar (vegetable), are
only in the emulsoid condition at a raised temperature. When cold they
set to form a " gel," in which the particles of the dispersed phase are no
longer separate but united to make a solid. Silicic acid, the best known
inorganic emulsoid, also sets to a gel on standing, either spontaneously
or on addition of electrolytes. It is of classical interest since it was the
substance largely used by Graham, the first worker on colloids.

Expt. 6. Preparation of gels, (a) Agar. Weigh out 2 gms. of agar and put it
to soak in 100 c.c. of distilled water for an hour or two. Then boil : the agar gives a
thick opalescent solution (sol) which sets to a gel on cooling. On warming, the gel
again becomes a sol, and, on cooling, again sets to a gel. Thus the change is a
reversible one. Agar is a mucilage which is obtained from certain genera of the
Rhodophyceae (see p. 51). (b) Silicic acid. Weigh out 20 gms. of commercial "water-
glass" syrup ( a concentrated solution of sodium silicate) and dilute willh 100 c.c. of
freshly boiled distilled water (free from carbon dioxide). Pour 75 c.c. of this solution
into a mixture of 25 c.c. of concentrated hydrochloric acid and 75 c.c. of water.
Dialyze the mixture in a parchment dialyzer against running water for 3-4 hours.
If to the dialyzed liquid a little very dilute ammonia is added, a gel will be formed
in the course of a few hours. In this case, however, the process is irreversible, that
is the gel cannot be reconverted again into the sol.

An interesting point in connexion with the colloidal state is that
emphasized by Ostwald, i.e. that this condition is a state, not a type,
of matter. Further, substances in the colloidal state do not constitute
a definite class. It is reasonable to suppose that all substances which
exist in the colloidal state can, under suitable conditions, also exist in
the crystalline state, and vice versa. Further, the continuous phase is
not always water. Sodium chloride, which is a very definite crystalloid,
can be obtained in the colloidal state in petroleum ether. Most metals,
even the alkali metals, have been obtained in colloidal solution : also a
great many metallic oxides, hydroxides and sulphides.

II] THE COLLOIDAL STATE 15

The colloidal phases so far dealt with can be tabulated as follows^:

disperse continuous

liquid solid gels

solid liquid suspensoids

liquid liquid emulsoids

Some of the properties of colloidal solutions may now be considered.
A point that has already been emphasized in the previous chapter is
that the surface of particles in the colloidal state is very great in pro-
portion to their mass. Such particles, moreover, unlike ions and small
molecules in true solution, possess the properties of the surfaces of matter
in mass, as, for instance, those connected with surface tension, electrical
charge, etc., and these are especially marked on account of the propor-
tionately large surfaces involved. Other properties are their inability,
as a rule, to exert an osmotic pressure, to raise the boiling point, and to
lower the freezing point of water. Some of the metallic suspensoids are
characterized by their colour, this being red, purple or blue as in the case
of gold sols.

An apparatus, by means of which the colloidal state can be demon-
strated ocularly, is the ultramicroscope. This is a special form of micro-
. scope in which apowerful beam of light is directed upon a colloidal solution,
which is then seen to contain a number of particles in rapid motion.
When analyzed by special methods, this motion has been found to be
identical with that shown by much larger, though still microscopic,
particles, which has been termed Brownian movement.

Expt. 7. Demonstration of Brownian movement of microscopic particles. Mount a
little gamboge in water and examine under the high power of a microscope. The
particles will be seen to be in rapid motion.

It has been shown that Brownian movement is the outcome of the
movement of the molecules of the liquid in which the particles are
suspended. This movement is one of the factors which keeps the sol
stable and prevents the particles from " settling " as in the case of a
true suspension.

Another factor tending to keep the sol stable is the electrical charge
borne by the particles. It is commonly known that there is usually a
difference of potential between the contact surfaces of phases. If the

1 There are also the following combinations (Bayliss, 1) :
disperse continuous

gas liquid foam

liquid gas fog

solid gas tobacco smoke

solid solid ruby glass (colloidal sol of gold in glass) .

16 THE COLLOIDAL STATE [ch.

particles in a colloidal solution all have the same charge, then they will
tend to repulse one another mutually. It is found that the particles
are charged, but the origin of the charge is not always clear. Sometimes
if the substance in colloidal state is capable of electrolytic dissociation,^
the charge may arise in this way. Substances, however, as already
mentioned, which are not dissociated may also bear a charge, and most
frequently it is a negative one. It follows, then, that when an electrolyte
is added to a colloidal solution, the charges on the colloidal particles are
neutralized by the oppositely charged ions of the electrolyte, and they
coalesce together and are precipitated.

As regards their behaviour to electrolytes the two classes, suspensoid&
and emulsoids, are very different. The suspensoids are very sensitive to-
traces of electrolytes, and, as they usually have a negative charge, it is
the cation of the electrolyte which is the active ion ; and of such, less of
a divalent ion, than of a monovalent ion, is needed for precipitation and
still less of a trivalent ion.

The emulsoids are far less sensitive to electrolytes in solution than
the suspensoids ; in fact, electrolytes, such as neutral alkali salts, must
be added in very large quantities to emulsoids before precipitation takes-
place. Also, as a rule, whereas the precipitation of suspensoids is irre-
versible, that of emulsoids is reversible, that is, they pass into solution
again on addition of water. In the case of an emulsoid in neutral solu-
tion this form of precipitation, unlike that of the suspensoids, may be
regarded as consisting of two processes. First, a process analogous to
that of " salting out " of soaps, esters, etc., in organic chemistry, which is,
in effect, a withdrawal of water from one phase into another. Secondly,
the precipitation is also affected to some extent by the valency of the
ions of the salt used in precipitation.

When, however, a neutral solution of such an emulsoid as protein is
made either slightly acid or alkaline, its behaviour towards neutral salts
becomes altered. The precipitating power of salts in acid or alkaline
medium is now in accordance with that on suspensoids. In alkaline
solution the coagulating power of a salt depends on the valency of the
cation ; in an acid solution it depends on the valency of the anion.

The behaviour of proteins in acid and alkaline media is undoubtedly
due to the fact that they are built up of amino-acids containing both
amino and carboxyl groups. Such molecules may behave either as an
acid or a base with the formation of salts. These are subject to electro-
lytic dissociation and hence acquire an electric charge. Such substances
have been termed " amphoteric electrolytes " (see p. 134).

II] THE COLLOIDAL STATE 17

Expt. 8. Precipitation of suspensoid sols by electrolytes. The sols of gold, silver
and arsenious sulphide carry an electro-negative charge : hence they are most readily
precipitated by di- or tri-valent positive ions, such as Ba" or Al'". Add a few drops
of barium chloride solution to the three sols (Expt. 3) respectively, and note that
they are precipitated, though some time may elapse before the precipitation is
complete. The ferric hydroxide sol, on the contrary, carries a positive charge. Hence
it is most readily precipitated by a sulphate or phosphate. If a drop of sodium
sulphate solution is added while the sol is hot, it is immediately precipitated.

Expt. 9. Precipitation of emulsoid sols by electrolytes. Saturate the starch, protein
and soap solutions prepared in Expt. 4 with solid ammonium sulphate. Precipitation
takes place in each case, and it is seen how large a quantity of electrolyte is needed
for the "salting out" of emulsoid sols. Filter off the protein precipitate and suspend
in distilled water. It will go into solution again, showing that the reaction is
reversible.

REFERENCES

1. Bayliss, W. M. Principles of General Physiology. London, 1920. 3rd ed.

2. Burton, E. P. The Physical Properties of Colloidal Solutions. London,
1916.

3. Hatschek, B. An Introduction to the Physics and Chemistry of Colloids.
London, 1919. 3rd ed.

4 Philip, J. C. Physical Chemistry: its Bearing on Biology and Medicine.
London, 1913. 2nd ed.

5. Taylor, W. W. The Chemistry of Colloids. London, 1915.

o.

CHAPT^E III

PLANT ENZYMES

Some indication has been given in the previous chapter of the large
number of complex processes which take place in the plant, and it has
been mentioned that many of these are controlled by enzymes.

The most remarkable feature in connexion with the chemical pro-
cesses of plant metabolism is the ease and rapidity with which, at ordi-
nary temperatures, chemical reactions take place, when under artificial
conditions they need a much longer time and higher temperatures.

It has been found that many of the chemical reactions in the plant
can be brought about in vitro on addition of certain substances which
can be extracted from the plant. These substances are known as enzymes.
It is the property of enzymes that they are able to accelerate reactions
which, in their absence, would only take place very slowly. The enzyme
cannot initiate these reactions and does not form part of their final
products.

Some inorganic substances have the same property of accelerating
reactions, and such substances are termed catalysts. For example, when
water is added to ethyl acetate, the latter begins to be decomposed slowly
into ethyl alcohol and acetic acid :

ethyl acetate + water — >- ethyl alcohol -|- acetic acid,

but if, in addition, some hydrochloric acid is added, hydrolysis takes place
with much greater rapidity, and at the end of the reaction the hydro-
chloric acid is found unchanged. Hence in this case hydrochloric acid
is an inorganic catalyst. Many other similar instances are known as, for
example, the catalyzing effect of a small quantity of manganese dioxide
which brings about the liberation of oxygen from potassium chlorate at
a much lower temperature than by heat alone.

By analogy, therefore, an enzyme may be defined as an organic
catalyst produced by the plant.

Another point in connexion with the above-mentioned reaction of
water with ethyl acetate, is the fact of its being representative of the
type known as reversible. After a certain amount of acetic acid and
ethyl alcohol has been formed, these recombine to form ethyl acetate
until in time a certain point of equilibrium is reached. Since the same

CH. Ill] PLANT ENZYMES 19

point of equilibrium is reached whether hydrochloric acid is used or not,
it is obvious that the hydrochloric acid accelerates the reaction in both
directions :

ethyl acetate + water :^ ethyl alcohol + acetic acid.

Such a reaction is termed a reversible one. Many of the processes
accelerated by enzymes in the plant are reversible, and there is reason
to believe that the enzyme accelerates the reaction in both directions.

The substance upon which, the enzyme acts is termed the substrate,
and it is supposed that some kind of loose combination occurs between
these two substances. The enzyme is unaltered when the reaction is
complete, unless it is affected by the products formed.

The enzymes are very widely distributed and form constituents of
all living cells, though all tissues do not necessarily contain the same
enzymes.

There is no doubt that many enzymes are specific, in which case
an enzyme can only accelerate one reaction, or one class of reaction.
We cannot be sure that any enzyme is specific and different from all
others, until it has been proved that it accelerates one process which is
incapable of being accelerated by any other enzyme. It is possible that
some enzymes, to which separate names have been given, are really
identical.

Most of the plant enzymes are soluble in water and dilute glycerol
and sometimes in dilute alcohol. Some can be extracted by simply
macerating the tissues with water; others are more intimately connected
with the protoplasm, and are only extracted if the protoplasm is killed
by certain reagents, of which those most frequently employed are toluol
and chloroform. These substances kill the protoplasm and do not, in
many cases, affect the enzyme. After the death of the protoplasm, the
enzymes are more readily extracted from the cell. From aqueous solu-
tions enzymes can usually be precipitated by adding strong alcohol.

The majority of enzymes are destroyed by raising the temperature
above 60° C. In vitro their reactions are generally carried out most
rapidly between the temperatures of 35-45° C.

In performing experiments with enzymes in vitrOy it is always neces-
sary to add an antiseptic, otherwise the reaction to be studied will be
masked or entirely superseded by the action of bacteria unavoidably
present. Toluol and chloroform mentioned above, as well as thymol, may
be used. These reagents prevent any bacterial action from taking place.
Some enzymes, however, are susceptible to chloroform, as, for instance,
maltase.

20 PLANT ENZYMES [ch.

The chemical nature of enzymes is at present unknown, because it
is difficult to purify them without destroying them, and hence to obtain
them of sufficient purity for chemical analysis. They were originally
thought to be proteins, but with the improvements in methods for puri-
fication, it has been found that the protein reactions disappear, although
the enzyme activity does not decrease. In solution they exist in the
colloidal condition.

The questions as to their origin and their relation to the protoplasm
cannot yet be answered with any certainty. It is also impossible to say
whether the majority of chemical processes in the plant are catalyzed by
enzymes.

A feature of enzyme action which is of considerable interest and
which has already been mentioned is the question as to whether enzymes
catalyze a reaction in both directions. Thus, in the case of hydrolytic
enzymes which constitute by far the greater number of known enzymes,
do they control the synthetic as well as the hydrolytic process ? There
is evidence that this is so, since, in many cases, the hydrolysis is not
complete. If the enzyme were a catalyst in one direction only, the
reaction would be complete. Further evidence is supplied by the fact
that, under suitable conditions, i.e. strong concentration of the substances
from which synthesis is to take place, certain syntheses have been carried
out in vitro. As an example may be quoted the synthesis of maltose
from a concentrated solution of glucose by maltase (Bayliss, 2).

In the living cell it is supposed that the hydrolysis and synthesis
are balanced. On the " death " of the protoplasm, which may be caused
by mechanical injury, vapour of chloroform or toluol, etc. (Armstrong,
7, 8), the reactions catalyzed by enzymes cease to be balanced and pro-
ceed almost always in the direction of hydrolysis and the splitting up of
more complex into simpler substances. This phenomenon is obvious when
any of the products can be recognized by smell or colour, as, for instance,
the smell of benzaldehyde on injuring leaves of plants containing cyano-
genetic glucosides (see p. 161), or the production of coloured oxidation
products when some of the aromatic glucosides are decomposed (see
p. 124).

If plant tissues are disintegrated, and the mass is kept at a tempera-
ture of about 38° C, the above-mentioned hydrolytic processes continue
to be catalyzed by the enzymes present until equilibrium is reached,
which will be near complete hydrolysis, especially if water has been added.
Such a process is termed " autolysis."

The chief plant enzymes may be classified to a certain extent accord-

Ill]

PLANT ENZYMES

21

ing to the reaction they catalyze, e.g. hydrolytic, oxidizing, etc., as
follows :

Hydrolysis
Enzyme Substrate Products

Lipase (p. 94)
„ (p. 99)

Chlorophyllase (p. 34)
Phytase (p. 102)
Gly cerophosphatase

(p. 99)
Diastase (p. 75)
Invertase (p. 78)
Maltase (p. 77)
Inulase (p. 60)
Cytase (p. 71)
Emulsin (p. 160)

Myrosin (p. 164)

Pepsin (p. 152)
Erepsin (p. 152)

Peroxidase (p. 122)
Oxygenase (p. 122)
Tyrosinase (p. 128)

Catalase (p. 129)
Reductase (oxido-re-
ductase) (p. 129)

Hexosephosphatase

(p. 22)
Zymase (p. 22)
Carboxylase (p. 22)

Urease (p. 181)
Pectase (p. 67)

Fats
Lecithin

Chlorophyll

Phytin

Glycerophosphoric acid

Starch

Cane sugar

Maltose

Inulin

Hemicellulose

Amygdalin

Sinigrin

Proteins
Peptones

Fatty acids and glycerol

Fatty acids, glycero-phosphoric acid

and choline
Chlorophyllide and phytol
Inositol and phosphoric acid
Glycerol and phosphoric acid

Dextrin and maltose

Dextrose and laevulose

Dextrose

Laevulose

Mannose and galactose

Benzaldehyde, prussic acid and

glucose
Allyl isothiocyanate, potassium,

hydrogen sulphate and glucose
Albuminoses and peptones
Polypeptides and amino -acids

Oxidation and reduction

Hydrogen peroxide
Catechol, etc
Tyrosine

Hydrogen peroxide
Water

Atomic oxygen
Peroxide

Melanin, ammonia and carbon di-
oxide
Molecular oxygen
Hydrogen and oxygen

Respiration (and fermentation)

Hexosephosphate

Hexose

Pyruvic acid, etc.

Hexose and phosphoric acid

Alcohol and carbon dioxide
Acetaldehyde and carbon dioxide

Other reactions

Urea
Soluble pectin

Ammonia and carbon dioxide
Cytopectic acid

Most of these various classes of enzymes will be dealt with in detail
in connexion with the chemical substances on which they react.

An excellent demonstration of the fact that a single cell may contain
all the various enzymes connected with the processes of metabolism is
afforded by the unicellular Fungus, Yeast (Saccharomyces), of which many

22 PLANT ENZYMES [ch.

species and varieties are known. The feature of special interest in con-
nexion with the Yeast plant is its power of fermenting hexoses, with the
formation of alcohol and carbon dioxide, the process being carried out
by means of an enzyme, zymase. The complete reaction is generally
represented as follows :

C6Hi206 = 2C02+2C2H50H

though there is little doubt that several stages are involved, including
oxidation, reduction and hydrolysis. It has been known for some time
that phosphates are essential to the action of zymase, and the first stage
is probably the formation of a hexosephosphate with the accompanying
production of ethyl alcohol and carbon dioxide :

2C6Hi2O6-i-2R2HPO4=C6Hi0O4(R2PO4)2-i-2C2H6OH + 2CO2 + 2H2O,

the hexosephosphate being continually decomposed by a hydrolytic en-
zyme, hexosephosphatase, yielding free phosphate again :

C6Hi0O4(R2PO4)2+2H2O = C6Hi2O6 + 2R2HPO4.

In addition to zymase and hexosephosphatase, yeast contains the
enzymes, invertase, protease, peroxidase, catalase, reductase, glycogenase,
carboxylase, a glucoside-splitting enzyme, and some form of diastatic
enzyme. The carboxylase decomposes a large number of aliphatic a-keto-
acids, of which the most important is pyruvic acid CHg • CO * CXDOH.
The reaction, which is also possibly concerned in fermentation, involves
the formation of the corresponding aldehyde with the evolution of carbon
dioxide :

CHa- CO • C00H = CH3- CHO + CO2.

Yeast also stores, as a reserve material, the polysaccharide, glycogen,
which occurs in animal tissues though it is rarely found in plants : this
is hydrolyzed by glycogenase into a monosaccharide. Finally, yeast con-
tains invertase, and most species, in addition, maltase, but from a few
species the latter enzyme is absent. Hence yeasts are able to ferment
the disaccharides, cane-sugar and maltose, since they can first hydrolyze
them to monosaccharides.

As in the case of the enzymes of other tissues, those of yeast can be
made to carry out their functions after the death of the living protoplasm.
The method of demonstrating this is to " kill " the cells by means of
drying at 25-30° C, by treatment with a mixture of alcohol and ether,
or by treatment with acetone and ether. In this way the protoplasm is
destroyed, but the enzymes remain uninjured. Yeast treated thus has
been termed " zymin."

in] PLANT ENZYMES 23

From zymin some of the enzymes, e.g. invertase and the glucoside-
splitting enzyme, can be extracted with w«ater: other enzymes, e.g. zymase
and maltase, are not so readily extracted. From the living cells the
enzymes are only obtained with difficulty, the extraction of yeast juice,
containing zymase and other enzymes, needing, by Buchner's method,
a pressure as great as 500 atmospheres.

In connexion with alcoholic fermentation by zymase, the following
point is of special interest. For carrying out this process, another sub-
stance is necessary in addition to the phosphate and enzymes already
mentioned, i.e. a thermostable co-enzyme of unknown nature. The sepa-
ration of zymase from the co-enzyme has been accomplished by filtering
expressed (Buchner) yeast juice through a special form of gelatine filter
under a pressure of 50 atmospheres,. The phosphate and co-enzyme can
also be removed from zymin by washing with water. The washed residue
is then found to be incapable of fermentation, as also are the washings.
If, however, the boiled washings are added to the washed residue, the
system is synthesized and can now carry out fermentation again. The
chemical nature of the co-enzyme, which is thermostable, and the precise
part played by it in the process, are as yet unknown (Harden, 4).

Expt. 10. Preparation of zymin. Take 50 gms. of bakers yeast and stir it into
300 c.c. of acetone. Continue stirring for 10 minutes, and filter on a filter-pump.
The mass is then mixed with 100 c.c. of acetone for 2 minutes and again filtered. The
residue is roughly powdered, well-kneaded with 25 c.c. of ether for 3 minutes, filtered,
drained and spread on filter-paper for an hour in the air. It can be finally dried at
45° C. for 24 hours.

Expt. 11. Action of zymase, (a) Detection of carbon dioxide. It has been shown
(Harden, 4) that the greater the volume of sugar solution used with a given weight
of zymin, the weaker is its action. To demonstrate its activity, therefore, it is best
to use not more than 5-10 c.c. of 10 7o glucose solution for every 2 gms. of zymin.
Place the mixture in a test-tube and fit it with a cork and glass tubing, the latter
dipping under a solution of lime water in a test-tube. Place the test-tube containing
the zymin and glucose solution in a beaker of water and warm to 35-40° C. Bubbles
of carbon dioxide will be evolved and will produce a precipitate of calcium carbonate
in the lime water. A control experiment should be made using boiled zymin.
(6) Detection of alcohol. Into a small flask put 8 gms. of zymin, 20 c.c. of 10 7o glu-
cose solution and a little toluol. Keep the flask in an incubator at 37-40° C. for
12 hours. Then filter through filter-paper (or linen) into a small distilling flask.
Distil over one half or two-thirds of the original volume. Add to the distillate in a
test-tube, 3-5 c.c. of iodine in potassium iodide solution and then 5 % caustic soda
until the colour vanishes. Shake up and warm gently in a beaker of water to 60° C.
A smell of iodoform will be detected and a yellow crystalline deposit of the same
substance will appear in the tube on cooling and standing. Examine the crystals
under the microscope and note their characteristic star-like shape.

24 PLANT ENZYMES [ch.

Expt. 12. Action of maltase. (Harden and Zilva, 12.) Into each of two small
flasks, put 20 c.c. of a 2 7o solution of maltose and 0*5 gm. of zymin. Boil the
contents of one flask. Then plug both flasks with cotton -wool, add a few drops of
toluol and place in an incubator at 38° C. for 12-24 hours. Filter the liquid from
both flasks and test by making the osazone (see p. 50), using at least 10 c.c. of the
filtrate in each case. Glucosazone will crystallize out from the unboiled, maltosazone
from the boiled, mixture.

Expt. 13. Action of carboxylase. (Harden, 10.) The action of carboxylase on
pyruvic acid is detected by the formation of carbon dioxide and acetaldehyde. Care-
fully prepared zymin will still respire, but, after washing, some constituent essential
to respiration is removed. Hence the zymin must be first washed and tested. Take
5 gms. of zymin and wash well on a filter with distilled water. Then suspend the
zymin in 50 c.c. of water in a flask and draw a slow current of air (previously passed
through two bottles of strong caustic soda and two bottles of saturated baryta
solution) through the suspension of zyniin into a receiving flask of baryta solution.
The flasks should be connected with pressure tubing and the apparatus must be air
tight. Continue to draw the current of air through until it ceases to produce a milki
ness in the receiving flask, due to any carbon dioxide in solution or to residual
respiration. Then add quickly to the suspension of zymin 50 c.c. of 1 % pyruvic acid
(by weight), 5 c.c. of normal caustic potash and 6 gms. of boric acid ; also a few drops
of caprylic alcohol to prevent frothing. Place the flask in a beaker of water at
30-40° C. and again draw a current of air. A copious precipitate of barium carbonate
will be formed in the receiving flask. The boric acid is used to prevent the solution
from becoming too alkaline owing to the formation of potassium carbonate, and,
being a weak acid, it has no inhibiting action on the enzyme.

The contents of the flask containing the zymin are filtered into a small distilling
flask and about 10 c.c. of distillate collected (cooled with ice if possible). To this
add 1-2 c.c. of a freshly made 1 ^Jq solution of sodium nitroprusside, followed by a
few drops of piperidine. A deep blue colour denotes the presence of acetaldehyde.

Expt. 14. Action of peroxidase (Harden and Zilva, 12.) Into four small
evaporating dishes, (a), (6), (c) and (c^), put the following :

(a) A suspension of 0*5 gm. of fresh yeast in 10 c.c. distilled water -I- 1 c.c. of
benzidine solution (1 o^ in 50% alcohol) + 2-3 drops of hydrogen peroxide (20 vols.).

(5) A suspension of 0*5 gm. of zymin in 10 c.c. of distilled water + 1 c.c. of
benzidine solution + 2-3 drops of hydrogen peroxide.

(c) A suspension of 0*5 gm. of washed zymin in 10 c.c. of distilled water+1 c.c.
of benzidine solution + 2-3 drops of hydrogen peroxide. (The zymin is washed by
putting it on a double folded filter-paper in a funnel and adding distilled water from
time to time. 50 c.c. of water should be used for 0*5 gm. of zymin.)

{d) A suspension of 0*5 gm. of washed zymin in 10 c.c. of washings + 1 c.c. of
benzidine solution + 2-3 drops of hydrogen peroxide.

A blue colour will develop in (a) showing that fresh yeast contains a peroxidase
(see p. 124). A blue colour will also develop in (c) but not in {h) and {d). This is
explained by assuming that the zymin contains an inhibitor, not present in fresh
yeast, but which is developed during the preparation of the zymin, and that this
inhibitor can be washed away by water. On adding the washings to the washed zymin
the reaction is inhibited again.

Ill] PLANT ENZYMES - 25

Expt. 15. Action of catalase. (Harden and Zilva, 12.) Completely fill a test-tube
with hydrogen peroxide (20 vols.) solution which has been diluted with an equal
volume of water and add 0*5-1 gm. of zymin. Place the thumb firmly over the mouth
of the tube, invert and place the mouth under water in a small basin, clamping the
tube in position. A rapid evolution of oxygen takes place. When the tube is about
three-fourths full of gas, close the mouth with the thumb while still under water and
remove the tube. Plunge a glowing splint into the gas and it will re-kindle to a flame.

Expt. 16. Action of protease. Weigh out 10 gms. of white flour, and allow it to
extract with 100 c.c. of distilled water for one hour, shaking from time to time. Then
filter on a filter-pump. The extract will contain the albumin, leucosin (see p. 138).
Into small flasks {a) and (6) put the following :

(a) 40 c.c. of the flour extract + 1 gm. of zymin -f 1 c.c. of toluol.

{h) 40 c.c. of flour extract -\- 1 gm. of boiled zymin -j- 1 c.c, of toluol.

Shake both flasks, plug with cotton-wool and place them in an incubator at 38° C.
for 48 hrs. After incubation, boil the liquid in both flasks, in order to coagulate un-
altered protein, and filter. Cool the filtrates from the respective flasks and add
bromine water drop by drop (see p. 153). A pink, or purplish-pink colour, due to the
presence of tryptophane, will be formed in tube (a). Hence hydrolysis of protein has
taken place. Tube (6) will show no colour or only that due to bromine. Add a little
amyl alcohol to both tubes and shake gently. The alcohol will be coloured pink or
purplish in the tube giving the tryptophane reaction.

Expt. 17. Action of reductase. (Harden and Norris, 11.) Take two test-tubes,
{a) and (6), provided with well-fitting corks and put in the following :

{a) 1 gm. of zymin -1- 20 c.c. of distilled water -\- 0-5 c.c. of methylene blue solu-
tion (made by diluting 5 c.c. of a saturated alcoholic solution to 200 c.c. with distilled
water).

(6) 1 gm. of boiled zymin -|- 20 c.c. of distilled water 4- 0*5 c.c. of methylene blue
solution.

Cork both tubes after adding a few drops of toluol and place in an incubator at
38° C. for 1-3 hours. The blue colour will practically disappear from tube {a) but
will remain in tube (6).

The methylene blue is reduced to a colourless leuco-compound which will become
blue again on re-oxidation.

Expt. 18. Enzyme actions of an aqueous extract of zymin. Weigh out 2 gms. of
zymin and place them. on a double folded filter-paper in a funnel and wash with 80 c.c.
of distilled water. With the filtrate make the following experiments.

(A) Action of invertase. (Harden and Zilva, 12.) Into two small flasks (a) and (b)
put the following :

{a) 10 c.c. of a 2 % solution of pure cane-sugar -f- 10 c.c. of the filtrate from zymin.

(6) 10 c.c. of the same solution of cane-sugar -h 10 c.c. of the boiled filtrate from
zymin.

Put both flasks in an incubator at 38° C. After 30 minutes add equal quantities
(about 1-2 c.c.) of Fehling's solution to both flasks and boil (see p. 54). Flask {a)
will show considerable reduction of the Fehling. Flask {h) will show comparatively
little reduction, that which does take place probably being due to the sugar previously
formed by the action of glycogenase on stored glycogen.

26 PLANT ENZYMES [ch. hi

(B) Action of the glucoside-splitting enzyme. (Caldwell and Courtauld, 9 ; Henry
and Auld, 13.) This enzyme will act upon the glucoside, amygdalin, which is present
in bitter almonds, with the production of glucose, benzaldehyde and prussic acid
(see p. 160). Into two small flasks (a) and (6) put the following:

(a) 20 c.c. of a 2 7o solution of amygdalin + 20 c.c. of the filtrate from zymin.

{h) 20 c.c. of the same solution of amygdalin + 20 c.c. of the boiled filtrate from
zymin.

Add a few drops of toluol to both flasks and then cork, inserting, with the cork,
a strip of paper which has been dipped in solutions of picric acid and sodium carbonate
(see p. 161). Put both flasks in an incubator at 38° C. for 12-24 hours. The picrate
paper in flask {a) will have reddened. Add a little Fehling's solution to the liquid in
the same flask and boil. The Fehling will be reduced. The liquid in flask (6) will
only reduce Fehling slightly [see Expt. A (6)] and the picrate paper will not be
reddened.

REFERENCES

Books

1. Abderhalden, B. Biochemisches Handlexikon, v. Berlin, 1911.

2. Bayliss, W. M. The Nature of Enzyme Action. London, 1919. 4th ed.

3. Euler, H. General Chemistry of the Enzymes. Translated by T. H. Pope.
New York and London, 1912.

4. Harden, A. Alcoholic Fermentation. London, 1914. 2nd ed.

5. Vernon, H. M. Intracellular Enzymes. London, 1908.

6. Wohlgemuth, J, Grundriss der Fermentmethoden. Berlin, 1913.

Papers

7. Armstrong, H. B., and Armstrong, B. P. The Origin of Osmotic Effiects.
III. The Function of Hormones in Stimulating Enzymic Change in Relation to
Narcosis and the Phenomena of Degenerative and Regenerative Change in Living
Structures. Proc. R. Sac, 1910, B Vol. 82, pp. 588-602. Ibid. IV. Note on the
Differential Septa in Plants with reference to the Translocation of Nutritive Materials.
Proc. R. Soc, 1912, B Vol. 84, pp. 226-229.

8. Armstrong, H. B., and Armstrong, B. P. The Function of Hormones
in regulating Metabolism. Ann. Bat., 1911, Vol. 25, pp. 507-519.

9. CaldTvell, R. J., and Courtauld, S. L. Studies on Enzyme Action.
IX. The Enzymes of Yeast: Amygdalase. Proc. R. Soc, 1907, B Vol. 79
pp. 350-359.

10. Harden, A. The Enzymes of Washed Zymin and Dried Yeast. I. Car-
boxylase. Biochem. J., 1913, Vol. 7, pp. 214-217. •

11. Harden, A., and Norris, R. V. The Enzymes of Washed Zymin and
Dried Yeast. II. Reductase. Biochem. J., 1914, Vol. 8, pp. 100-106.

12. Harden, A., and Zilva, S. S. The Enzymes of Washed Zymin and Dried
Yeast. III. Peroxydase, Catalase, Invertase and Maltase. Biochem. J., 1914, Vol. 8,
pp. 217-226.

13. Henry, T. A., and Auld, S. J. M. On the Probable Existence of Emulsin
in Yeast. Proc. R. Sac, 1905, B Vol. 76, pp. 568-580.

CHAPTER IV

CHLOROPHYLL

The fact has already been emphasized that the plant synthesizes all the
complex organic substances of which it is built from the simple com-
pounds, carbon dioxide, water and inorganic salts. The initial metabolic
process and the one from which all others have their starting-point is
that of a synthesis of a carbohydrate from carbon dioxide and water.
This synthesis can only be carried out in the light, and only in a green
plant, i.e. a plant containing chlorophyll. Chlorophyll may almost be con-
sidered the chemical substance of primary importance in the organic world,
for upon it depends the life of all plants and animals. Animals depend
for their existence on certain complex amino-acids, some of which they
are unable to synthesize for themselves, and which they derive from
plants. Plants in turn are unable to exist except by virtue of the pro-
perties of chlorophyll.

The property of chlorophyll which is so important is the power it
possesses of absorbing the radiant energy of the sun's rays and converting
it into chemical energy by means of which a carbohydrate is synthesized.
This summarizes the whole process, which, however, can scarcely be very
simple, and probably consists of several reactions at present undifferen-
tiated. If the formula for carbonic acid is compared with that of a simple
carbohydrate such as a tetrose, pentose or hexose, the following relation-
ship is seen :

H2CO3 -*■ (HaCO)^ where ^=4, 5 or 6,

that is, in the synthesis of a carbohydrate a reducing reaction must take
place.

Many hypotheses have been formulated as to the nature of these re-
actions. The one which has most frequently been advanced suggests
that formaldehyde is the first product of the synthesis from carbon dioxide
and water which takes place in the green plant ; that the reaction in-
volves reduction with elimination of oxygen :

H2C03=H2CO-}-02,

and that this product is later condensed to form a hexose,

6H2CO = C6Hi206.

28 CHLOKOPHYLL [ch.

As the concentration of sugar increases in the cell, further condensation
may take place to form starch :

X (C6Hi206) = (C6Hio05)^ + ^ H2O.

The facts in agreement with these views are : first, in most plants a
volume of oxygen is given off approximately equivalent to the volume
of carbon dioxide absorbed; secondly, in some plants starch, in others
sugar, is known to be produced during photosynthesis. The detection
of formaldehyde, either in the plant or in certain systems containing
chlorophyll, as a proof of its formation during photosynthesis, has been
shown to be invalid (see p. 37) (Jorgensen and Kidd, 2).

The value and significance of this reducing reaction is seen when it
is realized that, by oxidation of the carbohydrates synthesized, energy is
produced to supply the needs of the whole metabolism of the plant
(see p. 6).

In the chemical treatment of the subject of carbon assimilation, some
of the chemical properties of chlorophyll will first be considered, and,
later, its behaviour under certain conditons : the chemistry, however, of
the phenomenon itself is as yet unknown.

The following account, as far as it concerns chlorophyll, and the
accompanying experiments are taken from a resume (Jorgensen and
Stiles, 3) of the original work (Willstatter und Stoll, 1) upon which the
entire knowledge of the subject is based.

Chlorophyll.

Our knowledge of the chemistry of chlorophyll has, within recent
years, been set upon a firm experimental basis (Willstatter und Stoll, 1).
The results which have been arrived at may broadly be summarized as
follows :

In all plants examined the chloroplastids contain four pigments, of
which two (termed respectively chlorophylls a and h) are green, and
two are yellow. They occur in about the following proportions in fresh
leaves :

(Chlorophyll a . . . C5gH,2 05N4Mg ... 2 pts per 1000
^^^^"^ ]Chlorophyll6...C55H,oOeN,Mg...f „

Yellow 1^^^^*^^ •••• ^4oH56 i „

(Xanthophyll ... C4oH5«02 .:. J

A point of great interest in connexion with chlorophyll is that it
contains magnesium to the extent of 2'7 7o t>ut no other metal is present.
Chlorophyll a, when isolated, is a blue-black solid giving a green-blue

ly] CHLOROPHYLL 29

solution in the solvents in which it is soluble, i.e. ethyl alcohol, acetone,
chloroform, ether, carbon bisulphide, pyridine and benzene. Chlorophyll
b, when isolated, is a green-black solid giving a pure green solution : it
has much the same solubilities as chlorophyll a. The two chlorophylls,
however, can be separated by their different solubilities in methyl alcohol.
Both can be obtained in microscopic crystals.

Carotin crystallizes in orange-red crystals, and xanthophyll in yellow
crystals.

In the chloroplastids these pigments occur mixed with various colour-
less substances, fats, waxes, and salts of fatty acids.

When chlorophyll is spoken of, it will be understood to refer to the
green pigments and not to the yellow.

The pure pigments, when isolated, are readily soluble in acetone, ether
and benzene. When very thoroughly dried nettle leaves are treated with
pure acetone, no green colour is extracted, but if a few drops of water
are added, the extract becomes green. Also if acetone is poured on to
fresh leaves, the pigment is extracted. The explanation offered for these
phenomena is that chlorophyll is present in a colloidal condition in the
cell. This point will be considered again later (see p. 36).

The Common Nettle ( Urtica) is the plant which has been used for
material for the extraction of chlorophyll on a large scale, and it also
forms very useful material for extraction on a small scale. The pigment
has been found to be unaltered by drying, and, since dried leaves involve
far less bulk and dilution of solvents, material should be dried before
using. Some leaves (Elder and Conifers) are spoilt by drying. From
dried leaves pure solvents, such as petrol ether, benzene and acetone,
extract very little pigment for reasons which will be mentioned later,
but if the solvents contain a moderate amount of water, the pigment is
readily soluble. About 80 7o acetone is the best solvent. The nettle
leaves are removed from the stalks and laid on sheets of paper to dry.
When well air-dried they are finely powdered, and the powder further
dried at 30-40° C. in an incubator. The leaf-powder can be kept for a
considerable time in a well- stoppered bottle.

Expt. 19. Extraction of pigment. Two grams of leaf-powder are sucked to a filter-
paper on a small porcelain funnel and 2-3 c.c. of 85 % acetone are added. This is
allowed to soak into the powder for a few minutes. The fluid is then sucked through
with the pump, the flask disconnected and more acetone added. The operation is
repeated until 20 c.c. of the solvent have been added, when the powder is sucked dry.
A deep blue-green solution with a red fluorescence is obtained which contains all the
four pigments from the leaf. The acetone extract thus obtained is then poured into
double the quantity of petrol ether contained in a separating funnel. An equal

D

30 CHLOROPHYLL [ch.

quantity of distilled water is added, this being poured gently down the side of the
unnel in order to avoid the formation of emulsions. In the course of a few minutes,
the ether layer separates out and now contains the pigments. The lower layer, which
is slightly green, is run off. The addition of distilled water and subsequent removal
of the lower layer is repeated about four times, in order completely to remove the
acetone from the ether solution. If the ether solution should have become at all
emulsified, it can be cleared by shaking with anhydrous sodium sulphate and filtering.
The whole process should be repeated with another 2 gms. of leaf-powder and the
pigment transferred to ether^ since a solution in this solvent is required for later
experiments.
^ i/ Expt. 20. Demonstration of the presence of chlorophylls a and h. Of the petrol

ether solution from the last experiment, 10 c.c. are shaken with 10 c.c. of 92 7o methyl
alcohol. Two layers are formed of which the petrol ether layer contains chlorophyll
a, and the methyl alcohol layer chlorophyll h. The solution of chlorophyll a is blue-
green, while that of chlorophyll 6 is a purer green, but the colour difference between
them is diminished owing to the presence of the yellow pigments, of which carotin
is in the petrol ether, and xanthophyll in the methyl alcohol. Keep the two extracts
for Expt. 24.

As will be explained later, the green pigments of chlorophyll can be
saponified by alkalies and are then insoluble in ethereal solution. This
method can be adopted to separate the green from the yellow pigments,
xanthophyll and carotin.

y Expt. 21. Separation of green and yellow pigments. Shake 5 c.c. of an ether
solution of the pigments (Expt. 19) with 2 c.c. of 30 ^Iq caustic potash in methyl
alcohol (obtained by dissolving 30 gms. of potassium hydroxide in 100 c.c. of methyl
alcohol'}. After the green colour has reappeared, slowly add 10 c.c. of water and
then add a little more ether. On shaking the test-tube, two layers are produced, of
which the lower watery -alkaline one contains the saponified green pigments, while
the carotin and xanthophyll are contained in the upper ethereal layer,

Expt. 22. Separation of the two yellow pigments. The ether layer obtained in
the last experiment is washed with water in a separating funnel, and evaporated
down to 1 c.c. It is then diluted with 10 c.c. of petrol ether and next mixed with
10 c.c. 90 o/o methyl alcohol. The methyl alcoholic layer is removed and the petrol
ether layer is again treated with methyl alcohol and the methyl alcohol again
removed. This process is repeated until the methyl alcohol is no longer coloured.
The methyl alcohol contains the xanthophyll, the petrol ether the carotin.

Further accounts of the yellow pigments are given on p. 40.

The best known reactions of chlorophyll are those which take place
with acids and alkalies respectively.

Chlorophyll is a neutral substance, and, on treatment with alkalies,
it forms salts of acids, the latter being known as chlorophyllins. These
salts are soluble in water forming green solutions which are not however

1 The methyl alcohol must be very pure, otherwise the alcoholic potash solution will
become brown and discoloured.

IV] CHLOROPHYLL 31

fluorescent. Chlorophyll a may be represented as the methyl phytyl
-ester of an acid chlorophyllin (phytol is a primary alcohol, see p. 39):

.COOCH3 COOH

C32H3oON4Mg<f C32H3oON4Mg/

\COOC20H39 ^COOH

Chlorophyll a Chlorophyllin

On treatment in the cold with alkali, the ester is saponified, and the
alkali salt of chlorophyllin is formed. During saponification, there is a
change of colour in the pigment, the so-called brown phase, followed by
a return to green.

Expt. 23. Saponification of a mixture of the green pigments. Pour a little of the
ether solution obtained in Expt. 19 into a test-tube, and in a pipette take a little
30 % solution of potash in methyl alcohol. Place the lower end of the pipette at the
bottom of the test-tube and allow the potash to run in below the chlorophyll solu-
tion. At the interface between the solutions there appears immediately a brown-
coloured layer which diffuses on shaking. In about ten minutes it changes back
through an olive-green colour to pure green.

The chlorophyll has been saponified to the potassium salt of the acid chlorophyl-
lin. This salt is insoluble in ether, so if water is added to bring about a separation
of the two layers, the green colour is no longer present in the ethereal layer.

The change of colour on saponification is different for the two
chlorophylls, the brown phase produced in the above mixture of chloro-
phylls being due to a yellow phase produced by chlorophyll a, and a
brown-red phase produced by chlorophyll h. To demonstrate this the
phase test (Expt. 23) may also be carried out separately on the two
•chlorophylls.

vi Expt. 24. Saponification of chlorophylls a and b separately. The methyl alcohol
solution obtained in Expt. 20 is transferred to ether as in Expt. 19. Both the latter
and the petrol ether solution of chlorophyll a are saponified as in the previous
experiment.

As already demonstrated the potassium salts of the chlorophyllins
which are produced by saponification of the mixture of green pigments
in the cold are not fluorescent. By saponification of chlorophyll with
hot alkali, isochlorophyllins are formed (see Expt. 25 below) which are
fluorescent.

On heating chlorophyllins with concentrated alcoholic alkalies, a series
of decomposition products, phyllins (also acids) are obtained by removal
of carboxyl groups. The final phyllin has only one carboxyl group. When
this is removed, a substance, aetiophyllin, C3iH34N4Mg, is obtained which
contains no oxygen (see Scheme 1, p. 35).

Another difference between the results of treating chlorophyll with
hot and cold alkali is that in the former process the yellow pigments are

32 CHLOROPHYLL [ch.

destroyed. If then water is added after saponification with hot alkali,
and the solution is shaken up with ether, the ether will remain colourless^

When chlorophyll is treated with acids, a different reaction takes
place. The chlorophyll changes in colour to olive-green and loses most
of its fluorescence. The magnesium of the molecule is removed, being
replaced by hydrogen, and the resulting product is termed phaeophytin
(see Scheme 1, p. 35).

From phaeophytin a series of decomposition products have been
obtained, which fall into two groups, the phytochlorins and the phyto-
rhodins. The phytochlorins are olive-green in colour, and are derived
from chlorophyll a\ the phytorhodins are red, and are derived from
chlorophyll h. The phaeophytins from the two chlorophylls are indis-
tinguishable until the above decomposition products are obtained.
(The original discovery of two kinds of chlorophyll was brought about
by the differentiation of these decomposition products.)

A number of phytochlorins and phytorhodins have been identified
and are designated by letters a, h, etc. By employing a uniform method
of treatment, however, two of these products, phytochlorin e and phyto-
rhodin g, can be secured.

The phytochlorins and the phytorhodins are of course magnesium-free
compounds and can be obtained by the action of acid on the chlorophyllins
and isochlorophyllins. Phytochlorin e and phytorhodin g, in particular
are obtained by the action of acid on isochlorophyllins, i.e. they are
magnesium -free isochlorophyllins. They are formed by the addition of
acid to the products of saponification with hot alkali.

The separation of the various phytochlorins and phytorhodins can be
brought about by means of their different distribution between ether and
hydrochloric acid : each compound can be extracted from ether according
to the concentration of the acid used.

Expt. 25. The formation of phytochlorin and phytorhodin. 5 c.c. of an ether
solution containing both chlorophylls a and b are evaporated to dryness in a test-
tube in a water-bath, and the residue treated with 3 c.c. of boiling 30 % potash
solution in methyl alcohol, and boiled gently for half a minute. A liquid with a red
fluorescence is produced which consists of a solution of the potassium salts of
isochlorophyllins. The solution is diluted with double its volume of water, and
concentrated hydrochloric acid is added until the solution is just acid. The liquid is
then shaken with ether in a separating funnel : the dissociation products produced
by the previous treatment pass into the ether solution which thus acquires an
olive-brown colour.

The ether solution is shaken twice, each time with 10 c.c. of 4 % hydrochloric
acid (sp. gr. 1"02 i.e. 12-9 c.c. strong acid: 87*1 c.c. water), and the green-blue acid
layer is separated and neutralized with amomonia and shaken with more ether, which

IV] CHLOROPHYLL 33

then contains in solution phytochlorin e, the derivative of chlorophyll a. The phyto-
chlorin e gives to the ether an olive-green colour.

The ether layer remaining in the funnel, after the separation of the green-blue acid
layer, is now extracted with 10 c.c. of 12 o/q hydrochloric acid (sp. gr. 1-06 i.e. 38"1 c,c,
strong acid: 61*9 c.c. water). The green acid solution so obtained is diluted with
water and shaken with ether which then becomes coloured red and contains phyto-
rhodin ^, the derivative of chlorophyll h.

If the phyllins are acted upon by mineral acids, they lose their
magnesium in the same way as the chlorophyllins, and the series of sub-
stances obtained in this way are termed porphyrins. Thus aetiophyllin
will give aetioporphyrin C31H36N4 (see Scheme 1, p. 35).

The derivatives of chlorophyll which are free from magnesium, such
a.s phaeophytin, phytochlorin phytorhodin, the various porphyrins, etc.
combine readily with the acetates of some metals such as copper, zinc
and iron, and they form intensely coloured, stable compounds. The change
of colour is so noticeable that the smallest traces of certain metals can
be detected in this way. Hence it is very difficult to prepare the
magnesium-free chlorophyll unless the reagents are perfectly pure and
all contact with certain metals is avoided.

Ex'pt. 26. Substitution of copper for magnesium in chlorophyll. 2 c.c. of an ether
solution of chlorophyll are shaken with a little 20% hydrochloric acid (sp. gr. 1-10
i.e. 62*4 c.c. strong acid : 37*6 c.c. water), and then washed with water in a separating
funnel. If the ether tends to evaporate and deposit phaeophytin in the funnel^
a little more ether should be added. In this way is produced in ether solution the
magnesium-free chlorophyll derivative, phaeophytin. The solution is evaporated
down on a water-bath, and the residue dissolved in 5 c.c. of alcohol. The solution is.
olive-green in colour. This is heated and a grain of copper acetate or zinc acetate is-
added. The colour changes back to a brilliant green, but without fluorescence (if all
the chlorophyll has been converted into phaeophytin).

From the results of these recent investigations, it is now possible to
write formulae for the two chlorophylls as follows:

chlorophyll a (C32H30O N4Mg) (COOCH3) (COOC20H39)
chlorophyll h (C32H2802N4Mg) (COOCH3) (COOC20H39)

from which it will be seen that the phytol component amounts to one-
third of the weight of the chlorophyll. The structural formula for
chlorophyll is not completely known, but there is evidence that it contains
four pyrrole rings (cp. the pyrrolidine alkaloids, p. 175).

From the analyses of chlorophylls from different plants, it was found
that the phytol content varied, and plants which yielded little phytol
most readily produced "crystalline chlorophyll," a form of the pigment
which has been known for some considerable time to previous worker's^
The Cow Parsnip (Heracleum Sphondylium), Hedge Woundwort {Stachys

34 CHLOROPHYLL [ch.

sylvatica) and Hemp-nettle (Galeopsis Tetrahit) are plants which readily
give crystalline chlorophyll. In this connexion it has been suggested
that the chlorophyll in plants is accompanied by an enzyme, chlorophyl-
lase, which, in alcoholic media, brings about alcoholysis of the chlorophyll,
and replaces the phytyl by the ethyl radicle. The products, formerly
known as crystalline chlorophyll, are now termed chlorophyllides :

(C32H3oON4Mg) (COOCH3) (COOC2oH39) + C2H50H

= C2oH390H+(C32H3oON4Mg) (COOCH3) (COOC2H5).

Phytol Ethyl chlorophyllide

Similar chlorophyllides are produced by other alchohols. In aquedus
solutions chlorophyllase brings about hydrolysis and the free acid
chlorophyllide is formed (see Scheme 2, p. 35):

(C32H3oON4Mg) (COOCH3) (COOC2oH39) + H20

= C2oH390H+(C32H3oON4Mg)(GOOCH3)(COOH).

Chlorophyllide

Chlorophyllase is a very stable enzyme; it is not even destroyed by
boiling in alcohol for a short time, but if leaves are boiled in water, the
enzyme is destroyed.

Expt. 27. Microscopic examination of ethyl chlorophyllide. Prepare sections of
fresh Heracleum leaves and mount them in a drop of 90 % alcohol. Leave the slide
under a bell-jar containing a dish of alcohol. The section slowly dries in the course
of half a day or a day. It is then examined under the microscope when there will be
observed the characteristic triangular and hexagonal crystals of ethyl chlorophyllide
(crystalline chlorophyll).

Expt. 28. Production of methyl chlorophyllide in the leaf Sections may be used
as in the preceding experiment, or a piece of a leaf may be employed. In the latter
case a test-tube with 4 c.c. of 75 % methyl alcohol is taken and 1 gm. of fresh leaf
is added to it. The leaf first becomes a darker green and then during the course of
a few hours becomes yellowish. On holding the leaf to the light there can be
observed with the naked eye a number of black points. If sections of the leaf be cut
and examined under the microscope, these spots appear as aggregates composed of
rhombohedral crystals, occurring in certain cells.

Expt. 29. Extraction of ethyl chlorophyllide. Two grams of dry Heracleum leaf-
powder are left for a day in a test-tube containing 6 c.c. of 90 % alcohol. The extract
is then filtered through a small porcelain funnel and the powder on the filter washed
with a little acetone. The filtrate is mixed with an equal quantity of ether, and then
with some water. The ether solution is transferred to a separating funnel and
thoroughly washed wdth water, and then concentrated on a water-bath to | or 1 c.c,
and 3 c.c. of petrol ether are added. On standing, the ethyl chlorophyllide is pre-
cipitated in the form of crystalline aggregates. It is freed from yellow pigments by
shaking with a little ether, and can be further purified by redissolving in ether and
precipitating again with petrol ether.

IV]

CHLOROPHYLL

35

Expt. 30. The action of chlorophyllase. Fresh leaves of a species rich in chloro-
phyllase, e.g. Heracleum or Galeopsis^ are finely divided and put in a 70 % acetone
solution, 3 c.c. of solution being used for every gram of leaf. The chlorophyll, by
means of the chlorophyllase, is hydrolyzed into phytol and the acid chlorophyllide.
This can be demonstrated after about a quarter of an hour if the solution is diluted
with water, transferred to ether and shaken with 0'05 % sodium hydroxide. The
sodium hydroxide takes up more colouring matter the further the enzyme action
has progressed.

Expt 31. The destruction of chlorophyllase. If fresh leaves of a species rich in
chlorophyllase are first steeped in boiling water for a few minutes before they are
placed in the acetone solution, unaltered chlorophyll is extracted which does not
react with dilute alkali.

With acids — >■

(C32H3oON4Mg) (COOCH3) (COOC20H39) J. (C32H32ON4) (COOCH3) (COOC20H39)

chlorophyll a

phaeophytin

(C32H3oON4Mg) (COOH) (COOH)

chlorophyllin a
and isochlorophyllin a

interme

diate phyllins

C3iH34N4Mg

aetiophyllin

;C32H320N4) (COOH) (COOH)

phytochlorin e

and phytochlorins/and g

intermediate porphyrins

Scheme 1.

— ^ C31H36N4
aetioporphyrin

chlorophyll a
<MgN4C32H3oO)(COOCH3)(COOC2oH39)

with

meythl chlorophyllide a

<MgN4C32H3oO) (COOCH3) (COOCH3)

chlorophyllide a
(MgN4C32H3oO) (COOCH3) (COOH)

dilute acid

with

dilute acid

phaeophytin a
(N4C32H32O) (COOCH3) (COOC2

with

dilute acid
Scheme 2.

1H39)

03

methyl phaeophorbide a
(N4C32H32O) (COOCH3) (COOCH3)

§

phaeophorbide a
(N4C32H32O) (COOCH3) (COOH)

By treatment with acids, magnesium is removed from the chlorophyl-
lides, with the production of the corresponding phaeophorbides. Thus
methyl chlorophyllide a gives methyl phaeophorbide a, etc. (see Scheme 2,
above).

3—2

36 CHLOROPHYLL [ch.

It has been previously mentioned that water-free solvents, such as
acetone, ether and benzene, in which pure extracted chlorophyll is
soluble, will not extract the pigment from thoroughly dried leaves, but
if a little water is added, it readily goes into solution. From fresh leaves
also these solvents can extract the pigments.

As an explanation of the above phenomena, it has been suggested
that chlorophyll in the chloroplastid is in the colloidal state, and that^
when water is added to the dried leaf, a solution of mineral salts in the
leaf is formed which alters the colloidal condition of the chlorophyll and
makes it soluble. This view is supported by the fact that if a colloidal
solution of chlorophyll in water, made from the pure extracted pigment,
is shaken with ether, the ether remains colourless. If, however, a little
salt solution is added and the mixture shaken, the ethereal layer becomes
green. In preparing the colloidal solution the solvent, acetone, is replaced -•
by the medium, water, in which chlorophyll is insoluble.

The condition of chlorophyll is altered by plunging the leaves into
boiling water. The pigment is then much more readily soluble in ether,
etc., even when the leaves are subsequently dried. It is supposed that
the chlorophyll has diffused out from the plastids, and is in true solution
in accompanying waxy substances which have become liquid owing to
change of temperature.

A-j -^ Expt. 32. Preparation of a colloidal solution of chlorophyll. Take 10 c.c. of an

acetone extract of chlorophyll (Expt. 19) and pour this acetone solution into a large
volume of distilled water (100 c.c), the liquid being continually stirred. This opera-
tion can be most conveniently done by taking the acetone solution in a pipette and
allowing it to run out of the pipette while the latter is used as a stirring rod in the
water. Note the change in colour to a purer green, and the disappearance of
fluorescence.

^ Expt. 33. To demonstrate the difference between a true and a colloidal solution of
chlorophyll. Evaporate 10 c.c. of an acetone extract (Expt. 19) to complete dryness
and test its solubility in ether, petrol ether and benzene. It is soluble in all three
solvents. Now add these solvents to some of the colloidal solution prepared in the
last experiment, and note that the chlorophyll does not dissolve in any of these
solvents. If, however, some salt solution, e.g. a little magnesium sulphate, be added,
the chlorophyll is precipitated from its colloidal state and is now soluble in ether
and other solvents.

-*■ Expt. 34. To show that chlorophyll in the plant is probably in the colloidal

condition. Some nettle powder is carefully dried, e.g. by keeping it at 30-40° C. in an
oven, and then further drying in a vacuum desiccator over sulphuric acid. Small
quantities of this dry powder are put in test-tubes, and different pure water-free
substances such as acetone, ether, benzene and absolute alcohol are added. Note
that these solvents are not coloured by the chlorophyll. It can be demonstrated that

IV] CHLOROPHYLL 37

the extracted pigment is easily soluble in any of these substances. Repeat the experi-
ment with nettle powder moistened with a few drops of water, and note that the
solvents are immediately coloured.

Expt. 35. Pure solvents are able to extract chlorophyll from fresh leaves. Crush
10 gms. of fresh leaves of nettle, horse-chestnut or elder in a mortar with some clean
sand, and put the crushed material on a filter-paper in a porcelain funnel. Add
20 c.c. of pure acetone and suck it through by means of a water-pump. Repeat this
several times. The pure solvent is here able to extract the pigment.

Expt. 36. Treatment of fresh leaves with boiling water changes the condition of
the chlorophyll. Dry a quantity of leaves which have been put in boiling water and
examine their solubility as in Expt. 34. Note that the chlorophyll in this powder is
soluble in pure solvents.

There is finally another change which chlorophyll can undergo, namely
that of allomerization, which takes place in alcoholic solution. The
characteristic of allomerized chlorophyll is that it does not give the brown
phase when treated with alkali (see Expt. 23). Allomerization is accelerated
in alkaline solution but inhibited by small quantities of acid.

Expt. 37. To demonstrate that allomerized chlorophyll does not give the brown
phase test. Dissolve a little crude chlorophyll, obtained by evaporating an ether
solution, in absolute alcohol. To a sample of this add a little alkali, and perform the
phase test, from time to time, till at last the brown phase no longer appears.

Connexion of Chlorophyll with Formaldehyde.

In addition to the above, another chemical property of chlorophyll
of great interest, is that connected with the production of formaldehyde.
Those investigators, who have sought to confirm the formaldehyde
hypothesis of carbon assimilation, have based their evidence on tests for
formaldehyde both in the plant and in chlorophyll-containing systems
outside the plant. By exposing films, or solutions, of chlorophyll to light
in presence of carbon dioxide, they have detected formaldehyde as a
result (Usher and Priestley, 5).

The most recent investigations (Jdrgensen and Kidd, 2) have shown
that the experimental evidence is at present inadequate to support the
hypothesis, since formaldehyde arises from chlorophyll itself in the absence
of carbon dioxide.

In this later work (Jorgensen and Kidd, 2) on the behaviour of ex-
tracted chlorophyll in light, use has been made of a colloidal solution (see
p. 36) of pure chlorophyll (chlorophylls a and h) for experimental work.
The solution for this purpose must be prepared from pure chlorophyll,
which has been tested and shown to be free from yellow pigments, since

38 CHLOKOPHYLL [ch.

the latter absorb oxygen and may confuse the issue of the experiment.
The pure chlorophyll is prepared by extracting dried nettle leaves with
80-85 7o acetone in the usual way and transferring to petrol ether (p. 29).
The petrol ether extract is then washed with 80 % acetone to remove
colourless impurities, and with 80 7o methyl alcohol to remove xantho-
phyll. Finally all traces of acetone and methyl alcohol are removed by
washing with water. This renders the chlorophyll insoluble in petrol
ether, since it is only soluble in this solvent if traces of other solvents
are present. Hence the pigment is precipitated out as a fine suspension,
leaving the carotin in solution. The chlorophyll is filtered off through
powdered talc, taken up in ether, reprecipitated by petrol ether and
finally obtained as a blue-black micro-crystalline substance. The colloidal
solution or sol is made by dissolving 0*4 gm. of pure chlorophyll in 3 c.c.
of absolute alcohol and pouring into 300 c.c. of distilled water.

The advantage of using such a solution is that the experimental
conditions, in all probability, approach more nearly to the conditions in
the plant, and reactions with other substances take place more readily
than when the chlorophyll is used as a film. The use of pure, instead of
crude, chlorophyll is also important as by this means it is possible to
determine the changes taking place in chlorophyll itself without complica-
tions arising from the accompanying impurities. The discordant results
of various workers on this subject are doubtless due to the employment
of crude chlorophyll. Ethyl alcohol is the best solvent for preparing the
sol since it does not produce formaldehyde when exposed to light under
ordinary circumstances in glass vessels. Methyl alcohol and acetone
should be avoided as they themselves either contain or give rise to
formaldehyde.

The chlorophyll sol is electro-negative. It is stablized by weak
alkalies, but precipitated by weak acids.

Working with such a colloidal solution the results may be summarized
as follows.

When a chlorophyll sol is exposed to light in an atmosphere of
nitrogen in a sealed tube, no apparent change takes place in the chloro-
phyll, and no formaldehyde is produced.

When exposed in an atmosphere of carbon dioxide in a sealed tube,
the chlorophyll rapidly turns yellow- or brown-green. In the case of
sols of high concentration, the colour-change is preceded by precipitation
of the pigment. The same change takes place in the dark, only more
slowly. No formaldehyde is produced, and no absorption of carbon
dioxide could be detected. The yellow product has been shown to be the

IV] CHLOROPHYLL 39

magnesium-free derivative, phaeophytin, which is produced from the
pigment by the action of acids. The changes observed are explained by
the fact that the carbon dioxide, acting as a weak acid, first precipitates
the sol, if concentrated, and then acts, like other weak acids, on the
chlorophyll, producing phaeophytin. If the solution is kept neutral by
addition of sodium bicarbonate, there is no colour change. The identity
of phaeophytin was shown by the spectrum and by the restoration of
colour on adding a trace of copper acetate.

When exposed to light, and the atmosphere in the sealed tube is
replaced by oxygen or air, the chlorophyll turns yellow- or brown-green
as before and then bleaches. The change of colour from green to yellow
or brown is again due to the formation of phaeophytin, this being brought
about by the presence of an acid substance, which is produced during
bleaching, and increases throughout the process. Formaldehyde can be
detected in a very slight amount during bleaching, but is formed in
much greater quantity after bleaching is complete.

It is suggested that the formaldehyde is produced by the oxidation
and breaking down of the phytol component of the chlorophyll:

CH3— CH— CH— CH— CH— CH— CH— CH— C = C— CHgOH

I I i I I I I I- I

CH3 CH3 CH3 CH3 CH3 CH3 CH3 GH3 CH3

There is no reason for ascribing to any of the above reactions any
part in carbon assimilation. There is at present no hypothesis, supported
by satisfactory evidence, as to the process of carbon assimilation.

Expt. 38. Detection of formaldehyde as a product of oxidation of chlorophyll.
Extract 2 gms. of dried nettle leaf powder with 20 c.c. of 80 % acetone and transfer
it to petrol ether as in Expt. 19. Then shake the petrol ether extract four or five
times with an equal volume of 80 % acetone to remove colourless impurities. Next
the petrol ether extract is similarly shaken up with 80 % methyl alcohol which
removes the xanthophyll. This should be repeated until the methyl alcohol is
colourless. The petrol ether is finally washed repeatedly with water to remove traces
of acetone and methyl alcohol. The chlorophyll is in time precipitated as a fine
suspension, being insoluble in pure petrol ether. This suspension is filtered through
either kieselguhr or powdered talc on a small porcelain filter. The chlorophyll is
extracted from the powder on the filter with as small a quantity as possible of
absolute alcohol. This alcoholic solution is then poured, with constant stirring, into
100 c.c. of distilled water by which means a colloidal solution of chlorophyll is
obtained.

The test to be employed for formaldehyde is as follows (Schryver, 4). To 10 c.c.
of the liquid to be tested add 2 c.c. of a 1 o/o solution (freshly made) of phenylhydrazine
hydrochloride, 1 c.c. of a 5 % solution (freshly made) of potassium ferricyanide and
5 c.c. of concentrated hydrochloric acid. If formaldehyde is present a pink to magenta
colour is developed, either deep or pale, according to the quantity of formaldehyde.

40 CHLOROPHYLL [ch.

The reaction is due to the formation of a condensation product of formaldehyde and
phenylhydrazine, and this compound, on oxidation, yields a weak base forming a
coloured salt with concentrated hydrochloric acid. The salt is readily dissociated
again on dilution of the solution.

Two modifications (Schryver, 4) can be adopted in applying this test. First, in
testing for formaldehyde in pigmented solutions, the following course can be pursued.
The reaction mixture, after addition of phenylhydrazine, ferricyanide and hydro-
chloric acid, is diluted with water, and ether is added in a separating funnel. The
hydrochloride of the chromatogenic base is dissociated and the base is taken up by
the ether. The aqueous solution is run off, and on addition of strong hydrochloric
acid to the ether, the base passes into the acid as a coloured hydrochloride again.
By using a small quantity of acid, the sensitiveness of the test is increased, since the
colour is now distributed through a small quantity of liquid only.

The second modification consists in warming the solution to be tested for a short
time with the phenylhydrazine hydrochloride before adding the other reagents.
In this way, formaldehyde can also be detected if it should be in a polymerized
form.

As a control, 10 c.c. of the colloidal solution of chlorophyll should be tested,
vising both the above modifications. The remainder of the solution should be
exposed to simlight (or the light from either an arc or mercury vapour lamp) in a
loosely corked vessel, until it is completely bleached. The bleached solution, on
testing, will be found to give a positive test for formaldehyde.

The Yellow Plastid Pigments.

These have already been mentioned in connexion with the leaf
pigments (pp. 29 and 30). In addition, however, they have a further
significance in that they constitute the pigments, located in plastids,
of most yellow and orange flowers and fruits. Sometimes also they occur
in other organs, i.e. root of Carrot (carotin).

Carotin, C4oH5«, is an unsaturated hydrocarbon. It crystallizes in
lustrous rhombohedra which are orange-red by transmitted and blue by
reflected light. It is readily soluble in chloroform, benzene and carbon
bisulphide, but with difficulty in petrol ether and ether.

One of its most characteristic properties is that it readily undergoes
oxidation in air, and becomes bleached. With concentrated sulphuric
acid it gives a deep blue colour.

Xanthophyll, C40H56O2, also forms yellow crystals with a blue lustre.
It is soluble in chloroform and ether, but insoluble in petrol ether. It is
more soluble than carotin in methyl alcohol. It gives a blue colour with
sulphuric acid, and also oxidizes in air with bleaching.

IV] CHLOROPHYLL 41

The separation of the two pigments (see Expt. 22) is based on the
fact that in a mixture of petrol ether and methyl alcohol containing a
little water, the carotin passes entirely into the petrol ether, whereas the
greater part of the xanthophyll remains in the methyl alcohol layer.

REFERENCES

Books

1. Willstatter, R., und StoU, A. Untersuchungen liber Chlorophyll.
Methoden und Ergebnisse. Berlin, 1913.

Papers

2. Jorgensen, I., and Kidd, P. Some Photochemical Experiments with Pure
Ohlorophyll and their Bearing on Theories of Carbon Assimilation Proc. R. Soc,
1917, B Vol. 89, pp. 342-361.

3. Jorgensen, I., and Stiles, W. Carbon Assimilation. A Review of Recent
Work on the Pigments of the Green Leaf and the Processes connected with them.
New Phytologist, Reprint, No. 10. London, 1917.

4. Schryver, S. B. The Photochemical Formation of Formaldehyde in Green
Plants. Proc. R. Soc, 1910, B Vol. 82, pp. 226-232.

5. Usher, P. L., and Priestley, J. H. A Study of the Mechanism of Carbon
Assimilation in Green Plants. I. Proc. R. Soc, 1906, B Vol. 77, pp. 369-376. II.
Ibid. 1906, B Vol. 78, pp. 318-327. III. Ibid. 1912, B Vol. 84, pp. 101-112.

CHAPTER V

CARBOHYDRATES

The carbohydrates which occur in plants may be classified as follows :

Pentoses, C5H10O5 — Arabinose, xylose.
Methyl pentoses, C5H9O5 * CH3 — Rhamnose,.

isorhamnose.
Hexoses, CeHigO^ — Glucose, galactose,

mannose, laevulose.

Disaccharides (Sucrose, maltose, CiaHgaOn .

Trisaccharides (Raffinose and others.

Tetrasaccharides (Stachyose.

Monosaccharides .

Polysaccharides

Pentosans, (C6H804)n — Araban, xylan.
Starches, (CeHioOs),^ — Starch, dextrin, inulin.
Mannans, galactans, gums, mucilages,

pectic substances.
Celluloses, (C6Hio05)n.

The carbohydrates are widely distributed in plants and form most
important parts of their structure. Those most commonly found are :
cellulose, starch, pentosans, dextrin, glucose, sucrose, laevulose, and
maltose. Other sugars, especially trisaccharides, are known in addition
to those mentioned above, but they are somewhat restricted and specific
in their distribution.

As in the case of the proteins, so with the carbohydrates, the molecules
of the more simple and soluble crystalline compounds, such as the mono-
saccharides, are synthesized into more complex molecules which exist,
either in the colloidal (dextrin), or insoluble state (starch, cellulose).
The last-mentioned build up parts of the solid structure of the plant.
The resolution of the solid complex substances into simple ones is known
in many instances to be brought about in the plant by enzymes, and it
is highly probable that the synthesis of the complex from the simple is
also controlled by these enzymes.

CH. V] CARBOHYDRATES 43

The most commonly occurring sugars in plants are glucose, laevulose
sucrose and maltose : sucrose is hydrolyzed by the enzyme, invertase,
into one molecule of glucose and one molecule of laevulose : maltose by
the enzyme, maltase, into two molecules of glucose. Both invertase and
maltase are widely distributed. The connexion between various sugars
and photosynthesis, and their inter-relationships with each other in the
leaves, are reserved for another section.

Of the polysaccharides, cellulose is universally distributed in higher
plants and constitutes the greater part of the cell-walls. The pentosans,
galactans and mannans also, but to a lesser degree, are components of
their structure. Starch, in addition, is very widely distributed: it is
converted by the enzyme, diastase, into dextrin and maltose, and possibly
the same enzyme also controls its synthesis. In some plants no starch
is formed, and its place in metabolism is taken by inulin or cane-
sugar.

The various carbohydrates will first be dealt with in detail, and later
their inter-relationships will be considered.

Monosaccharides.

These are termed tetroses, pentoses or hexoses according to the number
of carbon atoms in the molecule. They contain primary (— CH2OH) or
secondary (= CHOH) alcohol groups, and either an aldehyde (- CHO)
group, as in glucose, or a ketone (= C = 0) group, as in laevulose. They
are, as a class, white crystalline substances, soluble in water and aqueous
alcohol, but insoluble in ether, acetone and many other organic solvents.
They are capable of certain characteristic chemical reactions which form
a basis for their detection and estimation. One of the most important
is that connected with the aldehyde and ketone groups, owing to which
they act as reducing agents, being themselves oxidized. The reducing
action usually employed is that which takes place with copper salts in
hot alkaline solution, whereby cuprous oxide is formed. Hence they are
termed " reducing " sugars. Another important reaction is the formation
of crystalline osazones (only in the case of sugars with aldehyde or
ketone groups), which, by virtue of their melting points and charac-
teristic crystalline forms, constitute, in several cases, valuable tests for the
presence of sugars.

A reaction exhibited by many of the monosaccharides is that of
forming a coloured product when heated with a phenol in presence of a

44 CARBOHYDEATES [ch.

strong acid. The reaction is due to the formation of a furfural compound
(see p. 46), and the colour depends on the particular sugar and phenol
used. Thus, with strong hydrochloric acid and orcinol, the colour is
violet-blue for pentoses and orange-red for hexoses; with the same acid
and phloroglucinol, the colour is red in both cases ; with a-naphthol and
strong sulphuric acid, the colour is purple in all cases. A variation of
this reaction provides a distinction between a ketone and an aldehyde
sugar. Thus, if hydrochloric acid diluted with its own volume of water
is used, a red colour is produced with resorcinol and a ketone sugar,
e.g. laevulose (Seliwanoff 's reaction). With an aldehyde sugar, e.g. glucose,
the colour is produced only by using concentrated acid.

Pentoses, Methyl Pentoses.

The pentoses contain five carbon atoms, and have the general formula
C5H10O5. They are said to be present in the free state to some extent in
leaves (Davis and Sawyer, 12). In plants they occur chiefly, however, as
condensation products formed with elimination of water. These products
are termed the pentosans, and are widely distributed ; on hydrolysis they
yield pentoses again. The various gums found in plants consist largely of
pentosans, and the pectins also contain pentose groups; both consequently
yield pentoses on hydrolysis (see pp. 63 and QQ). A pentose is also a
component of plant nucleic acid (see p. 141). It has recently been shown
(Spoehr, 33) that the metabolism of some succulent plants (Cactaceae)
is especially favourable to the production of pentoses. By condensation,
pentosan-mucilage is formed and this has the water-retaining properties
characteristic of succulents.

If we examine the structural formula of a pentose, as for example
arabinose :

H— C = O

I
HO— C*--H

H— C*— OH

I

H— C*— OH

I
H— C— H

I
OH

we see that each of the three carbon atoms marked * is united to four
different atoms or groups of atoms. Each of these carbon atoms is there-
fore asymmetric, and, with regard to it, there are two possible isomers

V]

CAKBOHYDRATES

45

(see p. 10, Cole, 5, for stereoisomerism). It will be found on examination
that there are eight possible isomers of the formulae given above :

CHO

I
HO— C— H

I
HO— C— H

I
HO— 0— H

I
CH2OH

^-Ribose

CHO

I
H—C— OH

HO— C— H

i
-0— H

HO-

CH2OH
^-Arabinose

CHO

i
H—C— OH

I
H—C— OH

i
H—C— OH

I
CH2OH

c?-Ribose

CHO

I
HO— C— H

I
H_C— OH

H—C— OH

I
CH2OH

c?-Arabinose

CHO

I
HO— C

H

H—C— OH

i
HO— C— H

I
CH2OH

^Xylose

CHO

I
H_C— OH

I
H—C— OH

HO

C— H

I
CH2OH

^-Lyxose
unknown

CHO

I
H—C— OH

I
HO— C— H

I
H—C— OH

I
CH2OH

o?-Xylose

CHO

I
HO— C— H

I
HO— C— H

H—C- OH

I
CH2OH

c?-Lyxose

Of these only seven have been isolated. The pentoses which occur
in plants are ^arabinose, c?-xylose^ and (Z-ribose. The two former, how-
ever, are known almost solely as condensation products, pentosans, in
gums, woody tissue, etc.; the latter only as a component of nucleic acid.
The pentoses form osazones (see p. 51 for reactions and composition).

Arabinose. This sugar occurs as the pentosan, araban, in various
gums, such as Cherry Gum, Gum Arabic, etc. (see p. 46).

Some of the properties and reactions of the pentoses are demonstrated
in the following experiments.

Expt. 39. Tests for arabinose. For reactions a-e use a 1 ^/o solution of arabinose :
for reaction / a 0*2 % solution.

If pure arabinose is not available, a solution for tests a, h and c can be prepared
from gum arable. Boil 5 gms. of the gum in 100 c.c. of water with 10 c.c. of con-
centrated hydrochloric acid for 5 minutes and then neutralize to litmus with alkali.
Such a solution is only suitable for the specific tests for arabinose, since it also
contains galactose (see p. 63). For tests a, 6 and c small pieces of solid gum arable
may even be used.

{a) Heat a few c.c. of the sugar solution in a test-tube with about half its volume
of concentrated hydrochloric acid. In the mouth of the test-tube place a piece of
filter-paper soaked with aniline acetate (made by mixing equal quantities of aniline,
water and glacial acetic acid). A pink colour will be produced in the paper. This is

1 Known formerly as Z-xylose.

46 CARBOHYDRATES [ch.

due to the fact that furfural is formed by the action of the acid on the pentose, and
the furfural then gives a red colour with aniline acetate solution :

JOH Hi

'1 1" CH = CH

CH-CH-iOHi ouo - I \o

CH— C<; '' CH=C

.! l>^=o \c=o

iOH >Hi I 1

Arabinose Furfural

This reaction, however, is also given by the hexoses but to a much less extent.

(6) Warm a few c.c. of the sugar solution with an equal volume of concentrated
hydrochloric acid in a test-tube, and add a small quantity of phloroglucinol. A bright
red coloration is produced.

(c) To a few c.c. of the sugar solution in a test-tube add an equal quantity of
concentrated hydrochloric acid, and then a little solid orcinol. Divide the solution
into two equal portions. Heat one portion. The solution becomes bluish changing
to reddish -violet and finally deposits a blue precipitate. To the other portion, after
heating for a time, add a few drops of 10% ferric chloride solution. A deep green
colour is at once produced. On the addition of a little amyl alcohol, the green
colour will be extracted by the alcohol.

(d) a-Naphthol reaction. Add to a little of the sugar solution a few drops of a
1 % solution of Q-naphthol in alcohol. Mix the two solutions and then run in about
5 c.c. of concentrated sulphuric acid down the side of the test-tube. A violet colora-
tion is produced at the junction of the two liquids. The coloration is due to a
condensation product of a-naphthol with furfural, the latter being formed by the
action of the acid on the carbohydrate. This reaction is likewise given by laevulose
and cane-sugar (since it yields laevulose, see p. 54), and less strongly by glucose and
maltose ; also by some proteins which contain a carbohydrate group.

(e) Boil a little of the arabinose solution with a few drops of Fehling's solution.
Keduction will take place.

(/) Make the osazone of arabinose following the instructions given for glucosazone
(see p. 50).

A solution of arabinose which will give the pentose reactions can also
be obtained by hydrolysis of Cherry Gum. The gum oozes from the bark
of various species of Frunus, such as the Cherry (Prunus Cerasus) and
the Bird Cherry (P. Padus),

Expt. 40. Preparation of arabinose solution from Cherry Oum. The gum is heated,
on a water-bath in a round-bottomed flask fitted with an air condenser \ with
dilute sulphuric acid (1 pt. by wt. of gum: 7 pts. by wt. of 4% sulphuric acid) for
about 5 hours. The solution is then neutralized with calcium carbonate and filtered.
Perform the tests a, h and c of Expt. 39 on the solution. A positive result is obtained
in each case. Since the solution contains other sugars as inpurities, it cannot con-

i.e. a wide piece of glass tubing about 3 ft. long passing through the cork.

V] CARBOHYDRATES 47

■clusively be used for tests d, e and /. If a considerable quantity of gum is available,
crystallization of arabinose should be attempted by concentrating the aqueous sugar
solution, extracting this with 90 ^/q alcohol and again concentrating in a desiccator
(see p. 55). If a very small quantity of gum only is available, the tests a, h and c
should be performed directly on a small piece of the gum in a test-tube.

A purer preparation of arabinose, which may be used for all the
tests of Expt. 39, can be obtained by the hydrolysis of araban (see
Expt. 48).

Xylose. This sugar occurs very widely distributed in woody tissue
a,s the pentosan, xylan (see p. 56). A solution of xylose which will give
the pentose reactions can be obtained from the hydrolysis of straw, or
the presence of xylan giving the pentose reactions can be directly de-
monstrated in straw, bran or sawdust (see Expt. 49).

A purer solution of xylose can be obtained from the hydrolysis of
xylan (see Expt. 51).

When xylose is oxidized with bromine, it yields xylonic acid which
has a characteristic cadmium salt. The formation of this salt is used as
a, method for identifying the sugar (see Expt. 51).

The methyl pentoses are pentoses in which one of the hydrogen
a-toms of the CH2OH group is replaced by the methyl group, CHg.

Rhamnose, C5H9O5CH3, occurs as the constituent of many glucosides
<see pp. 113, 159).

Hexoses.
If we examine the structural formula for a hexose, such as glucose :

H~C = 0
H— C*— OH
OH— C*— H
H— C*— OH

H— C»— OH

I
H— C_H

I
OH

we see that there are four carbon atoms marked * which are united to

48

CAKBOHYDRATES

[CH.

four different groups of atoms. It will be found in this case that there
are sixteen possible isomers, as against eight for pentose :

CHO

H_C— OH

I
H— C -OH

HO— 0— H

I
HO— 0— H

I
CH2OH

^-Mannose

CHO

I
H— C— OH

HO— C— H

I
H_C— OH

I
HO— C— H

I
CH2OH

Mdose

CHO

I
HO— C— H

I
H_C— OH

-OH

HO— C— H

I
CH2OH

Z-Galactose

CHO

I
HO— C— H

I
-C— H

I
HO— C— H

HO

HO— C— H

1
CH2OH

^-Allose
unknown

CHO

I
HO— C— H

I
HO— C— H

i
H_C— OH

H_C— OH

I
CH2OH

</-Mannose

CHO

I
HO— C— H

H-

.C— OH

I
HO— C-^H

H-

-OH

CH2OH
c?-Idosei

CHO

I
H_C— OH

I
HO— C— H

i

HO— C— H

I
H_C— OH

I
CH2OH

G?-Galactose

CHO

I
H_C— OH

!

H— C— OH

1
H_C— OH

I
H—C— OH

I
CH2OH

o?-Allose

CHO

I
HO— C— H

I
H_C— OH

I
HO— C— H

HO— C— H

I
CH2OH

^-Glucose

CHO

I
HO— C— H

I
HO— C— H

I
H—C— OH

HO— C— H

I
CH2OH

^-Gulose

CHO

H_C— OH

H-C— OH

■ I
H—C— OH

I
HO— C— H

I
CH2OH

^-Talose

CHO

I
H_C— OH

I
HO— C— H

I
HO— C— H

I
HO— C— H

CH2OH

Z-Altrose
unknown

CHO

H—C— OH

I
HO— C— H

I
H_C— OH

I
H—C— OH

I
CH2OH

o?-Glucose

CHO

I
H—C— OH

I
H—C— OH

HO— C— H

i
H_C— OH

i
CH2OH

fl^-Gulose^

CHO

I
HO— C^H

HO— C— H

I
HO— C— H

I
H—C— OH

I
CH2OH

c?-Talose

CHO

I
HO— C— H

I
H—C— OH

I
H—C— OH

H—C— OH

I

^ Known formerly as Z-Idose.

c?-Altrose
2 Known formerly as i-Gulose.

V] CARBOHYDRATES 49

Though many of the above sugars have been synthesized artificially,
only three are known to occur naturally, i.e. c?-glucose (dextrose or
grape-sugar), c?-mannose and d-galactose.

Since compounds containing asymmetric carbon atoms are optically
active, i.e. can rotate a plane of polarized light, it follows that the sugars
under discussion are optically active.

Glucose. This substance, which is also known as grape-sugar, is very
common and very widely distributed in plants. It occurs in the tissues
of leaves, stems, roots, flowers and fruits. It is produced as a result of
the hydrolysis of cane-sugar and maltose, and, in all probability, is the
first sugar synthesized from carbon dioxide and water. Its synthesis and
its relationships to other sugars will be discussed later (see p. 71). It
is a white crystalline substance, readily soluble in water and aqueous
alcohol, but only slightly soluble in absolute alcohol.

c?-glucose is dextro-rotatoi-y.

When either d- or Z-glucose is first dissolved in water, it is chemically
less active than would be expected of the aldehyde form depicted above.
This is explained by assuming that glucose, when first dissolved in water,
exists in the condition of a 7-lactone :

HO— C— H

CH2OH

In the above state the carbon atom marked * is also asymmetric so
that two forms of glucose are possible, a- and y8-glucose :

H— C— OH

CH2OH

iS-Glueose

y

50 CARBOHYDRATES [ch.

In solution, both the above forms pass by tautomerism into the
aldehyde form.

In the plant there are, as will be described later (p. 157), many aromatic
and other compounds containing one or more hydroxy 1 groups. These
hydroxyl groups of the aromatic substances are frequently replaced by
a glucose (or other sugar) molecule, and such compounds are termed
glucosides, as, for instance, salicin, the glucoside of salicylic alcohol which
occurs in Willow bark (see p. 167) :

HO

CH2OH
Salicin

These substances, moreover, may be classified either as a- or /8-
glucosides according to which of the above a or ^ forms of glucose has
combined with the residual part of the compound. Various glucosides
will be dealt with in Chaps, viii and X.

Expt. 41. Tests for glucose. Before dealing with the sugars actually isolated from
the plant, it is advisable that the following tests and reactions should be performed
with pure glucose using a 0'2^/q solution.

(a) Moore's test. Boil a little of the glucose solution with an equal volume of
caustic soda solution. A yellow colour is developed which is due to the formation of
a condensation product (caramel) of the sugar.

(6) Trommer's test. Add a few drops of a 1 % copper sulphate solution to 2-3 c.c.
of 5 0/0 caustic soda solution. A blue precipitate of cupric hydroxide is formed. Add
now 2-3 c.c. of the glucose solution, and the precipitate will dissolve. On boiling,
the blue colour disappears, and a yellow or red precipitate of cuprous oxide is formed.
If only a little sugar is present the blue colour will disappear, but no oxide may be
formed.

(c) Fehling's test. Boil a few c.c. of freshly made Fehling's solution in a test-tube
and note that it is unaltered. Then add an equal quantity of the glucose solution
and boil again. A red precipitate of cuprous oxide is formed.

(d) Osazone test. Take 10 c.c. of a 0*5 ^Iq solution of glucose in a test-tube and
add as much solid phenylhydrazine hydrochloride as will lie on a sixpenny piece,
at least twice as much solid sodium acetate and also 1 c.c. of strong acetic acid.

V] CARBOHYDRATES 51

Warm gently until the mixture is dissolved and filter into another test-tube. Then
place the tube in a beaker of boiling water for at least ^ hour, keeping the water
boiling all the time. Let the test-tube cool slowly, and a yellow crystalline deposit
of phenylglucosazone will separate out. Examine this under the microscope and it
will be found to consist of fine yellow needles variously aggregated into sheaves and
rosettes. Glucosazone melts at 204-205°C.

The osazone reaction takes place as follow^s :

CH20H(CHOH)4CHO-hH2NNHC6H6=CH20H(CHOH)4CH : N • NHCeHg-l-HaO.

Glucose phenylhydrazone

The phenylhydrazone is very soluble, but if an excess of phenyl-
hydrazine is used, a second hydrazine complex is introduced and an
insoluble osazone is formed :

CH2OH (CH0H)3— C— CH : N • NHCeHg

II
N-NHCgHs

Glucose reacts in this way by virtue of its aldehyde group. Phenyl-
hydrazine hydrochloride does not give an osazone when boiled with
glucose unless excess of sodium acetate be added. This acts on the
hydrochloride to form phenylhydrazine acetate and sodium chloride.

Galactose. Galactose rarely, if ever, occurs free in plants, though it
is fairly widely distributed in the form of condensation products, the
galactans, in combination with other hexoses and with pentoses (see
p. 62). These galactans form constituents of various gums, mucilages,
etc. Agar-agar, which is a mucilage obtained from certain genera of the
Red Seaweeds (Rhodophyceae), yields a high percentage of galactose on
hydrolysis with acids. Galactose also occurs as a constituent of some
glucosides from which it may be derived on hydrolysis.

One of the most important reactions of galactose is the formation of
mucic acid on oxidation with nitric acid. Mucic acid is practically in-
soluble in water and separates out as a crystalline precipitate on pouring
the products of oxidation into excess of water.

Expt. 42. Preparation of galactose from agar-agar. Weigh out 50 gms. of agar-
agar. Put it into a round-bottomed flask fitted with an air condenser (see p. 46).
Add 500 c.c. of 2 ^/q sulphuric acid and heat on a water-bath for 4 hrs. Neutralize
the solution with calcium carbonate and filter. Concentrate on a water-bath to a
syrup. On standing, crystals of galactose will separate out. Then add a little 50-75 o/q
alcohol and warm gently on a water-bath. By this means much of the dark-coloured
product will go into solution and can be poured off leaving the crystalline residue.
Take up this residue in a little hot water, boil well with animal charcoal to decolorize
the solution and filter. Concentrate again on a water-bath. On cooling, colourless
prisms of galactose will separate out.

4—2

52 CARBOHYDRATES [ch.

Expt. 43. Oxidation of galactose to mucic acid. Heat the galactose obtained in
the last experiment with nitric acid (1 gm. galactose to 12 c.c. of nitric acid of sp. gr.
1*15, i.e. 5 pts. of concentrated acid and 12 pts. of water) on a water-bath, until the
liquid is reduced to one-third of its bulk. Then pour the product into excess of
distilled water. On standing (for a day or two), a white sandy microcrystalline preci-
pitate of mucic acid will separate out.

Mannose. Mannose has not been detected free in many plants, but
is widely distributed as condensation products, the mannans, in certain
mucilages and in the cell-walls of the endosperm of various seeds (see
p. 61). From the mannans the sugar can be obtained by hydrolysis.
On adding phenylhydrazine hydrochloride and sodium acetate to a solu-
tion of mannose, the phenylhydrazone, which is nearly insoluble in water,
is formed almost immediately and hence constitutes a ready method for
the detection of the sugar.

Laevulose. This sugar, which is also termed fructose, is widely dis-
tributed in plants, in the tissues of leaves, stems, fruits, etc. It is formed,
together with glucose, in the hydrolysis by acids of cane-sugar. The
original cane^-sugar is dextro-rotatory, whereas laevulose is more laevo^
rotatory than glucose is dextro-rotatory; hence the mixture from the
hydrolysis is laevo-rotatory and is known as invert sugar, the change
being termed inversion. The same hydrolysis is brought about by the
widely distributed enzyme, invertase. The polysaccharide, inulin, also
yields laevulose on acid hydrolysis. Laevulose is a white crystalline sub-
stance, soluble in water and alcohol. Unlike glucose, it contains a ketone
instead of an aldehyde group :

CH2OH

I
c=o

1

HO— C— H

I
H— C— OH

I
H— C— OH

!
CH2OH

rf-Fructose

Laevulose reduces Fehling's and other copper solutions. It yields
the same osazone as glucose with phenylhydrazine hydrochloride and
sodium acetate. It also forms an osazone with methylphenylhydrazine
(m.p. 158° C), a reaction which constitutes a distinction from glucose
since the latter gives no osazone with this substance.

vj CARBOHYDRATES 53

Expt. 44. Tests for laevulose. The following tests should be performed with a
0"2 % solution of laevulose in the same way as for glucose (see p. 50).

(a) Moore's test. A positive result is obtained.

(6) Tromrner's test. A positive result is obtained.

(c) Fehling's test. Keduction takes place.

{d) Osazone test. Note that the crystals are identical with those formed from
glucose.

(e) a-Naphthol test (see ip. 4Q). A strong reaction is given.

(/) Seliwanoff's test. To 5 c.c. of Seliwaiioff's solution (prepared by dissolving
0'05 gm. of resorcinol in 100 c.c. of 1 in 2 hydrochloric acid) add a few drops of
laevulose solution and boil. A red coloration and a red precipitate are formed. Add
a little alcohol and the precipitate forms a red solution (see p. 44).

DiSACCHARIDES.

These sugars are formed from the monosaccharides by condensation
with elimination of water. By boiling with dilute acids, or by the action
of certain enzymes, they are hydrolyzed into monosaccharides. The two
most important disaccharides found in plants are maltose and cane-sugar.

Maltose. Maltose or malt-sugar, though it probably occurs in smaller
quantities than glucose and laevulose, is widely distributed in plant
tissues. It is formed in the hydrolysis of starch, and its relationships in
the plant to starch and to other sugars will be considered later. It is a
white crystalline substance soluble in water and alcohol. In constitution
it is a glucose-a-glucoside :

CeHnOs— O— Cr-H

H— C— OH

I
CH2OH

Maltose

It reduces Fehling's solution ; but less readily than glucose. With
phenylhydrazine hydrochloride and sodium acetate it forms an osazone
(m.p. 206° C), which is more soluble than glucosazone and crystallizes
in broader flatter needles. Maltose is dextro-rotatory.

Expt. 45. Tests for maltose. The tests a, 6, c and e, should be performed with a
0*2<^/o solution of maltose ; test d with a 2 7o solution (see also glucose, p. 50).
(a) Moore's test. A positive reaction is given.
(6) Trommer's test. A positive reaction is given.
(c) Fehling's test. Reduction takes place, but less strongly than with glucose.

54 CARBOHYDRATES [ch.

{d) Osazone test. Take 10 c.c. of the solution and treat as for glucosazone. The
crystals of maltosazone will be found to be much broader than those of glucos-
azone.

(e) Hydrolysis. Take 20 c.c. of the sugar solution and add 2 c.c. of concentrated
hydrochloric acid. Heat in a boiling water-bath for half an hour. Neutralize and test
for the osazone. Glucosazone will be formed.

Sucrose. Sucrose or cane-sugar is very widely distributed in plants,
in leaves, stems, roots, fruits, etc. It is a white substance which crystal-
lizes well, and is soluble in water and alcohol. As previously stated it is
hydrolyzed by dilute acids and by invertase into one molecule of glucose
and one molecule of laevulose. It is formed by the condensation of glucose
and laevulose with the elimination of water. Its constitution is in all
probability as follows :

CH2OH • C • (CH0H)2 • CH • CH2OH

O

/
CH • (CH0H)2 • CH • CHOH • CH.2OH

O

SO that both the ketone and aldehyde groups are rendered inactive. It
does not reduce Fehling's solution and does not form an osazone. It is
dextro-rotatory.

Expt. 46. Tests for cane-sugar. The following tests should be made with a 1 %
solution of pure crystalline cane-sugar (see also glucose, p. 50).

(a) Moore's test. A negative result is obtained.

(6) Fehling's test. No reduction talies place.

(c) a-Naphthol test. A positive result is given since sucrose yields laevulose.

{d) Hydrolysis. To a few c.c. of the solution add a drop of strong sulphuric acid
and boil for two minutes. Then neutralize with caustic soda using litmus as
indicator. Boil again and add Fehling's solution drop by drop. A reduction takes
place owing to the inversion of the cane-sugar by sulphuric acid.

(e) Seliwanoff^s test. A positive result is obtained owing to the liberation of
laevulose.

Tri- and Tetrasacch abides.
Several trisaccharides, condensed from various hexoses or pentoses
are known. Raffinose (fructose, glucose and galactose) has been isolated
from the seed of the Cotton Plant (Gossypium), from the Beet (Beta)
and other plants. Rhamninose (galactose and two molecules of rham-
nose) occurs in the fruit of Rhamnus infectoria, Gentianose (fructose

V] CARBOHYDRATES 55

and two molecules of glucose) has been isolated from the root of Gentian
(Gentiana). Melicitose (fructose and two molecules of glucose) occurs
in a manna ^ which exudes from the twigs of the Larch (Larix) and
Douglas Fir {Pseudotsuga).

A tetrasaccharide, stachyose (fructose, glucose and two molecules
of galactose) has been isolated from tubers of Stachys tubifera, from
White Jasmine (Jasminum) and other plants.

Polysaccharides.

These substances are formed by condensation, with elimination of
water, from more than three molecules of monosaccharides.

Pentosans,

It has already been mentioned that condensation products of the
pentoses, the pentosans, are widely distributed. The two most frequently
occurring pentosans are xylan and araban. No enzymes are known which
hydrolyze the pentosans. It is characteristic of xylan and araban that
they form copper compounds in Fehling's solution in presence of excess
of alkali.

Araban. This pentosan may be regarded as a condensation product
of arabinose as already indicated. It occurs in various gums (Gum
Arabic, Cherry Gum) frequently in combination with other substances.
On hydrolysis with acids, araban yields arabinose. (See also gums and
arabinose.)

Expt. 47. Preparation of araban from Gum Arabic. (Salkowski, 30.) Weigh out
20 gms. of gum arable and dissolve in 500 c.c. of warm water in a large evaporating
dish on a water-bath. Then add 200 c.c. of Fehling's solution and excess of strong
caustic soda solution. The araban will be precipitated as a white gummy mass which
will settle at the bottom of the dish. Filter off through muslin. Take up the preci-
pitate in the minimum quantity of dilute hydrochloric acid (1 pt. of acid : 1 pt. of
water), and then add alcohol. The araban separates out as a white precipitate. Wash
away the copper chloride with alcohol.

Expt. 48. Hydrolysis of araban. The araban from the last experiment is put
into a round-bottomed flask with about 200 c.c. of 2 ^/q sulphuric acid and heated on
a water-bath for 2 hours, the flask being fitted with an air condenser (see p. 46).

1 Manna is a name given to exudations from the branches of various trees and shrubs.
Sometimes the flow is assisted artificially as in the case of the Manna Ash {Fraxinus Ornus)
where the product, consisting almost entirely of the polyhydric alcohol, mannitol, is of
commercial value as a drug, etc. In other cases, the manna exudes as the result of the
attacks of insects. Mannas appear to be readily soluble in water to clear, non-sticky so-
lutions, thereby differing from gums and resins.

m CARBOHYDRATES [ch.

Then neutralize the liquid with calcium carbonate, filter from calcium sulphate, and
concentrate on a water- bath. Some of the solution of arabinose should be tested with
all the tests given in Expt. 39. The sugar can be extracted from the s}Tup with 90 ^/q
alcohol, but it crystallizes only with difficulty.

Xylan. This pentosan occurs in lignified cell-walls, and is the chief
constituent of " wood gum." It is found in the wood of many trees (not
Coniferae), in bran, in wheat and oat straw, in maize cobs, in the shells
of coconuts and walnuts, in the testa of the cotton (Gossypium) and in
many other tissues : also in some gums. On hydrolysis, xylan yields
xylose ; hence wood shavings, bran, straw, etc., will give the pentose reac-
tions on hydrolysis.

Bxpt. 49. Detection of pentose from pentosans in bran, sawdust and straw. Take
a small quantity of bran and boil it up several times with 98 % alcohol, filtering oft'
the alcohol after each treatment. This should remove any sugars or glucosides
present. Allow the alcohol to evaporate off from the bran, and then make the following
tests for pentoses (see Expt. 39) :

(a) Heat, for about one minute, a small quantity of the bran in a test-tube, with
sufficient concentrated hydrochloric acid to cover it. Care should be taken not to
char the material. Then add as much solid orcinol as will lie on the tip of a penknife.
Heat gently again for a few seconds. Then add one or two drops of strong ferric
chloride solution ; a green coloration will be produced. Add amyl alcohol and the
green colour will pass into the alcohol.

(6) Heat again another portion of the bran with the same quantity of concentrated
hydrochloric acid in a test-tube, but this time heat more strongly. After heating a
few minutes place a piece of filter- paper soaked in a solution of aniline acetate in the
mouth of the test-tube. A cherry-red coloration will denote the formation of
furfural.

The above method and tests with bran may be repeated in exactly the same way
using sawdust or straw.

Expt. 50. Preparation of xylan from sawdust. Extract one kilo of sawdust with
4 litres of 1-2% ammonia solution for 24 hrs. Then filter off" the ammoniacal solution
through muslin and repeat the extraction. The xylan is insoluble in ammoniacal
solution, and in this way colouring matters are removed. Finally wash the sawdust
well with water and press dry from the liquid. Then add to the sawdust sufficient
6% caustic soda solution to make a thick mush (about 1000-1500 c.c.) and allow it
to stand for 24 hrs. in a warm place. The alkaline solution is then pressed out
through calico and filtered through filter-paper. To the clear filtrate add an equal
volume of 96% alcohol which will precipitate the xylan as a sodium compound.
Filter off this precipitate, wash with alcohol, and decompose with alcohol to which
a little strong hydrochloric acid has been added to remove the sodium. The free
xylan is again washed with alcohol, and can be dried by washing with absolute
alcohol and ether and finally in a desiccator. It is a dirty-white powder which is
almost insoluble in wafer. Make the tests for pentoses (see Expt. 39) on a little of
the solid xylan. The reaction will be given in each

Y] CARBOHYDRATES 57

Expt. 51. Hydrolysis of xylan. Put the xylan obtained in the last experiment
in a round-bottomed flask fitted with an air condenser (see p. 46). Add 100 c.c. of
4 % sulphuric acid and heat on a water-bath for 4 hrs. NeutraHze the solution with
calcium carbonate, filter from calcium sulphate and concentrate on a water-bath.
Test a portion for pentoses (see Expt. 39) and a positive reaction will be obtained.
To a small quantity add also a few drops of Fehling's solution and boil. Reduction
will take place.

To the remainder of the xylose solution add bromine (see p. 47) gradually until
there is excess. Then remove the excess of bromine by warming on a water- bath.
Neutralize the solution, which contains xylonic acid, with cadmium carbonate and
evaporate on a water-bath. Extract the residue with alcohol and filter. On concen-
trating the alcoholic extract, white prismatic needles of cadmium xylonate separate
out.

It has been shown that pentosans, xylan and probably araban, occur
in leaves (Davis, Daish and Sawyer, 17). It is likely that the xylan is
widely distributed in all tissues since it forms a constituent of lignified
cell-walls.

Expt. 52. Detection of pentoses from pentosans in leaves. (Davis, Daish and
Sawyer, 17.) Take two large leaves of the Sunflower {Helianthus annuus). Tear into
small pieces and drop into boiling 98 % alcohol in a flask. Boil well and filter off" the
alcohol. Repeat until all the green colour is removed. Then dry oflf the alcohol and
grind up the leaf residue. Perform the test for pentoses (Expt. 39 a and c) on the
dry leaf tissue. It should give the above tests showing the presence of pentosans.

Leaves of the Violet ( Viola odorata) and Nasturtium ( Tropaeolum majus) may
•also be used.

Expt. 53. Method for determination of pentosans in tissues, hraii and leaves, etc.
Weigh, out 2 gms. of bran, put it into a round-bottomed flask, add 100 c.c. of 12 %
hydrochloric acid and fit the flask with a water condenser. Heat gently over wire
gauze and distil into a solution of phloroglucinol in 12 ^/^ hj'^drochloric acid. A green
precipitate of furfural phloroglucide is formed which eventually becomes almost black.
For accurate estimations of pentosans this is filtered off" and weighed on a Gooch
crucible. The same method may be used with leaf residue prepared as in Expt. 52.

Starches.

Starch. This is a very widely distributed substance in plants. It
occurs as solid grains throughout the tissues, in leaves, stems, roots, fruits
and seeds. It is absent, however, from a number of Monocotyledons,
e.g. Iris, Snowdrop (Galanthus), Hyacinthus, etc. (Blackman, 5). It forms
one of the chief reserve materials of plants, that is, it is synthesized from
sugar when carbon assimilation and carbohydrate synthesis are in pro-
gress, and is stored in the solid form in tissues as grains. In other
circumstances of the plant's existence, when material for metabolism is
not available from carbon assimilation, as for instance in germinating
seeds or growing bulbs or rhizomes, the starch is hydrolyzed into dextrin

58 CARBOHYDRATES [ch.

and soluble sugar, which is translocated and used as a basis for meta-
bolism. During the night in leaves there is also a similar hydrolysis of
the starch which has been temporarily stored from the excess of sugar
synthesized during the day.

Starch has a very large molecule and thus a high molecular weight.
It is insoluble in cold water. When heated with a little water it gives
starch paste, but on boiling with water it gives an opalescent " solution "
which really contains starch in the colloidal state as an emulsoid. In
this condition it does not diffuse through dialyzing membranes and does
not depress the freezing point of water. The " solution " cannot, strictly
speaking, be filtered, but generally, when hot, it passes to some extent
through ordinary filter-paper. Starch is insoluble in alcohol and is pre-
cipitated by it.

The most characteristic reaction of starch is the blue colour it gives
with iodine solution. This blue colour disappears on heating, but re-
appears again on cooling. Starch is precipitated from " solution " by
half saturation with ammonium sulphate : it does not reduce Fehling's
solution.

By boiling with dilute acids, starch is first converted into " soluble
starch " which still gives a blue colour with iodine. On further boiling,
various dextrins (see dextrins) are obtained which give either purple,
red or no colour with iodine. The final product, after prolonged boiling
with acids, is glucose. Hydrolysis with diastase yields dextrin and
maltose (see diastase, p. 75).

Expt. 54. Preparation of starch from Wheat. Starch may be prepared from a
cereal by the following method.

Take 25 gms. of flour and make it up into a dough with a little water. Allow it
to stand for half an hour. Then tie a piece of muslin over the top of a beaker which
is filled with water. Place the dough on the top of the muslin and rub it gently with
a glass rod. The starch will be separated from the gluten, and will be washed
through the muslin and on standing will sink to the bottom of the beaker. Allow
this to stand till the starch has settled, then decant off the bulk of the liquid. Filter
off the starch, and wash well with water, then with alcohol and finally with ether.
Dry in the steam-oven.

For the detection of starch in green leaves, see Expt. 77.

Expt. 55. Tests for starch. Take a small quantity of the starch prepared in the
previous experiment (or use commercial potato starch) and shake up with a little
cold water in a test-tube. Filter, and test the filtrate with a drop of iodine (in
potassium iodide) solution. No blue colour is obtained. Pour a drop of the iodine
solution on the residue in the filter. It turns deep blue.

Weigh out 2 gms. of the starch prepared in the last experiment, and mix it
into a thin cream with a little water. Boil rather more than 100 c.c. of water in an

V] CARBOHYDRATES 59

evaporating dish, and then gradually add to it the starch paste, keeping the water
boiling all the time. Ah opalescent "solution" is obtained. With a few c.c. of the
solution in each case make the following tests :

{a) Add 1-2 drops of iodine solution. A blue colour is obtained. Heat the solu-
tion : the blue colour disappears, but reappears on cooling.

(6) Add an equal volume of alcohol : the starch is precipitated.

(c) Add an equal volume of saturated ammonium sulphate solution : the starch
is precipitated, i.e. by half saturation with this salt.

{d) Add basic lead acetate solution : the starch is precipitated.

Expt. 56. Hydrolysis of starch. To 50 c.c. of the starch solution prepared in the
last experiment add 1 c.c. of strong sulphuric acid. Boil for 10-20 minutes in a
round-bottomed flask. Test a portion of the solution with iodine from time to time;
a purple, red or brown colour is formed due to the dextrin produced in hydrolysis.
To the remainder of the solution after neutralization, using litmus as indicator, add
some Fehling's solution and boil. Reduction takes place owing to the glucose formed
in hydrolysis.

Dextrins.

These compounds occur in the plant as transitory substances, since
they are formed as intermediate products of the hydrolysis of starch by
diastase. They are also formed on heating starch or by boiling it with
mineral acids (see previous experiment). The hydrolysis of starch to
dextrins is fairly rapid, but the conversion of dextrins into maltose is a
much slower process.

Both starch and dextrins have the same empirical formula. Various
forms of the latter have been identified, such as amylodextrin which gives
a blue colour with iodine, erythrodextrin which gives a brownish-red
colour with iodine, and achroodextrin which gives no colour with iodine.
The dextrins are readily soluble in water ; they are precipitated by
alcohol but not by basic lead acetate. On hydrolysis with acids, they
are converted into glucose.

Expt. 57. Preparation of dextrin hy hydrolysis of starch, (a) By diastase from
leaves of the Pea (Pisum sativum). Weigh out 10 gms. of commercial potato starch
and make it into a solution in 250 c.c. of boiling distilled water as in Expt. 55 and
cool. Then weigh out 10-15 gms. of fresh leaflets of the Pea {Pisum sativum) and
pound them well in a mortar. Add to the pounded mass 100 c.c. of distilled water
and a few drops of chloroform (see maltase, p. 77) and filter. The filtrate will contain
diastase (see also Expt. 78). Then add the diastase extract to the starch solution in
a flask, plug with cotton-wool and put in an incubator for 48 hrs. If a little of the
liquid is withdrawn from time to time and tested with iodine, it will be found that
the blue colour due to the starch gradually disappears and is replaced by the brownish-
red colour due to dextrin. After 48 hrs. there will be no trace of blue colour ; then
filter the liquid and concentrate the filtrate on a water-bath to a syrup. Treat the
residue with about 30 c.c. of 96-98 % alcohol and filter. A sticky mass of dextrin i

60 CARBOHYDRATES [ch.

left which should be extracted with a little hot alcohol and then reserved for the next
experiment To show the presence of maltose, the combined alcoholic extracts are
evaporated to dryness on a water-bath, the residue taken up in a little water and the
osazone test made (see p. 50) with the solution. Crystals of maltosazone will separate
out.

(6) By diastase from germinating Barley (Hordeum vulgare). Weigh out about
25 gms. of barley grains and allow them to germinate by soaking and spreading on
damp blotting-paper for 5-7 days. Pound the grains well in a mortar, add 100 c.c. of
water, allow to stand for 2-3 hrs. and filter. Precipitate the filtrate with alcohol and
allow to stand for 24 hrs. Filter off the precipitate, take up in water and add it to
the barley starch "solution," together with a few drops of chloroform. Proceed as
with {a) only the time for hydrolysis may be much shorter, i.e. 6-12 hrs.

Expt. 58. Tests for dextrin. Make a solution of the dextrin prepared in the last
experiment (or use commercial dextrin) and note that it is very soluble in water. With
the solution make the following tests :

{a) Add a little iodine solution. If erythrodextrin is present, a reddish-brown
colour is produced. Heat the solution and the colour will disappear. Cool again and
the colour will reappear. If only achroodextrin is present, no colour will be given
with iodine.

(6) Add an equal volume of strong alcohol. The dextrin is precipitated.

(c) Add an equal volume of saturated ammonium sulphate solution, i.e. half
saturation with ammonium sulphate. The dextrin is not precipitated.

id) Add some basic lead acetate solution : the dextrin is not precipitated.

Inulin.

Inulin. This substance occurs as a soluble " reserve material " in the
cell-sap of the underground stems, roots and also leaves of a number of
plants, especially members of the Compositae, e.g. Dahlia {Dahlia varia-
hilis), Jerusalem Artichoke (Helianthus tuber osus), Chicory (Cichorium
Intyhus) and the Dandelion {Taraxacum officinale). It is said to occur
also in the Campanulaceae, Lobeliaceae, Goodeniaceae, Violaceae and
many Monocotyledons {Hyadnthus, Iris, Muscari and Scilla).

Inulin is a condensation product of laevulose to which it bears much
the same relation as starch to glucose. It is a white substance, soluble
in water and insoluble in alcohol. It crystallizes out in the cells, in which
it occurs, in characteristic sphaero-crystals on addition of alcohol to the
tissues. It is hydrolyzed by mineral acids to laevulose : also by the
enzyme inulase which occurs in the plant.

£Jxpt. 59. Extraction of inulin. Cut off the tubers from two Dahlia (Dahlia
variabilis) plants, wash well, and put them through a mincing machine. Carefully
collect the liquid and the crushed tuber, and boil well with sufficient water to cover
the crushed material. Add also some precipitated calcium carbonate to neutralize
any free acids present. Then filter through fine muslin, and to the filtrate, which
should again be made quite hot, add lead acetate solution until a precipitate

V] CARBOHYDRATES 61

(of mucilaginous substances, etc.) ceases to be formed. Care should Tie taken to
avoid the addition of a large excess of lead acetate. Filter oflf the lead precipitate,
and saturate the filtrate with sulphuretted hydrogen till all excess lead is removed.
Filter off the lead sulphide, neutralize the filtrate to phenolphthalein with ammonia,
and evaporate to half bulk or less on a water- bath, when the inulin will probably
begin to deposit. Then pour into an equal volume of alcohol, and allow to stand for
one or two days. The crude precipitate of inulin is filtered off, dissolved in a small
amount of water, and reprecipitated with alcohol. It can be washed with alcohol and
ether and dried over sulphuric acid.

The Artichoke {Helianthus tuberosus) may also be used, about 12 tubers being
necessary.

Ba^pt. 60. Tests for inulin. Make a solution of some of the inulin prepared in
Expt. 59 in hot water. It will readily dissolve giving a clear solution. With the
solution make the following tests :

{a) Make a very dilute solution of iodine and add to it a drop or two of inulin
solution : the brown colour will be unaffected.

(6) Boil some inulin solution with a little Fehling : no reduction takes place.

If the inulin solution which is being used should reduce Fehling it indicates that
sugar is present as impurity. If this is the case, then a little of the solid inulin
should be washed free from sugar by means of alcohol before proceeding with the
following test.

(c) To 5 c.c. of Seliwanoff^s solution add a few drops of inulin solution and boil.
A red coloration is formed. This reaction is also due to the presence of laevulose
(see laevulose, p. 53),

Expt. 61. Hydrolysis of inulin. Some inulin is dissolved in very dilute hydrochloric
acid (about 0*5 %) and heated on a water-bath for half an hoiir in a round-bottomed
flask provided with an air condenser (see p. 46). The solution is then neutralized
with sodium carbonate and concentrated on a water-bath. With the concentrated
solution make the following tests :

(a) Boil with a little Fehling : the solution is rapidly reduced.

(6) Make the osazone test (see p. 50). Glucosazone crystals will be found to
be formed on microscopic examination. (Laevulose forms the same osazone as
glucose.)

(c) Make the test (c) of the last experiment. A positive result will be given.

M ANNANS.

The mannans which have already been mentioned (see p. 52) ar^
condensation products of the hexose, mannose. They occur most fre-
quently, either mixed, or in combination, with the condensation products
of other hexoses and pentoses (glucose, galactose, fructose and arabinose)
as galactomannans, glucomannans, fructomannans, mannocelluloses, etc.
Such mixtures or compounds of which mannans form a constituent are
widely distributed in the seeds of many plants, i.e. Palms (including the
Date-palm), Asparagus (Ruscus), Clover (Trifolium), Coffee Bean (Goffea

62 CARBOHYDRATES [ch.

arabica), Onion {Allium Cepa) and of members of the Leguminosae,
Rubiaceae, Coniferae and Umbelliferae. In seeds the mannans may con-
stitute, together with cellulose, the thickened cell-walls of the endosperm
and are included in the term " reserve- or hemi-cellulose " though they
are not strictly celluloses. " Vegetable ivory," which is the endosperm
of the Palm, Phytelephas macrocarpa, contains considerable quantities
of a mannan and is used as a source of mannose. Mannans, in addition,
form constituents of certain mucilages, as for instance those in Lily bulbs
(Lilium candidum, L. bulbiferum, L. Martagon and others) (Parkin, 25)
and tubers of various genera of the Orchidaceae : they are also found in
the roots of the Dandelion {Taraxacum), Helianihus and Chicory, Aspa-
ragus and Clover, and in the wood and leaves of various trees.

Many of the mannans, unlike true celluloses, are readily hydrolyzed
by dilute hydrochloric and sulphuric acids. The mannan in the Coffee
Bean, however, is hydrolyzed with difficulty.

Galactans.

These substances bear the same relationship to the hexose, galactose
as the mannans to mannose, that is, they are condensation products of
galactose (see p. 51). Similarly they frequently occur, together with the
condensation products of other sugars, as galactoaraban, galactoxylan,
galactomannan, etc. As such they form constituents of many gums and
mucilages and of the cell-walls of the reserve tissue of seeds, i.e. the
Coffee Bean {Coffea arabica), the Bean (Faba), the Lupin {Lupinus), the
Paeony {Paeonia), the Kidney Bean {Phaseolus), the Date {Phoenix),
the Pea (Ptswm), the Nasturtium {Tropaeolum) and many others (Schulze,
Steiger and Maxwell, 32).

Gums,

These substances occur widely distributed among plants, especially
trees. Some gums are wholly soluble in water giving sticky colloidal
solutions: others are only partially soluble. They are all insoluble in
alcohol. In the solid state they are translucent and amorphous.

Chemically the gums are varied in nature ; they may in general be
regarded as consisting of complex acids in combination with condensa-
tion products of various sugars, such as araban, xylan, galactan, etc.
On hydrolysis they give mixtures of the corresponding sugars, arabinose,
xylose, galactose, etc., in varying proportions, though in some cases one
sugar preponderates.

V] CARBOHYDRATES 63

Some of the best-known gums are the following :

Oum Arabic (arabin). This substance is obtained from an Acacia
{Acacia Senegal), a native of the Soudan. The gum exudes from the
branches. Other species of Acacia yield inferior gums. Gum arabic is
a mixture of the calcium, magnesium and potassium salts of arabic acid,
a weak acid of which the constitution is unknown, in combination with
araban and galactan.

Gum Tragacanth. This is a product from several Tragacanth shrubs
which are species oi Astragalus (Leguminosae), chiefly A, gummifer. It
is obtained by wounding the stem and allowing the gum to exude and
harden. On hydrolysis it gives a mixture of complex acids and various
sugars such as arabinose, galactose and xylose.

Cherry Gum (cerasin) occurs in the wood of the stems and branches
of the Cherry (Prunus Cerasus), the Bird Cherry (P. Padus), the Plum
(P. domestica), the Almond (P. Amygdalus) and other trees of the Rosa-
ceae. It exudes from fissures of the bark. On hydrolysis it yields almost
entirely arabinose.

Expt. 62. Reactions of Oum Arabic. Put a little gum arabic into an evaporating
dish and add a little water. Heat gently and stir. The gum will slowly dissolve,
giving a thick sticky solution which does not solidify or gel on cooling. Make the
following tests, using a little of the gum solution in a test-tube each time.

{a) Add a little alcohol. The gum is precipitated.

(6) Add a little Fehling's solution and boil. No reduction takes place.

The three following experiments show the presence of pentosan complexes in the
gum (see also Expt. 39, p. 45) :

(c) Add a little phloroglucinol to the guni and then strong hydrochloric acid. No
colour is produced. Now heat, and a cherry-red colour appears.

{d) Heat the gum solution with a little concentrated hydrochloric acid and then
add a trace of orcinol. Warm again and then add one or two drops of strong ferric
chloride solution. A green coloration will be produced.

(e) Heat the gum solution strongly with hydrochloric acid, and, after heating for
a few minutes, place a piece of filter-paper soaked in a solution of aniline acetate in
the mouth of the test-tube. A cherry-red coloration indicative of furfural will be
formed.

Expt. 63. Hydrolysis of Gum Arabic. Weigh out 10 gms. of gum arabic. Put it
into a round-bottomed flask and add 100 c.c. of water and 4 c.c. of strong sulphuric
acid. Warm gently until the gum goes into solution. Then fit the flask with an air
condenser (see p. 46) and heat on a water-bath for about 4 hrs. Cool the solution,
and neutralize with barium carbonate. Filter ofi'the barium sulphate and concentrate
the solution on a water-bath. Boil a drop or two of the syrup with Fehling's solution
and show that reduction takes place. (The original gum either does not reduce
Fehling at all, or, if so, only slightly.) Then add a httle nitric acid (sp. gr. 1-15, see

64 CARBOHYDRATES [ch.

Expt. 43) to the syrup and heat on a water-bath almost to dryness. Pour the residue
into about 100 c.c. of water and allow to stand. A microcrystalline precipitate of
mucic acid is formed showing the presence of galactose (see p. 52) as a product of
hydrolysis.

Mucilages.

The characteristic of these substances is that they swell up in water
and produce colloidal solutions which are slimy.

Mucilages are widely distributed and may occur in any organ of the
plant. Sometimes they are confined to certain cells, mucilage sacs or
canals. They are distinguished from the pectic substances by the fact
that they do not gelatinize. Some of the best known examples of muci-
lage-containing tissues are those in the root and flower of the Hollyhock
{Althaea rosea) : in succulent plants {Aloe, Euphorbia), in bulbs {Scilla^
Allium) and tubers {Orchis Morio) : in seeds of Flax or Linseed {Linum)
and in fruits of Mistletoe ( Viscum album).

The mucilages vary in composition. They appear to be largely, if
not wholly, condensation products of various sugars (galactose, mannose,
glucose, xylose, arabinose), similar constituents to those of many gum&
and hemicelluloses. On hydrolysis various mixtures of sugars are pro-
duced. Of the mucilages, that from linseed has been thoroughly inves-
tigated. It has been found on hydrolysis to give sugars only, e.g. arabinose,
xylose, glucose and galactose. In this respect mucilages differ from gums,
since the latter have always some other accompanying substance in
addition to sugars.

Expt 64. Preparation and properties of mucilage from Linseed (Linum) (Neville,
23). Take about 60 gms. of linseed and let it soak for 24 hrs. in 300 c.c. of water.
Then separate the slime from the seeds by squeezing through muslin, and add to the
liquid about twice its volume of 96-98 % alcohol. The mucilage is precipitated as
a thick slimy precipitate. Filter off the precipitate and wash with alcohol. By
washing with absolute alcohol and ether and finally drying in a desiccator, the mucilage
may be obtained as a powder.

Add water to some of the mucilage. It swells up and finally gives an opalescent
solution. Make with it the following tests :

(a) Add iodine. No colour is given.

(6) Add a little Fehling's solution and boil. No reduction takes place.

Expt. 65. Hydrolysis of Linseed mucilage. Put the remainder of the mucilage in
a round-bottomed flask and add 50 c.c. of 4 % sulphuric acid. Fit the flask with an
air condenser (see p. 46) and heat for at least four hours on a water-bath. Cool and
neutralize with barium carbonate. Filter off" the barium sulphate, and concentrate
the filtrate on a water-bath. With the concentrated solution make the following
tests :

V] CARBOHYDRATES 65

{a) Add a few drops to a little boiling Fehling solution. Reduction immediately
takes place.

(6) Make the phloroglucinol, orcinol and furfural tests for pentoses, using a small
quantity only of the hydrolysis mixture for the tests. A positive result will be given
in each case. The pentoses, arabinose and xylose, are responsible for these reactions.

(c) Add to some of the solution phenylhydrazine hydrochloride, sodium acetate
and a little acetic acid, and leave in boiling water for half an hour for the osazone test
[see Expt. 41 {d)]. A mixture of osazones will separate out, among which glucosazone
can be identified.

{d) Concentrate the remainder of the solution and then add some nitric acid of
sp. gr. 1"I5 (see Expt. 43). Evaporate down on a water-bath to one-third ol the bulk
of the liquid and then pour into about 100 c.c. of water. A white microcrystalline
precipitate of mucic acid will separate out, either at once or in the course of a day or
two. This demonstrates the presence of galactose.

^ PeCTIO SUBSTANCES.

These substances are considered at this point since they are said to
constitute, in more or less intimate connexion with cellulose, the middle
lamella of cell-walls in many tissues. The pectic substances are frequently
found in the juices of succulent fruits in which the tissues have dis-
integrated, such as red currants and gooseberries. They have been iso-
lated chiefly from fleshy roots, stems and fruits, as, for instance, from
turnips, beetroot, rhubarb stems, oranges, apples, cherries and straw-
berries ; quite recently, also, from cabbage, onions and pea-pods. Recent
investigations point to the fact that all these tissues contain the same
pectic material, and it is possible that all such substances may be
identical.

The chief pectic compound occurring in the cell-wall, probably in
combination with cellulose, is of aii acidic nature and has been provision-
ally termed pectinogen (Schryver and Haynes, 81). It is extracted, in
the form of the ammonium salt, by treating the tissue residue (after
expressing the juice) with warm dilute ammonium oxalate solution.
From this solution, either the salt, or, after acidification, pectinogen
itself, can be precipitated as a very bulky gelatinous mass by adding
alcohol. Pectinogen is an acid and is soluble in water giving a thick
opalescent solution ; its sodium, potassium, ammonium and calcium salts
are also soluble. Pectinogen solution, therefore, is not precipitated either
by acid or by dilute solutions of calcium salts.

In the case of juicy fruits, such as currants and gooseberries, the
pectinogen can be precipitated as a gelatinous precipitate by adding
alcohol to the expressed juice. In the case of fleshy fruits, stems and
roots, the juice, as a rule, contains but little pectinogen and the

o. 5

66 CARBOHYDRATES [ch.

procedure is as follows. The tissues are thoroughly disintegrated in a
mincing machine and pressed free from all juice in a powerful press.
The residue is then dried, finely ground, washed with water and finally
extracted with dilute ammonium oxalate solution in which pectinogen
is soluble. The extract is concentrated and the pectinogen precipitated
by alcohol. It may be purified by reprecipitation. When dried it forms
an almost colourless granular powder. Put into water it absorbs large
quantities of liquid and dissolves slowly, giving an opalescent solution
with a distinctly acid reaction.

When pectinogen solution is treated with normal caustic soda at
ordinary temperatures, the sodium salt is first formed and this is rapidly
changed into the salt of another substance termed pectin (cytopectic
acid) (Clayson, Norris and Schryver, 7). Cytopectic acid is insoluble in
water and is readily converted into a gel under certain conditions ; its
calcium salt is also insoluble. Thus, if a solution of pectinogen, made
alkaline with caustic soda, is allowed to stand for ten minutes, on adding
acid a gelatinous precipitate of cytopectic acid is formed, and on adding
calcium chloride solution, a gelatinous precipitate of the calcium salt of
cytopectic acid. A similar precipitate is also formed when lime water
is added in excess to a solution of pectin and it is allowed to stand.

If the tissue residue is first treated with caustic soda solution, the
pectinogen is changed in situ into the cytopectic acid which, though not
itself extracted with caustic soda, can be subsequently extracted by
ammonium oxalate solution and separated as a gel by addition of acid.

Analyses of pectin from various sources, i.e. apples, oranges, straw-
berries, cabbage, onions, pea-pods, rhubarb and turnips, have led to the
suggestion of C17H24O16 as its formula. There is also evidence that it
contains one pentose group. This can be detected and estimated by the
furfural phloroglucide method (see Expt. 53).

Expt. 66. Extraction and reactions of pectinogen. Take about half a pound of red
currants and squeeze out the juice through fine musHn into a large beaker. Then add
to the juice about 2-3 times its bulk of 96-98 % alcohol. A bulky gelatinous precipi-
tate of pectinogen will separate out. Allow the precipitate to stand for a time in the
alcohol and then filter off. Wash with alcohol and finally press free from liquid.
Dissolve the precipitate in as little water as will enable it to go into solution. To two
small portions of the solution add respectively (a) a few drops of strong hydrochloric
acid, (6) an excess of 5% calcium chloride solution. Note that no precipitate is
formed in either case.

Expt. 67. Conversion of pectinogen into pectin, and reactions of pectin. Take about
one-third of the pectinogen solution prepared in Expt. 66, make it alkaline with 4 7o
caustic soda, and let it stand for about 10-15 mmutes. Then divide the solution
into two parts and add respectively (a) sufficient strong hydrochloric acid to acidify,

V] CARBOHYDRATES 67

(6) excess of 5 % calcium chloride solution. In the first case a gel of pectin is formed :
in the second case a gelatinous precipitate of the calcium salt of pectin.

To a further quantity of the pectinogen add excess of lime water and let it stand.
The gelatinous calcium precipitate will separate out in a short time.

Expt. 68. Detection of the pentose group in pectinogen. Filter off the pectin gel
obtained in the last experiment and allow it to dry. Then test for the pentose group
by the orcinol, phloroglucinol and furfural tests (see Expt. 39). All results will be
found to be positive.

The extraction of pectinogen, etc. in the above experiments can equally well be
<;arried out with other material, e.g. ripe gooseberries, raspberries and strawberries,
using exactly the same methods.

Expt. 69. Preparation of pectinogen from Turnips. Take two full-sized turnips
and mince them finely in a mincing machine. Then wrap the mass in a piece of
strong unbleached calico and press out the juice as completely as possible in a press.
The juice contains little pectinogen and can be thrown away. The pressed mass is
then thrown into about 200 cc. of freshly prepared 0*5 ^1^ ammonium oxalate solution
heated to 80-90° C. on a water- bath and stirred to make a paste. The liquid is again
rapidly pressed out in the press. To the viscid extract an equal volume of 96%
alcohol is added, and the ammonium salt of pectinogen separates out as a voluminous
gelatinous precipitate. This is filtered off and, when pressed free from alcohol and
dried, can be used for tests as in the previous experiments.

The gelatinization of pectinogen can also be brought about by certain
enzymes termed pectases which are found in the juices of various plants,^
i.e. root of Carrot (Daucus Carota) and leaves of Lucerne (Medicago
^ativa), Lilac (Syringa vulgaris) and Clover (Trifolium pratense).

Expt. 70. Action of pectase on pectinogen. Make an extract of either Lucerne or
Olover leaves by pounding them in a mortar with a little water, and then filter. Add
the filtrate to some of the pectinogen solution prepared in Expt. 66 or 69. On
standing a gelatinous precipitate will be produced. Should the reaction be slow, it
may be accelerated by placing the mixture in an incubator.

Celluloses.
Celluloses are very important polysaccharides. They form constituents
of the structural part of all the higher plants. The cell-wall of the young
cell consists entirely of cellulose, but in older cells the walls may be
lignified, cuticularized, etc., i.e. the cellulose may be accompanied by
other substances such as lignin, cutin, mucilage, etc. In the light of
these facts the term cellulose is made to include:

1. Normal celluloses.

2. Compound celluloses.

(a) Ligno-celluloses.
(6) Pecto-celluloses.
(c) Adipo- or cuto-celluloses.

3. Pseudo- or Reserve celluloses.

5—2

68 CAKBOHYDRATES - [ch.

True or normal cellulose. Of this substance, as we have said,
many cell-walls are composed. The most familiar form of cellulose is
cotton, which consists of hairs, each being a very long empty cell, from
the testa or coat of the seed of the Cotton plant (Gossypium herbaceum).
Crude cotton (i.e. the hair cell-walls) is not quite pure cellulose, but
contains a small amount of impurity from which it is freed by treatment
first with alkali and subsequently with bromine or chlorine. All kinds
of cotton material, cotton-wool, and the better forms of paper (including
filter- paper) may be regarded as almost pure cellulose.

Pure cellulose is a white, somewhat hygroscopic, substance; It is
insoluble in water and all the usual solvents for organic substances. It
is, however, soluble in a solution of zinc chloride in hydrochloric acid in
the cold, and in a solution of zinc chloride alone on warming. It is also
soluble in ammoniacal cupric oxide (Schweizer's reagent).

In addition cellulose is soluble in concentrated sulphuric acid, which
on standing converts it first into a hydrate and then finally into glucose.
If, however, water is added to the sulphuric acid solution as soon as it is
made, the gelatinous hydrate of cellulose is precipitated. This substance
is termed "amyloid" since it gives a blue colour with iodine. Concentrated
nitric acid converts cellulose into nitrates, of which one is the substance,
gun-cotton. In 10% alkalies cotton fibres thicken and become more
cylindrical. This procedure has been employed by Mercer to give a
silky gloss to cotton, and the resultant product is called mercerized
cotton.

Expt. 71. The colour tests and solubilities of cellulose.

(a) Dip a little cotton- wool into a solution of iodine in potassium iodide. Then
put the stained wool into an evaporating dish and add a drop or two of concentrated
sulphuric acid. A blue coloration is given. This is due to the formation of the
hydrate "amyloid" mentioned above.

(6) Dip some cotton-wool into a calcium chloride iodine solution. (To 10 c.c. of
a saturated solution of calcium chloride add 0*5 gm. of potassium iodide and 0*1 gm.
of iodine. Warm gently and filter through glass-wool.) A rose-red coloration is
produced which eventually turns violet.

(c) Heat a strong solution of zinc chloride (6 pts. of zinc chloride to 10 pts. of
water) in an evaporating dish and add 1 part of cotton-wool. The cellulose will in
time become gelatinized, and if a little water is added from time to time, a solution
will eventually be obtained on continuous heating.

(d) Make a solution of zinc chloride in twice its weight of concentrated hydro-
chloric acid and add some cotton- wool. The wool will rapidly go into solution in the
cold.

(e) Add some cotton-wool to an ammoniacal copper oxide solution and note that
it dissolves. (To a strong solution of copper sulphate add some ammonium chloride
and then excess of caustic soda. Filter off the blue precipitate of cupric hydroxide,

V] CARBOHYDRATES 69

wash well, dry thoroughly, and dissolve in strong ammonia.) Add strong hydrochloric
acid and the cellulose is precipitated out again. Then add water and wash the
precipitate until it is colourless. Test the roughly dried precipitate with a little
iodine and strong sulphuric acid. A blue coloration is given.

All the above tests may be repeated with threads from white cotton material,
with filter-paper and good white writing paper.

Try tests {a) and (6) with newspaper, and note that they are not so distinct as
with writing paper owing to the presence of ligno-cellulose (see Expt. 73).

Expt. 72. Hydrolysis of cellulose hy acid. Dissolve as much filter-paper as possible
in 5 c.c. of concentrated sulphuric acid and when all is in solution pour into 100 c.c.
of distilled water. Boil the solution in a round-bottomed flask fitted with an air
condenser (see p. 46) and use a sand-bath for heating. After boiling for an hour,
cool and neutralize the solution with solid calcium cai-bonate. Add a little water if
necessary and filter. Test the filtrate with the following tests :

(a) Make the osazone [see Expt. 41 {d)^ Note that crystals of glucosazone are
formed.

(6) Add a little Fehling's solution and boil. Note that reduction takes place.

Instead of using filter-paper, the above experiment may also be carried out with
€otton-wool or threads from white cotton material.

Ligno-cellulose. As the cells in plants grow older the walls usually
become lignified, that is part of the cellulose becomes converted into
ligno-cellulose. The extreme amount of change is found in wood. The
least amount in such fibres as those from the stem of the Flax (Linum
iisitatissimum) which, when freed from such impurities, consist of cellu-
lose only and constitute linen. Other fibres, containing more ligno-
cellulose, are those of the stem of the Hemp plant {Cannabis sativa) and
the Jute plant (Cor chorus) from which string, rope, canvas, sacking and
certain carpets are made. The percentages of pure cellulose in these
various lignified tissues are as follows :

Cotton fibre 88-3%

Flax and Hemp fibre . . . 72-73 %

Jute 540/0

Beech and Oak wood . . . 35-38 %

The ligno-celluloses are generally regarded as consisting of cellulose
and two other constituents, of which one contains an aromatic nucleus
and the other is of the nature of a pentosan (see xylan, p. 56). Both
are sometimes classed together and termed lignin or lignon. The lignin
reactions (see below) depend on the presence of an aromatic complex.
It has been suggested that coniferin, vanillin and allied compounds
which are present in wood are probably the substances responsible for
the reaction (Czapek, 8).

70 CAEBOHYDRATES [ch.

Although the best paper is made from cellulose, cheaper forms of
paper are manufactured from ligno-cellulose, and, as a result, they, give
reactions for lignin and are also turned yellow by exposure to light.

Expt. 73, Reactions of lignin.

One of the most striking reactions of lignin (due as it is supposed to a furfural
grouping) is the magenta-red coloration given by phloroglucinol in the presence of
concentrated hydrochloric acid.

Soak the tissue to be experimented upon with an alcoholic solution of phloroglucinol
and then add a drop or two of strong hydrochloric acid. The magenta-red colour
will be produced.

As material, practically any lignified tissue may be used. Shavings from twigs
of any tree or shrub, e.g. pith and wood from the Elder {Samhucus nigra), will be
found useful : also shavings from a match ; straw, bran, coarse string, cheap white
paper, such as newspaper or white and pale-coloured papers used for wrappings.

Make the phloroglucinol test on good white writing paper. It should not give the
reaction since it is made from cellulose.

Other phenols (resorcinol, orcinol, catechol, pyrogallol, etc.) and their derivatives
will also give colour reactions with lignin in the presence of hydrochloric acid, but the
colorations in most cases are not so much developed as with phloroglucinol (Czapek, 8).

It should be noted that strong hydrochloric acid alone will sometimes
give a red colour with woody tissues: this is due to the presence of
phloroglucinol in the wood itself (see phloroglucinol, p. 102).

Pecto-cellulose. The non-cellulose constituents in this case belong
to the class of pectic substances which have already been considered (see
p. Q^). Such celluloses occur in the cell-walls of the tissues of many fleshy
roots, stems and fruits.

Adipo- or cuto-celluloses. These terms have been used for products
found in the walls of corky tissue (periderm) and cuticularized tissue
(cuticle). More correctly these substances should be termed respectively
suberin and cutin, and there is evidence (Priestley, 27) that cellulose is
absent from the actual layers of the cell-wall in which suberin and cutin
are present. Suberin may be regarded as an aggregate of various con-
densation products, or anhydrides, of certain organic acids (the suberogenic
acids), accompanied by small quantities of glycerides (true fats) of these
same acids. By saponification of the condensation products with alkali,
three suberogenic acids have been isolated in a more or less pure state,
i.e. phellonic acid, C22H43O3, phloionic acid, C22H40O7 and suberinic acid,
C17H30O3. The acids themselves are soluble in the usual solvents for
fats; phellonic acid or some of its salts may be soluble or have a tendency
to swell in water. The anhydrides, on the contrary, are insoluble in
solvents for fats and are totally unaffected by water.

V] CARBOHYDRATES 71

There is reason to believe that ciitin is an aggregate of similar
modifications of various "cutinogenic" acids. The suberin and cutin of one
plant probably differ from that of another in the kind and proportion of
the acids present.

Hemi-celluloses. These are not strictly celluloses since they are built
up of mannans, galactans and pentosans on lines which have already been
considered (see pp. 61 and 62). They frequently occur united with each
other, for instance as galacto-, gluco- and fructomannan, galactoaraban,
galactoxylan, etc. They are found in the cell-walls of the tissues of
many seeds, and are apparently hydrolyzed by certain enzymes, termed
cytases, during germination.

The Synthesis and Inter-relationships of Carbohydrates

IN THE Plant.

Now that the properties and characteristics of various carbohydrates
have been dealt with, their synthesis and their relationships, one to
another, may be considered.

In the previous chapter it has been shown how the plant synthesizes
a sugar from carbon dioxide and water by virtue of the chemical energy
obtained from transformation of radiant energy by means of chlorophyll.
When this sugar reaches a certam concentration in the cell, in the
majority of plants, starch is synthesized from it by condensation with
elimination of water. The starch is thus the first visible product of
assimilation and is temporarily "stored" in an insoluble form during the
day, when photosynthesis is active. During the night photosynthesis
ceases but the sugar is still translocated from the leaf, as it was in fact
during the day ; thus, since the supply ceases, the concentration in the
cell falls, and the "stored" starch is then hydrolyzed again into sugar,
and the process continues until the leaf is either starch- free, or contains
considerably less starch. During the next day, the starch formation is
repeated and so forth. The process of hydrolysis of starch is carried out
by the enzyme, diastase, with the formation of dextrin and maltose. In
all probability this same enzyme controls the synthesis of starch.

On the other hand, it has been shown that many plants do not form
starch at all in their leaves but only sugar. Examples are the adult
Mangold plant (Beta vulgaris) and many Monocotyledons {Allium,
Scilla).

72 CARBOHYDKATES [ch.

As to the question of which sugars are present in the leaf, there is
only evidence from accurate work on a few plants. Careful investiga-
tions have been made of the sugars in leaves of the Mangold (Beta
vulgaris) (Davis, Daish and Sawyer, 17), Garden Nasturtium (Tropaeo-
lum majus) (Brown and Morris, 6), the Snowdrop (Galanthus nivalis)
(Parkin, 26), the Potato (Solanum tuberosum) (Davis and Sawyer, 19)
and the Vine (Vitis vinifera). The general conclusions drawn from
these investigations are that sucrose, glucose, and laevulose are always
present in leaves: that maltose results from the hydrolysis of starch,
being absent from leaves which do not form starch. Maltose is not pre-
sent in appreciable quantity even in starch-producing leaves because it is
rapidly hydrolyzed into glucose by maltase. (In such cases where it has
been detected it has been due to diastase action during the drying of
leaves before extraction.) Other leaf carbohydrates are the pentoses
which have been found in a good many species examined and may be
widely distributed; the pentosans, their condensation products, also occur
as well as dextrin (Potato).

The next question to be considered is what sugar is first synthesized
in the leaf. Is it glucose, laevulose, sucrose or maltose ? It is known that
the enzymes, invertase and maltase, are commonly present in leaves and
that these enzymes respectively control the hydrolysis, of cane-sugar
into glucose and laevulose, and of maltose into glucose. It is also possible
that they respectively control the synthesis of sucrose and maltose.
Laevulose, likewise, as may be supposed, can be obtained from glucose.
Thus all the sugars can be readily converted one into another, but to
ascertain which is the first product of synthesis is not an easy problem.

In addition to the above-mentioned work on the nature of the sugars
present in leaves, a good deal of careful analysis has been made as to the
proportions in which the sugars occur relatively to each other during
stated periods of time, with a view to answering the question as to
which is the first-formed sugar. There are two possibilities: one, that
it is sucrose and that it is hydrolyzed into glucose and fructose: the
other, that it is glucose, from which fiructose is derived, and the two are
then synthesized to form sucrose.

Opinion is divided on this point and there is not at present sufficient
experimental evidence to decide the matter. The majority of investi-
gators regard sucrose as the first-formed sugar, and suggest that it is
inverted into hexoses for purposes of translocation, since the smaller
molecules would diffuse faster. There is experimental evidence that
there is an increase in hexoses in the conducting tissues. Others favour

V] CARBOHYDRATES 73

the view that glucose is the first-formed sugar, and bring forward
evidence to this effect. There is however no reason why hexoses should
not be formed first and then converted into cane-sugar and temporarily
stored as such, being again reinverted into hexoses for translocation.
Nor is there any reason for supposing that the first formed sugar is always
the same in every plant.

There appears to be very little doubt that maltose is formed in
the hydrolysis of starch, and also that starch is a temporary reserve
material in the leaves, but whether formed direct from sucrose or fi-om
hexoses cannot be stated.

There is some evidence in favour of the view that glucose is more
readily used in respiration than laevulose, for under circumstances when
neither can be increased, the glucose tends to disappear.

From the leaf the various sugars are translocated to other organs of
the plant, e.g. root, stem, flower, fruit and seed. In some cases starch
is synthesized from the sugars and "stored" in roots, tubers, tuberous
stems, fruits and seeds. In other cases the sugars themselves may be
"stored," as, for instance, in the root of the Beet (Beta vulgaris), or they
may have a biological significance, as in sweet fruits. It must also be
borne in mind that sugars are employed throughout the plant in re-
spiration and in the synthesis of more complex substances, i.e. cellulose,
gums, pentosans, mucilage, aromatic substances, fats and to a certain
extent proteins: in fact they or their precursors constitute the basis from
which all organic compounds are synthesized.

The following experiments can be performed with either the Garden
Beet or the Mangold Wurzel, both of which are varieties of Beta vulgaris,
the Common Beetroot. The sugars in the leaves and petioles of the
Mangold have been investigated (Davis, Daish and Sawyer, 17) and
sucrose, laevulose and glucose have been found. Starch is absent in
the adult plant and also maltose. The opinion is held that sucrose is
the first-formed sugar of photosynthesis and that this is hydrolyzed for ,
translocation on account of the greater rate of diffusion of the smaller
molecules of glucose and laevulose. These are again synthesized in the
root to form sucrose where the latter is stored, and hexoses are almost
absent from this organ. Though the facts concerning the distribution
of the sugars stated above are reliable, it is not certain that the deduc-
tions are permissible. The leaf contains the enzymes, invertase, maltase
and diastase (Robertson, Irvine and Dobson, 28).

In connexion with the occurrence of various sugars in leaves it is of
interest to note that glucose, fructose and mannose can pass over into

u

CARBOHYDRATES

[CH.

one another in alkaline aqueous solution. This has been explained by
their conversion into the enolic (unsaturated) form common to all three
hexoses :

CHO

I

HCOH

I
HOCH

I
HCOH

I
HCOH

I
CH2OH

Glucose

CHO

I
HOCH

i
HOCH

I
HCOH

HCOH

I
CH2OH

Mannose

CH2(OH)

I
CO

I

HOCH

I
HCOH

I
HCOH

I
CH2OH

Fructose

CH(OH)

II
COH

I
HOCH

I
HCOH

I
HCOH

I
CH2OH

Enolic form

Expt. 74. Formation of laevulose and mannose from glvxiose hy alkalies. Into a
small flask put 50 c.c. of a 5% solution of glucose and add 5 c.c, of a 10% solution
of potash. Cork the flask and leave it in an incubator at 35° C. for 24 hours. Cool
and neutralize the alkali with a few drops of acetic acid. Test a few drops for laevulose
with Seliwanoff's reaction (see p. 53). Then add 5 c.c. of a solution of phenylhydrazine
acetate (5 gms. of phenylhydrazine dissolved in 5 c.c. of glacial acetic acid) and shake
well. After a few seconds, the solution becomes turbid and a precipitate of mannose-
hydrazone is formed (see p. 52). Examine under the microscope and note the charac-
teristic crystalline spheroids.

It is not known whether the pentoses are formed de novo in carbon,
assimilation or whether they arise from the hexoses. A relationship of
interest in this connexion is that between the pentoses and the hexoses
actually occurring in plants, as will be seen by comparing the formula of
c?-xylose with that of c?-glucose and the formula of Z-arabinose with that
of c?-galactose. An additional interest lies in the fact that galactose and
arabinose occur so frequently together in gums, while other polysac-
charides give glucose and xylose on hydrolysis.

Expt. 75. To show the presence of both hexoses and sucrose in the Zea/ (Davis, Daish
and Sawyer, 17). Take about 5 gms. of fresh leaf of either the Beet or Mangold.
i» [Leaves of the Garden Nasturtium {Tropaeolum majus) and Wild Chervil {Chaero-
phyllum sylvestre) may also be used.] Tear them into small pieces and drop them
into boiling 90-98 % alcohol in a flask on a water-bath. In this way the enzymes of
the leaf are killed, and no changes will occur in the carbohydrates present. After
boiling for a short time, the alcohol is filtered off and the extraction repeated.
Evaporate the filtrate to dryness in an evaporating dish on a water-bath. The
filtrate will contain chlorophyll and various pigments, sugars, glucosides, aromatic
compounds and other substances according to the plant used. Then add about 20 c.c.
of water and at intervals a few drops of basic lead acetate until it ceases to form
a precipitate. By this means all hexoses combined with aromatic substances as
glucosides (see p. 157) are precipitated as insoluble lead salts. The precipitate is
filtered off and the lead in the filtrate removed by 1 % sodium carbonate, avoiding

V] CARBOHYDRATES 75

excess. Filter again and the filtrate will contain the sugars. Boil the latter and add
Fehling's solution drop by drop till reduction ceases. Filter off the copper oxide and
then boil the solution with dilute sulphuric acid for a few minutes and make neutral
to litmus. Reduction will occur on adding more Fehling and boiling, owing to the
inversion of the cane-sugar present.

Expt. 76. To show the presence of hexoses in the leafhy means of the formation of
glucosazone. Leaves of Beta, Chaerophyllum sylvestre, or Tropaeolum may be used.
Extract as in the previous experiment and precipitate the glucosides with the minimal
amount of basic lead acetate. Test for osazone in the filtrate as in Expt. 41 {d).

Expt. 11. To obtain starch from green leaves. Weigh out 25 gms. of leaflets of the
Pea {Pisum sativum). The leaves should have been picked in the evening after a
sunny day, and it does not matter if the cut leaves are left overnight. Dip the leaf-
lets for a moment into boiling water, remove excess of water and drop them into
200 c.c. of 96-98% alcohol and boil till the chlorophyll is extracted: then filter.
Take the residue of leaves and pound (but not finely) in a mortar and then wash
thoroughly with distilled water. Filter through muslin and press free from water
(this process extracts most of the protein). Boil the residue with 100 c.c. of water and
filter. To the filtrate add iodine. At first the colour may disappear owing to the
presence of protein in solution in addition to the starch. When more iodine is added
a deep blue coloration is formed.

Plant Enzymes which hydrolyze Carbohydrates.

Diastase. In the plant starch may be regarded as a reserve product.
It is synthesized from sugar, and may be again hydrolyzed into sugar.
It can be shown experimentally that starch is converted into glucose by
boiling with acids, but in the plant the hydrolysis of starch is catalyzed
by the enzyme, diastase. Although the reaction is doubtless of con-
siderable complexity, it may, broadly speaking, be represented as
follows:

(C6Hio05)„-}-H20 >■ (C6Hio05)a; + Ci2H220ii

Dextrin Maltose

Thus the final products under these conditions are dextrin and the
disaccharide, maltose; and not glucose.

It is reasonable to assume that cells which contain starch also either
contain, or are capable of producing, diastase. But the amount of
diastase present, or at any rate capable of being extracted, varies in
different tissues. Diastase, like most enzymes, is soluble in water. In
many cases, however, a water-extract from fresh crushed tissues in which
diastase occurs, will not contain any appreciable amount of enzyme.
This is sometimes due to the fact that the protoplasm does not readily
yield up the enzyme until it has been killed. If the tissues are dried at
a moderate temperature (30-40° C.) both the powdered leaves them-
selves and a water extract are fairly rich in diastase ; or, if the living

76 CARBOHYDRATES [ch.

tissues are macerated and extracted with water to whicli chloroform
has been added, the cells die more rapidly and yield up the enzyme to
the solvent. From such a water extract, a crude precipitate containing
the enzyme may be obtained by addition of alcohol. For obtaining the
maximum results with diastatic activity in leaves, a water extract should
be made after they have been killed, either by drying, or by the action
of toluol or chloroform.

It has been shown (Brown and Morris, 6) that in leaves which con-
tain tannin, the presence of the latter largely inhibits the action of the
enzyme and may be the cause, in such cases, of an entire lack of activity
in the extract.

The diastatic activity of leaves appears to vary largely in different
genera and species. The subject has been investigated (Brown and
Morris, 6) and a list of their relative activities has been drawn up as
follows.

[The numbers represent the amount of maltose, expressed in grams, which 10
gms. of air-dried leaf will produce from soluble-starch (starch treated with dilute
hydrochloric acid) by hydrolysis in 48 hrs. at 30° C]

Pisum sativum 240*30 Helianthus annuus 3-94

Phaseolus multiflorus 110'49 H. tuberosus 378

Lathyrus odoratus 100*37 Funkia sinensis 5*91

L. pratensis 34*79 Allium Cepa 3*76

Trifolium pratense 89*66 Hemerocallis fulva 2*07

T. ochroleucum 56*21 Populus sp 3*79

Vicia sativa 79*55 Syringa vulgaris 2*53

V. hirsuta 53*23 Cotyledon Umbilicus 4*61

Lotus corniculatus 19*48 Humulus Lupulus 2*01-9*60

Lupinus sp 3*51 Hymenophyllum demissum ... 4*20

with Clover 27*92 Hydrocharis Morsus-ranae ... 0*267

Tropaeolum majus 3*68-9*64

From the above table it is seen that the leaves of genera of the
Leguminosae are apparently very rich in diastase. Whether this is so,
or whether in other plants the diastatic activity is inhibited by other
substances, has not yet been ascertained. As mentioned above, tannins
inhibit the action of diastase, and hence leaves rich in tannin, e.g. Hop
{Hamulus), cannot be expected to yield good results.

The tissues of germinating barley (Hordeum vulgare) also contain
large quantities of diastase, and this material can be used to demonstrate
the solubility, isolation and activity of the enzyme.

The action on starch of diastase from the leaf of the Common Pea
(Pisum sativum) and from germinating barley grains has already been

V] CARBOHYDRATES 77

demonstrated [see Expt. 57 (a) and (b)] in connexion with dextrin. The
following experiments have special reference to the enzyme.

jEJxpt. 78. To demonstrate the activity of diastase from germinating barley. {See
also Expt. 57.)

Pound up 2-3 gins, of germinated barley grains in a mortar and extract the mass
with 50 c.c. of water. Filter, and take two equal portions in two test-tubes. Boil one
tube. To both tubes add an equal quantity of the starch solution prepared as in
Expt. 55. Place the tubes in a beaker of water at 38-40° C. From time to time
withdraw a drop from each tube with a pipette and test with iodine solution on a
white tile. The starch in the unboiled tube will gradually give the dextrin reactions
(see p. 59) ; that in the boiled tube will remain unchanged.

This simple method may also be adopted for showing the diastatic activity of
leaves. Instead of germinating barley, a few leaflets of the Pea {Pisum sativum) or
Clover {Trifolium pratense) should be pounded up in a mortar and extracted with
50 c.c. of water and filtered.

Maltase. This enzyme hydrolyzes maltose into two molecules of
glucose:

C12H22O11 + H2O = 2C6H12O6.

Investigations upon maltase have, until recently, produced rather
contradictory results, but later work (Davis, 14: Daish, 15, 16) has led
to more satisfactory conclusions. The latter show that maltase is most
probably present in all plants in which hydrolysis of starch occurs. It
has been detected in leaves of the Nasturtium {Tropaeolum), the Potato
{Solarium), the Dahlia, the Turnip (Brassica), the Sunflower (Helianthus)
and the Mangold (Beta), and it is most probably widely distributed in
foliage leaves. Its detection is not easy for various reasons which are
as follows. It is not readily extracted from the tissues by water : it is
unstable, being easily destroyed by alcohol and chloroform. Its activity
is also limited or even destroyed at temperatures above 50° C. Hence
the extraction of maltase, by merely pounding up tissues with water,
does not yield good results : moreover, as an antiseptic, toluol must be
used and not chloroform. Finally, if the enzyme is to be extracted from
dried material, this must not be heated at too high a temperature previous
to the extraction.

Maltase occurs in quantity in both germinated and ungerminated
seeds of cereals. If, in kilning, malt has not been heated at too high a
temperature, the maltase may not be destroyed, and, in such cases, malt
extract will contain both diastase and maltase. This would explain the
fact that glucose, instead of maltose, has sometimes been obtained by
the action of malt diastase on starch. In other cases, when a higher
temperature has been employed, the maltase will be destroyed. Maltase

78 CARBOHYDRATES [ch.

itself, of course, does not act directly upon starch but only on maltose.
The use of chloroform, as an antiseptic, by some observers explains how
they came to overlook the presence of maltase, thus obtaining maltose,
and not glucose, as an end product in hydrolysis by malt extracts. The
optimum temperature for the maltase reaction is 39° C.

The presence of maltase in leaves is not readily shown for the
following reasons. Since maltase is destroyed by alcohol, the prepara-
tion of a crude precipitate of the enzyme by precipitating a water
extract of the leaves is not satisfactory. If the water extract is added
directly to maltose, and incubated, hydrolysis may be demonstrated by
determining the reducing power of the sugars formed. A control
experiment must, however, be made by incubating the water extract
alone, and subsequently determining the reducing power of any sugars
present.

Invertase. This enzyme hydrolyzes cane-sugar into one molecule of
glucose and one molecule of laevulose :

C12H22O11+ H20 = C6Hi206-f-C6Hi206.

Invertase is probably very widely distributed in plants. Its presence
has been demonstrated in the leaves and stem, though not in the root,
of the Beet (Beta vulgaris) (Robertson, Irvine and Dobson, 28). Also in
the leaves of a number of other plants (Kastle and Clark, 22). Its de-
tection, by its action on sucrose, is not easy on account of the presence
of other enzymes and reducing sugars in leaf extracts.

The absence of invertase from the root of the Beet raises a difficulty
as to how the cane-sugar is synthesized from the hexoses supplied from
the leaves (see p. 73). Some observers (Robertson, Irvine and Dobson,
28) incline to the view that cane-sugar is synthesized in the stems and
travels as such to the roots. Others (Davis, Daish and Sawyer, 17)
maintain that the cane-sugar is synthesized in the root, even though
invertase is absent.

V] CARBOHYDRATES 79

REFERENCES

Books

1. Abderhalden, E. Biochemisches Handlexikon, ii. Berlin, 1911.

2. Armstrong, B. P. The Simple Carbohydrates and the Glucosides. London,
1919. 3rded.

3. Atkins, W. R. G. Some Recent Researches in Plant Physiology. London,
1916.

4. Mackenzie, J. E. The Sugars and their Simple Derivatives. London, 1913.

Papers

5. Blackman, F. P. The Biochemistry of Carbohydrate Production in the
Higher Plants from the Point of View of Systematic Relationship. N. Phytol.^ 1921,
Vol. 20, pp. 2-9.

6. Bro"wn, H. T., and Morris, G. H. A Contribution to the Chemistry and
Physiology of Foliage Leaves. J. Chem. Soc, 1893, Vol. 63, pp. 604-677.

7. Clay son, D. H. P., Norris, P. W., and Schryver, S. B. The Pectic
Substances of Plants. Part II. A Preliminary Investigation of the Chemistry of the
Cell- Walls of Plants. Biochem. J., 1921, Vol. 15, pp. 643-653.

8. Czapek, P. Ueber die sogenannten Ligninreactionen des Holzes. Zs.
physiol. Chem., 1899, Vol. 27, pp. 141-166.

9. Davis, W. A., and Daish, A. J. A Study of the Methods of Estimation
of Carbohydrates, especially in Plant-extracts. A new Method for the Estimation
of Maltose in Presence of other Sugars. J, Agric. Set., 1913, Vol. 5, pp. 437-468.

10. Davis, W. A., and Daish, A. J. Methods of estimating Carbohydrates. II.
The Estimation of Starch in Plant Material. The Use of Taka-Diastase. J. Agric.
Sci, 1914, Vol. 6, pp. 152-168.

11. Daish, A. J. Methods of Estimation of Carbohydrates. III. The Cupric
Reducing Power of the Pentoses— Xylose and Arabinose. J. Agric. Sci., 1914, Vol. 6,
pp. 255-262.

12. Davis, W. A., and Sawyer, G. 0. The Estimation of Carbohydrates.
IV. The Presence of Free Pentoses in Plant Extracts and the Influence of other
Sugars on their Estimation. J. Agric. Sri., 1914, Vol. 6, pp. 406-412.

13. Davis, W. A. The Hydrolysis of Maltose by Hydrochloric Acid under the
Herzfeld Conditions of Inversion. A Reply to A. J. Kluyver. J. Agric. Sci.^ 1914,
Vol. 6, pp. 413-416.

14. Davis, W. A. The Distribution of Maltase in Plants. I. The Function of
Maltase in Starch Degradation and its Influence on the Amyloclastic Activity of
Plant Materials. Biochem. J., 1916, Vol. 10, pp. 31-48.

15. Daish, A. J. The Distribution of Maltase in Plants. II. The Presence of
Maltase in Foliage Leaves. Biochem. J., 1916, Vol. 10, pp. 49-55.

16. Daish, A. J. The Distribution of Maltase in Plants. III. The Presence of
Maltase in Germinated Barley. Biochem,. J., 1916, Vol, 10, pp. 56-76.

17. Davis, W. A., Daish, A. J., and Sawyer, G. C. Studies of the Forma-
tion and Translocation of Carbohydrates in Plants. I. The Carbohydrates of the
Mangold Leaf. J. Agric. Sci., 1916, Vol. 7, pp. 255-326.

80 CARBOHYDKATES

18. Davis, W. A. Studies of the Formation, etc. II. The Dextrose-Laevulose
Ratio in the Mangold. J. Agrie. Sci., 1916, Vol. 7, pp. 327-351.

19. Davis, W. A., and Sa-wyer, G. C. Studies of the Formation, etc. III.
The Carbohydrates of the Leaf and Leaf Stalks of the Potato. The Mechanism of
the Degradation of Starch in the Leaf. J. Agric. Set., 1916, Vol. 7, pp. 352-384.

20. Davis, W. A. The Estimation of Carbohydrates. V. The supposed Pre-
cipitation of Reducing Sugars by Basic Lead Acetate. J. Agric. Sci., 1916, Vol. 8,
pp. 7-15.

21. Haynes, D. The Gelatinisation of Pectin in Solutions of the Alkalies and
the Alkaline Earths. Biochem. J., 1914, Vol. 8, pp. 553-583.

22. Kastle, J. H., and Clark, M. B. On the Occurrence of Invertase in
Plants. Amer. Chem. J., 1903, Vol. 30, pp. 421-427.

23. Neville, A. Linseed Mucilage. J. Agric. Sci., 1913, Vol. 5, pp. 113-128.

24. Parkin, J. Contributions to our Knowledge of the Formation, Storage and
Depletion of Carbohydrates in Monocotyledons. Phil. Trails. R. Soc, B Vol. 191,
1899, pp. 35-79.

25. Parkin, J. On a Reserve Carbohydrate which produces Mannose, from the
Bulb oiLilium. Proc. Camb. Phil. Soc, 1900-1902, Vol. 11, pp. 139-142.

26. Parkin, J. The Carbohydrates of the Foliage Leaf of the Snowdrop
{Galanthus nivalis), and their Bearing on the First Sugar Of Photosynthesis. Biochem.
J., 1911, Vol. 6, pp. 1-47.

27. Priestley, J. H. Suberin and Cutin. N. Phytol., 1921, vol. 20, pp.17-29.

28. Robertson, R. A., Irvine, J. 0., and Dobson, M. B. A Polarimetric
Study of the Sucroclastic Enzymes in Beta vulgaris. Biochem. J., 1909, Vol. 4,
pp. 258-273.

29. Salko"Wski, B. Ueber die Darstellung des Xylans. Zs. physiol. Chem.
1901-2, Vol. 34, pp. 162-180.

30. SalkovTSki, B. Ueber das Verhalten des Arabans zu Fehling'scher Losung.
Zs. physiol. Chem., 1902, Vol. 35, pp. 240-245.

31. Schryver, S. B., and Haynes, D. The Pectic Substances of Plants.
Biochem. J., 1916, Vol. 10, pp. 539-547.

32. Schulze, E., Steiger, B., und Max-well, W. Zur Chemie der Pflanzen-
zellmembranen. I. Abhandlung. Zs. physiol. Chem., 1890, Vol. 14, pp. 227-273.

33. Spoehr, H. A. The Carbohydrate Economy of Cacti. Carnegie Institution
of Washington Publication, 1919, No. 287.

34. Tutin, P. The Behaviour of Pectin towards Alkalis and Pectase. Biochem.
J., 1921, Vol. 15, pp. 494-497.

CHAPTER VI

THE VEGETABLE ACIDS

Though the name "vegetable acids" might strictly be applied to all
acids found in plants, it is, as a rule, restricted to certain acids and
hydroxy-acids of the methane, ethylene and acetylene series.

We may take first the acids of the methane series which biologically
fall into two groups, the simpler members associated with fundamental
metabolism and the more complex ones associated with fat formation.
The first six members, at least, may be included among the vegetable
acids in the narrow sense. They are liquids, readily volatile in steam,
and several of them, without doubt, are closely involved in some of the
most fundamental and important reactions of plant metabolism. In fact
their relationships to certain of the amino-acids which are constituents
of most proteins, cannot be too strongly emphasized. The higher
members (with ten and more carbon atoms) are solids insoluble in water.
The glycerol esters of certain of these higher members are important
constituents of the plant fats and will be considered in the following
chapter. The first six representatives of the series are :

Acids of the methane series Corresponding amino-acids

Formic acid H • COOH

Acetic acid CH3 • COOH amino-acetic acid or glycine

Propionic acid CH3 • CH2 * COOH amino-propionic acid or alanine

Butyric acid CHg- CHg •CH2- COOH

Valeric acid CH3 • CH2 ' CH2 * CH2 ' COOH amino-iso-valeric acid or valine

Caproic acid CH3 • CH2 * CHg • CH2 • CH2 ' COOH amino-iso-caproic acid or leucine

Formic acid can be obtained by submitting plants to steam distil-
lation. This indicates that it probably exists in the free state in plants,
though there is the possibility of its being formed from other substances
during distillation. There is good evidence (Dobbin, 1), however, that it is
present in the stinging hairs of the Nettle ( Urtica dioica). It is a liquid
which is volatile with steam and can be readily reduced to formaldehyde
with nascent hydrogen.

o. 6

82 THE VEGETABLE ACIDS [ch.

Expt. 79. Tests for formic acid. Make a solution of formic acid (1 c.c. acid :
100 c.c. water) and perform the following tests :

{a) Acidify 10 c.c. with a few drops of strong hydrochloric acid and add a little
magnesium powder. The formic acid will be reduced to formaldehyde. Filter and
test for the latter by Schryver's test (see p. 39).

(6) Neutralize a few c.c. of the solution with dilute caustic soda and add a few
drops of 5 "/o mercuric chloride solution and heat. The mercuric salt is reduced to
mercurous chloride which is precipitated, being insoluble.

Expt. 80. Detection of formic acid in the Nettle (Urtica dioica). Take a strong
filter-paper (about 10 cms. in diameter) of the best quality and soak it in a concen-
trated solution of barium hydroxide. Allow the paper to dry in air, whereby the
barium hydroxide is converted into carbonate. Take at least 200 Nettle leaves, and,
with gloved hands, carefully blot both sides of the leaves between the folded paper.
Break up the paper in about 40 c.c. of distilled water, warm and filter on the pump.
Wash with 10 c.c. of hot water. To the filtrate containing barium formate add
0'5 gra. of glacial phosphoric acid and distil with a water condenser. Add about
20 drops of strong hydrochloric acid to the distillate and then magnesium powder.
When hydrogen is no longer evolved, filter,and test for formaldehyde by Schryver's
reaction. A positive result will be obtained.

Acetic acid has been found to occur in plants, both in the free state
and as salts and esters. Possibly, however, in some cases it may have
arisen from the decomposition of other substances during distillation.

Propionic acid has rarely been detected in plants. Butyric, isobutyric
and caproic acids have been detected in a few plants.

Isovaleric acid has been isolated from various plants, notably species
of Valerian ( Valeriana).

Esters of the above acids form important plant constituents since
they are responsible for many fruit odours. Amyl acetate, for instance,
occurs in the fruit of the Banana (Musa sapientum): amyl formate,
acetate and caproate are probably present in the fruit of the Apple
(Pyrus Malus), etc. Such compounds are frequently classed with the
"essential oils" (see p. 108).

The next group to be considered are the monohydroxy-acids of the
methane series. Of these glycollic acid may be mentioned.

GlycoUic acid, or hydroxy-acetic acid, CHg'OH'COOH, has been
isolated from unripe fruit of the Grape and from the leaves of the Virginian
Creeper (Ampelopsis hederacea). Also from the Sugar-cane (Saccharum
ojfficinarum), the Lucerne (Medicago sativa) and the Tomato (Lycoper-
sicum esculentum). Its relationship to the amino-acid, glycine (see p. 134),
should be borne in mind.

VI] THE VEGETABLE ACIDS 83

The dibasic acids of the methane series contain several important
members :

Dibasic acids Corresponding amino-acids

Oxalic acid (C00H)2
Malonicacid CH2-(COOH)2

Succinic acid CH2 * CH2 * (C00H)2 amino-succinic or aspartic acid

Glutaric acid CH2 ' CH2 * CH2 * (C00H)2 amino-glutaric or glutaminic acid

Adipic acid CH2 • CH2 • CHg ' CH2 • (C00H)2

Oxalic acid occurs very frequently and widely distributed in plants,
usually as the calcium salt, and apparently less frequently as the sodium
and potassium salts. It has rarely been detected as the free acid. It is
especially abundant in spp. of Oxalis, in the Rhubarb {Rheum Rhaponticum)
and Sorrel (Rumeoo Acetosa). The calcium salt is precipitated on adding
calcium acetate to a solution of the acid. Calcium oxalate is insoluble
in acetic acid, but soluble in dilute mineral acids.

Ea;pt. 81. Tests for oxalic acid. Take a 2% solution of oxalic acid, neutralize
with caustic soda (or use a soluble oxalate) and make the following tests :

(a) To 5 c.c. add a few drops of 5 % calcium chloride solution. A white pre-
cipitate of calcium oxalate is formed. Divide the precipitate into two portions. To
one add an equal quantity of strong acetic acid : the precipitate is insoluble even
on heating. To the other add strong hydrochloric acid drop by drop : the precipitate
is soluble. Hence the free acid can be precipitated with calcium acetate but not with
calcium chloride.

(6) To 5 c.c. add a few drops of 5 % lead acetate solution. A white precipitate of
lead oxalate is formed. Add an equal quantity of strong acetic acid and warm ; the
precipitate is insoluble.

Ba:pt. 82. Preparation of calcium oxalate from leaves of the Sorrel (Rumex Acetosa).
Take 100 gms. of fresh leaves of the Sorrel. Boil them in an evaporating dish with
200 c.c. of water and squeeze the boiled mass through linen. Boil the filtrate again
and filter on a pump. Acidify the filtrate with acetic acid, and add a concentrated
solution of calcium acetate until no more precipitate is formed. The precipitate
cannot readily be filtered off" so that it should be allowed to settle for 12 hours. Then
decant ofl:' the liquid and boil up the precipitate in the minimum amount of 10 ^/^
hydrochloric acid. On cooling, calcium oxalate will separate out in characteristic
crystals. On examining under the microscope, these will be seen to be octahedra,
giving the appearance of a square with a diagonal cross (envelope form). Leaves of
Rhubarb {Rheum Rhaponticum) can also be used, taking about 250 gms. in 500 c.c.
of water.

It is stated that there is an enzyme widely distributed in plants
(Staehelin, 3) which has the power of decomposing oxalic acid with the
production of carbon dioxide.

6—2

84 THE VEGETABLE ACIDS [ ch.

Malonic acid has been isolated from the Sugar Beet {Beta vulgariif
var. Rapay, It forms insoluble calcium and lead salts.

Succinic acid is probably widely distributed in plants. It has been
isolated from the unripe Grape, from fruit of the Gooseberry, Currant,
Apple and Banana, from Rhubarb {Rheum Rhaponticum), Greater
Celandine {Chelidonium majus) and other plants. Succinic acid crystallizes
well in rhombic prisms or plates. It is not very readily soluble in cold
water, though more so in hot. Its salts with the alkali metals are readily
soluble. Calcium succinate is deposited as acrystalline precipitate on adding
calcium chloride to fairly concentrated solutions of the acid after neutrali-
zation (or of a soluble succinate), but from a dilute solution it is not
precipitated except on addition of alcohol. Barium succinate comes
down as a crystalline precipitate even from dilute solutions. Ferric
succinate is insoluble and its formation is used in the detection of the
acid.

The relationship of succinic acid to aspartic, or a-amino-succinic, acid
which is an abundant constituent of many proteins (see p. 134) should be
noted.

Expt. 83. Tests for succinic acid. A. Take a 1 % solution of succinic acid>
neutralize with caustic soda (or use a soluble succinate) and make the following tests :

(a) To 5 c.c, add a few drops of 5 ^q calcium chloride solution. A slight precipitate
is formed, especially on rubbing the sides of the tube with a rod. To another 5 c.c.
add again calcium chloride solution followed by an equal volume of 96 % alcohol.
A white precipitate of calcium succinate is formed.

(6) To 5 c.c. add a few drops of 5 % barium chloride solution. A crystalline
precipitate of barium succinate is formed and, again, its appearance is hastened by
rubbing the sides of the tube.

(c) To 5 c.c. add a few drops of 5 7o lead acetate solution. A white precipitate of
lead succinate is formed. Add an equal quantity of strong acetic acid. The pre-
cipitate is soluble.

{d) To 10 c.c. add about 1-2 c.c. of 5% ferric chloride solution. A red-brown
gelatinous precipitate of ferric succinate is formed. Filter oflf the precipitate, wash
well and boil with about 20 c.c. of dilute ammonia. Filter off the ferric hydroxide,
and to the filtrate, after boiling off any excess of ammonia, add 5 % barium chloride
solution. A crystalline precipitate of barium succinate is formed. This test con-
stitutes a method for identifying succinic acid.

B. Make a cold concentrated solution of succinic acid, neutralize (or use a soluble
succinate) and add 5 ^Jq calcium chloride solution. A crystalline precipitate of calcium
succinate will separate out. Its appearance may be hastened by rubbing or shaking.

1 It should be noted that an exceptionally large number of chemical substances have
been isolated from the Sugar Beet on account of their accumulation in the waste products
from sugar manufacture. There is little doubt that the same substances could be isolated
from other plants if sufficient quantity of material were employed.

Yi] THE VEGETABLE ACIDS 85

Glutaric and adipic acids have been detected in extracts from the
root of the Sugar Beet (Beta vulgaris var. Rapa). It is probable that
they also occur in other plants. The relationship of glutaric acid to
glutaminic acid is important (see p. 134).

Of the monohydroxy-dibasic acids, malic acid is the best known.

Malic acid. It should be noted that in constitution malic acid is a
hydroxy -succinic acid. It is widely distributed in plants, being found
in many fruits, such as those of the Apple, Pear, Cherry, etc. ; also in
leaves and vegetative parts, especially in some succulents (Crassulaceae,
Mesembryq^nthemum).

Malic acid crystallizes in colourless needles which are very deli-
quescent and hence difficult to obtain. Its salts with the alkali metals
are soluble. Calcium malate is only precipitated from a very concentrated
solution of the acid (after neutralization) or of a soluble malate. Very
few well-defined tests can be made for malic acid.

Bxpt. 84. Tests for malic acid. A. Take a 2 o/q solution of malic acid, neutralize
with caustic soda (or use a soluble malate) and make the following tests :

(«) Add a few drops of 5 o/o calcium chloride solution. No precipitate is formed,
but the addition of an equal volume of 96 o/q alcohol will bring down a precipitate of
calcium malate.

(6) Add a few drops of 5 % lead acetate solution. A white precipitate of lead
malate is formed. Add a little acetic acid and warm. The precipitate dissolves.

B. Heat a little solid malic acid in a dry test-tube. It melts and then gives oflf
fumes of maleic acid which condense in white crystals on the cooler parts of the
tube.

Expt. 85. Preparation of malic acid from apples. Take six apples (total weight
from 500-700 gms.). Cut them into thin slices and drop the slices as quickly
as possible into the minimum amount of boiling alcohol in a conical flask. In
this way the oxidizing enzymes are destroyed, and brown oxidation products are
avoided. After well boiling, filter through paper. Neutralize the filtrate to litmus
with sodium hydroxide solution, and add concentrated calcium chloride solution
until a precipitate ceases to be formed. Allow the precipitate of calcium malate to
settle and then add alternately a few drops of calcium chloride solution and a
little alcohol to ensure complete precipitation. Decant, and filter off the calcium
malate. Dissolve the malate in the minimum amount of hot water, filter and add
concentrated lead acetate solution until a precipitate of lead malate ceases to be
formed. Filter off the lead malate, suspend it in a minimum amount of water, and
pass in sulphuretted hydrogen until the malate is decomposed. Filter and concen-
trate in a crystallizing dish on a water-bath. Crystals of malic acid are deposited.
Test as in Expt. 84.

86 THE VEGETABLE ACIDS [ch.

Of the dihydroxy-dibasic acids, tartaric acid is the best known. It
should be noted that tartaric acid is dihydroxy-succinic acid. Thus the
three acids are related as follows:

Succinic acid COOPI • CHg • CHg ' COOH
Malic acid COOH -CHOH 'CHa 'COOH
Tartaric acid COOH • CHOH • CHOH • COOH

Tartaric acid is widely distributed in plants, often in the form of
the calcium or potassium salts. It occurs in many fruits, as for instance,
those of the Grape (Vitis vinifera), Tomato (Lycopersicum esculentum).
Mountain Ash (Pyrus Aucuparia) and Pineapple {Ananas sativus); it
has also been detected in the leaves and vegetative parts of many plants.
Tartaric acid is easily soluble in water from which it crystallizes in colour-
less prisms. Calcium tartrate is only slightly soluble in cold water, though
more so in hot. On adding calcium chloride to a soluble tartrate, calcium
tartrate is precipitated, more or less rapidly according to the strength of
the solution, and sometimes as a crystalline precipitate. The crystals may
occur as characteristic rhombic prisms with octahedral faces or as needles.
The precipitate is soluble in acetic acid. The acid potassium salt of
tartaric acid is soluble with difficulty in water and hejice is used in
identification of the acid.

Racemic acid, which is a combination of dextro- and laevo-tartaric
acids, is also found in certain varieties of the Grape. Calcium racemate
is insoluble in acetic acid. It is soluble in hydrochloric acid from which
it separates out rapidly in a crystalline state on neutralizing with ammonia
(tartrate only separates slowly).

Expt. 86. Tests for tartaric acid. A. Take a 1 % solution of tartaric acid,
neutralize with caustic soda (or use a soluble tartrate) and make the following tests r

(a) Add a few drops of 5 % calcium chloride solution. A white precipitate of
calcium tartrate is formed. Add an equal volume of glacial acetic acid and warm ;
the precipitate dissolves.

(6) Add a few drops of 5 % lead acetate solution. A white precipitate of lead
tartrate is formed. Add acetic acid and warm ; the precipitate dissolves.

(c) To 2-3 c.c. in a test-tube add a few drops of ferrous sulphate solution. Place
the test-tube in a beaker of cold water, and add a few drops of hydrogen peroxide
followed by an excess of caustic soda solution. A deep violet or blue colour is obtained.
The colour is due to the formation of dihydroxymaleic acid and the reaction of this
with the ferric salt present.

{d) To one drop of tartrate add 2 drops of a 2 % solution of resorcinol and then
3 c.c. of strong sulphuric acid. Heat gently. A rose colour is formed which deepens
to a violet-red.

VI] THE VEGETABLE ACIDS 87

B. Take 2-3 cm. of a strong solution of tartaric acid, acidify with glacial acetic
acid and add a little potassium acetate solution. A white crystalline precipitate of
potassium hydrogen tartrate will be formed.

Expt. 87. Identification of tartaric acid in grapes. Take 150-200 gms. of unripe
gi-apes (early July) and boil them well with the minimum amount of water in an
evaporating dish. As they soften they should be well stirred and crushed. Then
filter and squeeze the mass through strong linen. Neutralize the filtrate with caustic
soda, heat to boiling and filter on a pump. Cool the filtrate, and add 2-3 c.c. of
saturated calcium chloride solution. Allow the mixture to stand for 24 hours.
A crystalline precipitate will separate out. Under the microscope this will be seen
to consist of needles and octahedra. The needles are a double salt of ci?-tartaric and
^-malic acid (Ordonneau, 2) ; the octahedra consist either of tartaric acid or racemic
acid or a mixture of both. Filter off" this precipitate and heat in 50 ^Jq acetic acid.
The double salt and the tartaric acid will dissolve, but octahedra of racemic acid (if
present) will remain undissolved. Filter and make the following tests with the filtrate :

(a) Add to a small quantity in a test-tube, resoreinol and sulphuric acid as in
Expt. 86 A{d); a positive result is given.

(6) Evaporate down the remainder on a water-bath and add potassium acetate
and acetic acid as in Expt. 86 ^ ; potassium hydrogen tartrate crystallizes out.

If octahedra are left undissolved after treating with 50% acetic acid, racemic
acid is present. Heat this residue with dilute hydrochloric acid. It will go into
solution. Neutralize a portion with ammonia, and the acid will crystallize out at
once. Test another portion with resoreinol as in (a) ; a positive result will be given.

Of the tribasic acids, citric acid, C3H4 • OH * (C00H)3, is the most
important.

Citric acid occurs in large quantities in fruits of the genus Citrus, i.e.
in the Orange, Lime, Lemon, etc. Also in many other fruits, such as the
Gooseberry, Currant, Tomato, etc.

Expt. 88. Tests for citric acid. A. Take a 1 % solution of citric acid, neutralize it
with caustic soda (or use a soluble citrate) and make the following tests :

(a) Add 5 o/^ calcium chloride solution. No precipitate is given. Heat to boiling
and a white precipitate of calcium citrate is formed. Calcium citrate is soluble in
cold water but insoluble in hot water.

(6) Add 5 «/o lead acetate solution. A white precipitate of lead citrate is formed.
Add an equal quantity of acetic acid and warm : the precipitate is soluble.

B. Take 5 c.c. of a 2 % solution of citric acid and add 3 c.c. of Denig^s' reagent
(prepared by dissolving with the aid of heat 1 gm. of mercuric oxide in a mixture of
4 c.c. of strong sulphuric acid and 20 c.c. of distilled water). Boil, and add a 2 %
solution of potassium permanganate drop by drop. The permanganate is at first
decolorized, but, on further cautious addition, the colour persists. Finally the liquid
becomes turbid and a white precipitate forms. This is due to a mercury compound
of acetone-dicarboxylic acid, resulting from the oxidation of citric acid by the per-
manganate.

88 THE VEGETABLE ACIDS [ch. vi

C. Heat gently a few crystals of citric acid for some time with an equal weight
of resorcinol and a few drops of concentrated sulphuric acid. Add excess of alkali ;
the solution shows a fine blue fluorescence due to the presence of a product, resocyan.

Expt. 89. Preparation of citric acid from lemons. Squeeze the juice from three
lemons and filter through muslin. Measure the volume of the juice, and add strong
caustic soda solution, carefully, until the reaction is slightly alkaline. Filter and for
every 10 c.c. of juice, add 5 c.c. of a 10% solution of calcium chloride. No precipitate
is formed. Now heat to boiling and a copious precipitate of calcium citrate is formed.
Filter off, while hot, on a filter-pump, wash with a little boiling water, drain well and
dry in the air. Weigh and add the requisite amount of sulphuric acid (1 gm. of
citrate=15 c.c. of normal sulphuric acid). Allow the mixture to stand for a short
time, filter and concentrate the filtrate in a glass dish on a water bath. Crystals of
citric acid separate out on concentrating considerably. (If, for any reason, insufficient
sulphuric acid has been added, some calcium citrate may separate out first on con-
centrating. If so, add a few drops of sulphuric acid, filter and continue to concen-
trate.) Drain off the citric acid on a filter-pump, dissolve in water and make the
tests in Expt. 88.

The acids of the ethylene series have not as yet been very widely
detected.

Fumaric acid, COOH • CH = CH * COOH, occurs in the Fumariaceae
(Fumaria, Corydalis) and Papaveraceae (Glaucium).

Aconitic acid, COOH • CH^ * C • COOH • CH • COOH, is found in the
Monkshood (Aconitum) and other genera of the Ranimculaceae.

The best known acid of the acetylene series is sorbic acid, found in
berries of the Mountain Ash {Pyrus Aucuparia).

REFERENCES

1. Bobbin, L. On the Presence of Formic Acid in the Stinging Hairs of the
Nettle. Proc. Roy. Soc, Edinburgh, 1920, Vol. 39, pp. 137-142.

2. Ordonneau, Ch. De I'acidit^ des raisins verts et de la preparation de
I'acide malique. Bull, de la soc. ckim., 1891, Vol. 6, pp. 261-264.

3. Staebelin, M. Die RoUe der Oxalsaure in der Pflanze. Enzymatischer Abbau
des Oxalations. Biochem. Zeitschr., 1919, Vol. 96, pp. 1-49.

CHAPTER VII

FATS AND ALLIED SUBSTANCES

A FAT may be defined as an ester or glyceride of a fatty acid. Just
as an inorganic salt, such as sodium chloride, is formed by the reaction
of hydrochloric acid with sodium hydroxide, so a fat is formed by the
reaction of the trihydric alcohol, glycerol, and a fatty acid.

The word fat is not a familiar one in botanical literature, the term
oil being more commonly used. It is generally met with in connexion
with the reserve products of seeds. The oils of seeds are, however, true
fats. The term oil may be misleading to some extent, because a fat
which is liquid at ordinary temperatures is usually spoken of as an oil,
and yet there are also many other substances, of widely differing chemi-
cal composition, which have the physical properties of oils, and which are
known as such.

Most of the vegetable fats are liquid at ordinary temperatures but
some are solids.

The best-known series of acids from which fats are formed is the
series CnHgnOa of which formic acid is the first member. The other
members of the series are:

Formic acid
^ Acetic acid

Propionic acid
^ Butyric acid

Valeric acid
1 Caproic acid

(Enanthylic acid
1 Caprylic acid

Pelargonic acid
^ Capric acid

Undecylic acid
1 Laurie acid

Tridecylic acid

H-COOH

CH3-C00H

CaHs-COOH

CsHr-COOH

C4H,-C00H

C5Hn • COOH

CeHis-COOH

C7H15 • COOH

CgHn-COOH

CgHig-COOH

CioHgi-COOH
CnHsa'COOH
CiaHae-COOH

1 Myristic acid
Isocetic acid

1 Palmitic acid
Daturic acid

1 Stearic acid
Nonadecylic acid

1 Arachidic acid

1 Behenic acid

2 Lignoceric acid]
2 Carnaiibic acidj

Hyaenic acid
2 Cerotic acid
2 Melissic acid

C13H27 •
G14H29
CisHsi

C17H35
C18H37

C19H39
C21H43

C23H47

C24H49
C25H61
CtMriKQ

COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH

COOH

COOH
COOH
COOH

1 Occur in fats.

Occur in waxes.

90 FATS AND ALLIED SUBSTANCES [ch.

Another series is the oleic or acrylic series CnHgn-aOa of which the
members are:

Tiglic acid CsHyOa
Oleic acid C18H34O2
Elaidic acid C18H34O2
Iso-oleic acid C18H34O2
Erucic acid C22H42O2
Brassidic acid C22H42O2

Of these, oleic acid (as glyceride) is the most widely distributed.

Yet other series are:

The linolic CnH2„_402

The linolenic C„H2,i_602
The clupanodonic C„H2„_802
The ricinoleic C„H2„_203

The fat which occurs in an oil-containing seed is not composed of the
glyceride of one acid, but is a mixture of the glycerides of several, or
even a large number of different acids, often members from more than
one of the above series. Thus the fat of the fruit of the Coconut (Cocos
nucifera) consists of a mixture of the glycerides of caproic, caprylic,
Capric, lauric, myristic, palmitic and oleic acids. Linseed oil from the
seeds of Linum usitatissimmn again, is a mixture of the glycerides of
palmitic, myristic, oleic, linolic, lin"6lenic and isolinolenic acids. Similar
mixtures are found in other fruits and seeds.

Since glycerol is a trihydric alcohol, it would be possible for one or
more of the three hydroxyls to react with the acid to form mono-, di- or
tri-glycerides. All these cases occur and, sometimes, one hydroxyl is
replaced by one acid, and another hydroxyl by a different acid.

When the distribution of fats among the flowering plants is con-
sidered, they are found to be more w^idely distributed than the botanist
is generally led to suppose.

The following is a list of some of the plants especially rich in fats as
reserve material in the fruits or seeds. It represents only a selection of
the better known genera, since many other plants have fatty seeds. An
approximate percentage of oil present in the fruit or seed is given.

Gramineae: Maize {Zea Mays) 4 7o-

Palmaceae : Oil Palm {Elaeis guinensis) 62 "/^^ : Coconut Palm (Cocos
nucifera) 65 7o-

Juglandaceae : Walnut (Juglans regia) 52 Yo-
Betulaceae: Hazel {Gorylus AvelUma) 55 "/o-
Moraceae : Hemp {Cannabis sativa) 33 7o-
Papaveraceae : Opium Poppy (Papaver somniferum) 47 ^/o-

VII] FATS AND ALLIED SUBSTANCES 91

Cruciferae : Garden Cress (Lepidium sativum) 25 "/o : Black Mustard
(Sinapis nigra) 20 7o : White Mustard {Sinapis alba) 25 7o : Colza
(Brassica rapa var. oleifera) 33 7o : Rape (Brassica napus) 42 Yo-

Rosaceae : Almond {Prunus Amygdalus) 42 7o '- Peach (P. Persica)
35 Vo : Cherry (P. Gerasus) 35 Vo ; Plum (P. domestica) 27 7„.

Linaceae : Flax (Linum usitatissimum) 20-40 °/o-

Euphorbiaceae : Castor-oil (Ricinus communis) 51 Vo*

Malvaceae: Cotton {Gossypium herhaceum) 24%-

Sterculiaceae : Cocoa (Theobroma Gacao) 54 Yo-

Lecy thidaceae : Brazil Nut (Bertholletia excelsa) 68 "/o-

Oleaceae: OMve {Glea euroimea) 20-1 0^1 q-. Ash (Fraooinus excelsior)
27%.

Rubiaceae: Coffee (Goffea arabica) 12*'/o-

Cucurbitaceae: Pumpkin (Gucurbita Pepo) 41 °/o-

Compositae: Sunflower {Helianthus annuus) 38 "/o-

The conclusion must not be drawn from the above list that the seeds
of the plants mentioned have exclusively fats as reserve materials. In
many cases fat may be the chief reserve product, but in others it may
be accompanied by either starch or protein or both.

Some of the best-known examples of fat-containing seeds which yield
"oils" of great importance in commerce, medicine, etc., are Ricinus
(castor oil), Brassica (colza oil), Gossypium (cotton-seed oil), Gocos
(coconut oil), Elaeis (palm oil), Glea (olive oil).

In the plant the fats are present as globules in the cells of the fat-
containing tissues.

Plant fats may vary from liquids, through soft solids, to wax-like
solids which generally have low melting-points. They float upon water
in which they are insoluble. They are soluble in ether, petrol ether,
benzene, chloroform, carbon tetrachloride, carbon bisulphide, etc.: some
are soluble in alcohol. With osmic acid fats give a black colour, and they
turn red with Alkanet pigment which they take into solution.

Expt. 90. Tests for fats. Weigh out 50 gms. of Linseed {Linum usitatissimum)
and grind in a cofFee-mill. Put the linseed meal into a flask, cover with ether, cork
and allow the mixture to stand for 2-12 hrs. Filter off the ether into a flask, fit with
a condenser and distil off the ether over an electric heater. (If a heater is not avail-
able, distil from a water-bath of boiling water after the flame has been turned out.)
When the bulk of the ether is distilled off", pour the residue into an evaporating dish
on a water-bath and drive off the rest of the ether. With the residue make the
following tests in test-tubes :

(a) Try the solubilities of the oil in water, petrol ether, alcohol and chloroform
It is insoluble in water and alcohol, but soluble in petrol ether and chloroform.

92 FATS AND ALLIED SUBSTANCES [ch.

(6) Add a little 1 o/o solution of osmic acid. A black colour is formed. (This re-
action is employed for the detection of fat in histological sections.)

(c) Add to the oil a small piece of Alkanet (Anchusa ojicinalis) root, and warm
gently on a water-bath. The oil will be coloured red. Divide the oil into two portions
in test-tubes. To one add a little water, to the other alcohol. The coloured oil will
rise to the surface of the water in one case, and sink below the alcohol in the other.
The Alkanet pigment being insoluble in both water and alcohol, these liquids remain
uncoloured.

Keep some of the linseed oil for Expt. 91.

It is well known that the hydrocarbons of the unsaturated ethylene

series CJi^n will combine directly with the halogens, chlorine, bromine

and iodine to give additive compounds, thus:

C2H4-|-Br2 = C2H4Br2

ethylene bromide

The acids of this series also behave in the same way, and since many
plant fats contain members of the series, the fats will also combine with
the halogens.

FxpL 91, To show the presence of unsaturated groups in a fat. To a little of the
linseed extract add bromine water. Note the disappearance of the bromine and the
formation of a solid product.

. One of the most important chemical reactions of fats is that known
as saponification. When a fat is heated with an alkaline hydroxide the
following reaction takes place :

CnHgsCO-O— CHg

I
C17H35CO • O— CH +3K0H =3Ci7H35COOK+CH20H • CHOH • CHgOH

I glycerol

CnHssCOO— CH2

tristearin

The potassium salt, potassium stearate, of the fatty acid, stearic acid,
is termed a soap. The ordinary soaps used for washing are mixtures of
such alkali salts of the various fatty acids occurring in vegetable and
animal fats, and are manufactured on a large scale by saponifying fats
with alkali. The soaps are soluble in water, so that when a fat is heated
with a solution of caustic alkali, the final product is a solution of soap,
glycerol and excess of alkali. The soap is insoluble in saturated salt
(sodium chloride) solution, and when such a solution is added to the
saponified mixture, the soap separates out and rises to the surface of the
liquid. This process is known as "salting out." If the saponified mixture
is allowed to cool without salting out, it sets to a jelly-like substance.
When caustic potash is used for saponification and the product is allowed
to set, a ''soft" soap is formed. Hard soaps are prepared by using caustic
soda and salting out.

VII] FATS AND ALLIED SUBSTANCES 93

The properties of soaps in solution are important. When a soap goes
into solution, hydrolysis takes place to a certain extent with the for-
mation of free fatty acid and free alkali. The free fatty acid then forms
an acid salt with the unhydrolyzed soap. This acid salt gives rise to an
opalescent solution and lowers the surface tension of the water with the
result that a lather is readily formed.

The property of soaps of lowering surface tension is the reason for
their producing very stable emulsions when added to oil and water (see
chapter on colloids, p. 12).

Expt. 92. Hydrolysis of fat with alkali. Take 12 Brazil nuts, the seeds of Berthol-
letia (Lecythidaceae). Crack the seed coats and pound the kernels in a mortar. Put
the pounded nut in a flask, cover it with ether, and allow the mixture to stand for
2-12 hrs. Filter into a weighed or counterpoised flask and divstil off the ether as in
Expt. 90. Weigh the oil roughly and add 4-5 times its weight of alcoholic caustic
soda (prepared by dissolving caustic soda in about twice its weight of water and
mixing the solution with twice its volume of alcohol). Heat on a water-bath until
no oil can be detected when a drop of the mixture is let fall into a beaker of water.
Then add saturated sodium chloride solution. The soaps will rise to the surface.
Allow the soaps to separate out for a time and then filter. Press the soap dry with
filter-paper, and test a portion to see that it will make a lather. Neutralize the
filtrate from the soap with hydrochloric acid and evaporate as nearly as possible to
dryness on a water-bath. Extract the residue with alcohol and filter. Test the
filtrate for glycerol by means of the following tests :

{a) To a Uttle of the solution add a few drops of copper sulphate solution and
then some sodium hydroxide. A blue solution is obtained owing to the fact that
glycerol prevents the precipitation of cupric hydroxide.

(6) Treat about 5 c.c. of a 0*5 % solution of borax with suflBcient of a 1 7o solution
of phenolphthalein to produce a well-marked red colour. Add some of the glycerol
solution (which has first been made neutral by adding acid) drop by drop until the
red colour just disappears. Boil the solution : the colour returns. The reaction is
probably explained thus. Sodium borate is slightly hydrolyzed in solution and boric
acid, being a weak acid, is only feebly ionized, and therefore the solution is alkaline.
On adding glycerol, glyceroboric acid (which is a strong acid) is formed and so the
reaction changes to acid. On heating, the glyceroboric acid is hydrolyzed to glycerol
and boric acid, and the solution again becomes alkaline.

(c) Heat a drop or two with solid potassium hydrogen sulphate in a dry test-tube ;
the pungent odour of acrolein (acrylic aldehyde) should be noted,:

C3H803=C2H3-CHO-f-2H20.

In addition to Brazil nuts, the following material can also be used :
endosperm of Coconut, ground linseed, almond kernels and shelled seeds
of the Castor-oil plant (Ricinus): about 50 gms. should be taken in each
case.

94 FATS AND ALLIED SUBSTANCES [ch.

Expt. 93. ReactioTis of soaps, (a) Take some of the soap which has been filtered
off and shake up with water in a test-tube. A lather should be formed. (6) Make
a solution of a little of the soap in a test-tube and divide it into three parts. To each
add respectively a little barium chloride, calcium chloride and lead acetate solutions.
The insoluble barium, calcium and lead salts will be precipitated. (The curd which
is formed in the case of soap and hard water is the insoluble calcium salt.) Thirdly,
take the remainder of the soap and acidify it with dilute acid in an evaporating dish,
and warm a little on a water-bath. The soap is decomposed and the fatty acids are
set free and rise to the surface.

JExpt. 94. Reactions of fatty acids, (a) Try the solubilities in ether and alcohol
of the acids from the previous experiment. They are soluble, (b) Shake an alcoholic
solution of the fatty acids with dilute bromine water. The colour of the bromine is
discharged owing to the bromine forming additive compounds with the unsaturated
acids.

The question of the metabolism of fats in the plant is a very com-
plicated one and has not yet been satisfactorily investigated. All plants
may have the power of synthesizing fats, and a great number, as we
have seen, contain large stores of these compounds in the tissues of the
embryo, or endosperm, or both. The point of interest is that of tracing
the processes by which these fats are synthesized, and are again hydro-
lyzed and decomposed. The products of decomposition may serve for the
synthesis of other more vital compounds as the embryo develops, and
before it is able to synthesize the initial carbohydrates, and to absorb
the salts requisite for general plant metabolism.

One fact seems fairly clear, namely that when fat-containing seeds
germinate, an enzyme is present in the tissues which has the power of
hydrolyzing fats with the formation of fatty acids and glycerol. Such
enzymes are termed lipases.

The lipase which has been most investigated is that which occurs
in the seeds of the Castor-oil plant (Ricinus communis). It has been
shown that if the germinating seeds are crushed and allowed to auto-
lyze (p. 20) in the presence of an antiseptic, the amount of fatty acid
increases, whereas in a control experiment in which the enzyme has
been destroyed by heat, no such increase takes place (Reynolds Green,
13, 14).

Investigation has shown the enzyme to be present also in the resting
seed, but in an inactive condition as a so-called zymogen (Armstrong,
4, 5, 6, 7). The zymogen is considered to be a salt and, after acidifica-
tion with weak acid, the salt is decomposed, and the enzyme becomes
active. After the preliminary treatment with acid, however, the enzyme
is most active in neutral solution. The effect of acid on the zymogen

VII] FATS AND ALLIED SUBSTANCES 95

may be demonstrated by autolyzing the crushed seed with a little dilute
acetic acid; the increase of acidity will be found to be much greater
l-han in the case of a control, experiment in which acid has not been
added.

It has not been found possible to extract the enzyme from the resting
seed. An active material can be obtained by digesting the residue, after
extraction of the fat, with dilute acetic acid and finally washing with
water. This material can then be used for testing the hydrolytic power
of the enzyme on various fats.

There is little doubt that lipase catalyzes the synthesis of fats as
well as the hydrolysis; the reaction, in fact, has been carried out to a
certain extent in vitro.

Expt. 95. Demonstration of the existence of lipase in ungerminated Ricinus seeds.

A . Remove the testas from about two dozen Ricinus seeds and pound the kernels
up in a mortar. Into three small flasks («), {h) and (c), put the following:

{a) 2 gms. of pounded seed + 10 c.c. of water.

(6) 2 gms. of pounded seed + 10 c.c. of water + 2 c.c. of N/10 acetic acid.

(c) 2 gms. of pounded seed + 10 c.c. of water + 2 c.c. of N/10 acetic acid, and
boil well.

Add a few drops of chloroform to all three flasks, plug them with cotton-wool,
and allow them to incubate for 12 hours at 37° C. Then add 2 c.c. of N/10 acetic
acid to flask (a), and 25 c.c. of alcohol to all three flasks. Titrate the fatty acids
present with N/10 alkali, using phenolphthalein as an indicator. A greater amount
of fat should be hydrolyzed in (6) than in (a), and also slightly more in {a) than in
(c). The addition of alcohol checks the hydrolytic dissociation of the soap formed on
titration.

B. Pound up about 15 gms. of Ricinus seeds which have been freed from their
testas, and let the pounded mass stand with ether for 12 hrs. Then filter, wash with
ether and dry the residue. Weigh out three lots, of 2 gms. each, of the fat-free meal
and treat as follows :

(a) Grind up the 2 gms. of meal in a mortar with 16 c.c. of N/10 acetic acid
(i.e. 8 c.c. of acid to 1 gm. of meal), and let it stand for about 15 minutes. Then
wash well with water to free from acid, and transfer the residue to a small flask.
Add 5 c.c. of castor oil, 2 c.c. of water and a few drops of chloroform.

{h) Treat the 2 gms. of meal as in (a), but, after washing, and before transferring
to the flask, boil well with a little distilled water. Add 5 c.c. of oil, 2 c.c. of water
and a few drops of chloroform.

(c) Put the 2 gms. of meal into the flask without treatment and then add 5 c.c. of
oil, 2 c.c. of water and a few drops of chloroform.

Incubate all three flasks for 12 hours, and then titrate with N/10 caustic soda,
after addition of alcohol as in ^. A certain amount of acetic acid is always retained
by the seed residue, and this is ascertained from the value for flask (6). Flask (c) will
act as the control.

96 FATS AND ALLIED SUBSTANCES [ch.

Another question to be considered is the mode of synthesis in the
plant of the complex fatty acids which form the components of the fats.
No conclusive work has been done in this direction, but many investi-;
gators have held the view that the fats arise from carbohydrates, notably
the sugars. In fact, it has been shown that in Paeonia and Ricinus, as
the seeds mature, carbohydrates disappear and fats are formed.

The sequence of events, however, in the synthesis of fatty acids from

sugars is very obscure. If we examine the formulae, respectively, of a

hexose :

CH2OH CHOH CHOH -CHOH -CHOH CHO

and a fatty acid, e.g. myristic acid :

H3C — CH2 * CH2 ' CH2 ' CH2 ' CH2 * CH2 * CH2 * CH2 ' CH2 ' CH2 ' CH2 * CH2 ' COOH

it is seen that three main changes are concerned in the synthesis of such
a fatty acid from sugar, i.e. reduction of the hydroxyl groups of the sugar,
conversion of the aldehyde group into an acid group, and finally the
condensation or linking together of chains of carbon atoms. An inter-
esting fact in connexion with this point is that all naturally occurring
fatty acids have a straight, and not a branched, carbon chain and also
contain an even, and not an odd, number of carbon atoms. It has been
suggested (Smedley, etc., 15-17) that acetaldehyde and a ketonic acid,
pyruvic acid, may be formed from sugar. By condensation of aldehyde
and acid, another aldehyde is formed with two more carbon atoms. By
repetition of the process, with final reduction, fatty acids with straight
chains are produced.

WAXES

Waxes differ from fats in that they are esters of fatty acids with
alcohols of high molecular weight of the methane series in place of
glycerol. Such alcohols are cetyl alcohol, CigHgaOH, carnaiibyl alcohol,
C24H49OH, ceryl alcohol, CaeHggOH, and melissyl (or myricyl) alcohol,
CgoHeiOH, etc.

Waxes occur as a deposit on the leaves, fruits and stems of many
plants: they constitute, for instance, the "bloom" on the Grape, the
Plum and the leaves of Aloe, Mesembryanthemum, etc., though they
rarely occur in sufficient quantity to be readily collected. Nevertheless,
the waxes of various plants have been isolated and analysed. The
following are well known since they occur in considerable amounts:

Carnatiba wax is produced by the leaves of a Brazilian Palm (Gopernicia
cerifera). The leaves are detached and beaten, and the particles of wax
collected and melted. About 2000-4000 leaves produce 16 kilos of wax.

VII] FATS AND ALLIED SUBSTANCES 97

Palm wax is obtained from the stem of the Wax Palm (Ceroxylon
andicolum), a native of the Andes, and Raphia wax from the leaves of
another palm {Raphia Ruffia). Pisang wax is produced by the leaves of
a variety of the Banana {Musa Cera).

Waxes from different plants contain mixtures of various esters, of
which the component alcohols have been mentioned above. The most
commonly occurring acids are myristic, lignoceric, carnatibic, cerotic and
melissic acids (see p. 89).

Expt. 96. Tests for wax. Take some commercial carnaiiba wax and make the
following experiments :

(a) Warm a small piece with alcohol in a test-tube. It goes into solution and
separates out on cooling as a white crystalline deposit. Examine the crystals under
the microscope.

(6) Warm a small piece with ether. It is soluble.

(c) Heat a small piece of wax with solid potassium hydrogen sulphate in a test-
tube. There is no smell of acrolein, since glycerol is absent [see Expt. 92 (c)].

Phytosterols or Plant Sterols.

These substances are unsaturated monohydric alcohols of high
molecular weight of which the structural formulae are unknown. They
are probably present in all parts of plants but the members most fully
investigated have chiefly been obtained from seeds. The sterols are
always found accompanying vegetable fats, and this connection is ac-
centuated by the fact that they are soluble in the solvents used in fat
extraction. When the fat is saponified, the sterols remain unaltered and
are said to form the "unsaponifiable residue" of fats.

Various sterols have been isolated from different plants: many are
isomeric and a usual formula is C27H45OH. One of the best defined
sterols is sitosterol which occurs in the grain of the Wheat {Triticum
vulgare) and Rye (Secale cereale): also in seeds of the Flax (Linum
usitatissimum) and the Calabar Bean {Physostigma venenosum).

Expt. 97. Detection of sterol in the grain of the Wheat. Weigh out 300 gms. of
grains and grind them in a coffee mill. Add 350 c.c. of ether to the ground mass in
a flask, and allow it to stand for 24 hrs. Filter the extract through a pad of asbestos
or glass wool in a funnel. Then wash the residue with another 150 c.c. of ether and
filter. The ether extract is then saponified with sodium ethylate which is prepared
as follows. Weigh out 2 gms. of metallic sodium, cut it into small pieces and add it
slowly to 20 c.c. of 96-98% alcohol. When it has dissolved, add the solution of
sodium ethylate to the ether extract in a separating funnel, shake well and allow the
mixture to stand for at least 24 hours. Saponification takes place in the cold, and
soap separates out. Filter, and shake up the filtrate several times with water in a
o. 7

98 FATS AND ALLIED SUBSTANCES [ch.

separating funnel to remove alkali. Then evaporate off the ether in an evaporating
basin on a water-bath after turning out the flame. Dissolve the unsaponifiable
residue in a small quantity of hot 96-98 ^/o alcohol and cool. A crystalline deposit
of sterol will separate out. Examine under the microscope and note the elongated
six-sided plates. Make 5 c.c. of a chloroform solution of some of the unsaponifiable
residue and test for sterols as follows :

{a) To 2 c,c. of the chloroform extract add 2 c.c. of concentrated sulphuric acid.
The chloroform layer develops a reddish-yellow to blood-red colour according to the
amount of sterol present. The sulphuric acid layer shows a very characteristic green
fluorescence. Pipette off the chloroform into a basin ; it shows a play of colours, blue,
green and yellow due to absorption of water.

(6) To 2 c.c. of the chloroform extract add 20 drops of acetic anhydride and then
concentrated sulphuric acid drop by drop. A violet-pink colour appears which later
changes to blue and green.

Lecithins.

These substances are probably present in all living cells. True
(pure) lecithin can be isolated from the animal, but preparations from
the plant have hitherto always been mixtures with other substances.
Various plant lecithins with such impurities have been isolated from
seeds of the Wheat (Triticum vulgare), Castor-oil Plant {Ricinus com-
munis), Pea (Pisum sativum), Lupin (Lupinus) and others: also from
leaves of the Horse Chestnut (Aesculus Hippocastanum) and root of the
Carrot {Daucus Carota).

Lecithin is a complex substance in which one hydroxyl of the glycerol
of a fat forms an ester with phosphoric acid, the latter being also combined
with the base, choline (see p. 170).

CHg • OOC • R

I
CH -OOCR

i
CH2— o

I

HO— P = 0
/

o

/

C2H4

N = (CH3)3
OH

Lecithins are yellowish wax-like substances which, on exposure to air,
rapidly darken and become brown. They are hydrolysed by boiling with
alkalies with the production of glycero-phosphoric acid, fatty acids and

VII] FATS AND ALLIED SUBSTANCES 99

choline. The same decomposition is effected by lipase. An enzyme,
glycerophosphatase, which decomposes glycero-phosphoric acid into
phosphoric acid and glycerol has been shown to be present in bran and
the seed of the Castor-oil Plant {Ricinus communis). Unlike lipase it is
soluble in water (Plimmer, 12).

Expt. 98. Tests for lecithin. With commercial lecithin make the following tests :
(a) Test its solubility in ether, chloroform, benzene and carbon disulphide. It is
soluble in all these solvents. To the ether solution add acetone ; the lecithin is pre-
cipitated.

{b) Boil a little lecithin with alcohol in a test-tube. It is soluble.

(c) To the alcoholic solution from (6), add an alcoholic solution of cadmium
chloride. A white precipitate of a double salt of lecithin and cadmium chloride
separates out. Filter this oflf and test its solubilities in chloroform, benzene, etc. It
is soluble. The double cadmium salt has been employed in the preparation and
purification of lecithin.

(d) Heat a little lecithin with some strong caustic soda solution in a test-tube.
Trimethylamine is evolved which can be detected by its smell. Acidify, and the
fatty acids will separate out.

(e) Test for phosphoric acid in the following way. Weigh out 0*1 gm. of lecithin
a,nd mix it well with 1*4 gm. of potassium nitrate and 0*6 gm. of sodium carbonate.
Incinerate the mixture in a porcelain crucible until it is coloiu-less. Then dissolve
the residue in the minimum amount of hot water, neutralize with hydrochloric acid,
acidify with a few drops of concentrated nitric acid and pour the solution into an
equal volume of boiling 3 % ammonium molybdate solution. A yellow precipitate
of ammonium phosphomolybdate is produced,

REFERENCES

Books

1. Abderhalden, E. Biochemisches Handlexikon, in. Berlin, 1911.

2. Allen's Commercial Organic Analysis. Vol. 2. London, 1910.

3. Leathes, J. B. The Fats. London, 1910.

4. Le'wko'witsch, J. Chemical Technology and Analysis of Oils, Fats and
Waxes. 6th ed. London, 1921.

5. Maclean, H. Lecithin and allied Substances. The Lipins. London, 1918.

Papers

6. Armstrong, H. B. Studies on Enzyme Action. Lipase. Froc. R. Soc,
1905, B Vol. 76, pp. 606-608.

7. Armstrong, H. E., and Ormerod, B. Studies on Enzyme Action. Lipase.
II. Proc. R. Soc, 1906, B Vol. 78, pp. 376-385.

8. Armstrong, H. B., and Gosney, H. W. Studies on Enzyme Action.
Lipase. III. Proc. R. Soc, 1913, B Vol. 86, pp. 586-600.

9. Armstrong, H. E., and Gosney, H. W. Studies on Enzyme Action.
Lipase. IV. The Correlation of Synthetic and Hydrolytic Activity. Proc. R. Soc,
1915, B Vol. 88, pp. 176-189.

7-2

100 FATS AND ALLIED SUBSTANCES [ch. vii

10. Ellis, M. T. Contributions to our Knowledge of the Plant Sterols. Part I.
The Sterol Content of Wheat {Triticum sativum). Biochem. J"., 1918, Vol. 12, pp.
160-172.

11. Miller, B. C. A Physiological Study of the Germination of Belianthus
annuus. Ann. Bot, 1910, Vol. 24, pp. 693-726. Ihid. 1912, Vol. 26, pp. 889-901.

12. Plimmer, R. H. A. The Metabolism of Organic Phosphorus Compounds.
Their Hydrolysis by the Action of Enzymes. Biochem. J.^ 1913, Vol. 7, pp. 43-71.

13. Reynolds Green, J. On the Germination of the Seed of the Castor-oil
Plant (Ricinus communis). Proc. R. Soc, 1890, Vol. 48, pp. 370-392.

14. Reynolds Green, J., and Jackson, H. Further Observations on the
Germination of the Seeds of the Castor-oil Plant {Ricinus communis). Proc. R. Soc.y
1906, B Vol. 77, pp. 69-85.

15. Smedley, I. The Biochemical Synthesis of Fatty Acids from Carbohydrate.
J. Physiol., 1912, Vol. 45, pp. xxv-xxvii.

16. Smedley, I., and Lubrzynska, B. The Biochemical Synthesis of the
Fatty Acids. Biochem. ./., 1913, Vol. 7, pp. 364-374.

17. Lubrzynska, B., and Smedley, I. The Condensation of Aromatic
Aldehydes with Pyruvic Acid. Biochem. «/., 1913, Vol. 7, pp. 375-379.

CHAPTER VIII

AROMATIC COMPOUNDS

The aromatic compounds may be defined as substances containing the
benzene carbon ring or a similar ring. A very great number occur
among the higher plants but of these many are restricted in distribution,
and may only be found in a few genera or even in one genus: others, on
the other hand, are widely distributed. At present our knowledge of the
part they play in general plant metabolism is slight.

The more widely distributed aromatic plant products may be grouped
as:

1. The phenols, and their derivatives.

2. Inositol and phytin.

3. The aromatic acids, alcohols and aldehydes.

4. The tannins.

5. The "essential oils" and resins.

6. The flavone, flavonol and xanthone pigments, known as the soluble
yellow colouring matters.

7. The anthocyan pigments, known as the soluble red, purple and
blue colouring matters.

In connexion with the aromatic compounds it should be noted that
many of them contain hydroxyl groups, and one or more of these groups
may be replaced by the glucose residue, CeHnOg — , with elimination of
water and the formation of a glucoside, in the way already described
(see p. 50). The majority of such compounds are sometimes classed
together as a group — the glucosides — regardless of the special nature of
the substance to which the glucose is attached (this course has been
followed to some extent in Chapter x with compounds, the chief interest
of which lies in their glucosidal nature). In treating of the aromatic
substances in the following pages, mention will be made when they occur
as glucosides, this combination being in these cases only a subsidiary
point in their structure.

The various groups of aromatic substances will now be considered in
detail.

Phenols.

There are three dihydroxy phenols, resorcinol, catechol and quinol,
but only the two latter are known to exist in the £ree state in
plants. Resorcinol frequently occurs as a constituent of complex plant

102 AROMATIC COMPOUNDS [ch.

products, and may be obtained on decomposition of such complexes by

fusion with strong alkali, etc.

OH

/\

OH

OH ' OH OH

Resorcinol Catechol Quinol

Quinol has been found in the free state in the leaves and flowers
of the Cranberry ( Vaccinium Vitis-Idaea). As a glucoside, known as
arbutin, it occurs in many of the Ericaceae (see also p. 166).

Phloroglucinol is the only member of the trihydroxy phenols found
uncombined in plants. It is very widely distributed in the combined
state in various complex substances (Waage, 23).

/NoH

HO

K/

OH

Phloroglucinol

Inositol and Phytin.

Inositol is widely distributed in plants, especially in young leaves
and growing shoots. It has been isolated from leaves of the Walnut
(Juglans regia), fruit of the Mistletoe ( Viscmn album) and the unripe
seed-pods of various plants. It is a polyhydric alcohol derived from
benzene:

HOH

/^\

HOHC CHOH

HOHC CHOH
HOH

Inositol is soluble in water but crystallizes out on adding strong
alcohol. It occurs also in seeds as the compound, phytin. The latter
is an acid calcium and magnesium salt of inositol phosphoric acid which
is a condensation product of inositol with six molecules of phosphoric
acid (Plimmer and Page, 21). An enzyme, phytase, also occurring in
seeds is able to hydrolyze phytin into inositol and phosphoric acid
(Plimmer, 20).

VIII] AROMATIC COMPOUNDS

Aromatic Acids, Aldehydes and Alcohols.

103

There are two important series of these compounds found in the plant
which can be represented respectively by benzoic acid and cinnamic acid
and their derivatives :

COOH

V

Benzoic acid

CH=CHCOOH

Cinnamic acid

Salicylic acid, or o-hydroxy-benzoic acid, occurs, both in the form of
esters and in the free state, in various plants. The corresponding alcohol,
saligenin or salicylic alcohol, in the form of the glucoside, salicin, occurs
in the bark of certain species of Willow (Salix), and in the flower buds of
the Meadow-sweet (Spiraea Ulmaria). Salicin is hydrolyzed by an en-
zyme contained in the plant in which it occurs into saligenin and
glucose (see also p. 167). Salicylic aldehyde occurs in species of Spiraea
and other plants.

COOH

OH

V

CH=CH-COOH

OH

V

o-Coumaric acid

Salicylic acid

The corresponding derivative of cinnamic acid, i.e. o-coumaric acid
is widely distributed as the anhydride, coiimarin (see p. 165).

The relationship of cinnamic acid to phenylalanine and of ^-coumaric
acid to tyrosine (see p. 135) is important.

Protocatechuic acid is a dihydroxy-benzoic acid. It has been found
in the free state in a few plants, but is more widely distributed as a
constituent of many plant products. As will be shown later it forms the
basis of the series of tannins.

COOH

OH

CH=CHCOOH

OH

OH

Protocatechuic acid

OH

Caffeic acid

104 AROMATIC COMPOUNDS [ch.

The corresponding derivative of cinnamic acid, i.e. caffeic acid (see
also p. 123) is probably widely distributed. It is related to dihydroxy-
phenylalanine (see p. 152).

Coniferyl alcohol is related to caffeic acid (see p. 103). Coniferyl
alcohol, when oxidised, yields the aldehyde, vanillin (so much used for
flavouring) which occurs in the fruits of the Orchid (Vanilla plani-
folia). (See also p. 166.)

Gallic acid is a trihydroxy-benzoic acid :

COOH

HO

V

OH

OH

It occurs free in gall-nuts, in tea, wine, the bark of some trees and in
various other plants. It forms a constituent of many tannins. It is a
crystalline substance not very readily soluble in cold but more soluble in
hot water. In alkaline solution it rapidly absorbs oxygen from the air
and becomes brown in colour.

Expt. 99. The extraction and reactions of gallic acid. Take 100 gms. of tea, dry
in a steam oven and grind in a mortar. Put the powder into a flask and cover well
with ether. The preliminary drying and grinding can be omitted, but if carried out
will make the extraction more complete. After at least 24 hrs. filter off the extract,
and either distil or evaporate off the ether. The ether will be coloured deep green
by the chlorophyll present in the dried leaves, and a green residue will be left. Add
about 20 c.c. of distilled water to the residue, heat to boiling and filter. Heating
is necessary because the gallic acid is only sparingly soluble in cold water. Keep the
residue for Expt. 103. With the filtrate make the following tests ; for (a), (6) and (c)
dilute a few drops of the filtrate in a porcelain dish :

(a) Add a drop of 5% ferric chloride solution. A blue-black coloration is given.

(6) Add a drop or two of iodine solution. A transient red colour appears.

(c) Add a drop or two of lime water. A reddish or blue coloration will be given.

{d) To a few c.c. of the filtrate in a porcelain dish add a little 5 7o lead acetate
solution. A precipitate is formed which turns red on addition of caustic potash solu-
tion, and dissolves to a red solution with excess of potash.

(e) To a few c.c. of the filtrate in a test-tube add a little 1 ^/o potassium cyanide
solution. A pink colour appears, but disappears on standing. On shaking with air it
reappears.

(/) To a few c.c. of the filtrate in a test-tube add a few drops of 10% gelatine
solution. No precipitate is formed.

{g) To a few c.c. of the filtrate in a test-tube add a little 5 o/q lead nitrate solution.
No precipitate is formed.

VIII] AROMATIC COMPOUNDS 105

Tannins.

This is a large group of substances, many of which are of complex
composition. They arise in the plant from simpler compounds, such as
protocatechuic, gallic and ellagic acids. Their formation takes place in
various ways, either by condensation, accompanied by elimination of
water, or by oxidation, or both ; there may also be condensation with other
aromatic complexes.

The tannins are widely distributed in the higher plants and, although
no very systematic investigation has been made, it is obvious that some
plants are rich in these substances, others poor, and others, again,
apparently entirely without them. The tannins generally occur in
solution in the cells of tissues of the root, stem, leaf, fruit, seed and
flowers: sometimes they are confined to special cells, tannin-sacs, but
after the death of the cell, the cell-walls of the dead tisssue become
impregnated with the tannin. In tannin-producing plants, the tannin
is generally found throughout the plant, and it probably tends to
accumulate in permanent or dead tissues, such as the bark (dead cortex
and cork), woody tissue, underground stems, etc.

Tannins appear to be more frequent in woody than in herbaceous
plants, though in the latter they naturally only accumulate in the
persistent underground stems and root-stocks. In annuals, also, tannins
seem to be more rare: this may be due to the fact that in a short-lived
plant, comparatively little tannin is formed and is not so readily detected
as in the tissues of a perennial.

In certain plants which are highly tannin-producing and are also
woody perennials, the bark becomes very rich in tannins. These barks
are consequently of considerable commercial importance for tanning of
leather. As examples may be taken species of Caesalpinia, Spanish
Chestnut (Castanea), Eucalyptus, Oak (Quercus), Mangrove (Rhizophora),
Sumac (Rhus). Tannins also occur in quantity in galls, especially on
species of Quercus.

As a class, the tannins are non-crystalline and exist in the colloidal
state in solution. They have a bitter astringent taste. They have
certain properties and reactions in common, i.e. they precipitate gelatine
from solution, are themselves precipitated from solution by potassium
bichromate, and give either blue or green colorations with solutions of
iron salts. Many tannins occur as glucosides but this is by no means
always the case.

106 AROMATIC COMPOUNDS [ch.

It is possible to classify the tannins into two groups according to
whether they are complexes derived from protocatechuic acid or gallic
acid:

1. The pyrogallol tannins. These give a dark blue colour with ferric
chloride solution, and no precipitate with bromine water.

2. The catechol tannins. These give a greenish-black colour with
iron salts, and a precipitate with bromine water.

Expt. 100. Reactions of tannins. Take three oak galls (the brown galls formed by
species of Cynips on the Common Oak) and pound them finely in a mortar. Boil up
the powder well with a small amount of water in an evaporating basin and let stand
for a short time. Then filter. The filtrate will contain tannin together with im-
purities. Make the following tests with the extract :

(a) Put 2 c.c. of the tannin extract into a small evaporating dish, dilute with
water, and add a drop or two of 5 % ferric chloride solution. A deep blue-black
colour is produced.

(6) Put 2 or 3 drops of the tannin extract into a small evaporating dish, and
dilute with water: add a little dilute ammonia and then a few drops of a dilute
solution of potassium ferricyanide solution. A red coloration will appear.

(c) To 5 c.c. of the tannin solution in a test-tube add some strong potassium
dichromate solution. The tannin will be precipitated.

{d) To about 5 c.c. of the tannin extract in a test-tube add a little 5 ^(q lead acetate
solution. The tannin will be precipitated.

(e) Melt a little of a 10 ^j^ solution of gelatine by warming gently and then pour
drop by drop into a test-tube half full of tannin extract. The gelatine will be pre-
cipitated.

For the above tests, in addition to galls, the bark stripped from two or three year
old twigs of Quercus may also be used, and will give the same reactions. The bark
should be cut into small pieces for extraction.

It should be noted that although many tannins give the above
reactions, it does not necessarily follow that all tannins will give all the
reactions.

Expt. 101. To demonstrate the existence of pyrogallol and catechol tannins. The
existence of a pyrogallol tannin which gives a blue reaction with iron salts has been
illustrated in the last experiment on the Oak galls and the bark from Oak twigs.
The bark of the Sumac {Rhus Coriaria) and the fruit pericarp, leaves and bark of the
Sweet Chestnut {Castanea vulgaris) may be used as additional material for pyrogallol
tannins.

For an iron-greening tannin strip off* the outer bark from two to three year old
twigs of the Horse Chestnut {Aesculus Hippocastanum). Cut or tear the bark into
small pieces and boil well with a little water in an evaporating dish. Filter and test
the filtrate with ferric chloride solution as in Expt. 101. A green coloration will be
given. Iron-greening tannins may also be extracted from the bark of twigs of the
Walnut {Juglans regia) and of the Larch {Larix europaea).

In the case of both classes of tannins, in addition to the ferric chloride reaction,
the tests of Expt. 101 (c) and (e) should also be made on the extracts, in order to

VIII] AROMATIC COMPOUNDS 107

confirm the presence of tannin, since other substances, such as flavones, may give a
green colour with iron salts (see p. 111).

Some of the individual tannins will now be considered.

Gallotannic (or tannic) acid is one of the most important of the
pyrogallol tannins. It occurs in Oak galls and Oak wood, in tea, in the
Sumac (Rhus Coriaria), etc. According to recent investigations (Fischer
and Freudenburg, 8) tannic acid may be regarded as a compound of one
molecule of glucose with five molecules of digallic acid in which five
hydroxyls of the sugar are esterified by five molecules of acid:
CH2(0X) • CH(OX) • CH • CH(OX) • CH(OX) • CH(OX)

1-^ o !

where

X= —CO • C6H2(OH)2 • O • CO • C6H2(OH)3

Tannic acid is an almost colourless amorphous substance. It has an
astringent taste, is soluble in water and alcohol, only slightly soluble in
ether, and insoluble in chloroform. It" is decomposed, by boiling with
2 Yo hydrochloric acid, into gallic acid.

Expt. 102. Extraction and reactions of tannic {or gallotannic) add. By a crude
method a solution of gallotannic acid can be obtained from tea. About 5 gms. of the
residue, after the extraction with ether in Expt. 100, is again extracted with ether
once or twice which will remove all but traces of gallic acid. Boil up the residue from
ether with a little water and filter. With the filtrate make the following tests which
differentiate between gallic and gallotannic acid :

(a) To about 10 c.c. add a little IO^Iq gelatine. The gelatine is precipitated.

(6) To a little of the filtrate add a few drops of 5 % lead nitrate solution. The
tannic acid is precipitated.

The remaining tests are given in common with gallic acid. If the extract is too
coloured, dilute with water.

(c) Dilute a few drops of the filtrate with water in a porcelain dish and add a
drop of 5 % ferric chloride solution. A blue-black colour is given.

{d) Dilute a few drops of the filtrate with water in a porcelain dish and add a
drop or two of iodine solution. A transient red colour is formed.

(e) To a little of the filtrate in a test-tube add a few drops of 1 7o potassium
cyanide solution. A reddish-brown colour is formed which changes to brown but
becomes red again on shaking with air.

In addition to tannic acid, a great many other tannins are known,
but their constitution is obscure.

Expt. 103. To demonstrate that in tannin-containing plants the tannin may he
also present in the leaves. Take about two dozen leaves of the Common Oak {Quercus
Rohur) and pound them in a mortar. Then boil the crushed mass in an evaporating
dish with a little water. Filter, and with the filtrate make the tests for tannin.
Leaves of other trees also may be used, e.g. the Wig Tree {Rhus Cotinus), Sweet
Chestnut {Castanea vulgaris).

108 AROMATIC COMPOUNDS [ch.

Expt. 104. To demonstrate that tannins ma.y he present in herbaceous as well as
woody plants. Extract some leaves, as in the last experiment, of Scarlet Geranium
{Pelargonium zonale) and test for tannin.

Expt. 105. To demonstrate that tannins may he present in petals and fruits, in
addition to other parts of the plant. Extract and test for tannins as in the last experi-
ment, using petals of Pelargonium zonale. Common Paeony {Paeonia officinalis) or
Kose (any garden variety), inflorescence of Flowering Currant {Rihes sanguineum),
flowers of Horse Chestnut {Aesculus Hippocastanum) or pericarp of Sweet Chestnut
(Castanea).

The " Essential Oils" and Resins.

When plant tissues are suspended in water, a current of steam passed
through the suspension, and the distillate collected, a mixture of volatile
substances will be found in the distillate and these can be separated from
the water by various methods. Such a mixture of organic volatile pro-
ducts constitutes an "essential oil." The classification is purely arti-
ficial, as the mixture is heterogeneous and contains substances of very
different chemical constitution. Since, however, the majority of "oils"
consist largely of aromatic compounds, they are included in the present
chapter. In many cases the " essential oil " contains some product of
commercial value. About two hundred and fifty plants, representing
between fifty and sixty Natural Orders, provide definite " oils," most of
which are prepared commercially.

The chemical substances found in "essential oils" can be broadly
classed as follows (see also p. 82).

1. The terpenes, which are complex, unsaturated (usually aromatic)
hydrocarbons frequently of the formula, CjoHig, e.g. pinene, limonene,
caryophyllene and phellandrene.

2. Alcohols derived from the terpenes, e.g. borneol, menthol, citro-
nelloP, geraniol^ and linaloP; corresponding aldehydes, e.g. citronellal^
and other aromatic aldehydes, e.g. cinnamic aldehyde.

3. Esters of the above alcohols, e.g. bornyl acetate, geranyl acetate,
linalyl acetate and menthyl acetate ; also esters of other aromatic acids,
e.g. methyl salicylate.

4. Phenols of high molecular weight, e.g. thymol, carvacrol and
eugenol.

The following provide some examples of " essential oils " :
" Oil of turpentine," from species of Pinus, Larix and Abies, contains
pinene.

The compound is aliphatic.

VIII] AKOMATIC COMPOUNDS 109

*' Lavender oil," from Lavandula vera (Labiatae), contains limonene,
linalyl acetate, linalol and others.

" Peppermint oil," from Mentha piper ata (Labiatae), contains menthol,
menthyl acetate and others.

" Clove oil," from Eugenia caryophyllata (Myrtaceae), contains
eugenol and caryophyllene.

" Cinnamon oil," from Cinnamomum zeylanicum (Lauraceae), contains
cinnamic aldehyde, eugenol and phellandrene.

"Lemon oil," from Citrus Limonum (Rutaceae), contains limonene,
citronellol and citral.

" Thyme oil," from Thymus vulgaris (Labiatae), contains thymol and
carvacrol.

"Rose oil," from Rosa centifolia (Rosaceae), contains citronellol,
geraniol and others.

Camphor is a ketone derived from a solid terpene, camphene. The
former occurs in the Camphor Tree (Cinnamomum Camphora), a genus
of the Lauraceae.

The resins are oxidation products of the terpenes. They are
differentiated into balsams and hard resins. The former consist of resins
dissolved in, or mixed with, liquid terpenes, e.g. Canada balsam and crude
turpentine. Copal and dammar are examples of hard resins.

£Ia:pt. 106. Preparation of ^^ clove oil" from cloves (Wester, see p. 10). Cloves
are the dried flower-buds of Eiigenia caryophyllata (Myrtaceae). Take 100 gms. of
cloves, pound them in a mortar and put the mass into a two litre flask one third full
of water. Pass a current of steam through the flask, and collect the distillate cooled
by a water condenser. The " essential oil " of Eugenia consists chiefly of the phenol,
eugenol, C6H4(OH)(OCH3)CH2CH=CH2, together with small quantities of the
terpene, caryophyllene. The latter distills over first, but cannot be isolated unless
much larger quantities of material are used. The eugenol settles out as an "oil" at
the bottom of the watery distillate. Continue the distillation for four hours, or more,
till all the eugenol has distilled over. Then add 25 gms. of sodium chloride for each
100 c.c. of the distillate, and shake up the mixture in a separating funnel with small
quantities of petrol ether until no more eugenol can be extracted. The petrol ether
extract is then distilled on a water bath (after the flame has been removed) to 25 c.c.
Then extract it three times with 20 c.c. of 5 7o sodium hydroxide solution in a
separating funnel, whereby the sodium salt of the eugenol is formed and passes into
the alkaline solution turning it yellow. The petrol ether now contains only the small
quantity of the hydrocarbon, caryophyllene. Traces of the latter are now removed
from the alkaline phenolate by extracting again with 20 c.c. of petrol ether. Then
add dilute sulphuric acid to the phenolate. The eugenol separates out as a milky
suspension, which gradually collects together as a yellow "oil." Then neutralise again
with sodium carbonate solution (which does not form a phenolate), and extract the
eugenol with petrol ether. Distil ofi' the ether, and the eugenol remains.

no AROMATIC COMPOUNDS [ch.

The Flavone and Flavonol Pigments.
These yellow colouring matters are very widely distributed in the
higher plants (Shibata, Nagai and Kishida, 22). They are derived from
the mother substances, flavone and flavonol, the latter only differing
from the former in having the hydrogen in the central 7-pyrone ring
substituted by hydroxyl :

A.^°^P / \ /\/°N

L CH K ,

COH

Flavone Flavonol

The naturally occurring pigments, however, have additional hydro-
gen atoms replaced by hydroxyl groups, that is they are hydroxy-flavones
and flavonols, and the various members differ among each other in the
number and position of these hydroxyl groups. Some of the members
are widely distributed, others less so. Quite often more than one repre-
sentative is present in a plant.

The flavone and flavonol pigments are yellow crystalline substances,
and as members of a class they have similar properties. They occur in
the plant most frequently as-glucosides, one or more of the hydroxyl
groups being replaced by glucose, or, sometimes, by some other hexose,
or pentose. In the condition of glucosides, they are much less coloured
than in the free state, and, being present in the cell-sap in very dilute
solution, they do not produce any colour effect, especially in tissues con-
taining chlorophyll. Occasionally they give a yellow colour to tissues,
as in the rather rare case of some yellow flowers {Antirrhinum) where
colour is due to sqluble yellow pigment.

In the glucosidal state, the flavone and flavonol pigments are, as a
rule, readily soluble in water and alcohol, but not in ether. In the non-
glucosidal state they are, as a rule, readily soluble in alcohol, somewhat
soluble in ether, but soluble with difficulty in water.

The flavone and flavonol pigments can be easily detected in any
tissue by the fact that they give an intense yellow colour with alkalies
(Wheldale, 24). If plant tissues be held over ammonia vapour, they
turn bright yellow, showing the presence of flavone or flavonol pigments:
the colour disappears again on neutralization with acids. (The reaction
is especially well seen in tissues free from chlorophyll, such as white
flowers.) This reaction will be found to be almost universal, showing
how wide is their distribution. With iron salts, solutions of the pigments

VIII] AROMATIC COMPOUNDS 111

give green or brown colorations. With lead, insoluble salts are formed.
Several of the members are powerful yellow dyes, and hence some plants
in which they occur, such as Ling {Erica cinerea), Dyer's Weld or
Rocket (Reseda luteola), have been used for dyeing purposes. The value
of these colouring matters as dyes has led to their chemical investigation,
and as a result the constitution, etc., of the hydroxy-flavones and flavonols
is well established.

Ejcpt. 107. Demonstration of the presence offlavone or flavonol pigments in tissues
without chlorophyll. Take flowers of any of the undermentioued species and put them
in a flask with a few drops of ammonia. They will rapidly turn yellow owing to the
formation of the intensely yellow salt of the flavone or flavonol pigments present
in the cell-sap. If the flowers are next treated with acid the yellow colour will dis-
appear.

Also make an extract of some of the flowers with a little boiling water. Filter,
cool and add the following reagents :

{a) A little alkali. A yellow colour is produced.

(h) A little ferric chloride solution. Either a green or brown coloration is produced.

(c) A little basic lead acetate solution. A yellow precipitate of the lead salt of
the flavone or flavonol pigment is formed.

The flowers of the following species can be used : Snowdrop {Galanthus nivalis)^
Narcissus {Narcissus poeticus), white variety of Lilac {Syringa vulgaris)^ Hawthorn
{Crataegus Oxyacantha), White Lily {Lilium candidum\ white var. of Phlox, double
white Pink, white Stock {Matthiola) etc., etc., in fact almost any species with white
flowers or a white variety.

Expt. 108. Demonstration of the presence of flavone or flavonol pigments in tissues
containing chlorophyll. Make a hot water extract of the leaves of any of the under-
mentioned species. Make with it the same tests as in the previous experiment.

Almost any green leaf may be used, but the following are suggested : Snowdrop
{Galanthus nivalis), Dock {Rumex ohtusifolius), Goutweed {Aegopodium Podagraria\
Dandelion ( Taraxacum officinale), Violet ( Viola odorata), Eibwort Plantain {Plantago
lanceolata), Elder {Samhucus nigra).

The most important flavone pigments are apigenin, chrysin and
luteolin.

Apigenin has not yet been found to be widely distributed. Its
formula is :

HoA^/°^

OH

OH

It occurs in the Parsley {Garum Petroselinum) (Perkin, 12) and in
the flowers of the ivory-white variety of Snapdragon (Antirrhinum
majus) (Wheldale and Bassett, 25).

112

AROMATIC COMPOUNDS

[CH.

Expt. 109. Extraction of apiin, the glucoside of apigenin^ from the Parsley (Carum
Petroselinum). Take some Parsley leaves and boil in as little water as possible.
Filter off the extract and make the following tests for apigenin :

{a) Add alkali. A lemon yellow coloration is given.

(6) Add basic lead acetate solution. A lemon yellow precipitate is formed.

(c) Add ferric chloride solution. A brown colour is produced.

{d) Add ferrous sulphate solution. A reddish-brown colour is produced.

Apiin frequently separates out in a gelatinous condition from aqueous
and dilute alcoholic solutions.

Chrysin is a flavone occurring in the buds of various species of
Poplar {Populus). It has the formula :

HO

A^^''^

CH

OH

Luteolin does not appear to be widely distributed, though possibly
it occurs in many plants in which it has not yet been demonstrated. Its
formula is represented as :

HO

/\/''\

OH

K

OH

XO

OH

It occurs in the Dyer's Weld or Wild Mignonette {Reseda luteola)
(Perkin, II), Dyer's Greenweed or Broom (Genista tinctoria) (Perkin, 17)
and in the yellow variety of flowers of the Snapdragon {Antirrhinum
majus) (Wheldale and Basse tt, 27). It has been much used as a yellow
dye: hence the names of the first two plants (Perkin and Horsfall, 14).

The most important flavonol pigments are quercetin, kaempferol,
myricetin and fisetin.

Quercetin is apparently one of the most widely distributed of the
whole group of yellow pigments, and has the formula :

HO

/\^''\

P»

OH

OH

^'

OH

VIII] . AROMATIC COMPOUNDS 113

It occurs, either free, or combined with various sugars (glucose,
rhamnose) as glucosides, in many plants, as for instance the following :
in the bark of species of Oak (Quercus), in berries of species of Buck-
thorn (Rhamnus), in flowers of Wallflower (Gheiranthns Gheiri), Haw-
thorn (Grataegus Oxyacaniha) (Perkin and Hummel, 16), Pansy {Viola
tricolor) (Perkin, 13) and species of Narcissus : in leaves of Ling {Galluna
erica) (Perkin, 17), and the outer scale leaves of Onion bulbs (Perkin
and Hummel, 15).

Expt. 110. Preparation of a glucoside of quercetin fro7n flowers of either a species
of Narcissus or the Wallflower (Cheiranthus Cheiri). The most suitable species of
Narcissus is N. Tazetta, but N. incomparabilis or any of the common yellow trumpet
varieties such as the Daffodil {N. Pseudo-Narcissus) can be used. Take about 50
flowers of Narcissus Tazetta or about 20 gms. of petals of the Wallflower of either
the brown or the yellow variety. The brown colour is due to a mixture of yellow
plastid and of soluble purple (anthocyan) pigment in the sap. Pound the flowers in a
mortar and then extract in a flask with boiling alcohol. Filter off the alcoholic
extract and evaporate to dryness on a water-bath. Then add a little water and ether
to the residue and transfer the whole to a separating funnel. The ether takes up
the yellow plastid pigments, but the flavone and, in the case of the brown Wallflower,
the anthocyan pigment remain in the water. Very soon, however, at the plane of
separation of the liquids, the glucoside separates out as a crystalline deposit. This
can be filtered off ; with a dilute solution in alcohol make the following tests :

(a) Add a little alkali. The yellow colour is intensified, but the intensification
disappears on adding acid.

(6) Add a little lead acetate solution. An orange precipitate of the lead salt is
formed.

(c) Add a little ferric chloride solution. A green coloration is produced.

{d) Heat some of the alcoholic solution on a water-bath, acidify with strong
hydrochloric acid and add zinc dust. A pink or magenta colour is produced
(see p. 121).

Kaempferol occurs in the flowers of a species of Larkspur {Delphi-
nium consolida) (Perkin and Wilkinson, 19) and Pr units (Perkin and
Phipps, 18) and in the leaves or flowers of several other plants. It has
the formula:

HO

^^^""^

\/

■rC

OH

HO ^°

\„^°"

O.

114 AKOMATIC COMPOUNDS • [ch.

Myricetin and fisetin are two other flavones which have been found
in [species of Sumac {Rhus) and other plants. They have respectively
the formulae :

HO

^^\/''\

HO

OH HO.^^/ \.

OH

\

OH

OH

Myricetin Fisetin

The Anthocyan Pigments.

These pigments are the substances to which practically all the blue,
purple and red colours of flowers, fruits, leaves and stems are due
(Wheldale, 3). They occur in solution in the cell-sap and are very
widely distributed, it being the exception to find a plant in which they
are not produced. As members of a group, they have similar properties,
but differ somewhat among themselves, the relationships between them
being much the same as those between the various flavone and flavonol
pigments. They occur in solution in the cell-sap but occasionally they
crystallize out in the cell. They are present in the plant in the form of
glucosides, and in this condition they are known as anthocyanins ', as
glucosides they are readily soluble in water and as a rule in alcohol
[except blue Columbine (Aquilegia), Cornflower (Gentaurea Cyanus) and
some others] but are insoluble in ether and chloroform. The glucosides
are hydrolyzed by boiling with dilute acids, and the resulting products,
which are non-glucosidal, are termed anthocyanidins (Willstatter and
Everest, 30). The latter, in the form of chlorides, are insoluble in ether,
but are generally soluble in water and alcohol. The anthocyanins can
be distinguished from the anthocyanidins in solution by the addition of
amyl alcohol after acidification with sulphuric acid. The anthocyanidins
pass over into the amyl alcohol, the anthocyanins do not. The antho-
cyanins and anthocyanidins themselves (with one exception) have not
yet been crystallized, but of both classes crystalline derivatives with
acids have been obtained (Willstatter and Everest, 30).

In considering the reactions of anthocyan pigments the difference
between those given by crude extracts and those of the isolated and
purified substances must be borne in mind. With acids the anthocyan
pigments give a red colour: with alkalies they give, as a rule, a blue or
violet colour when pure, but if flavone or flavonol pigments are present

vm] AKOMATIC COMPOUNDS 115

(as may be the case in a crude extract) they give a green colour, due to
mixture of bhie and yellow. In solution in neutral alcohol and water
many anthocyan pigments lose colour, and this is said to be due to the
conversion of the pigment into a colourless isomer which also gives a
yellow colour with alkalies (Willstatter and Everest, 30); hence even a
solution of a pure anthocyan pigment may give a green coloration with
alkali due to mixture of blue and yellow. The isomerization can be
prevented or ^lessened by addition of acids, or of neutral salts which form
protective addition compounds with the pigment. With lead acetate
anthocyan pigments give insoluble lead salts, blue if the pigment is
pure, or green, as in the case of alkalies, if it is mixed with flavone or
flavonol pigments, or the colourless isomer.

When anthocyan pigments are treated with nascent hydrogen, the
colour disappears but returns again on exposure to air. It is not known
what reaction takes place.

Expt. 111. The reactions of anthocyanins and anthocyanidins. Extract petals of
the plants mentioned below with boiling alcohol in a flask. Note that the anthocyan
colour may disappear in the alcoholic extract. Filter off some of the alcoholic extract
and make the following tests {a) and (6) with it :

{a) Add a little acid and note the bright red colour.

(6) Add a little alkali and note the green colour.

The decolorized petals, after filtering off the extract, should be warmed with a
little water in an evaporating dish. The colour is brought back if pigment is still
retained by them.

Evaporate the remainder of the alcoholic extract to dryness and note that the
anthocyan colour returns. Dissolve the residue in water and continue the following
tests, taking a little of the solution in each case :

(c) Add ether and shake. The anthocyan pigment is not soluble in ether.

(o?) Add acid. A bright red colour is produced.

(e) Add alkali. A bluish-green or green colour is produced which may pass to
yellow.

(/) Add basic or normal lead acetate solution. A bluish-green or green precipitate
is produced.

{g) Add a little sulphuric acid and then amyl alcohol and shake ; the latter does
not take up any of the red colour, indicating that the pigment is in the anthocyanin
(glucosidal) state.

(A) Heat a little of the solution on a water-bath with dilute sulphuric acid and
then cool and add amyl alcohol. The colour will pass into the amyl alcohol, indi-
cating that the pigment is now in the anthocyanidin (non-glucosidal) state.

{i) Acidify a little of the solution with hydrochloric acid and add small quantities
of zinc dust. The colour disappears. Filter off the solution and note that the colour
rapidly returns again.

For the above reactions it is suggested that the following flowers be used as

8—2

116 AROMATIC COMPOUNDS [ch.

material : magenta Snapdragon {Antirrhinum majus)^ brown Wallflower {Cheiranthus
Cheiri), crimson Paeony {Paeonia ojfficinalis\ magenta " Cabbage " Rose, Violet
( Viola odorata\ but the majority of coloured flowers will serve equally well.

Though the above represent the reactions and solubilities given by
the greater number of anthocyan pigments, it will be found that all are
not alike in these respects. Thus, for instance, the pigments of certain
blue flowers, e.g. blue Larkspur {Delphinium), Cornflower {Centaur ea
Cyanus) and blue Columbine {Aquilegia) are neither soluble nor lose
their colour in alcohol, but are soluble in water.

There is a small group of plants belonging to some allied natural
orders, of which the anthocyan pigments give chemical reactions still
more different from the general type already described, though they
nevertheless resemble each other. Such, for instance, are the pigments
of various genera of the Chenopodiaceae [Beet {Beta), Orache {Atriplex)\
Amarantaceae {Amaranthus and other genera), Phytolaccaceae {Phyto-
lacca) and Portulacaceae {Portulaca). These anthocyan pigments are
insoluble in alcohol but soluble in water : they give a violet colour with
acids, red to yellow with alkalies, and a red precipitate with basic lead
acetate.

Anthocyan pigments may also occur in leaves, and this is very obvious
in red-leaved varieties of various species such as the Copper Beech, the
Red-leaved Hazel, etc.

Expt. 112. Extraction of anthocyan pigment from the Red-leaved Hazel. Extract
some leaves of the Blood Hazel {Corylus Avellana var. rubra) with alcohol. Filter off
and evaporate the solution to dryness. Add water. Pour a little of the crude mixture
in the dish into a test-tube and add ether. There will be a separation into a green
ethereal layer containing chlorophyll, and a lower water layer containing anthocyan
pigment. Filter the extract remaining in the dish and with the filtrate make the
tests already given in Expt. Ill ic)-{i).

The leaves of the Copper Beech {Fagus sylvatica var. purpurea) can also be
used.

In many flowers, the cells of the corolla may contain, in addition to
anthocyan, yellow plastid (see p. 40) pigments. The colour of the petals
is in these cases the result of the combination of the two, and is usually
some shade of brown, crimson or orange-red, as in the brown-flowered
variety of Wallflower {Cheiranthus Cheiri), the bronze or crimson
Chrysanthemum, the brown Gaillardia and the orange-red flowers of
Nasturtium {Tropaeolum majus). The presence of the pigments can be
demonstrated by their different solubilities (see Expt. 110).

Anthocyanins and anthocyanidins have been isolated from various

VIIl]

AROMATIC COMPOUNDS

117

species. The pigments themselves with one exception have not been
obtained in the crystalline state, but crystalline compounds with acids
have been prepared both of the glucosidal and non-glucosidal forms.

All the pigments so far described appear to be derived from three
fundamental compounds, pelargonidin, cyanidin and delphinidin, of which
the chlorides are represented thus :

CI

CI

HO

OH

HO

OH H

Pelargonidin chloride

PH
OH

OH H

Cyanidin chloride

CI

HO

/N^

PH

OH
OH

C-OH

V^c/

OH H

Delphinidin chloride

It has been suggested, at least in the case of cyanidin, the pigment
of the Cornflower {Centaurea Cyanus), that the pigment itself is a neutral
substance, purple in colour and of the following structure ( Willstatter,
28,31):

/N^

PH
OH

C~OH

\^C^
OH H

Further, that the blue pigment of the flower is the potassium salt of the
purple, and the red acid salt, cyanidin chloride, depicted above, is a so-
called oxonium compound of the purple.

Pelargonidin, moreover, has been prepared synthetically (Willstatter
and Zechmeister, 33)

The above three pigments, either as glucosides or in the form of
methylated derivatives, are found in a number of plants which are listed
below (Willstatter, etc., 29, 32). The sugar residues or methyl groups
may, of course, occupy different positions, thus giving rise to isomers :

118

AROMATIC COMPOUNDS

[CH.

Pelargonidin.

Callistephin Monoglucoside of pelargo-
nidin

Pelargonin Diglucoside of pelargoni-

din

Flowers of Aster {Callistephus chinen-
sis)

Flowers of Scarlet Geranium {Pelar-
gonium zonale), pink var. of Corn-
flower (Centaurea Cyanus) and cer-
tain vars. of Dahlia {D. variabilis).

Asteriu

Chrysanthemin
Idaein
Cyanin

Mekocyanin
Keracyanin

Peonin

Cyanidin.

Monogliicoside of cyanidin

Monoglucoside of cyanidin

Monogalactoside of cyani-
din
Diglucoside of cyanidin

Diglucoside of cyanidin
Rhamnoglucoside of cya-
nidin
Diglucoside of peonidin
(cyanidin monoethyl
ether)

Flowers of Aster {Callistephus chinen-

sis) ■ ■ .-

Flowers of Chrysanthemum {C. indi-

cum)
Fruit of Cranberry ( Vaccinium Vitis-

Idaea)
Flowers of Cornflower {Centaurea

Cyanus\ Rosa gallica and certain

vars. of Dahlia {D. variabilis)
Flowers of Poppy {Papaver Rhoeas)
Fruit of Cherry {Primus Cerasus)

Flowers of Paeony {Paeonia oficinxdis)

Delphinidin.

Violanin
Delphinin

Ampelopsin

Myrtillin

Althaein

Petunin

Malvin

Oenin

Rhamnoglucoside of del- I Flowers of Pansy ( Viola tricolor)
phinidin

Flowers of Larkspur {Delphinium con-
solida)

Diglucoside of delphini-
din -f- jo-hydroxybenzoic
acid

Monoglucoside of ampe-
lopsidin (delphinidin
monomethyl ether)

Monogalactoside of myr-
tillidin (delphinidin mo-
nomethyl ether)

Monoglucoside of myrtilli-
din

Diglucoside of petunidin
(delphinidin monome-
thyl ether)

Diglucoside of malvidin
(delphinidin dimethyl
ether)

Monoglucoside of oenidin
(delphinidin dimethyl
ether)

Fruit of Virginian Creeper {Ampelop-
sis quinq%iefolia)

Fruit of Bilberry ( Vaccinium Myrtillus)

Flowers of deep purple var. of Holly-
hock {Althaea rosea)

Flowers of Petunia {P. violacea)
Flowers of Mallow {Malva sylvestris)
Fruit of Grape ( Vitis vinifera)

VIIl]

AROMATIC COMPOUNDS

119

Of the methylated compounds, myrtillidin and oenidin may be re-
presented thus:

CI

Ho

fY^^

PH

OH
OH

HO

fV\

^^^ C-OCH.

HO H

Myrtillidin

PH

OCH3
OH

H

HP

Oenidin

Expt, 113. Preparation and reactions of pelargonin chloride. Extract the flowers
from two or three large bosses of the Scarlet Geranium {Pelargonium zonale) in a
flask with hot alcohol. Filter ofi* and concentrate on a water-bath. Then pour the
hot concentrated solution into about half its volume of strong hydrochloric acid. On
cooling, a crystalline precipitate of pelargonin chloride separates out. Examine under
the microscope and note that it consists of sheaves and rosettes of needles. Filter oflF
the crystals, take up in water and make the following experiments with the solution :

{a) Add alkali. A deep blue-violet colour is produced.

(6) Take two equal quantities of solution in two evaporating dishes. To one add
as quickly as possible some solid sodium chloride. The colour in the solution without
salt will rapidly fade owing to the formation of the colourless isomer in neutral
solution : this change is prevented to a considerable extent in the solution containing
salt owing to the formation of an addition compound of the pelargonin with the
sodium chloride which prevents isomerization (see p. 115). To portions of the water
solution (without sodium chloride) which has lost its colour add respectively acid
and alkali. The red colour returns with acid owing to the formation of the red acid
oxonium salt : with alkali a greenish-yellow colour will be produced due to the
formation of the salt of the colourless isomer. If alkali is added to the portion of the
pigment solution containing the sodium chloride, it will be found that it still gives
a violet colour.

(c) Add sulphuric acid and amyl alcohol. The alcohol does not take up the
colour. Add amyl alcohol after acidifying another portion of the solution with
sulphuric acid and heating on a water-bath. The alcohol now abstracts some of the
colour. This shows that the glucoside pelargonin exists in the first case, but is de-
composed into the non-glucosidal pelargonidin after heating with acid.

{d) Acidify with hydrochloric acid and add zinc dust : the colour disappears and
returns again after filtering.

Expt. 114. Preparation of the acetic acid salt of pelargonin. Make an alcoholic
extract of petals as in Expt. 113. Evaporate down and pour into glacial acetic acid
instead of hydrochloric acid. The crystals of the salt formed are smaller and more
purple in colour than those of the chloride.

In considering the anthocyan pigments, the question now arises —
What is the chemical significance of the various shades in the living
plant? Apparently the same pigment may be present in two flowers
of totally different colours, as in the blue Cornflower and the magenta

120 AROMATIC COMPOUNDS [ch.

Rosa gallica. It has been suggested that in such cases the pigment is
modified by other substances present in the cell-sap: thus it may be
present in one flower as a potassium salt, in another as an oxonium salt
of an organic acid, and in a third in the unaltered condition. But exactly
how these conditions are brought about is not clear. In one or two cases,
moreover, where there is a red or pink variety of a blue or purple flower,
the variety, when examined, has been found to contain a different pigment
and one less highly oxidized than that in the species itself The above
phenomena are exemplified in the Cornflower (Centaurea Gyanus). The
flowers of the blue type contain the potassium salt of cyanin, the purple
variety, cyanin itself, while those of the pink variety contain pelargonin.
The mode of origin of anthocyan pigments in the plant is as yet
obscure. It has been suggested ( Wheldale, 24) that they have an intimate
connexion with the flavone and flavonol pigments, which can be seen at
once by comparing the structural formula of quercetin with that suggested
for cyanidin:

Wo

^^ .O^ ^ .OH I ^ A , ,OH

OH

^" OH H

Quercetin Cyanidin

All the anthocyan pigments so far isolated, however, have been found
to contain the flavonol, and not the flavone, nucleus.

Just as in the case of the flavone and flavonol pigments, some of the
anthocyan pigments are specific, while others, on the contrary, are common
to various genera and species. Also more than one anthocyan pigment
may be present in the same plant.

It will be pointed out later that small amounts of a substance iden-
tical with cyanidin are said to be formed by reduction of quercetin with
nascent hydrogen, but this does not necessarily prove that the formation
of anthocyan pigments in the plant takes place on the same lines. If
we compare the formulae for a number of anthocyan with flavone and
flavonol pigments, it is seen that they may be respectively arranged in
a series, each member of which differs from the next by the addition of
an atom of oxygen :

Luteolin, kaempferol and fisetin CigHioOa Pelargonidin C15H10O5
Quercetin C16H10O7 Cyanidin CisHjoOe

Myricetin CieHioOs Delphinidin C15H10O7

VIII] AROMATIC COMPOUNDS 121

The relationship between these two classes of substances in the plant
can only be ascertained by discovering which flavone, flavonol and an-
thocyan pigments are present together^ and then to determine whether
the relationship is one of oxidation or reduction, a problem which has
not yet received adequate attention (Everest, 7).

A reaction which is of interest in connexion with the relationship
between the above two classes of pigments is that which takes place
when solutions of some flavone or flavonol pigments are treated with
nascent hydrogen. If an acid alcoholic solution of quercetin is treated
with zinc dust, magnesium ribbon or sodium amalgam, a brilliant magenta
or crimson solution is produced, and this solution gives a green colour
with alkalies (Combes, 6). The red substance thus produced has been
termed "artificial anthocyanin" or allocyanidin. The product is not a
true anthocyan pigment but has, it is suggested, an open formation
(Willstatter, 31):

9-\__ >o"

OH H

It is said, however, to contain small quantities of a substance iden-
tical with natural cyanidin from the Cornflower (Willstatter, 31). The
fact that small quantities of a natural anthocyan pigment can be obtained
artificially from a hydroxyflavonol by reduction does not necessarily imply
that one class is derived from the other in the living plant.

From the above reaction of quercetin the result follows that when
many plant extracts [most plants (see p. 110) contain flavone or flavonol
pigments] are treated with nascent hydrogen, artificial anthocyan pig-
ment is produced. Moreover, it seems probable that if the yellow^
pigments acted upon are in the glucosidal state, and if the reduction
takes place in the cold, allocyanin (the glucoside of allocyanidin) is
formed and the product is not extracted from solution by amyl alcohol.
But if the flavone is non-glucosidal, or if the solution is boiled before or
after reduction, then allocyanidin (non-glucosidal) is formed and is
extracted by amyl alcohol.

Ea^pt 115. Formation of allocyanidin from quercetin. Make an alcoholic solution
of a little of the glucoside of quercetin prepared from %\ih.QY Narcissus or Gheiranthus

1 The only two satisfactory cases known are Delphinium consolida, which contains
kaempferol and delphinidin, and Viola tricolor, which contains quercetin and delphinidin.
Neither of these confirms the hypothesis of simple reduction.

122 AROMATIC COMPOUNDS [ch.

(see Expt. 110). Acidify with a little strong hydrochloric acid and heat on a water-
bath in an evaporating basin. Add a little zinc dust from time to time. A brilliant
pink or magenta colour due to allocyanidin is produced. To a little of this solution
add some alkali : a green colour is produced. If the alcohol and hydrochloric acid
are evaporated off, and a little water and sulphuric acid added, on shaking up with
amyl alcohol, all the allocyanidin passes into the amyl alcohol. (The distribution of
the allocyanidin in the amyl alcohol is greater with aqueous sulphuric acid than with
aqueous hydrochloric acid.)

Expt. 116. Formation of allocyanin from quercetin. Make a suspension of the
glucoside of quercetin from Cheiranthus or Narcissus (see Expt. 110) in about 2N
sulphuric acid, and then add zinc dust (or a drop of mercury about the size of a pea
and a little magnesium powder) in the cold. The pink or magenta colour is gradually
developed. Divide the coloured solution into two parts in two test tubes. Boil one
for 5-10 minutes. Then add amyl alcohol to each. In the unboiled test-tube the
amyl alcohol extracts no colour, since allocyanin is present. In the boiled test-tube
allocyanidin is taken up by the amyl alcohol as in Expt. 115.

Expt. 117. Formation of allocyanin and allocyanidin from plant extracts. For
this purpose the yellow varieties "Primrose" or "Cloth of Gold" of the Wallflower
{Cheiranthus Cheiri) can be used. The flowers are pounded in a mortar, extracted
with cold water, the water extract acidified with sulphuric acid, and zinc dust (or
mercury and magnesium powder as above) added. A red coloration is slowly
developed. To some of the red solution add amyl alcohol. The colour is not
abstracted (allocyanin). Boil another portion. The allocyanin is thus converted into
allocyanidin which is then taken up on addition of amyl alcohol.

Oxidizing Enzymes.

There are certain enzymes in the plant which are concerned with
processes of oxidation and reduction (Chodat, 1). They are considered at
this point since we have most information of them in their connexion
with aromatic substances.

Peroxidases. A peroxidase is practically always present in the tissues
of the Higher Plants. These enzymes are able to decompose hydrogen
peroxide with the formation of "active" or atomic oxygen:
H2O2 -f peroxidase = H2O -f O.

The tests for peroxidases will be considered later.

Oxidases (synonymous with laccases or phenolases) are only present
in about 63 "/o of the Higher Plants. A plant oxidase, moreover, is made
up of three components, i.e. (1) an enzyme, termed an oxygenase, (2) an
aromatic substance containing an ortho-dihydroxy grouping such as that
in catechol and (3) a peroxidase as above described (Wheldale Onslow, 9).

There are a number of substances with the catechol grouping, that

VIII]

AROMATIC COMPOUNDS

123

is two hydroxy! groups in the ortho position, found in plants, such as
catechol, protocatechuic acid, caffeic acid, hydrocaffeic acid, etc.,

COOH CH=CH— COOH

/\

OH

/\

OH

OH

OH

Catechol

OH

Protocatechuic acid

\/'

OH

CafFeic acid

When solutions of such substances are left in air, they slowly aut-
oxidize with the production of brownish oxidation products, accompanied,
at the same time, by the formation of peroxides, probably hydrogen
peroxide (since organic peroxides . tend to decompose in the presence
of water with the production of hydrogen peroxide). The oxygen in this
form — O — O — can be detected by chemical tests in the solutions. In
plants, moreover, which contain catechol compounds, there are present
certain enzymes, the oxygenases, which catalyze the autoxidation of the
catechol compounds, and these only, with rapid production of a brown
colour and of a peroxide \ Since peroxidases are also universally present,
these may decompose the peroxide with production of active oxygen:

catechol substance + oxygenase + molecular oxygen — ►peroxide
peroxide + peroxidase — »► active oxygen.

This system, which constitutes an oxidase, is therefore capable of
transforming molecular into active oxygen, and may in this way bring
about oxidations in the plant which would not otherwise occur.

Catechol substances with the accompanying oxygenases are only
present, as mentioned above, in about 63 °/o of the higher plants. The}^
are present in about 76% of the Monocotyledons, in about 84^0 of the
Sympetalae but only in about 50 "/o of the Archichlamydeae examined.
Usually the genera of an order are all of one kind, either oxidase plants
or peroxidase plants without the oxygenase and catechol elements. A
few examples of oxidase orders are Gramineae, Umbelliferae, Labiatae,
Boraginaceae, Solanaceae and Compositae: of peroxidase orders, Liliaceae,
Cruciferae and Crassulaceae : of mixed orders, Ranunculaceae, Rosaceae
and Leguminosae.

After death by inj ury, chloroform vapour, etc., the tissues of oxidase
plants usually turn brown or reddish-brown in air, e.g. fruit of Apple,
petals oi Anemone, Rosa, etc.; peroxidase plants, on the contrary, do not

1 The term oxygenase was originally applied by Bach and Chodat to ferment like com-
pounds which form peroxides.

124 AROMATIC COMPOUNDS [ch.

show this phenomenon. Since the oxidase provides an active oxidizing
system, it is probable that a general oxidation of aromatic and other
substances {in addition to catechol) takes place after death, in many
cases leading to the production of dark pigments, e.g. the blackening of
lacquer from latex of the Lacquer tree {Rhus verniciferay. In Schenckia
hliimenaviana (Rubiaceae), also, the whole plant turns bright red in
chloroform vapour, and blue pigments are formed in flowers of an Orchid
(Phajus) after death.

Tests for peroxidases are based on the property of a number of sub-
stances (benzidine, a-naphthol, guaiacum, pyrogallol, etc.) of giving highly
coloured oxidation products in presence of active oxygen. Hence solutions
of the above substances in the presence of hydrogen peroxide provide
tests for peroxidases:

XD'

HoN<r V-< >NH

OH NH2

a-Naphthol Benzidine p-Phenylenediamine

Expt. 118. Demonstration of the presence of a peroxidase. Pound' up a little
Horse-radish root {Cochlearia Armoracia) with water. Filter and, taking a few c.c.
each time in a small evaporating dish, make the following tests :

{a) Add a few drops of a 10 % solution of guaiacum. No colour is developed.
Add a few drops of hydrogen peroxide : a deep blue colour appears.

Guaiacum gum is obtained from two West Indian species of Guaiacum trees,
G. offi^cinale and O. sanctum^ partly as a natural exudation and partly by means of
incisions. It gives a yellow solution with alcohol which contains guaiaconic acid, and
the latter, on oxidation, yields guaiacum blue. As far as possible, inner portions of
the resin lumps should be used, as the resin oxidizes in air, and then may give un-
reliable results. It is best to make the tincture freshly before use, and, as a precaution,
to boil it on a water-bath with a little blood charcoal (preferably Merck's) and filter.
Guaiacum gum tends to form peroxides on exposure to air, and these are removed
by the above treatment.

(6) A 1 0/0 solution of a-naphthol in 50 % alcohol, followed by a few drops of
hydrogen peroxide. A lilac colour is developed.

(c) A 1 ^Iq solution of benzidine in 50 % alcohol followed by a few drops of
hydrogen peroxide. A blue colour is developed.

{d) A\ ^/q solution of jo-phenylenediamine hydrochloride in water followed by a
few drops of hydrogen peroxide. A greenish colour is developed.

Repeat the above experiments with an enzyme extract that has been boiled. No
colour is given, showing that the enzyme has been destroyed by boiling. Other

1 The chief constituent of the latex, however, is a catechol derivative.

VIII] AROMATIC COMPOUNDS 125

material which may be used for the above tests is fruit of the Melon and Cucumber
and root of the Kadish and Turnip.

Of the above substances only guaiacum, as a rule, is sufficiently
sensitive to be oxidized by the amount of active oxygen produced by the
plant oxidase. The juices and water extracts of oxidase plants will usually
blue guaiacum immediately. If considerable quantities of sugars or
tannins are present in the tissues, they may inhibit the guaiacum
test.

Another test which may be used is the following. A solution of
dimethyl -jo-phenylenedidmine hydrochloride and a-naphthol in presence
of dilute sodium carbonate gives a deep violet-blue colour in the presence
of an oxidase.

Expt. 119. Demonstration of the presence of an oxidase. Cut two or three thin
slices from a fresh tuber of the Potato, pound well in a mortar, add a little water
and filter. With a few c.c. of the extract in an evaporating dish make the following
tests :

(a) Add a few drops of 10 ^/o solution of guaiacum. A blue colour appears.

(6) Add 2*5 c.c. of a 0*14 % solution of a-naphthol and 2 '5 c.c. of a 0*17 7o solution
of dimethyl -jo-phenylenediamine hydrochloride and 5 c.c. of 0*1 °l^ solution of sodium
carbonate. A deep violet-blue colour appears.

Control experiments should be performed by using boiled enzyme extract. Other
material which may be used is fruit of the Pear, Plum and Cherry.

Expt. 120. To show the distribution of oxidases and peroxidases in various plants^
and the correlation between the presence of oxidase and browning on injury or in
chloroform vapour. Take a selection of the plants given below, and in each case grind
up a portion of the plant in a mortar with a little water and filter. Divide the filtrate
into two parts in small porcelain dishes. Allow one part to stand in air, and note the
darkening in colour in cases where an oxidase is present. To the other add a few
drops of guaiacum. To extracts containing a peroxidase only, after 5-10 minutes,
add in addition a few drops of hydrogen peroxide. Further, small pieces of the plants
to be tested should be placed in a corked flask containing a few drops of chloroform,
and the development of browning noted in the case of plants containing an oxidase.
For demonstration of oxidases the following plants may be used : Christmas Rose
{Helleborus niger)^ Dandelion {Taraxacum offi^cinale), Forget-me-not {Myosotis),
Hawthorn {Crataegus) and White Dead Nettle {Lamium album). For peroxidases :
Arabis, Aubrietia, Pea {Pisum sativum), Stock {Matthiola\ Wallflower {Cheiranthus
Chdri) and Violet ( Viola).

The peroxidases, like other enzymes, can be extracted either with
water or dilute alcohol and precipitated from solution by strong alcohol.

Expt. 121. Preparation of peroxidase from Horse-radish (Cochlearia) roots. Mince
up the Horse-radish roots in a mincing machine. The product is allowed to stand for
24 hrs. to enable the glucoside, potassium myronate, to be hydrolyzed by the enzyme,
myrosin. Then extract with 80 % alcohol. The alcohol is decanted off, and the

126 . AKOMATIC COMPOUNDS [ch.

residue pressed free from alcohol in a press. The residue is next extracted with 40 %
alcohol for 48 hrs., filtered and precipitated with 90 % alcohol. The precipitate,
which contains the peroxidase, is filtered off". Dissolve up in water and make the
test for peroxidases (Expt. 118).

Peroxidase from the Horse-radish has been prepared on a large scale
and very carefully purified (Willstatter and Stoll, 34). The purified

t product was found to consist chiefly of a nitrogenous glucoside, a result
which does not throw much light on its catalyzing properties.

The oxidation of pyrogallol, in the presence of a peroxidase and
hydrogen peroxide, has been used as a method for estimating the activity
of these enzymes. Solutions of known strength of pyrogallol and hydro-
gen peroxide are used, and to the mixture a solution of a known weight
of prepared peroxidase is added. An oxidation product, termed purpuro-
gallin is formed. After a definite time, the reaction is stopped by adding
acid, and the purpurogallin extracted by ether. The ether extract is
colorimetrically compared with an extract containing a known amount
of purpurogallin (Willstatter and Stoll, 34).

Expt. 122. Outline of method for estimating peroxidase hy formation of purpuro-
gallin. Make a solution of 0*5 gm. of pyrogallol in 200 c.c. of distilled water, and
add to it 1 c.c. of 5 o/o hydrogen peroxide. Then add about 5 c.c. of a solution of
Horse-radish peroxidase from Expt. 121. After 5 minutes add to half the mixture
25 c.c. of dilute sulphuric acid and extract the purpurogallin with ether in a
separating funnel. The purpurogallin will be extracted by the ether, giving a yellow
solution. Allow the other half of the mixture to stand. The colour will deepen, and
a reddish deposit of purpurogallin will be precipitated. Examine a little of the
deposit under the microscope. It will be found to consist of sheaves of crystals.

A solution of peroxidase from Alyssum leaves [Expt. 124 (6)] can also be used.

The fact that an oxidase contains an oxygenase and catechol substance
may be demonstrated as follows. The tissue of an oxidase plant is rapidly
pounded under alcohol (to avoid oxidation) and extracted several times
with cold alcohol, by which the. catechol substance is removed. The two
enzymes, oxygenase and peroxidase, remain in the tissue residue. This
residue or its water extract will give no (or very little) reaction with
guaiacum, since one of the components for producing the peroxide has
been removed. If now a little catechol is added followed by guaiacum,
a blue colour immediately appears. Moreover, from an alcoholic extract
of the tissues the catechol substance can be precipitated as a lead salt,
the lead removed as insoluble sulphate, and the aromatic compound set
free again in solution. If the enzyme extract is then added to the solu-
tion of the catechol substance, a brown colour is produced together with
a peroxide, and the mixture will give a blue colour with guaiacum.

viii] AROMATIC COMPOUNDS 127

Expt. 123. Resolution of the components of the oxidase in the Potato tuber. (A)
Separation of peroxidase and oxygenase. Cut a few thin slices from a peeled potato
and put them in a mortar which contains sufficient 96 % alcohol to prevent, as far
as possible, exposure to the air, and pound them thoroughly. Filter quickly on a
filter-i)ump, and repeat the process several times until a colourless powder, consisting
of cell-residues, starch, etc. is obtained. The enzymes (including the peroxidase and
oxygenase) of the cells are precipitated by the alcohol and remain in the cell-residue.
Make a water extract of the white powder and filter. To a portion of the filtrate add
a few drops of guaiacum tincture ; no blue colour is given. Add further a few drops
of dilute hydrogen peroxide: a blue colour appears. (B) Separation of the aromatic
substance. Take about 500 gms. of freshly peeled potato tuber, cut it into thin slices
and drop them as rapidly as possible into a flask containing 250 c.c. of boiling 96 <)/o
alcohol on a water-bath. Continue boiling for 15 mins,, and then filter. Evaporate
off the alcohol from the filtrate, take up the residue in a little water, warm and filter.
To the filtrate add concentrated lead acetate solution until a precipitaoe ceases to be
formed. Filter off the precipitate, which is pale yellow in colour, stir up in a little
water and add 10 % sulphuric acid drop by drop until the yellow colour is destroyed,
and the lead is converted into lead sulphate. Filter off the lead sulphate : the filtrate
contains the aromatic substance in solution. Neutralize the solution carefully with
1 ^/o caustic soda and make the following tests with separate portions in small
evaporating dishes :

(a) Add a drop of ferric chloride solution : a deep green colour appears. Add
further a few drops of 1 o/^ sodium carbonate solution. The green colour changes to
a bluish- and finally, a reddish-purple. This reaction is characteristic of aromatic
compounds containing the catechol grouping, i.e. two hydroxyl groups in. the ortho
position (see p. 123).

(6) Add a little of the enzyme solution prepared in (A). The mixture will
gradually turn brown owing to the oxidation of the aromatic by the oxygenase.

(c) To (6) add a few drops of guaiacum tincture. A blue colour is given owing to
the presence of the peroxide formed in (6), the oxidase system being now complete.

Expt. 124. Actio7i of oxygenase on catechol, (a) The oxygenase of the Potato
tuber {or Pear fruit). Make a 1 o/q solution of catechol in distilled water. To some
of this solution, in a small evaporating dish, add a little of the enzyme solution from
Expt. 123 (A). Note that the catechol solution gradually turns brown. Add further
a few drops of guaiacum tincture. A blue colour appears, (b) Enzyme extract of
Alyssum leaves. Pound up 2-3 Alyssum leaves in a mortar with some 96 % alcohol,
and filter on a filter-pump. Repeat the process until the residue is practically
colourless. Extract the residue with a little distilled water and filter. Proceed as in
(a). No browning of catechol takes place and no blue colour is formed on the sub-
sequent addition of guaiacum.

For section (a) the following material may also be used: fruits of Apple and
Greengage, flowers of Horse Chestnut (Aesculus) and leaves of Pear, the method of
preparation in (6) being employed. For section (6) flowers of white Arabis may also
be used.

If in the preparation of the enzymes from the Potato tuber, the
tissue is allowed to brow^n before extracting with alcohol, the cell-residue

128 AROMATIC COMPOUNDS [ch.

is tinged with brown and, on extraction with water, the filtrate will give
an oxidase reaction with guaiacum. This is to be explained by the fact
that the peroxide has been adsorbed by the tissue residue. This pheno-
menon is probably the explanation of the preparation of some oxidases
called "laccases." Such enzymes have been obtained by the precipita-
tion with strong alcohol of the expressed juices (containing peroxide
since they were obtained by crushing the tissues) of plants which brown
on injury. The enzyme and other organic matter is precipitated and
carries with it the peroxide. Such a product will readily oxidize phenols
with other groupings, e.g. pyrogallol, quinol, etc.

Tyrosinase. This enzyme is widely distributed in plants. It occurs
in the Banana (Musa sapientum), Wheat {Triticum vulgar e), Beet {Beta
vulgaris), Oriental Poppy (Papaver orientale), Lacquer tree (Rhus
vernicifera), Potato (Solarium tuberosum) and Dahlia (Dahlia variabilis).
It has been demonstrated in about 16 natural orders and 21 genera.

Tyrosinase oxidizes tyrosine with the evolution of carbon dioxide
and ammonia and the production of a pink colour which darkens through
red to black. The final black pigments are known as melanins. A solu-
tion of ^-cresol

CH3

V

OH

can be used as a delicate test for the enzyme. If the enzyme is present,
a yellowish or orange-red colour is formed.

Tyrosinase of the Potato tuber can be precipitated from a water
extract with absolute alcohol: or if the potato tissue is extracted with
cold 96 7o alcohol, the enzyme is precipitated and remains in the tissue
residue, as does the peroxidase (Expt. 123(A)], but the tyrosine is
almost entirely washed away.

£Jxpt. 125. Demonstration of the presence of tyrosinase in the Potato. Take about
half a potato and proceed as in the preparation of peroxidase [see Expt. 123 (A)].
Roughly dry the powder left on the filter and then add about 100 c.c. of water and
allow to stand for 15 mins. Filter, and divide the filtrate into four portions a, 6, c
and d. Make a suspension of a little tyrosine in water (tyrosine is only slightly
soluble in cold water).

To a add 5 c.c. of tyrosine suspension.

To h add 5 c.c. of tyrosine suspension and boil.

To c add some p-cresol.

To d nothing is added.

VIII] AROMATIC COMPOUNDS 129

Plug all the tubes with cotton-wool, put in an incubator at 38° C. for 2-3 hrs.
Note that tube a fairly rapidly turns red, then brown and finally black. Tube d may
darken a little owing to the action of tyrosinase and oxygenase on the traces of
plant aromatics left in the tissue. Tube h remains unaltered. Tube c gives an
orange-red colour.

It is probable that tyrosinase is a mixture of enzymes, of which an
oxidase is one component. It appears to be a fact that the plants which
give the tyrosinase reactions are always oxidase, and not peroxidase,
plants.

Reductases. (Oxido-reductases.) These enzymes (Bach, 4) catalyze
the decomposition of water into hydrogen and oxygen, provided sub-
stances are present which will accept the hydrogen and oxygen re-
spectively. Such an enzyme has been shown to be present in the tuber
of the Potato. It will reduce nitrates to nitrites, provided acetaldehyde
is present, the latter being oxidized to acetic acid.

Expt. 126. Demonstration of the presence of a reditctase in the Potato. Prepare a
crude enzyme extract of the tuber as in [Expt. 123 (A)]. Take 10 c.c. of a 4 %
solution of sodium nitrate in a test-tube, heat it in a beaker of water to 60° C. and
then add 10 c.c. of the enzyme extract, followed by 3 drops of IOo/q acetaldehyde
solution. Prepare a control tube with boiled enzyme extract. Keep the tubes at
60° C. for 2-3 minutes. Test for nitrite with a few drops of an alcoholic solution of
indole and a few drops of strong hydrochloric acid. The unboiled tube should give
a red colour.

Catalases. These enzymes are probably present in all plants. They
decompose hydrogen peroxide with the formation of molecular oxygen
(see Expt. 15).

The function of the peroxidases, reductases, catalases and tyrosinase
in the living cell is not known. It would appear that the oxidase reaction
(as detected by guaiacum, etc.) is the outcome of post-mortem changes
after the death of the cell. It is probable, however, that the processes
giving rise to it may take place to some extent, though under control,
in the living cell and it has been suggested, in fact, that oxidases play
a part in respiration (Palladin, 10). There is certainly reason to believe
that the first stages of respiration in plants involve a fermentation of a
hexose similar to that taking place in yeast. The enzymes, zymase and
carboxylase have been shown to be present in the tuber of the Potato
and the root of Beet (Bodnar, 5). Hexosephosphatase has also been
demonstrated in the bran of Wheat and seeds of the Castor-oil Plant
(Ricinus communis) (Plimmer, 20). Whether oxidases act upon the
products formed by the preliminary action of zymase remains an open
question. The fact that they are not universally present in plants
presents a difficulty.

o. 9

130 AROMATIC COMPOUNDS [ch.

REFERENCES

Books

1. Chodat, R. Darstelkmg von Oxydasen und Katalasen tierischer und pflanz-
licher Herkunft, Methoden ihrer Anwendung. Handbuch der biochemischen
Arbeitsmethoden. E. Abderhalden, Berlin, 1910, Vol. 3 (1), pp. 42-74.

2. Perkin, A. G., and Everest, A. B. The Natural Organic Colouring
Matters. London, 1918.

3. Wheldale, M. The Anthocyanin Pigments of Plants. Cambridge, 1916.

Papers

4. Bach, A. Zur Kenntnis der Reduktionsfermente. IV. Mitteilung. Pflanz-
liche Perhydridase. Biochem. Zs., 1913, Vol. 52, pp. 412-417.

5. Bodnar, J. Ueber die Zymase und Carboxylase der KartofFel und Zuckerriibe.
Biochem. Zs., 1916, Vol. 73, 193-210.

6. Combes, R. Sur la presence, dans des feuilles et dans des fleurs ne formant
pas d'anthocyane, de pigments jaunes pouvant dtre transform^s en anthocyane.
C. R. Acad. scL, 1914, Vol. 158, pp. 272-274.

7. Everest, A. E. The Production of Anthocyanins and Anthocyanidins.
Part III. Proc. R. Soc, 1918, B Vol. 90, pp. 251-265.

8. Fischer, E., und Preudenberg, K. Ueber das Tannin und die Synthese
ahnlicher Stoffe. Ber. D. chem. Ges., 1912, Vol. 45, pp. 915-935.

9. Onslo-W, M. Wheldale. Oxidising Enzymes. II. The Nature of the
Enzymes associated with certain Direct Oxidising Systems in Plants. Biochem. J.
1920, Vol. 14, pp. 535-540. IV. The Distribution of Oxidising Enzymes among the
Higher Plants. Bioch. J., 1921, Vol. 15, pp. 107-112.

10. Palladin, W. Ueber das Wesen der Pflanzenatmung. Biochem. Zs., 1909,
Vol. 18, pp. 151-206.

11. Perkin, A. G. Luteolin. Part I. J. Chem. JSoc, 1896, Vol. 69, pp. 206-212.
Part II. Ibid., 1896, Vol. 69, pp. 799-803.

12. Perkin. A. G. Apiin and Apigenin. J. Chem. Soc, 1897, Vol. 71, pp. 805-
818. Ibid., 1900, Vol. 77, pp. 416-423.

13. Perkin, A. G. Robinin, Violaquercetin, Myrticolorin and Osyritrin.
J. Chem. Soc, 1902, Vol. 81, pp. 473-480.

14. Perkin, A. G., and Horsfall, L. H. Luteolin. Part III. J. Chem. Soc,
1900, Vol. 77, pp. 1314-1324.

15. Perkin, A. G., and Hummel, J. J. Occurrence of Quercetin in the Outer
Skins of the Bulb of the Onion. J. Chem. Soc. 1896, Vol. 69, pp. 1295-1298.

16. Perkin, A. G., and Hummel, J. J. The Colouring Matters occurring in
various British Plants. Part I. ./. Chem. Soc, 1896, Vol. 69, pp. 1566-1572.

17. Perkin, A. G., and Newbury, P. G. The Colouring Matters contained
in Dyer's Broom {Genista tinctoria) and Heather {Calluna vulgaris). J. Chem. Soc,
1899, Vol. 75, pp. 830-839.

18. Perkin, A. G., and Phipps, S. Notes on some Natural Colouring Matters.
J. Chem. Soc, 1904, Vol. 85, pp. 56-64.

19. Perkin, A. G., and Wilkinson, E. J. Colouring Matter from the
Flowers oi Delphinium Consolida. J. Chem. Soc, 1902, Vol. 81, pp. 585-591.

VIII] AROMATIC COMPOUNDS 131

20. Plimmer, R. H. A. The Metabolism of Organic Phosphorus Compounds.
Their Hydrolysis by the Action of Enzymes. Biochem. t/., 1913, Vol. 7, pp. 43-71.

21. Plimmer, R. H. A. and Page, H. J. An Investigation of Phytin.
Biochem. J., 1913, Vol. 7, pp. 157-174.

22. Shibata, K., Nagai, I., and Kishida, M. The Occurrence and Physio-
logical Significance of Flavone Derivatives in Plants. J. Biol. Chem., 1916, Vol. 28,
pp. 93-108.

23. Waage, T. Ueber das Vorkommen uud die RoUe des Phloroglucins in der
Pflanze. Ber. D. hot. Ges., 1890, Vol. 8, pp. 250-292.

24. Wheldale, M. On the Nature of Anthocyanin. Froc. Camb. Phil. Soc.^
1909, Vol. 15, pp. 137-168.

25. Wheldale, M., and Bassett, H. LI. The Flower Pigments oi Antirrhinum
majus, II. The Pale Yellow or Ivory Pigment. Biochem. J., 1913, Vol. 7, pp. 441-
444.

26. Wheldale, M., and Bassett, H. LI. The Flower Pigments oi Antirrhi-
num majus. III. The Red and Magenta Pigments. Biochem. J., 1914, Vol. 8,
pp. 204-208.

27. Wheldale, M., and Bassett, H. LI. The Chemical Interpretation of
some Mendelian P'actors for Flower-Colour. Proc. R. Soc, 1914, B Vol. 87,
pp. 300-311.

28. Willstatter, R. Ueber die FarbstofFe der Bliiten und Frlichte. SitzBer.
Ak. Wiss., 1914, pp. 402-411.

29. W^illstatter, R., Bolton, E. K., Mallison, H., Martin, K., Mieg,
W., Nolan, T. S., und Zollinger, B. H. Untersuchungen Uber Anthocyane.
Liebigs Ann. Chem., 1915, Vol. 408, pp. 1-162.

30. Willstatter, R., und Everest, A. E. Ueber den Farbstoff der Korn-
blume. Liehigs Ann. Chem., 1913, Vol. 401, pp. 189-232.

31. Willstatter, R., und Mallison, H. Ueber die Verwandtschaft der
Anthocyane und Flavone. SitzBer. Ak. Wiss., 1914, pp. 769-777.

32. Willstatter, R., und Weil, F. J. Untersuchungen iiber Anthocyane.
Liebigs Ann. Chem. 1916, Vol. 412, pp. 113-251.

33. Willstatter, R., und Zechmeister, L. Synthese des Pelargonidins.
mzBer. Ak. Wiss., 1914, pp. 886-993.

34. Willstatter, R., und StoU, A. Ueber Peroxydase. Liebigs Ann. Chem.,
1918, Vol. 416, pp. 21-64.

9—2

CHAPTER IX

PROTEINS AND AMINO-ACIDS

No class of compounds is of more fundamental significance than the
proteins. The matrix of protoplasm largely consists of proteins in the
colloidal state, and, without doubt, they occur to some extent in the
same condition in the cell-sap. They are also found in the cell in the
solid state, in the form of either amorphous granules, termed aleurone,
or crystalline or semi-crystalline bodies, termed crystalloids. Both solid
forms constitute "reserve material" and are often found in seeds, tubers,
bulbs, buds and roots.

Plant proteins may be classified on the following plan:

1. The simple proteins.

(a) Albumins.

(b) Globulins.

(c) Prolamins (Gliadins).

(d) Glutelins

2. Conjugated proteins.

(a) Nucleoproteins.

3. Derived proteins.

{a) Metaproteins.

(b) Proteoses ( Albumoses).

(c) Peptones.

(d) Polypeptides.

Although they are present in every cell in all parts of plants, little,
however, is known of plant proteins, except of those in seeds, because
of the difficulties of obtaining them in sufficiently large quantities, and
of separating them from each other.

Proteins are in the colloidal state when in so-called solution, and are
unable to diffuse through parchment membranes. The proteoses and
peptones, however, which have simpler molecules, can diffuse through
such membranes.

The vegetable proteins are soluble in various solvents according to
the nature of the protein; some are soluble in water, others in dilute
salt solutions, others, again, in dilute alkalies, and a few in dilute alcohol.
Vegetable albumins are coagulated from solution on boiling, but most

CH. IX] PKOTEINS AND AMINO- ACIDS 133

of the globulins, unlike the corresponding animal products, are only
imperfectly coagulated on heating and some not at all. The precipitate
formed when coagulation is complete will not go into solution again
either in water, acid, alkali or salts. Alcohol precipitates the proteins;
in the case of animal proteins, the precipitate becomes coagulated and
insoluble if allowed to remain in contact with the alcohol but this does
not appear to be so with plant proteins.

In addition, certain neutral salts, the chlorides and sulphates oi
sodium, magnesium and ammonium, have the property of precipitating
proteins (except peptones) from solution when added in sufficient quan-
tity. The protein is quite unchanged in precipitation and can be made
to go into solution again. The various proteins are precipitated by
different concentrations of these salt solutions (see p. 138).

The salts of calcium and barium and the heavy metals produce
insoluble precipitates with the proteins, and in this case the reaction is
irreversible.

In regard to chemical composition, the proteins contain the elements
carbon, hydrogen, nitrogen, oxygen and sulphur. There is every reason
to believe tfiat the protein molecule is constituted of amino-acids con-
densed, with elimination of water, on the plan which may be depicted
as follows:

Ri Rii Riii

NH2— CH— COjOH HjNH— CH— COiOH H:NH— CH— COiOH HiNH-

-COjOH H;NH— CH— COOH

Conversely, when the proteins are acted upon by hydrolyzing en-
zymes, a series of hydrolytic products are formed which have smaller
molecules than the original proteins. They may be enumerated as:

1. Albumoses.

2. Peptones.

3. Amino-acids.

In the same way when proteins are boiled with acids, a number of
the amino-acids are obtained as an end-product.

The above amino-acids may be either aliphatic or aromatic, and they
are characterized by having one or more hydrogen atoms, other than
those in the carboxyl groups, replaced by the group — NHg. Thus they
are acids by virtue of the carboxyl groups, and bases by virtue of the
— NH2 groups: towards strong acids they act as bases, and towards

134 PROTEINS AND AMINO-ACIDS [ch.

strong bases as acids. The amino-acid, alanine, for instance, forms salts,

sodium amino-propionate with a base, and alanine hydrochloride with

an acid :

CH3— CH— COONa CH3— CH— COOH

I I

NH2 NHg-HCI

Substances behaving in this way have been termed "amphoteric"
electrolytes (see also p. 16).

In the proteins, which are formed by condensation, as explained above,
there are always some NHg and COOH groups left uncombined. Hence
a protein must, in the same way, have the properties of both an acid
and a base.

The amino-acids which are obtained by the hydrolysis of plant pro-
teins may be classified as follows:

Aliphatic compounds.

Mono-carboxylic mono-amino acids :

Glycine or a-amino-acetic acid

CH2(NH2)COOH

Alanine or a-aniino-propionic acid

CH3-CH(NH2)-COOH

Valine or a-amino-iso-valeric acid

CH3^

>CHCH(NH2)C00H

CH3

Leucine or a-amino-iso-caproic acid

CH3V

■ >CH • CH. • CH(NH2) • COOH
CH3^

Iso-leucine or a-amino-jS-methyl-^-ethyl-propionic acid

CH3.

^CH •CH(NH2) COOH
C2H6

Serine or a-amino-jS-hydroxy-propionic acid

CH20HCH(NH2)COOH

Dicarboxylic mono-amino acids :

Aspartic acid or a-amino-succinic acid

COOH • CH2 • CH(NH2) • COOH
Glutaminic acid or a-amino-glutaric acid

COOH •CH2-CH2 •CH(NH2)- COOH

ixj PROTEINS AND AMINO- ACIDS 135

Mono-carboxylic di-amino acids :

Arginiiie or S-giianidine-a-araino- valeric acid
NH2

HN=:C— NH • CH2 • CH2 • CH2 • CH(NH2) • COOH

Lysine or a-f-di-aniino-caproic acid

CHaCNHa) ' CHg ' CHg • CH2 ' CH(NH2) ' COOH

Dicarboxylic di-amino acid :

Cystine (dicysteine) or di-/3-thio-a-amino-propionic acid

CH2 — S — S — CH2

I I

CH(NH2) CH(NH2)

COOH COOH

Aromatic compounds.

Mono-carboxylic mono-amino acids :

Phenyl-alanine or /3-phenyl-a-aniino-propionic acid
C6H5-CH2CH(NH2)COOH
Tyrosine or jo-hydroxy-phenyl-alanine

OH • C6H4 • CH2 • CH(NH2) • COOH

Heterocyclic compounds.
Proline or a-pyrrolidine-carboxylic acid

CH2 CH2

I I

CH2 CHCOOH

^NH^

IJistidine or /3-iminazole-alanine
CH

NH N

CH=C— CH2 • CH(NH2) ' COOH
Tryptophane or ^-indole-alanine CgHoN • CH2 ' CH (NH9) ' COOH

C'CH2CH(NH2)COOH

CH

Different proteins are formed by various combinations of the above
acids and hence give different amounts on hydrolysis.

There are certain properties and chemical reactions by means of
which proteins can be detected. These are illustrated in the following
experiment.

136 PKOTEINS AND AMINO- ACIDS [ch.

Expt. 127. Tests for proteins. Weigh out about 10 gms. of dried peas (Pisum),
grind them in a coffiee-mill and then add 100 c.c. of water to the ground mass.
Allow the mixture to stand for an hour. Filter, and make the following tests with
the filtrate (see ix 147).

(a) The xanthroproteic reaction. To a few c.c. of the protein solution in a test-tube
add about one-third of its volume of strong nitric acid. A white precipitate is
formed (except in the case of proteoses, peptones, etc.). On boiling, the precipitate
turns yellow, and may partly dissolve to give a yellow solution. Cool under the tap,
and add strong ammonia till the reaction is alkaline. The yellow colour becomes
orange. The precipitate is due to the fact that metaprotein (see p. 143) is formed
by the action of acid on albumins or globulins, and this metaprotein is insoluble in
strong acids. The yellow colour is the result of the formation of a nitro-compound
of some aromatic component of the protein, such as tyrosine, tryptophane and
phenylalanine.

(6) MUIotHs reaction. To a few c.c. of the protein solution add about half its
volume of Millon's reagent^. A white precipitate is formed. On warming, the preci-
pitate turns brick-red, or disappears and gives a red solution. The white precipitate is
due to the action of the mercuric nitrate on the proteins. The reaction is character-
istic of all aromatic substances which contain a hydroxyl group attached to the
benzene ring. The aromatic complex in the protein to which the reaction is due is
tyrosine.

(c) The glyoxylic reaction {Hopkins and Cole). To about 2 c.c. of protein solution
add an equal amount of "reduced oxalic acid 2." Mix the solutions, and then add an
equal volume of concentrated sulphuric acid, pouring it down the side of the tube.
A purple ring forms at the junction of the two liquids. If the liquids are mixed by
shaking the tube gently, the purple colour will spread throughout the solution. The
substance in the protein molecule to which the reaction is due is tryptophane. If
carbohydrates are present in the liquid to be tested, the colour is not good, owing to
blackening produced by the charring with the strong sulphuric acid.

{d) The biuret reaction. To a few c.c. of the protein solution add about 1 #.0. of
40 0/0 sodium hydrate apd one drop of 1 % solution of copper sulphate. A violet or
pink colour is produced. The reaction is given by nearly all substances containing
two CONH groups attached to one another, to the same nitrogen atom, or to the
same carbon atom. The cause of the reaction with proteins is the presence of one
or more groupings formed by the condensation of the carboxylic group of an amino-
acid with the amino group of another amino-acid (see p. 133).

1 This reagent is made by dissolving 30 c.c. of mercury in 570 c.c. of concentrated
nitric acid and then adding twice its bulk of water. It contains mercurous and mercuric
nitrates, together with excess of nitric acid and a little nitrous acid.

2 Keduced oxalic acid is prepared as follows: (a) Treat 500 c.c. of a saturated solution
of oxalic acid with 40 gms. of 2 % sodium amalgam. When hydrogen ceases to be
evolved, the solution is filtered and diluted with twice its volume of distilled water. The
solution contains oxalic acid, sodium binoxalate and glyoxylic acid (COOH • CHO).
(6) Put 10 gms. of powdered magnesium into a flask and just cover with distilled water.
Add slowly 250 c.c. of saturated oxalic acid, cooling under the tap. Filter off the insoluble
magnesium oxalate, acidify with acetic acid and dilute to a litre with distilled water.

IX] PROTEINS AND AMINO-ACIDS 137

(e) The sulphur reaction. Boil a few c.c. of the protein solution with an equal
quantity of 40 o/q sodium hydrate for two minutes, and then add a drop or two of
lead acetate. The solution turns black (or brownish, if only a small amount of
protein is present). This reaction is due to the formation of sodium sulphide by
the action of the strong alkali on the sulphur of the protein. On addition of the
lead salt, either a black precipitate, or dark colour, due to lead sulphide is formed.
The sulphur in the protein molecule is mainly present as cystine.

For the following tests, a purified protein solution is necessary, since the reactions
may also be given by accompanying aromatic substances, carbohydrates, etc. For
this purpose take 40 gms. of ground peas, add to the meal about 200 c.c. 10 ^jq sodium
chloride solution, and allow the mixture to stand, with occasional stirring, for 3-12
hrs. (see p. 147). Then filter off the extract, first through muslin, and, subsequently,
through filter-paper. Put the extract to dialyze for 24 hrs. in a collodion dialyzer^
until the protein is well precipitated. (Toluol should be added to the liquid in the
dialyzer.) Then filter ofi" the protein. Reserve half, and dissolve the other half in
about 50 c.c. of 5 o/q sodium nitrate solution. With this solution (after reserving a
portion for Expt. 129) make the following tests :

(/) Precipitation hy alcohol. To a few c.c. in a test-tube, add excess of absolute
alcohol. The protein is precipitated.

{g) Precipitation hy the heavy metals. Measure out a few c.c. of the protein
solution into three test-tubes, and add respectively a little (1) 5% copper sulphate
solution, (2) 5 o/^ lead acetate solution, (3) 5 o/o mercuric chloride solution : the protein
is precipitated in each case.

The following test cannot be demonstrated on the Pea protein, since carbohydrates
are absent in this case. It can, however, be demonstrated in later experiments (see
p. 145.

{h) Molisch's reaction. To a few c.c, of the protein solution add a few drops of a
1 % solution of a-naphthol in alcohol. Mix, and then run in an equal volume of
strong sulphuric acid down the side of the tube. A violet ring is formed at the
junction of the two liquids. The reaction signifies the existence in a protein of a
carbohydrate group which gives rise, on treatment with acid, to furfural. The latter
then condenses with a-naphthol to give a purple colour (see also Expts. 39, 44, 46).
(i) Precipitation by salts of alkaline earth metals. To a few c.c. of the protein
solution add a little 5 <Yo barium chloride solution. A precipitate is formed on standing.
(./) Precipitation by neutral salts. Saturate a few c.c. of the protein solution with
finely powdered ammonium sulphate. The protein is precipitated or "salted out."

Since from a neutral salt solution the pea globulin is precipitated by acid (see
p. 139), the tests {k)-{m) should be carried out with a solution of the protein in dilute
acid. Dissolve, therefore, t|ie remainder of the solid pea globulin in about 40 c.c. of
lOo/o acetic acid, filter, and make the following tests :

{k) Precipitation by tannic acid. Add a little 3 <>/o tannic acid solution : the
protein is precipitated.

1 The collodion solution is made by adding 75 c.c. of ether to 3 gms. of well-dried
pyroxylin, allowing it to stand for 10-15 minutes and then adding 25 c.c. of absolute
alcohol. The dialyzers are prepared by coating the inside of a large test-tube with the
solution and then filling with water, after the film is sufficiently dried so as not to be
wrinkled by touching with the finger. The sac can then be withdrawn from the tube.

138 PROTEINS AND AMINO-ACIDS [ch.

(l) Precipitation hy Eshach's solution ^ Add a little Esbach's solution : the pro-
tein is precipitated.

{m) PrecipitatiQ7i hy phosphotungstic acid. Add a little 2% solution of phos-
photungstic acid in 5 % sulphuric acid : the protein is precipitated.

The substances used in the tests ik)—{m) are termed "alkaloidal re-
agents" because they also cause precipitation of alkaloids (see Chap. xi).

We are now in a position to deal with the different groups of pro-
teins in detail:

Simple Proteins.

Albumins. Very few vegetable albumins have been investigated.
They can be best defined as proteins which are soluble in water and are
coagulated by heat. Animal albumins are distinguished by the fact
that they are not precipitated by saturating their neutral solutions with
sodium chloride or magnesium sulphate; nor are they precipitated by
half-saturation with ammonium sulphate. This distinction cannot be
applied to vegetable proteins, since some are precipitated by the above
treatment. It is often not easy to determine whether a plant protein is
an albumin, on account of the difficulty of removing traces of salts, acids
or bases which cause solubility, and also of separating the albumins from
the globulins with which they occur. Albumins are however probably
widely distributed in plant tissues.

The best-known albumins are:

Leucosin, which occurs in the seeds of Wheat (Triticum vulgare),
Rye (Secale cereale) and Barley {Hordeum vulgare).

Legumelin, which occurs in seeds of the Pea (Pisum sativum), Broad
Bean (Vicia Faba), Vetch {Vicia sativa), Lentil (Ervuni Lens) and some
other Leguminous seeds.

Phaselin, which occurs in the Kidney-bean {Phaseolus vulgaris).

Ricin, which occurs in the Castor-oil Bean (Ricinus communis).
Expt. 128. Demonstraiion of the presence of an albumin {leucosin) in wheat or
barley jiour {see also Expt. 135). Weigh out 10 gins, of wheat or barley flour, add
100 CO. of distilled water and allow to stand, with occasional stirring, for 2-6 hrs.
Then filter off the solution. Slowly heat the solution to boiling, and note that a
precipitate of coagulated protein is formed.

Globulins. These may be defined as the proteins which are in-
soluble in water but soluble in dilute salt solutions, the concentration of
the salt solution necessary for complete solution (see p. 139) varying
with the salt or protein under consideration. It should be noted that,

1 Esbach's solution is prepared by dissolving 10 gms. of picric acid and 10 gms. of citric
acid in water and making the solution up to a litre.

IX] PROTEINS AND AMINO-ACIDS 139

in making z(;a^er-extracts of plant tissues, it may happen that globulins
pass into solution to some extent owing to the presence of inorganic
salts in the tissues themselves. This has also already been illustrated
in Expt. 127 in which an extract of the globulin of the Pea was obtained
by treating ground Pea seeds with distilled water only.

It is characteristic of animal globulins that they are precipitated by
saturation of their solutions with magnesium sulphate. Many of the
vegetable globulins cannot be precipitated by the above means, though
they are all, as far as tested, precipitated by sodium sulphate at 33" C.
Many also (like animal globulins) are precipitated by half-saturation
with ammonium sulphate, though others are not precipitated until their
solutions are nearly saturated with this salt [see Expt. 127 (j)].

Unlike animal globulins, vegetable globulins are, as a rule, only
imperfectly coagulated by heat, even on boiling.

Bxpt. 129. Demonstration of the coagulation of globulin. Heat a few c.c. of the
solution of dialyzed Pea globulin (from Expt. 127) in a test-tube. Note that the
protein is largely precipitated, but the solution does not become quite clear.

One very important characteristic of the vegetable globulins is the
ease with which a number of them can be obtain ed in crystalline form.
This result may be achieved by dialyzing a salt solution of the globulin.
The salt passes out through the membrane, and the protein is deposited
in the form of crystals. An alternative method is to dilute the saline
solution of globulin with water at 50 — 80° C. until a slight turbidity
appears. Then warm further until this goes into solution, and cool
gradually, when the protein will separate in crystals. The globulin,
edestin, from seeds of the Hemp {Cannabis sativa) crystallizes very
readily (see Expt. 139) and crystals can also be obtained of the globulins
from the seeds of the Brazil nut {Bertholletia excelsa), the Flax or Linseed
(Linum usitatissimum), the Oat {Avena sativa) and the Castor-oil plant
(Ricinus communis); other globulins separate out on dialysis as spheroids,
sometimes mixed with crystals.

The solubilities of plant globulins are further complicated by the
fact that some of these substances form acid salts which have different
solubilities from the proteins themselves. Thus edestin is insoluble in
water, but soluble in either dilute salt solution or acid. In the presence
of acid it forms salts which are insoluble in dilute salt solutions. Thus
edestin in dilute acid solution is precipitated by a trace of salt, or in
dilute salt solution by a trace of acid (see Expt. 130). Legumin, on the
other hand, from the Pea and other Leguminosae is soluble in water in

140 PROTEINS AND AMINO- ACIDS [ch.

the free state; combined with a small amount of acid as a salt, it is
insoluble in water but soluble in neutral salt solution, that is, it has the
solubilities of a globulin (see p. 147).

Expt. 130. The formation of salts hy edestin. Grind up 5 gms. of seeds of the
Hemp {Cannabis saiiva) in a coffee-mill. Extract with 50 c.c. of warm (not above
60° C.) 10% sodium chloride solution and filter. Add a drop of strong hydrochloric
acid to the filtrate. Edestin chloride, which is insoluble in salt solutions, is precipi-
tated. Filter and drain off all the liquid, wash once and then suspend the precipitate
in distilled water. Add 1 or 2 drops of hydrochloric acid carefully and stir till most
or all of the precipitate goes into solution. Filter, and to the filtrate add a few drops
of saturated sodium chloride solution. The edestin acid salt is again precipitated.

The following is a list of the principal known globulins (Osborne, 2):

Pea (Pisum sativum).

Legumin, in seeds of

Broad Bean {Vicia Faha).
Vetch ( Vicia sativa).

I Lentil (Ervum Lens),

Vignin, in seeds of Cow Pea ( Vigna sinensis).

Glycinin, in seeds of Soy Bean {Glycine hispida).

r Kidney Bean (Phaseolus vulgaris).
Phaseolin (crystalline), in seeds of -| Adzuki Bean (P. radiatus).

'^Lima Bean (P. lunatus).
Conglutin, in seeds of Lupin (Lupinus).

rPea (Pisum sativum),
Vicilin, in seeds of ^ Broad Bean ( Vicia Faha).

ILentil (Ervum Lens).
Corylin, in seeds of Hazel Nut (Corylus Avellana).

'Almond (Pruniis Amygdalus).

Amandin, in seeds of

Peach (P. Persica).
Plum (P. domestica).

, Apricot (P. Armeniaca).

^European Walnut (Juglans regia).
Juglansin, in seeds of - American Black Walnut (/. nigra).

lAmerican Butter-nut (J. cinerea).
Excelsin (crystalline), in seeds of Brazil Nut (Bertholletia excelsa).

Edestin in seeds of. Hemp (Cannabis sativa).

Avenalin, in seeds of Oat (Avena sativa).

Castanin, in seeds of Sweet Q\iQ^tnVit(Gastaneavulgaris).

Maysin, in seeds of Maize (Zea Mays).

Tuberin, in tubers of Potato (Solanum tuberosum).

IX] PROTEINS AND AMINO- ACIDS 141

Crystalline globulins have also been isolated from the following seeds
but have as yet no distinctive names: Flax {Liniim usitatissimurn), Squash
(Gucurbita maxima), Castor-oil Bean {Ridnus communis), Coconut (Cocos
nucifera), Cotton-seed {Gossypium herbaceum), Sunflower {Helianthus
annuus), Radish (Raphanus sativus), Peanut (Arachis hypogaea), Rape
(Brassica campestris) and Mustard {Brassica alba).

It will be seen that the majority of reserve proteins of seeds are
globulins. It is probable that native and artificial crystalline proteins
are identical in many cases.

Prolamins. These proteins are characterized by the fact that they
are insoluble in water and dilute saline solutions, but are soluble in
70-90 Yo alcohol. Such proteins are peculiar to plants, and are formed
to a considerable extent in the seeds of cereals. The principal ones which
have been isolated are :

Gliadin found in the seeds of Wheat (Triticum vulgare).

„ „ „ Rye (Secale cereale).

Hordein „ „ Barley (Hordeum vulgare).

Zein „ „ Maize {Zea Mays).

The properties of the gliadins are demonstrated in Expts. 135, 136
and 137).

Glutelins. The proteins of this group are insoluble in water, dilute
saline solutions and in alcohol, but they are soluble in dilute alkalies.
Glutenin of wheat is the only well-characterized member of this class
which has so far been isolated, though other cereals most probably
contain similar proteins. A protein of this nature has also been obtained
from seeds of Rice {Oryza sativa). The properties of the glutelins are
demonstrated in Expts. 135 and 136.

Conjugated Proteins.

Nucleoproteins. Though these proteins probably form constituents
of all cells, the only members of the class investigated are those of the
wheat embryo. This has been possible since nuclei form a large propor-
tion of the tissue of the embryo. They may be regarded as protein salts
of nucleic acid, i.e. protein nucleates. On hydrolysis with acids or enzymes
they split up into various proteins and nucleic acid. The nucleoproteins
are also connected with the purines (see p. 179).

Nucleic acid. Plant nucleic acids have so far only been investigated
from two sources, namely from the embryo of Wheat and from the Yeast
cell. These two products appear to be identical, and, on analogy with

142 PROTEINS AND AMINO- ACIDS [ch.

animal nucleic acids, it is probable that all plant nucleic acids may prove
to have the same composition. The nucleic acid investigated is a complex
substance formed by the condensation of four nucleotides, each of which
consists of phosphoric acid, a pentose sugar and a purine. Thus yeast
nucleic acid is represented as:

HOv

O^P-
HQ/

-0

• C5H7O2

•C5H4N50

1

guanine group

0

HOx

1

o=p-

HO/

-0

• C5H6O •

■C4H4N3O

1

cytosine group

0

HO^

1

\

1

So/"

-0

• C5H6O

■ C5H4N5

1

adenine group

0

HOv

1

O^P-
HO/

-0

• C5H702

•C4H3N2O2

uracil group

On hydrolysis, nucleic acid yields phosphoric acid, rf-ribose and the
four purines as ultimate products. Nucleic acid is insoluble in water
but soluble in dilute alkalies: owing to the difficulty of obtaining other
suitable material, nucleic acid is usually prepared from Yeast.

Expt. 131. Preparation of nucleic acid from Yeast (from Bertrand, see p. 10).
Take 40 gms. of baker's yeast and add 30 c.c. of 30 % caustic soda solution. Break
up the mass thoroughly and allow it to stand for fifteen minutes. Then add
20 c.c. of water, shake well and at the same time add 10-20 c.c. of 10 ^/q solution of
ferric chloride which will produce a gelatinous precipitate. The mass, which should
be homogeneous, is drained upon a cloth placed in. a funnel, so that the almost clear
liquid can be collected in a beaker. The residue is washed with 50 c.c. of warm
water (at 60-70° C.) and again drained on a cloth. The brownish filtrate is added
to an equal volume of alcohol and enough hydrochloric acid is added to render the
mixture slightly acid. A precipitate of nucleic acid is produced. The liquid should
be allowed to stand until the precipitate has settled well. The supernatant fluid
is then decanted, and the precipitate filtered off on a small porcelain funnel using,
if possible, a hardened filter-paper. The precipitate is washed with a little alcohol
and dissolved in the minimum amount of 10 % caustic soda solution. This is re-
precipitated by pouring into acid alcohol and finally collected on a small funnel,
again using hardened filter paper.

The nucleic acid is tested for the pentose (ribose) and the phosphoric acid com-
ponents as follows :

(a) A portion of the precipitate is shaken up with a few c.c. of strong hydro-
chloric acid in a test tube, a little orcinol is added and the liquid tested for pentoses
(see Expt. 39). i

IX] PROTEINS AND AMINO- ACIDS 143

(6) The remainder of the precipitate is boiled for a few minutes with dilute nitric
acid (1 part acid : 1 part water) in a test-tube. Then add an equal volume of
30 **/y solution of ammonium nitrate and 3-5 drops of concentrated nitric acid. Heat
to boiling and add 2 c.c. of a 3 ^/q solution of ammonium molybdate. A yellow pre-
cipitate of phosphomolybdate is produced.

Derived Proteins.

Metaproteins. These are hydrolytic products of albumins and glo-
bulins formed by the action of water or dilute acid or alkali. They are
insoluble in water, strong mineral acids and all solutions of neutral salts,
but are soluble in dilute acids and alkalies in the absence of any large
amount of neutral salt.

Expt. 132. Reactions of metaprotein. Dissolve about 1 gm. of edestin (see Expt. 139)
in 50 c.c. of a 2^0 hydrochloric acid and keep on a boiling water-bath for 2 hrs.
Neutralize with dilute sodium carbonate solution. A copious precipitate of meta-
protein separates out which is insoluble in water. Filter off the precipitate and wash.
Make with it the following tests :

{a) Dissolve up some of the precipitate again in 0*4% hydrochloric acid. To
portions of the solution add : (i) Dilute sodium carbonate : the metaprotein is pre-
cipitated again and redissolves in excess, (ii) Concentrated hydrochloric acid : the
metaprotein is precipitated, (iii) Boil some of the acid solution. No coagulum is
formed : the metaprotein is not precipitated by boiling when in solution, and can
still be precipitated by neutralizing with sodium carbonate.

(6) Suspend some of the precipitate in water and boil. Cool and add 0'4o/o
hydrochloric acid : the precipitate is now insoluble, since the metaprotein is coagu-
lated when boiled in suspension.

(c) To some of the precipitate suspended in water, add gradually saturated
ammonium sulphate solution : the metaprotein is insoluble in all concentrations of
the salt.

Proteoses (albumoses) and peptones. These substances are formed
as products of hydrolysis by enzymes. When present in extracts from
seeds, however, it is sometimes uncertain whether they formed original
constituents of the seeds or resulted from hydrolysis.

As a result of the enzyme hydrolysis of proteins a mixture of several
proteoses is usually produced which can be separated by various methods,
such as different solubilities in ammonium sulphate, alcohol, etc. The
albumoses are soluble in water, salt solutions, dilute acids and alkalies.
They are all precipitated by complete saturation with ammonium
sulphate, and some by half-saturation with the same salt. On the whole,
they give the general colour reactions of the proteins, and are precipitated
by the protein precipitants, though some groups of proteoses show certain
exceptions. Their solutions are not coagulated on boiling.

144 PROTEINS AND AMINO- ACIDS [ch.

The peptones are the only proteins not precipitated by complete
saturation with ammonium sulphate. They give the protein colour
reactions and are precipitated by tannic acid and lead acetate.

Expt. 133. Separation and reactions of proteoses. Prepare about 20 gms. of gluten
from 50 gms. of flour as in Expt. 135 {d). Put the gluten into a small flask, add
100 c.c. of 0*2 % hydrochloric acid and 0*5 gm. of commercial pepsin : add also a
little toluol, shake and plug with cotton-wool. Leave in an incubator at 38° C. for
two days. (A control experiment should also be made with 100 c.c. of 0*2 % hydro-
chloric acid and 0*5 gm. of pepsin. Since pepsin itself gives a biuret reaction, a
control is necessary for comparison in the next experiment.) After two days, the
incubated mixture is neutralized to litmus with dilute sodium carbonate solution,
filtered and saturated while boiling with solid ammonium sulphate. A precipitate of
proteoses is formed, which can be gradually collected together as a sticky mass and
removed with a glass rod. Dissolve the precipitate i u some hot water, filter and make
the following tests :

{a) Xanthoproteic reaction. A positive result is given. A modification of this
reaction is characteristic of most proteoses. Add a few drops of nitric acid : a white
precipitate is formed which disappears on heating gently and reappears on cooling.

(6) MillonJs reaction. A positive result is given.

(c) Glyoxylic reaction. A positive result is given.

id) Biuret reaction. A pink or pinkish-violet colour is given.

(e) Sulphur reaction. A positive result is given.

(/) Add a little 3 ^Iq tannic acid solution. A precipitate is formed.

{g) Add a drop of 5 % copper sulphate solution. A precipitate is formed.

(A) Add a drop of strong acetic acid and then a couple of drops of 5 «/o potassium
ferrocyanide. A precipitate is formed which disappears on heating gently and re-
appeifcrs on cooling. ^ ^'

(^) Boil some of the solution. No coagulum is formed.

Expt. 134. Detection of peptone. The saturated solution, from which the proteoses
have been precipitated, is then filtered and to a measured quantity (about 5 c.c.)
twice the volume of 40 % sodium hydroxide is added and a drop of 1 % copper
sulphate solution. A pink colour appears, due to the presence of peptone. A test
should be made with the control solution containing hydrochloric acid and pepsin
only. An adequate amount should be saturated with ammonium sulphate, filtered
and 5 c.c. tested for peptone. The reaction is less marked than in the actual hydro-
lytic product. Concentrate the remainder of the peptone solution on a water-bath
and pour off from the excess of ammonium sulphate crystals. Filter and make
the following tests : (i) Xanthoproteic, (ii) Millon's, (iii) Glyoxylic, (iv) Tannic acid.
A positive result is obtained in each case.

The Seed Proteins of Certain Plants.

The proteins present in the seeds of certain genera and species, upon
which special investigations have been made, may now be considered.

It should be borne in mind that there are always several proteins
present in the seed. Some are reserve proteins of the cells of the

IX] PROTEINS AND AMINO- ACIDS 145

endosperm or of the storage tissue of the cotyledons : others are proteins
of the protoplasm and nuclei of the tissues of the embryo and of the
endosperm.

Proteins of Cereals {Gramineae).

As far as investigations have gone it may be said that the starchy
seeds of cereals are poor in albumins and globulins. The chief reserve
proteins belong to the peculiar group of prolamins, and a considerable
portion also consists of glutelins.

The grain of Wheat (Triticum vulgare) contains some proteose and
a small percentage of an albumin, leucosin. A globulin occurs only
in very small amount. The bulk of the protein consists of gliadin (a
prolamin) and of glutenin (a glutelin). Nucleoproteins are present in
the embryo, but there is no gliadin or glutenin (Osborne and Voorhees, 16).

The gliadin of wheat has the peculiar property of combining with
water to form a sticky mass which binds together the particles of
glutenin, the whole forming what is termed gluten. It is this phenomenon
which gives the sticky consistency and elastic properties to dough.

JSxpt. 135. Extraction of the proteins of the Wheat grain, (a) Extraction of
albumin {leucosin) and proteose. Take 100 gms. of white flour (the same quantity of
wheat grains which have been ground in a coflfee-mill may be used, but the extraction
in this case is slower), put the ground mass in a large flask or beaker and add
250 c.c. of distilled water. Let the mixture stand for 1-4 hrs., shaking occasionally.
Filter off some of the liquid, first through muslin and then on a filter-purap. Reserve
the residue on the filter and test the filtrate for proteins [Expt. 127, (a)-(fl?)].

Boil a second portion of the filtrate. A precipitate of the albumin, leucosin, is
formed. Filter off this precipitate, cool the filtrate and make the protein tests again.
All the above tests are given by the proteose in solution. Also make the following
special tests for proteoses (Expt. 133). (i) Add a few drops of strong nitric acid. A
white precipitate is formed which disappears on heating gently and reappears on
cooling, (ii) Add one drop of strong acetic acid and two drops of 5 7o potassium
ferrocyanide solution. A white precipitate is formed which disappears on heating
gently and reappears on cooling.

(6) Extraction of the globulin. Take the residue of ground wheat and drain on
a filter-pump. Then extract with 250 c.c. of 10 % sodium chloride solution for
12-24 hrs. Filter off, first through muslin, and then through paper on a filter-pump.
Put the extract to dialyze in a collodion dialyzer for 24 hrs. (toluol should be added
to the liquid in the dialyzer). Filter off the precipitate, which will be very slight,
and dissolve it in a little 10 7o sodium chloride. (Though so little globulin is present,
the experiment is instructive for comparison with the large amount of globulin ob-
tained from many other seeds.) Make the tests for protein [Expt 127, (a)-(ciO] with
the solution (Millon's cannot be used on account of the presence of chlorides). Also
try the effect of (i) boiling the sodium chloride solution : coagulation is not complete,
(ii) adding a little acid : a precipitate is formed as in the case of edestin.

o. 10

146 PROTEINS AND AMINO-ACIDS [ch.

(c) Extraction of gliadin. Take 100 gms. of flour (or ground wheat) and add
125 c.c. of 70 % alcohol. Warm on a water- bath and filter. Repeat the process with
another 125 c.c. of alcohol. Evaporate the filtrates, which contain gliadin, on a
water-bath (or better distil off" the alcohol in vacuo). When reduced to about half its
bulk, take a little of the filtrate and filter. Divide this filtrate into two parts in test-
tubes. To one add water : to the other absolute alcohol. A white precipitate of
gliadin is formed in each case, since it is insoluble in both water and strong alcohol,
though soluble in dilute alcohol. The remainder of the gliadin extract is evaporated
almost to dryness, and then poured slowly into a large volume of distilled water.
A milky precipitate of gliadin is formed which may be made to settle by adding a
little solid sodium chloride and stirring. Filter off the gliadin and dissolve in 10 ^Jq
acetic acid. With the solution make the tests for protein [Expt. 127, (a)-(o?)J.

{d) Extraction of glutenin. Take 100 gms. of flour, make it into a firm dough
with water in an evaporating dish and allow this to stand for half an hour. The
dough consists of gluten (gliadin and glutenin) to which the starch adheres. Then
put the dough into a piece of fine muslin and knead and wash thoroughly in a
stream of water until all the starch is removed. Collect some of the washings in a
beaker and to this suspension of starch add a few drops of iodine solution. It will
turn a deep blue-black colour. When the starch is completely washed away, an
elastic rubbery mass of gluten will remain.

Take about 10 gms. of the gluten, divide it into small pieces and heat it in a
flask on a water-bath with small quantities of 70 % alcohol until the extract gives
no, or very little, milkiness (due to gliadin) on pouring into water. Decant ofl" the
alcohol from the residue of the glutenin, as it can only be filtered with difiiculty.
Dissolve the glutenin in 0*2 % caustic potash solution. Neutralize a portion of this
solution with deci-normal sulphuric acid, drop by drop. A precipitate of glutenin is
formed. Test the remainder for proteins [Expt. 127 {a)-{d)'\.

Expt. 136. To demonstrate the fact that gluten formation depends on the presence
of gliadin. Repeat Expt. 135 {d) with flour that has been extracted with 70 o/q alcohol
for two or three days. (The alcohol is allowed to stand on the flour in the cold. It
is then poured ofl", and more added, and the process repeated. The flour is now dried
again, first in air, then in the steam-oven and finally is ground in a mortar.) No gluten
will be formed on account of the absence of gliadin.

In the Barley (Hordeum vulgare) grain, small percentages of an
albumin, apparently identical with leucosin, and of a globulin, barley
edestin, are present, together with some proteose. The main protein is
a prolamin, hordein, very similar to, but not identical with, gliadin.
There is no well-defined glutelin (Osborne, 11).

In the Rye (Secale cereale) grain there are small percentages of
proteose, and of leucosin and edestin. The greater part of the protein
is gliadin, said to be identical with that in wheat.

In the Maize {Zea Mays) grain there is apparently no true albumin,
though there is some proteose. There are small quantities of globulin,
but the greater part of the protein is a prolamin, termed zein, and a
glutenin (Osborne, 12).

IX] PROTEINS AND AMINO- ACIDS 147

Expt. 137. Extraction of the prolamin^ zein, of the Maize grain. Grind up 100 gms.
•of maize grains in a coffee-mill, or preferably use maize meal. Add 250 c.c. of hot
95 o/o alcohol. Filter, and concentrate the filtrate, which contains the zein, on a
water-bath (or, better, distil in vacuo). Pour a few drops of the concentrated extract
into (1) absolute alcohol, (2) distilled water. As in the case of gliadin and hordein,
a precipitate of zein will be formed. Then pour the whole extract, after evaporating
to a small bulk, into excess of distilled water, and add a little sodium chloride. The
precipitate of zein will slowly settle, and can be filtered off. Zein is not readily
soluble in acids and alkalies. Hence Millon's and the xanthoproteic tests should be
made on the solid material. Zein does not contain the tryptophane nucleus. To
demonstrate this, the glyoxylic reaction should be made by shaking up some solid
jzein in reduced oxalic acid and adding sulphuric acid and mixing. No purple colour
is formed.

Proteins of Leguminous Seeds {Leguminosae).

In the Leguminosae, which are starchy seeds, the chief reserve
proteins, as contrasted with those of cereals, are globulins. The various
proteins occurring may be enumerated as:

Legumin. A globulin which forms the chief protein in the seeds of
the Broad Bean (Vicia Faba), the Pea (Pisum sativum), the Lentil
{Ervum Lens) and the Vetch (Vicia sativa). Legumin itself is soluble
in water, but occurs as salts which are insoluble in water and soluble
in saline solutions. Some portion can be extracted from the seed by
water only.

Vicilin. A globulin occuring in smaller quantities than legumin and
found only in the Pea, Bean, and Lentil seeds.

Phaseolin. A globulin forming the bulk of the protein of the Kidney
Bean (Phaseolus vulgaris).

Conglutin. A globulin forming the bulk of the protein in Lupin
{Lupinus lute us) seeds.

Legumelin. An albumin found in small quantities in the Pea,
Broad Bean, Vetch and Lentil.

Phaselin. An albumin found in small quantity in the seeds of the
Kidney Bean (Phaseolus vulgaris).

Small quantities of proteoses are found in most of the above seeds.

Expt. 138. Extraction of the proieiris of the Pea (Pisum sativum) (Osborne and
Campbell, 13, 14 ; Osborne and Harris, 15). As we have seen (Expt. 127), a certain
amount of protein, including globulin, goes into solution when ground peas are ex-
tracted with water. A more complete method of extraction is as follows. Grind in
a coffee-mill 20-30 gms. of peas, add to the ground mass 50-60 c.c. of 10 % sodium
chloride solution and allow the mixture to stand for 1-2 hrs. Then filter off and

10—2

148 PROTEINS AND AMINO- ACIDS [ch.

saturate the filtrate with solid ammonium sulphate. The globulins, legumin and
vicilin, are precipitated out. Filter off" the precipitate, and then take up in dilute
ammonium sulphate (y^Q saturated) and add saturated ammonium sulphate in the
proportion of 150 c.c. to every 100 c.c. of the solution {^jj saturation). The legumin
is precipitated and can be filtered off". Saturate the filtrate with ammonium sulphate :
the vicilin is precipitated and can be filtered off. Dissolve up a little of each preci-
pitate in 10 °/o sodium chloride, and boil. The vicilin is coagulated, but the legumin is
not. Then dissolve up the remainder of the precipitates in dilute ammonium sulphate,
and test both the solutions for protein by the usual reactions [Expt. 127, {a)-{d)].

The albumin, legumelin, which occurs only in small quantities in the seeds, can
be obtained by dialyzing a water extract of the ground peas until all the globulin is
precipitated. On filtering and heating the filtrate, a coagulum of legumelin is formed.

Proteins of Fat- containing Seeds.

Of the seeds which contain fat as a reserve material, those investi-
gated have been found, in contrast to the cereals, to contain largely
globulin as reserve protein. In many cases these globulins have been
obtained in crystalline form after extraction from the plant.

The Hemp-seed (Cannabis sativa) contains one of the best-known
crystalline globulins, namely edestin. Pure neutral edestin is insoluble
in water but soluble in salt solutions. In the presence of acid, however^
edestin forms salts which are insoluble in salt solutions. Hence a solution
of edestin in sodium chloride is precipitated by even small quantities of
acids, and, conversely, a solution of edestin in acid is precipitated by
small quantities of salt (Osborne, 10).

Expt. 139. Extraction and crystallization of edestin from Hemp-seed. Take 50 gms.
of hemp*seed and grind in a cofiee-mill. Put the ground seed in a large evaporating
dish and add 200 c.c. of 5 ''/o sodium chloride solution. Heat with a small flame and
stir constantly. A thermometer should be kept in the dish, and the liquid must not
rise above 60° C. Filter oflf, in small quantities at a time, keeping the solution in the
dish warm. On cooling, the edestin separates out from the filtrate more or less in
crystals. To obtain better crystals, filter off the edestin that has been deposited, and
pour the filtrate into a dialyzer; add a little toluol, and suspend the dialyzer in
running water. As soon as it is cloudy, examine the dialyzed solution for crystals
under the microscope. Add a little 5% sodium chloride solution to the original
precipitate of edestin in the filter. Make with the filtrate the following tests:
(i) The tests for proteins [Expt. 127, {a)-{d\ except Millon's]. (ii) Boil a little of the
solution: it is imperfectly coagulated, (iii) Add a little acid: edestin chloride i&
precipitated.

In the Castor- oil seed {Ricinus communis) there is also present a
globulin which can be obtained in a crystalline form by the method of
Expt. 139. In addition, there is present an albumin, ricin, which has-
peculiar toxic properties (Osborne, 10).

IX] PROTEINS AND AMINO- ACIDS 149

A well-crystallized globulin can be obtained from the Linseed {Linum
usitatissimum) (Osborne, 9, 10), and a globulin, excelsin, from the Brazil nut
{Bertholletia excelsa) (Osborne, 10) also in crystalline or semi-crystalline
form. Similar globulins can be extracted from a number of other seeds,
i.e. Coconut (Cocos nucifera), Sunflower (Helianthus annuus), Cotton-seed
{Gossypium herbaceum), Mustard-seed (Brassica alba) and many others.
The fat is first removed from the ground seed by either ether or benzene;
the residue is then extracted with dilute sodium chloride and the extract
dialyzed.

The Amino- Acids.

There is every reason to believe, since they always arise in hydrolysis
of proteins, that amino-acids are universally distributed in the plant.
It is, however difficult to isolate and detect them, except in certain special
cases, as, for instance, in germinating seeds when a large store of protein
is being rapidly hydrolyzed and translocated.

A point of interest in connection with amino-acids is the high per-
centage of glutaminic acid in many proteins especially those of the
Oramineae (35-40 7o) and Leguminosae (15-20 7o)- Moreover, since
glutaminic and aspartic acids have two carboxyl groups, only one will be
combined in the peptide linkage, the other being free. It appears that
the free carboxyl groups of these acids are, even in the protein, combined
with ammonia forming an amide, — CONHg. Consequently, when pro-
teins containing a high percentage of glutaminic acid are hydrolyzed
they yield a correspondingly high percentage (18-23 "/o) of "amide"
nitrogen, as ammonia, compared with other proteins (6-7 "/o)- Moreover,
as a result of hydrolysis in the plant itself the respective amides, glu-
tamine and asparagine, are formed and not the free acids.

The following is a short account of the occurrence of some of the
amino-acids in the free state (see also p. 134).

Valine has been isolated from seedlings of the Vetch ( Vicia), Lupin
(Lupinus) and Kidney Bean (Pltaseolus). It is present in larger amounts
in etiolated seedlings of Lupin than in the green plants.

Leucine is widely distributed. It has been isolated from, seedlings
of Vicia, Vegetable Marrow (Cucurbita), Lupinus, Pea (Pisum) and
Goosefoot (Chenopodium), It has also been found in Phaseolus, Water
Ranunculus {Ranunculus aquatilis), buds of Horse Chestnut (Aesculus
Hippocastanum) and in small quantities in Potato tubers and other
plants.

150 PROTEINS AND AMINO- ACIDS [ch,

Isoleucine has been extracted from seedlings of Vicia sativa.
Aspartic acid. The amide of this acid, i.e. asparagine,
CONHa'CHg-CHNHg-COOH
is widely distributed in plants. It is present in shoots of Asparagus
from which it derives its name. It has also been extracted in very con-
siderable quantities from etiolated seedlings of Vicia, Lupin, and from
various plants such as Potato, Dahlia, Garden Nasturtium (Tropaeolum),.
Cucurhita and Sunflower {Helianthus).

Expt. 140. Preparation of asparagine from shoots of Asparagus (Asparagus
officinalis). Weigh out 500 gms. of shoots of asparagus and pound them in a mortar.
Put the mass in a large evaporating dish, add 500 c.c. of water and heat on a water-
bath. Squeeze the mass through linen and heat the fluid to boiling in a dish. Filter
off" the coagulated protein and to the filtrate add tannic acid (to precipitate the
remaining proteins, proteoses and peptones) until no more precipitate is formed.
Filter and remove any excess of tannic acid by adding a concentrated solution of lead
acetate drop by drop. Filter oflf the precipitate and remove any excess of lead acetate
with dilute sulphuric acid. Again filter and finally precipitate the asparagine by
adding a concentrated solution of mercuric nitrate (acidify the solution when making
with a few drops of nitric acid) until no further precipitate is formed. Filter off" the
mercury precipitate, suspend it in 150-200 c.c. of water, warm slightly and pass sul-
phuretted hydrogen through until the precipitate is decomposed. Filter oflF the
mercuric sulphide, and suck air through the solution until it ceases to smell of
sulphuretted hydrogen. Neutralize the solution and concentrate on a water-bath to
a small bulk. Then add about an equal volume of 98 'Yo alcohol. Crystals of asparagine
will separate out. Filter off" these on a small conical porcelain funnel, wash with
alcohol and dry.

Make a solution of the asparagine (or use the commercial substance) in water and
perform the following tests :

(a) Add a saturated solution of copper acetate. A blue crystalline precipitate of
the copper salt of asparagine separates out. Its appearance is hastened by shaking
or rubbing.

(6) Boil 2-3 c.c. of the solution with one c.c. of 40% caustic soda solution.
Ammonia is evolved and may be detected by holding red litmus paper in the mouth
of the test-tube. Fumes of ammonium chloride will also be formed by introducing
a glass rod moistened with strong hydrochloric acid into the tube.

Glutaminic acid. The amide, again, of this acid, i.e. glutamine,

CONH2CH2'CH2-CHNH2COOH

is widely distributed. It has been isolated from seedlings of Cucurhita^
Lupinus, Helianthus, Castor-oil plant (Ricinus), Spruce Fir (Picea
excelsa) and a number of Cruciferae.

Bxpt. 141. Preparation of glutaminic acid from gluten (from Cole, see p. 10).
Prepare gluten from 100 gms. of flour. This should give about 20 gms. of the dry
product. Divide the gluten into small pieces and dissolve it in 150 c.c. of concen-

IX] PROTEINS AND AMINO-ACIDS 151

trated hydrochloric acid in a round bottomed flask heated on a water-bath. Then add
10 gms. of good blood charcoal (Merck's if possible) and boil on a sand-bath with a
reflux condenser for six hours. Filter, and evaporate the filtrate in vacuo to about
75 c.c. Put the residue into a narrow cylinder, stand this in ice and saturate with
dry hydrochloric acid gas. (This is prepared by slowly dropping strong sulphuric
acid from a separating funnel fitted into a flask containing strong hydrochloric acid,
and then passing the gas evolved through a second flask of strong sulphuric acid.)
Keep the liquid in a cool place for 24 hours, then cool with ice. Crystalline gluta-
minic hydrochloride will separate out. Add an equal volume of ice-cold alcohol and
allow the mixture to stand. Filter on a porcelain funnel through hardened filter-
paper or linen. Wash with ice-cold strong hydrochloric acid. Dry in a desiccator
over potash and sulphuric acid. Glutaminic acid can be prepared from the hydro-
chloride by dissolving this in the minimal amount of water and adding 5*3 c.c. of
normal caustic soda solution for every gram of product taken. If the solution is
then evaporated and cooled, glutaminic acid will separate out.

Arginlne has been isolated from seedlings of Lupinus, Gucurhita^
Vicia, and Pisum. It is especially abundant in the seedlings of some
Coniferae, i.e. Picea eoocelsa, Silver Fir (Abies pectinata) and Scotch Fir
(Pinus sylvestris). It also occurs in roots and tubers, as for instance in
those of the Turnip (Brassica campestris), Artichoke (Helianthus tubero-
sus), Chicory (Cichorium Intybus), Beet (Beta vulgaHs), Potato and
Dahlia, and in the inner leaves of the Cabbage (Brassica oleracea).

Lysine has been isolated from seedlings of Lupinus, Vicia and Pisum.
Also from the inner leaves of the Cabbage and tubers of the Potato.

Phenylalaline has been isolated from seedlings of Lupinus luteus,
Vicia sativa and Phaseolus vulgaris.

Tyrosine is very widely distributed. It is present in seedlings of
Vicia sativa, Gucurbita, Lupinus, Tropaeolum and tubers of Potato,
Turnip, Dahlia, Beet and Celery. Also in berries of Elder (Sambucus),
in Clover (Trifolium), Bamboo (Bambusa) shoots and other plants.

Proline has been isolated in very small quantities from etiolated
seedlings of Lupinus albus.

Histidine has been isolated from seedlings of Lupinus and tubers of
Potato.

Tryptophane is an important amino-acid and is the one most readily
detected on account of the characteristic pink or magenta colour given
in its free state with bromine water. The glyoxylic reaction (see p. 136) is
given by tryptophane in either the combined state in the protein molecule
or in the free state. It has been isolated from seedlings of Lupinus albus
and Vicia sativa.

152 PROTEINS AND AMINO- ACIDS [ch.

Dihydroxy phenylalanine. This amino-acid, which contains two
hydroxyl groups in the ortho position, has not been detected as a con-
stituent of proteins. It occurs in the free state in all parts of the plant
of the Broad Bean ( Vicia Faba) (Guggenheim, 8) and it has also been
found in the Velvet Bean (Stizolobium). It readily oxidizes in air and is
doubtless responsible for the intense black coloration which appears in
all parts of the Broad Bean plant after death of the tissues.

Bxpt. 142. Extraction of dihy droxyphenylalanine fromthe Broad Bean (Vicia Faba).
Take one kilo, of green pods of the bean and put them through a mincing machine.
Put the minced mass immediately into boiling water, boil for a few minutes and
filter through linen, squeezing the residue thoroughly. Then add lead acetate solution
to the filtrate until no further precipitate (consisting of lead compounds of proteins,
amino-acids, flavones, etc.) is formed, avoiding an excess of acetate. Filter off and
discard this precipitate. Then add ammonia to the filtrate until it is distinctly
alkaline to litmus. A yellow precipitate of the lead compound of dihydroxy-
phenylalanine comes down. Filter, and suspend the precipitate in 500 c.c. of water
and pass in sulphuretted hydrogen until the precipitate is decomposed. Filter, and
evaporate the filtrate to a small bulk in vacuo preferably in a current of carbon
dioxide. Crystals of dihydroxyphenylalanine will separate out. Make a solution of
the crystals and perform the following test. Add 5 ^j^ ferric chloride solution. A
green colour is formed. Then add a little 1 % sodium carbonate solution ; the
green colour changes to violet.

The Proteases.

We have seen in the previous pages that proteins can be hydrolyzed
artificially with the intermediate production of proteoses and peptones,
and the final production of a number of amino-acids. There is no doubt
that this process of hydrolysis takes place in the living plant, and it is
believed that the converse process, the synthesis of these proteins from
amino-acids, also takes place in the cell.

There is evidence that this hydrolysis of proteins is catalyzed by
certain enzymes which have been termed proteases. On analogy with
other enzymes, we may suppose that these enzymes also catalyze the
synthesis of the proteins.

It seems highly probable that the proteases are of two types:

1. Pepsin-like enzymes, which catalyze the hydrolysis of proteins to
peptones, and, in all probability, the reverse process.

2. Erepsin-like enzymes, which catalyze the hydrolysis of albumoses
and peptones to amino-acids, and, in all probability, the reverse process.

We now turn to the evidence for the existence of proteases. In
autolysis (see p. 20) the hydrolytic activity of many enzymes is un-
controlled, and in the case of the proteins, the amino-acids are formed

IX] PROTEINS AND AMINO- ACIDS 153

as end-products. Amino-acids are rarely present in plants in sufficient
quantity to be detected readily, at any rate in small quantities of material,
but if the tissues are put to autolyze at temperatures of 38-40° C, the
^mino-acids then accumulate and can be detected. Of all the amino-acids
the one which is most readily identified is tryptophane. If the autolyzed
product is boiled, acidified and filtered to remove the remaining proteins,
and, to the filtrate, bromine is added, drop by drop, the formation of a
pink or purple colour will indicate the presence of free tryptophane, and
hence it may be assumed that protein-hydrolysis has taken place.
Probably the formation of amino-acids in autolysis is a universal property
of plant tissues, for tryptophane has been detected on autolysis of many
different parts of plants. Examples are the germinating seeds of the
Bean (Vicia Faba), Scarlet B,unner (Phaseolus rrmltiflorus), Pea (Pisuvi
sativum), Lupin (Lupinus hirsutus) and the Maize {Zea Mays): and in
ungerminated seeds of the above, though less readily. It is also said to
be formed on autolysis of leaves of Spinach (>Sfpi?iacm), Cabbage (Brassica),
Nasturtium (Tropaeolum majus), Scarlet Geranium {Pelargonium zonale),
Dahlia (Dahlia variabilis) and others: also of fruits of Melon {Cucumis
Melo), Cucumber {Cucumis sativus), Banana {Musa sapientum), Tomato
(Lycopersicum esculentum) and others: of bulbs of the Tulip (Tulipa),
Hyacinth {Hyacinthus orientalis) and underground roots of Turnip
(Brassica), Carrot {Daucus Carota) and Beet {Beta vulgaris) (Vines,
17-19; Blood, 3; Dean, 5, 6).

Expt. 143. The formation of tryptophane on autolysis of resting seeds. Grind up
in a coffee-mill 15 gms. of Mustard {Brassica alba) seed. Transfer to a flask, and
add 100 c.c. of distilled water and about 2 c.c. of toluol. Plug the mouth of the flask
with Qotton-wool and put in an incubator for 3 days. Then filter off" the liquid, boil
the filtrate and add a few drops of acetic acid. Filter off" any precipitate formed,
cool the filtrate and add bromine water slowly and carefully drop by drop, shaking
well after each drop. A pink or purple colour denotes the presence of tryptophane.
Excess of bromine will destroy the colour. Then shake up gently with a little amyl
alcohol. The purple colour will be extracted by the amyl alcohol which will rise to
the top of the water solution. A control experiment should be made using 10 gms.
of seed which has been well boiled with water in an evaporating dish.

It has been assumed that the formation of amino-acids from proteins
on autolysis is the outcome of two .processes, the hydrolysis of proteins
to peptones by pepsins, and the hydrolysis of peptones to amino-acids
by erepsins.

The next point to be considered is the possibility of detecting these
two classes of enzymes separately. If either the pulp, or water extract,
of various plant tissues be added to peptone solution and allowed to

154 PKOTEINS AND AMINO- ACIDS [ch.

incubate at 38° C, tryptophane can be readily detected after a day or
two. This has been found to be true for the tissues of many seeds^
seedlings, roots, stems, leaves and fruits (such as those already mentioned
above and others); the result indicates the wide distribution of an erepsin
type of enzyme. The detection of this enzyme is facilitated by the
addition of the artificial supply of peptone.

Expt. 144. The detection of erepsins in plants.

(a) In resting seeds. Grind up 10 gms. of seeds in a coffee-mill, and add 100 c.c.
of water, 0'2 gm. of Witte's peptone ^ and a little toluol. Incubate for 2-3 days. The
following seeds may be used : Hemp {Cannabis sativa\ Castor-oil {Ricinus communis)^
Pea {Pisum sativum), Scarlet Runner {Phaseolus multifiorus\ Broad Bean {Vicia
Faha) and fruit of Wheat ( Triticum vulgare). Test for tryptophane. Controls should
be made in these and the following cases.

(6) In germinating seeds. Take 10 germinating peas, pound in a mortar, add
100 c.c. of distilled water, 0-2 gm. of Witte's peptone, and a little toluol. Incubate
for 3 days. Test for tryptophane.

(c) In leaves. Pound up a small cabbage leaf, add 100 c.c. of water, 0*2 gm. of
Witte's peptone and a little toluol. Incubate for 3 days. Test for tryptophane.

{d) In roots. Pound up about 20 gms. of fresh carrot root. Add about 100 c.c.
of water, 0-2 gm. of Witte's peptone and a little toluol. Incubate for 3 days. Test
for tryptophane.

The pepsin type of enzyme is less readily detected. It has long been
known that the pitchers of the Pitcher-plant {Nepenthes) secrete an
enzyme which digests fibrin. A few other cases of protein-digesting
enzymes are well known, such as the so-called "bromelin" from the fruit
of the Pine-apple {Ananas sativus), "cradein" from the latex and fruit
of the Fig {Ficus) and "papain" from the fruit and leaves of the Papaw
tree {Carica Papaya). Such enzymes were formerly termed "vegetable
trypsins" as they were thought to be of the type of animal trypsin which,
alone, hydrolyzes proteins to amino -acids. On analogy with the results of
research with other enzymes, it seems likely that "papain," "cradein"
and "bromelin" are all mixtures of pepsin and erepsin. In addition to
these better known cases, it has also been stated that fibrin is digested
by extracts or pounded pulp of the fruits of the Cucumber and the
Melon, the "germ" (embryo) of Wheat, the bulbs of Tulip and Hyacinth,
the seedlings of the Bean, Pea, Scarlet Runner, Lupin and Maize, and
the ungerminated seeds of the Pea, Lupin and Maize. These have also
been shown to contain erepsin.

^ Is prepared from fibrin and consists of a mixture of proteoses and peptone. It is free
from tryptophane.

IX] PROTEINS AND AMINO- ACIDS 155

A separation of pepsin from erepsin has been achieved in the case
of the seeds of the Hemp (Cannabis sativa) by means of the different
solubilities of the two enzymes in water and salt solutions.

Expt. 145. ^The extraction and the separation of the two enzymes^ erepsin and
pepsin^ from Hemp-seed (Cannabis sativa) ^ Weigh out 50 gms. of hemp-seed, grind
it in a coffee-mill and extract with 250 c.c. of 10 % sodium chloride solution. Allow
the mixture to stand all night and then filter. Both operations should be carried
out at as low a temperature as possible. Measure the filtrate, and add acetic acid
to the extent of 0*2 %. A dense precipitate is formed. Filter again, keeping as cool
as possible.

The acid filtrate contains the erepsin, but not the pepsin. Measure out 40 c.c.
into each of three small flasks, and add the following : (i) 0*2 gm. of Witte's peptone,
(ii) the same, only boil the whole solution, (iii) 0*2 gm. of carmine fibrin 2. Add a
little toluol to all three flasks, plug with cotton-wool, and incubate for three to four
days. Test for tryptophane in flasks (i) and (ii) ; the first gives a marked reaction,
the second little or no reaction. The fibrin in (iii) will remain unaltered.

The precipitate produced by the acetic acid is then washed on the filter twice
with 100 c.c. of 10 % sodium chloride solution, containing 0*2 % acetic acid, to
remove traces of erepsin. The precipitate is then treated with about 70 c.c. of
water, allowed to stand for a time, and then filtered. The filtrate is divided into
three equal portions. Add the following respectively : (i) O'l gm. of carmine fibrin,
(ii) the same, but the solution is boiled, (iii) 0'2 gm. of Witte's peptone. Add a little
toluol to all three flasks, plug with cotton-wool and incubate for 3-4 days. The
fibrin will be seen to digest slowly in flask (i) : (ii) will show no digestion, and
(iii) will give no tryptophane reaction.

REFERENCES

Books

1. Abderhalden, B. Biochemisches Handlexikon, iv. Berlin, 1911.

2. Osborne, T. B. The Vegetable Proteins. London, 1909.

Papers

3. Blood, A. F. The Erepsin of the Cabbage [Brassica oleracea). J. Biolog,
Chem., 1910-1911, Vol. 8, pp. 215-225.

4. Chibnall, A. C, and Schryver, S. B. Investigations on the Nitrogenous
Metabolism of the Higher Plants. Part I. The Isolation of Proteins from Leaves.
Biochem. J., 1921, Vol. 15, pp. 60-75.

5. Dean, A. L. On Proteolytic Enzymes. I. Bot. Gaz., 1905, Vol. 39,
pp. 321-339.

1 Vines, S. H. Ann. Bot., 1908, Vol. 22, pp. 103-113.

2 Freshly washed and finely chopped fibrin is placed in carmine solution (1 gm. carmine,
1 c.c. of ammonia, 400 c.c. of water) for 24 hrs. Then strain off and wash in running
water till washings are colourless.

156 PROTEINS AND AMINO- ACIDS [ch. ix

6. Dean, A. L. On Proteolytic Enzymes. II. Bot. Gaz., 1905, Vol. 40,
pp. 121-134.

7. Fisher, B. R. Contributions to the Study of the Vegetable Proteases.
Biochem. J., 1919, Vol. 13, pp. 124-134.

8. Guggenheim, M. Dioxyphenylalanine, eine neue Aminosaure aus Vicia
faha. Zs. physiol. Chem. 1913, Vol. 88, pp. 276-284.

9. Osborne, T. B. Proteids of the Flax-seed. Amer. Chem. J., 1892, Vol. 14,
pp. 629-661.

10. Osborne, T. B. Crystallised Vegetable Proteids. Amer. Chem. J., 1892,
Vol. 14, pp. 662-689.

11. Osborne, T. B. The Proteids of Barley. J. Amer. Chem. Soc., 1895, Vol. 17,
pp. 539-567.

12. Osborne, T. B. The Amount and Properties of the Proteids of the Maize
Kernel. J. Amer. Chem. Soc., 1897, Vol. 19, pp. 525-532.

13. Osborne, T. B., and Campbell, G. F. Proteids of the Pea. J. Amer.
Chem. Soc, 1898, Vol. 20, pp. 348-362.

14. Osborne, T. B., and Campbell, G. F. The Proteids of the Pea, Lentil,
Horse Bean and Vetch. J. Amer. Chem. Soc., 1898, Vol. 20, pp. 410-419.

15. Osborne, T. B., and Harris, I. F. The Proteins of the Pea {Pisum
sativum). J. Biol. Chem., 1907, Vol. 3, pp. 213-217.

16. Osborne, T. B., and Voorhees, C. G. The Proteids of the Wheat-
" Kernel. Amer. Chem. J., 1893, Vol. 15, pp. 392-471.

17. Vines, S. H. Tryptophane in Proteolysis. Ann. Bot., 1902, Vol. 16,
pp. 1-22.

18. Vines, S. H. Proteolytic Enzymes in Plants. I. Ann. Bot., 1903, Vol. 17,
pp. 237-264. II Ibid. pp. 597-616.

19. Vines, S. H. The Proteases of Plants. I-VII. Ann. Bot., 1904-1910
Vols. 18-24.

CHAPTER X

GLUCOSIDES

Attention has been drawn (Chapters v and viii) to the fact that in the
plant, compounds containing hydroxyl groups often have one or more of
these groups replaced by the CeHnOg — residue of glucose. Such com-
pounds are termed glucosides. The substances in which this substitution
most frequently occurs are of the aromatic class, and the glucosides may
be regarded, on the whole, as ester-like compounds of carbohydrates with
aromatic substances. The non-sugar portion of the glucoside may vary
widely in nature, and may be, for instance, an alcohol, aldehyde, acid,
phenol, flavone, etc. The sugar constituent is most frequently glucose,
but pentosides, galactosides, mannosides and fructosides are also known.
Sometime^ more than one monosaccharide takes part in the composition
of the glucoside. (These various relationships are shown in the accom-
panying table.) The inclusion of all glucosides in a class is in a sense
artificial: the character held in common (with very few exceptions) is
that, on boiling with dilute acids, or, by the action of enzymes, hydrolysis
takes place, and the glucoside is split up into glucose (or other sugar)
and another organic constituent. A number of compounds occurring as
glucosides have already been dealt with, for example, the tannins and
flavone, flavonol and anthocyan pigments, but, in these cases, the sig-
nificance of the compounds lies rather in the nature of their non-sugar
constituents than in the fact of their being glucosides.

There are, however, a number of glucosides which have been grouped
together and are more readily classified in this way than in any other.
Some of them, doubtless, have come into prominence as glucosides on
account of their association with well-known and specific enzymes, as,
for instance, the glucoside amygdalin associated with the enzyme emul-
sin, and the glucoside sinigrin with the enzyme myrosin.

The hydrolyzing enzymes are by no means always specific, for in
vitro one particular enzyme may be able to hydrolyze several glucosides.
Many glucoside- splitting enzymes have been described, though there is
no reason to suppose that each glucoside is only acted upon by an
enzyme specific to that glucoside. It is likely moreover that some of
the different enzymes described will probably prove to be identical.

In some cases where more than one monosaccharide is attached to

158

GLUCOSIDES

[CH.

the glucoside, the different sugar groups are removed separately b}^
different enzymes (see later, emulsin, p. 160).

The glucosides as a whole (except flavone, flavonol and anthocyan
pigments) are colourless crystalline substances. When extracting them
from the plant, it is usually necessary to destroy the accompanying
enzyme by dropping the material into boiling alcohol or some other
reagent (see autolysis, p. 20).

In Chapter v it has already been mentioned that c?-glucose exists in
two stereoisomeric forms, the a and the /3 form.

It was also pointed out that the glucosides can be classed either as
a- or y8- glucosides, according to whether the a or the yS form of glucose
combines with the non-glucose residue.

RO— Cr- H H— C^^OR

H

HO

H

C— CH

I
C— H

0

I
H_C— OH

I

CH2OH CH2OH

a-glucoside /3-glucoside

Maltose, for instance, is regarded as an a-glucoside of cZ-glucose. It
has been further shown that the enzyme maltase can only hydrolyze
a-glucosides, whereas other enzymes, e.g. the prunase component of
emulsin, only act on yS-glucosides.

The various glucosides considered in detail in this chapter together
with some others are grouped under the following headings (Arm-
strong, 3):

Products of hydrolysis

Alcohols
Glucose 4- coniferyl alcohol

Glucose + saligenin + benzoic acid
Glucose + saligenin
Glucose + syringenin

Aldehydes
Glucose -f benzaldehyde -|- prussic

acid
Glucose 4- parahydroxy benzaldehyde

4- prussic acid
Glucose -H acetone + prussic acid

Glucoside

Coniferin

Populin

Salicin

Syringin

Amygdalin

Dhurrin

Linamarin

Plant in which commonly
found

(Coniferae, Beta^ Asparagus^

Scorzonera) .
{Populus)
(Salix, Populus)
{Ligustrum, Syringa^ Jasmi-

num)

{Prunus^ Pyrus)
{Sorghum)
{Linum^ Phaseolus)

^]

GLUCOSIDES

159

Glucoside

Prulaurasin
Prunasin
Sambunigrin
Vicianin

Oaultherin
Strophanthin

Arbutin
Hesperidin
Naringin
Phloridzin

Aesculin
Fraxin

Olucotropaeolin

Sinalbin

Sinigrin

Apiin

Isoquercitrin

Lotusin

Myricitrin

Quercitrin

Kobinin

Rutin

•Cyanin
Delphinin

Malvin
Oenin
Peonin
Pelargonin

Aucubin
Digitalin
Indicaa

Plant in which commonly
found

(Prunus)

{Cerasus^ Prunus)
(Sambucus)
( Vicia)

(Gaultheria^ Spiraea)
(Strophanthus)

(Ericaceae)
{Citri(s)
{Citrus)
(Rosaceae)

{Aesculus)
\Fraxinus)

{Tropaeolum, Lepidi
{Brassica alba)
{Brassica nigra)

(Carum)

{Oossypium)

{Lotus)

{Myrica)

{Quercus, Fraxinus, Thea)

{Robinia)

{Ruta^ CappariSj Polygonum)

{Centaurea^ Rosa)
{Delphinium)

{Malva)
{ Vitis)
{Paeonia)
Pelargonium, Centaurea)

{Aucuba, Plantago)

{Digitalis)

{Iiydigofera)

Products of hydrolysis

Aldehydes (cont.)

Glucose + benzaldehyde + prussic
acid

Glucose + benzaldehyde + prussic
acid

Glucose + benzaldehyde + prussic
acid

Vicianose + benzaldehyde + prussic
acid

Adds

Glucose + methyl salicylate

Mannose -|- rhamnose + strophanthi-
din

Phenols

Glucose + quinol

Glucose + rhamnose -f hesperetin

Glucose + rhamnose + narigenin

Glucose + phloretin

Coumarin derivatives
Glucose + aesculetin
Glucose + fraxetin

Miistard-oils
Glucose + benzyl isothiocyanate +

potassium hydrogen sulphate
Glucose + sinapin acid sulphate -|-

acrinylisothiocyanate
Glucose + allyl isothiocyanate +

potassium hydrogen sulphate

Flavone andflavonol pigments
Apiose 1 4- apigenin
Glucose + quercetin
Glucose + prussic acid+lotoflavin
Rhamnose + my ricetin
Rhamnose + quercetin
Rhamnose + galactose + kaempferol
Glucose + rhamnose + quercetin

Anthocyan pigments
Glucose + cyanidin
Glucose + oxy benzoic acid + delphi-

nidin
Glucose + malvidin
Glucose + oenidin
Sugar + peonidin
Glucose + pelargonidin

Various constituents
Glucose + aucubigenin
Glucose + digitalose + digitaligenin
Glucose + indoxyl

1 An abnormal sugar, C5H10O5 , containing a branched chain of carbon atoms.

160 GLUCOSIDES [ch.

Cyano PHOBIC Glucosides.

The characteristic of these substances is that they yield prussic acid
as one of the products of hydrolysis. They are fairly widely distributed:
the following list (Greshoff, 15) includes most of the natural orders in
which such glucosides occur: Araceae, Asclepiadaceae, Berberidaceae>
Bignoniaceae, Caprifoliaceae, Celastraceae, Compositae, Convolvulaceae,
Cruciferae, Euphorbiaceae, Gramineae, Leguminosae, Linaceae, Myrta-
ceae, Oleaceae, Passifloraceae, Ranunculaceae, Rhamnaceae, Rosaceae,
Rubiaceae, Rutaceae, Saxifragaceae, Tiliaceae and Urticaceae.

Amygdalin. This is one of the most important of the cyanophoric
glucosides. It occurs in the seeds of the bitter Almond (Prunus
Amygdalus) but it appears to be almost entirely absent from the sweet
or cultivated Almond. It also occurs in the seeds of the other species of
Prunus — the Plum (P. domestica), the Peach (P. Perdca), etc. — of the
Apple {Pyrus Malus) and the Mountain Ash (P. Aucuparia). It occurs
sometimes in leaves, flower and bark.

By the action of an enzyme, originally termed emulsin, which occurs
in both the bitter and the sweet varieties of Almond, the glucoside is
broken up as follows in two stages:

CaoHarNOn + H2O = CeHiaOg + C14H17NO6

mandelonitrile gkicoside (prunasin)

C14H17NO6 + H.O = CgHiaOe + HON + CgHgCHO

benzaldehyde

It should be noted that the sweet Almond contains emulsin although
it is almost entirely free from amygdalin.

Recently (Armstrong, Armstrong and Horton, 8) emulsin has been
shown to consist of two enzymes, amygdalase and prunase: amygdalase
hydrolyzes amygdalin with formation of mandelonitrile glucoside and
glucose, whereas prunase hydrolyzes mandelonitrile glucoside (prunasin)
with formation of benzaldehyde, prussic acid and glucose. On the basis
of these reactions amygdalin is represented as:

I ° — I i

CH2OH CHOH CH CHOH CHOH CH • O ' CH2CHOH CH CHOH CHOH CH • O • CH

I o I I

CN

Prunasin occurs naturally in the Bird Cherry {Cerasus Padus), and
it is found that prunase may exist in a plant, e.g. Cherry Laurel
(P. Laurocerasus), which does not contain amygdalase.

X] GLUCOSIDES 161

Prulaurasin {laurocerasin) is a glucoside occurring in the leaves of
the Cherry Laurel (Prunus Laurocerasus). It has been represented as
racemic mandelonitrile glucoside, prunasin being the dextro form.

Sambunigrin is a glucoside occurring in the leaves of the Elder
(Sambucus nigra). It has been represented as laevo mandelonitrile
glucoside.

When tissues containing cyanophoric glucosides and their corre-
sponding enzymes are submitted to autolysis, injury, or the action of
chloroform, hydrolysis takes place (see autolysis, p. 20). A rapid method
(Mirande, 17; Armstrong, 5) for detecting the prussic acid is to insert
paper dipped in a solution, of sodium picrate into a tube containing the
plant material together with a few drops of chloroform. In the presence
of prussic acid the paper becomes first orange and finally brick-red owing
to the formation of picramic acid.

In addition to those previously mentioned there are other British
plants, the leaves of which give off prussic acid on autolysis (presumably
fi-om cyanophoric glucosides), as for example the Columbine (Aquilegia
vulgaris). Arum (Arum maculatum), Hawthorn {Crataegus Oxyacantha),
Reed Poa {Glyceria aquatica), Bird's-foot Trefoil (Lotus corniculatus).
Alder Buckthorn (Rhamnus Frangula), Black and Red Currant and
Gooseberry (Rihes nigrum, R. rubrum, R. Grossularia), Meadow Rue
(Thalictrum aquilegifolium) and the Common and Hairy Vetches (Vida
sativa and V. hirsuta).

It has been shown (Armstrong, 7) that of the species L. corniculatus
there is a variety (L. uliginosus) (taller and growing in moister situations)
which does not produce cyanophoric substances and hence does not give
off prussic acid on autolysis.

Ea^pt. 146. Method of detection of cyanophoric glvx;osides in the plant. Take three
flasks : in one put a whole leaf of the Cherry Laurel {Prunus Laurocerasus) : in the
second a leaf which has been torn in pieces and then either pricked with a needle or
pounded in a mortar : in the third a leaf with a few drops of chloroform. Cork all
three flasks, inserting with the corks a strip of sodium picrate paper. (The paper is
prepared in the following way : strips of filter-paper are dipped in a 1 % solution of
picric acid, are then suspended on a glass rod and allowed to dry in air. Before
using, the paper is moistened with 10% sodium carbonate solution and is suspended
in the moist condition just above the material to be examined. In the presence of
prussic acid, the paper first becomes orange-yellow, then orange and finally brick-red.)
In a short time the paper in the flask containing the leaf and chloroform will turn
red : in the flask with the injured leaf, the reddening will take place rather more:
slowly, whereas in the case of the entire leaf, the paper will remain yellow.
o. 11

162 GLUCOSIDES [ch.

The above experiment may also be carried out, usually with success, on leaves of
the Columbine {Aquilegia vulgaru)^ the Arum {Arum maculatum) and plants of the
Bird's-foot Trefoil {Lotus corniculatus) : also with bitter almonds and apple pips, and
young shoots of Flax {Linum perenne). In the case of the seeds, these may be used
crushed, both with and without chloroform, the uninjured seed being used as a
control.

Expt. 147. Preparation of amygdalin. Weigh out 100 gms. of bitter almonds.
Kemove the testas by immersing them for a short time in boiling water. Then
pound up the almonds well in a mortar and transfer to a flask. Add about
200-300 c.c. of ether and allow the mixture to stand for 2-12 hours. Filter off the
ether and extract again with fresh ether. The greater part of the fat will be removed
in this way. Then dry the residue from ether and, as rapidly as possible, extract
twice or three times with boiling 90-98% alcohol which removes the amygdalin.
The residue, after ether extraction, contains both amygdalin and emulsin, and, if
allowed to stand, the emulsin will hydrolyze the amygdalin : hence the necessity for
rapid extraction with alcohol. Evaporate the filtered alcoholic extract on a water-
bath or, better, distil in vacuo to a small bulk. Then add an equal volume of ether
and allow the mixture to stand for a time. The amygdalin separates out on standing.
Filter off the precipitate, dissolve in a little hot water and allow to crystallize in a
desiccator.

Expt. 148. Preparation of emulsin (Bourquelot, 10). Weigh out 25 gms. of
sweet almonds. (Bitter almonds can also be used. The sweet variety is preferable ;
since from them the emulsin can be more readily prepared free from amygdalin.)
Plunge them for a moment into boiling water and remove the testas. Pound
thoroughly in a mortar, and extract the bulk of the oil with ether as in the last
experiment. Then grind up the residue with 50 c.c. of a mixture of equal parts of
distilled water and water saturated with chloroform and allow the whole to stand
for 24 hours. Filter by means of a filter-pump, and to the filtrate add glacial acetic
acid (1 drop to 15 c.c. of the filtrate) whereby the protein is precipitated. Again
filter, and to the filtrate add 3-4 times its volume of 96-98 o/^, alcohol. The emulsin
is deposited as a white precipitate. Filter off the precipitate and dissolve it in about
100 c.c. of cold distilled water.

Expt. 149. (a) To demonsti^ate the hydrolysis of amygdalin by emulsin. Into
each of two flasks put 50 c.c. of a 1-3 % solution of amygdalin. To one flask add
25 c.c. of the emulsin solution prepared in the last experiment. To the other flask
add 25 c.c. of enzyme solution after it has been well boiled, and again boil the
mixture after adding the enzyme. Fit each flask with a cork and sodium picrate
paper. The paper in the flask containing the unboiled enzyme will rapidly turn red,
the control remaining yellow. Unless both the enzyme and the amygdalin solution
are well boiled in the case of the control, the paper may show reddening in time on
account of traces of prussic acid present in both solutions.

(6) Simplified method for extraction of amygdalin and emulsin^ and demonstration
of hydrolyds of amygdalin by emulsin. Take 12 bitter almonds. Remove the testas
by immersing them for a short time in boiling water. Then pound up the almonds
well in a mortar and transfer to a flask. Add about 50 c.c. of alcohol and heat to
boiling on a water-bath. Filter off the extract, and evaporate it to dryness on a
water-bath. The residue will contain amygdalin.

X] GLUCOSIDES 163

Take six sweet almonds and remove the testas as before. Pound in a mortar and
transfer to a flask. Add a little ether and allow to stand for a short time. Pour off
the ether, and add a little more which should again be poured off. This removes some
of the fat and makes extraction of the emulsin easier. Then extract the residue
with about 40c.c. of distilled water and filter. The filtrate contains the enzyme
emulsin.

Take lOc.c. of the emulsin solution, and divide it into two portions in two test-
tubes. Boil one well (see Expt. 149 a), and to both add equal quantities of a water
extract of the amygdalin prepared above. Cork the tubes and insert picric paper
with the cork in each case.

It has been found, as previously mentioned, that emulsin can
hydrolyze other glucosides, as for instance, salicin (see pp. 50, 167). On
hydrolysis, salicin splits up into salicylic alcohol (saligenin) and glucose.
Salicin, itself, gives no colour with ferric chloride but saligenin gives a
violet colour, and by means of this reaction the course of the hydrolysis
can be followed.

Expt. 150. To demonstrate the hydrolysis of salicin by emulsin. To 10 c.c. of a
1 ^/o solution of salicin in a test-tube add 10 c.c. of the emulsin solution prepared in
Expt. 148 or 149. As a control, boil in a second test-tube another 10 c.c. of the
emulsin solution and add 10 c.c. of salicin solution. After about an hour, add to
both test-tubes a few c.c. of strong ferric chloride solution. A purple colour will be
given in the first test-tube but no colour in the control. The process of hydrolysis
will be accelerated by placing the tubes in an incubator.

A modification can be made as follows. A second pair of test-tubes should be
prepared as before and to both sufficient ferric chloride should be added to give a
faint yellow tinge. The unboiled mixture will gradually acquire a purple colour at
ordinary temperature.

Other cyanophoric glucosides are dhurrin, phaseolunatin (linamarin),
lotusin and vicianin.

Dhurrin occurs in seedlings of the Great Millet {Sorghum vulgare).
On hydrolysis it yields glucose, prussic acid and parahydroxybenzaldehyde
{C6H4 • OH • CHO). It is hydrolyzed by emulsin.

Phaseolunatin occurs in seeds of the wild plants of Phaseolvs lunatus
and in seedlings of Flax (Linum). It is associated with an enzyme which
hydrolyzes it into acetone, glucose and prussic acid.

Lotusin occurs in Lotus arabicus. On hydrolysis by an accompanying
enzyme (lotase) it gives glucose, prussic acid and a yellow pigmen'*
lotoflavin.

Vicianin occurs in the seeds of a Vetch {Vicia angustifolia). It is
hydrolyzed by an accompanying enzyme into prussic acid, benzaldehyde
and a disaccharide, vicianose.

11—2

164: GLUCOSIDES [ch.

Mustard-oil Glucosides.

These are glucosides containing sulphur and they have been found
chiefly among the Cruciferae. Sinigrin and sinalbin, the glucosides of
mustard, have been most investigated.

Sinigrin. This glucoside occurs in the seed of Black Mustard
(Brassica nigra) and other species of Brassica. Also in the root of the
Horse-radish (Cochlearia Armoracia). Sinigrin is hydrolyzed by the
enzyme, myrosin (Guignard, 16; Spatzier, 18) (which occurs in the plant
together with the glucoside), into allyl isothiocyanate, potassium hydrogen
sulphate and glucose:

C10H16O9NS2K + H2O = C3H5NCS + CeHisOe + KHSO4

Expt. 151. Extraction of sinigrin from Black Mustard. Weigh out 100 gms. of
Black Mustard seed. Grind the seed in a coffee-mill and afterwards pound in a
mortar. Heat 175 c.c. of 85 % alcohol to boiling in a flask on a water-bath and add
the pounded mustard, and after boiling about \ hour, filter and press out the alcohol.
Then put the dried cake of residue into 300 c.c. of water and allow the mixture to
stand for 12 hours. Press out the liquid and after filtering and neutralizing with
barium carbonate, concentrate in vacuo to a syrup. Then extract with 90 % alcohol
and filter. On concentrating and exposing in a crystallizing dish, the sinigrin
separates out in white needles.

Sinalbin occurs in the seeds of White Mustard {Sinapis alba). By
myrosin it is hydrolyzed to p-hydroxybenzylisothiocyanate, acid sinapin
sulphate and glucose:

C3oH420i5N2S2 + H2O = CgHiaOe + C^H.ONCS -f- CJ6H24O5NHSO4

Expt. 152. Extraction of sinalbin from White Mustard. Weigh out 100 gms. of
White Mustard seed. Grind and pound well and extract the fat with ether. Then
extract with twice its weight of 85-90 % alcohol several times and well press out the
alcohol. The extract is evaporated to half its bulk and filtered. On cooling, the
sinalbin separates out in crystals.

Expt. 153. Preparation of myrosin. Weigh out 50 gms. of White Mustard seed
and grind in a coffee-mill. Add 100 c.c. of water and allow the mixture to stand for
12 hours. Then filter and allow the filtrate to run into 200 c.c. of 95-98 % alcohol.
A white precipitate is formed which contains the myrosin. Filter off the precipitate
and wash on the filter with a little ether.

Expt. 154. Action of myrosin on sinigrin. Put into two test-tubes equal quantities
of a solution of the sinigrin prepared in Expt. 151. Dissolve some of the myrosin
prepared in the last experiment in water and divide the solution into two parts.
Heat one part to boiling and then add the two portions respectively to the two test-
tubes of sinigrin. Plug both test-tubes with cotton-wool. After about ^ hour a
strong pungent smell of mustard oil, allyl isothiocyanate, will be detected in the
unboiled tube.

A more simple method of demonstrating the action of myrosin is as follows.

X] GLUCOSIDES 165

Pound about 5 gms. of Black Mustard seed in a mortar and then boil with water.
Some mustard oil will be formed before the myrosin is destroyed, so that boiling
should be continued until no pungent odour can be detected. Then filter and cool
the solution and divide into two parts. To one add some myrosin solution. To the
other an equal quantity of boiled enzyme solution. After h hour the smell of allyl
isothiocyanate should be detected in the unboiled tube.

Saponins.

These substances are very widely distributed, being found in the
orders: Araliaceae, Caprifoliaceae, Combretaceae, Compositae, Cucurbi-
taceae, Gramineae, Guttiferae, Lecythidaceae, Leguminosae, Liliaceae,
Loganiaceae, Magnoliaceae, Myrtaceae, Oleaceae, Piperaceae, Pitto-
sporaceae, Polemoniaceae, Polygalaceae, Primulaceae, Proteaceae,
Ranunculaceae, Rhamnaceae, Rosaceae, Rutaceae, Saxifragaceae,
Thymelaeaceae and the majority of the orders of the cohort Centro-
spermae. On hydrolysis with dilute mineral acids the saponins yield
various sugars — glucose, galactose, arabinose, rhamnose — together with
other substances termed sapogenins.

The saponins are mostly amorphous substances readily soluble in
water (except in a few cases) giving colloidal solutions. These solutions
froth on shaking, and wdth oils and fats they produce very stable
emulsions. By virtue of this property they have been used as substitutes
for soap. The Soapwort (Saponaria) owes its name to the fact that the
root contains a saponin.

COUMARIN GlUCOSIDES.

These substances are hydroxy derivatives of coumarin, which itself
may be represented as:

CH=:CH— CO

Ao I

V .

AescTilin is one of the best known of these glucosides. It occurs in
the bark of the Horse Chestnut (Aesculus Hippocastanum). On hydro-
lysis with dilute acids it yields glucose and aesculetin, the latter being

represented as:

CO

J

166 GLUCOSIDES [ch.

Aesculin is characterized by giving in water solution a blue fluor-
escence which can be detected even in great dilution. The fluorescence
is increased in alkaline, and decreased in acid, solution.

Expt. 155. DemoTistration of the presence of aesculin in Aesculus hark. Strip off
the bark from some young twigs of Aesculus and boil in a little water in an evaporating
dish. Filter and pour the filtrate into excess of water in ^a large vessel. A blue
fluorescent solution will be formed.

Glucosides of Flavone, Flavonol and Anthocyan Pigments.

These substances have already been considered in Chapter viii.

Glucosides of vakious Composition,

Coniferin. This glucoside (see also p. 104) occurs in various members
of the Coniferae and also in Asparagus. On hydrolysis with mineral acids
or emulsin, it breaks up as:

/X

Coniferin

OH

Coniferyl alcohol

Arbutin. This glucoside is found in the leaves of the Bearberry
(Arctostaphylos Uva-ursi), Pyrola, Vaccinium, and other Ericaceae and
also of the Pear (Pyrus communis).

On hydrolysis with acids arbutin yields quinol and glucose:

C12H16O7 + HaO;^ CfiHeOa + CeHjoOe

The same hydrolysis is brought about by the enzyme emulsin.

It has been suggested that the darkening of leaves of the Pear
(Bourquelot and Fichtenholz, 11, 12, 13) either on autolysis or injury, or
at the fall of the leaf, is due to the hydrolysis of the arbutin by a gluco-
side-splitting enzyme in the leaf, and subsequent oxidation of the
hydroquinone so formed by an oxidase.

Expt. 156. Extraction of arhutin from leaves of the Pear (Pyrus communis).
Weigh out 100 gms. of fresh leaves (without petioles). Tear the leaves into small
pieces and drop them as quickly as possible into about 500 c.c. of boiling 96-98 0/
alcohol in a flask. Boil for about 20 mins., adding more alcohol if necessary. Then
filter off the alcohol and pound up the leaf residue in a mortar and extract again with
boiling alcohol. Filter and distil off the alcohol from the extract in vacuo. Extract
the residue with 100-200 c.c. of hot water and filter. Warm the filtrate and precipitate
with lead acetate solution until no more precipitate is formed. This removes flavones»

X]

GLUCOSIDES

167

tannins, etc. but the arbutin is not precipitated. Filter and pass sulphuretted
hydrogen into the filtrate to remove any excess of lead acetate. Filter and concentrate
the filtrate in vacuo to a syrup. Then extract twice with small quantities of ethyl
acetate. Concentrate the ethyl acetate on a water-bath and cool. A mass of crystals
of arbutin will separate out. This should be filtered off on a small filter, and re-
crystallized from ethyl acetate. Take up a little of the purified glucoside in water
and add a drop or two of ferric chloride solution. A blue coloration will be given.

Salicin. This substance occurs in the bark of various species of
Willow {Salix) and Poplar (Populus): also in the flower-buds of the
Meadow- Sweet (Spiraea Ulmaria). On hydrolysis with acids, or on
treatment with emulsin, salicin is decomposed into saligenin or salicylic
alcohol and glucose :

CisHiaOv + H2O = CgHpH • CH,OH + C^HiaOe

Saligenin gives a violet colour with ferric chloride solution and in this
way the progress of the reaction can be demonstrated (see also p. 168).

Indican. This glucoside occurs in shoots of the so-called "Indigo
Plants," Indigofera Anil, I. erecta, I. tinctoria, I. sumatrana: also in
the Woad (Isatis tinctoria), in Polygonum tinctorium and species of the
Orchids, Phajus and Galanthe. When boiled with acid or hydrolyzed by
an enzyme contained in the plant, it gives glucose and indoxyl:

/\

-c-o

.^\

+ H,0

CH
Indican

-C'OH

+ CeHisOe

CH

Indoxyl

The colourless indoxyl can be oxidized either artificially or by an
oxidase contained in the plant to a blue product, indigotin or indigo.

/\

C'OH HO-C-

+ 20+ II

,. n CH

Indoxyl

"^ ,Ay

/\

2HoO +

-CO OC

/S

NH'

'^NH^^

\NH

Indoxyl Indigo

The relationship between indoxyl and tryptophane (see p. 135) should
be noted.

V

168 GLUCOSIDES [ch. x

REFERENCES

Books

1. Abderhalden, B. Biochemisches Handlexikon, ir. Berlin, 1911.

2. Allen's Commercial Organic Analysis. Glucosides (E. F. Armstrong), Vol. 7,
1913, pp. 95-135.

3. Armstrong, B. P. The Simple Carbohydrates and the Glucosides. London,
1919. 3rded.

4. Van Rijn, J. J. L. Die Glykoside. Berlin, 1900.

Papers

5. Armstrong, B. P. The Rapid Detection of Emulsin. J. Physiol.^ 1910,
Vol. 40, p. xxxii.

6. Armstrong, H. B., Armstrong, B. P., and Horton, B. Studies on
Enzyme Action. XII. The Enzymes of Emulsin. Proc. R. Soc, 1908, B Vol. 80,
pp. 321-331.

7. Armstrong, H.B., Armstrong, E.P., and Horton, B. Herbage Studies.
L Lotus cornkulatus, a Cyanophoric Plant. Proc. R. Soc, 1912, B Vol. 84, pp. 471-484.
II. Variation in Lotus cornicidatus and Trifolium repens (Cyanophoric Plants).
Proc. R. Soc, 1913, B Vol. 86, pp. 262-269.

8. Armstrong, H. E., Armstrong, B. P., and Horton, E. Studies on
Enzyme Action. XVI. The Enzymes of Emulsin. Proc R. Soc, 1912, B Vol. 85.
(i) Prunase, the Correlate of Prunasin, pp. 359-362. (ii) Distribution of /3-Enzymes
in Plants, pp. 363-369. (iii) Linase and other Enzymes in Linaceae, pp. 370-378.

9. Armstrong, H. B., and Horton, B. Studies on Enzyme Action. XIII.
Enzymes of the Emulsin Type. Proc R. Soc, 1910, Vol. 82, pp. 349-367.

10. Bourquelot, B. Sur I'emploi des enzymes comme reactifs dans les re-
cherches de laboratoire. J. pharm. chim., 1906, Vol. 24, pp. 165-174 ; 1907, Vol. 25,
pp. 16-26, 378-392.

11. Bourquelot, B., et Pichtenholz, A. Sur la presence d'un glucoside dans
les feuilles de poirier et sur son extraction. J. pharm. chim., 1910, Vol. 2, pp. 97-104.

12. Bourquelot, B., et Pichtenholz, A. Nouvelles recherches sur le gluco-
side des feuilles de poirier : son rdle dans la production des teintes automnales de ces
organes. J. fharm. chim., 1911, Vol. 3, pp. 5-13.

13. Bourquelot, B., et Pichtenholz, A. Sur le glucoside des feuilles de
poirier. C. R. Acad, sci., 1911, Vol. 153, pp. 468-471.

14. Dunstan, W., and Henry, T. A. The Chemical Aspects of Cyanogenesis
in Plants. Rep. Brit. Ass., 1906, pp. 145-157.

15. Greshoflf, M. The Distribution of Prussic Acid in the Vegetable Kingdom.
Rep. Brit. Ass., 1906, pp. 138-144.

16. Guignard, L. Sur quelques proprietds chimiques de la myrosine. Bui.
soc hot., 1894, Vol. 41, pp. 418-428.

17. Mirande, M. Influence exercee par certaines vapeurs sur la cyanogenese
vegetale. Precede rapide pour la recherche des plantes k acide cyanhydrique.
C. R. Acad, sci., 1909, Vol. 149, pp. 140-142.

18. Spatzier, W. Ueber das Auftreten und die physiologische Bedeutung des
Myrosins in der Pflanze. Jahrh. wiss. Bot., 1893, Vol. 25, pp. 39-77.

19. Winterstein, B., und Blau, H. Beitrage zur Kenntnis der Saponine.
Zs.physiol. Chem., 1911, Vol. 75, pp. 410-442.

CHAPTER XI

PLANT BASES

There are present in plants a number of substances which form a group,
and which may be termed nitrogen bases, or natural bases. These sub-
stances are of various constitution but they have the property in common
of forming salts with acids by virtue of the presence of primary, secondary,
or tertiary amine groupings. Such groupings confer a basic property
upon a compound and, as a result, salts are formed with acids on analogy
with the formation of ammonium salts :

NH3+ HCI = NH4CI (NH3 • HCI)

CH3NH2+ HCI = CH3NH2 • HCI
methylamine

(CH3)2 NH + HCI = (CH3)2 NH • HCI
diraethjlamine

(CHgJg N + HCI = (CH3)3 N • HCI
trimethylamine

The hydrogen atoms of ammonia can also be replaced by groups of
greater complexity, as will be seen below.

Complex ring compounds in which nitrogen forms part of the ring
are termed heterocyclic, such as the alkaloids, purines and some amines,
for instance pyrrolidine (see below).

The plant bases can be conveniently classified into four groups and
this is also to a large extent a natural grouping. They are:

1. Amines ) ci- 1 .11

^ ^ . . y bimpler natural bases.

2. i3etainesj ^

3. Alkaloids.

4. Purine bases.

The first two groups have been termed the simpler natural bases.
They are much more widely distributed in the vegetable kingdom than
the alkaloids and purines, since they have probably much more significance
in general metabolism. The isolation of the simpler bases is a matter of
much greater difficulty than that of the alkaloids: the former are soluble
in water but insoluble in ether and chloroform, and so are not readily
separated from other substances. The alkaloids, however, occur in the
plant as salts of acids and if the plant material is made alkaline the free
bases can be extracted with ether or chloroform.

170 PLANT BASES [ch.

The betaines are ainino-acids in which the nitrogen atom is com-
pletely methylated, and, with one or two exceptions, this grouping does-
not occur in the true alkaloids. The betaines have only feebly basic
properties.

The alkaloids, in contrast to the simpler natural bases, are rather
restricted in their distribution, many being limited to a few closely
related species or even to one species.

The purine bases are a small group of substances intimately related
to each other and to uric acid.

Amines.

Methylamine, CHg * NHg, occurs in the Annual and Perennial Dogs-
Mercury {Mercurialis annua and M.perennis) and in the root of the Sweet
Flag {Acorus Calamus).

Trimethylamine, (6113)3 " N, occurs in leaves of the Stinking Goose-
foot (Chenopodium Vulvaria), in flowers of the Hawthorn (Crataegus
Oxyacantha) and Mountain Ash (Pyrus Aucuparia), and in seeds of
Mercurialis annua.

Putrescine, NH2(CH2)4*NH2, occurs in the Thorn Apple {Datura)
and tetramethylputrescine in a species of Henbane (Hyoscyamus muticus),

Hordenine occurs in germinating Barley grains. It is represented as :

H0<<; 3>CH2'CH2' N(CH3)2

Pyrrolidine is said to occur in small quantities in leaves of the
Carrot (Daucus Carota) arid Tobacco (Nicotiana) leaves. It is repre-
sented as:

CH2 CH2

I I

CH2 CH2
\nh/

Other amines occur among the lower plants (Fungi).

Choline is sometimes classified with the betaines. It is however
intimately connected with lecithin (see p. 98) which is not the case with
the betaines. It may be represented as:

.OH
(CH3)3: N<(

XHo-CHoOH

XI] PLANT BASES 171

Choline is very widely distributed in plants. It is a constituent of the
phosphatide, lecithin, and is probably thereby a constituent of all living
cells. It has been found in seeds of the Bean ( Vicia Faba), Pea (Pisum
sativum), Strophanthus spp., Oat {Avena sativa), Cotton (Gossypium
herbaceam), Beech {Fagus sylvatica). Fenugreek {Trigonella Foenum-
graecum) and Hemp {Cannabis sativa): in seedlings of Lupins, Soy
beans, Barley and Wheat: in Potatoes and Dahlia tubers and in the
subterranean parts of Cabbage (Brassica napus), Artichoke (Helianthus
tuberosus), Scorzonera hispanica, Chicory (Cichorium Intybus), Celery
(Apium graveolens) and Carrot {Daucas Carota); aerial parts of Meadow
Sage (Salvia pratensis) and Betony {Betonica officinalis)^ and many other
tissues. It can only be isolated in very small quantity.

Betaines.

The betaines, as previously stated, are amino-acids in which the
nitrogen atom is completely methylated. Most betaines crystallize with
one molecule of water; thus betaine itself in this condition probably has
the following constitution, from which its relationship to glycine or
aminoacetic acid is indicated:
OH

(CH3)3| N<' HsN-CH.-COOH

XHa'COOH
Betaine or hydroxytrimethyl- Aminoacetic acid

aminoacetic acid

When dried above 100^ C, the betaines lose water and are represented
as cyclic anhydrides; thus betaine becomes:

(CHj),: N CO

\ch/

The individual betaines, probably on account of their close connexion
with proteins, are more widely distributed than the individual alkaloids.
Further investigation may show an even more general distribution of
betaines.

Betaine or trimethylglycine occurs in all species of Chenopodiaceae
so far examined including the sugar Beet {Beta vulgaris) from which it
derives its name: in some genera only of the Amarantaceae : in the "Tea
Plant" {Lycium barbarum): in seeds of Cotton {Gossypium herbaceum),
Sunflower {Helianthus annum) and Oat {Avena sativa): in tubers of
Artichoke {Helianthus tuberosus), shoots of Bamboo {Bambusa), leaves of
Tobacco {Nicotiana Tabacum) and in malt and wheat germs.

172 PLANT BASES [ch»

Stachydrine, though a betaine, is included by most writers among
the alkaloids, and this classification has been followed here (see p. 176);
it is probably a derivative of proline (see p. 135).

Betonicine, C7H13O3N, is also, like stachydrine, found in the Betony
(Betonica officinalis). It is a derivative of oxyproline.

Hypaphorine or trimethyltryptophane, C14H18O2N2, occurs in the
seeds of a tree, Erythrina Hypaphorus, which is grown for shade in
Coffee plantations.

Trigonelline, like stachydrine, is usually classed with the alkaloids
(see p. 175) but it should probably be included among the betaines on
account both of its structure and of its wide distribution.

Other betaines, trimethylhistidine, ergothioneine, occur in the
Fungi.

Alkaloids.

The plant alkaloids, so-called because of their basic properties, have
attracted considerable attention on account both of their medicinal
properties and, in many cases, their intensely poisonous character. They
were also the plant bases to be first investigated. As previously men-
tioned they are not widely distributed, some being, as far as is known,
. restricted to one genus, or even species. Moreover, several closely related
^alkaloids are frequently found in the same plant. The orders in which
they" largely occur are the Apocynaceae, Leguminosae, Papaveraceae,
Ranunculaceae, Rubiaceae and Solanaceae.

The alkaloids may be present in solution in the cell-sap in the young
tissues, but in older and dead tissues they may occur in the solid state ;
they may be found throughout the plant or more abundantly in the seed,
fruit, root or bark (quinine).

The alkaloids are, as a rule, insoluble in water, but soluble in such
reagents as alcohol, ether, chloroform, etc. The majority are crystalline
solids which are not volatile without decomposition, but a few, for
example coniine, nicotine, which contain no oxygen, are volatile liquids.

The alkaloids occur in the plant as a rule as salts of various organic
acids, such as malic, citric, succinic and oxalic, and sometimes with an
acid peculiar to the alkaloid with which it is united (e.g. quinic acid in
quinine and meconic acid in opium). Artificial salts, i.e. sulphates,
chlorides and nitrates, are easily prepared and are readily soluble in
water, and from these solutions the free base is precipitated again on
addition of alkali.

XI] PLANT BASES 173

The alkaloids themselves belong to various classes of compounds,
though the basic character always preponderates. Thus, for example,
piperine is an amide and can be hydrolyzed into the base piperidine and
piperic acid : atropine is an ester made up of the base tropine and tropic
acid.

Various methods are employed for the extraction of alkaloids but
the exact course of events depends on the alkaloid in question. On the
whole the method is either to treat the plant material with alkali and
then extract the free alkaloid with ether or chloroform and finally purify
by making a salt again; or to extract the alkaloid from the plant with
dilute acid, set free the insoluble, or difficultly soluble, base with alkali,
and then prepare a salt of the base.

Though individual alkaloids have distinctive reactions, the group as
a whole has certain reactions in common, namely the precipitation by
the so-called "alkaloidal reagents." These reagents are tannic, phospho-
tungstic, phosphomolybdic and picric acids, also potassium-mercurio-
iodide solution and iodine in potassium iodide solution.

Expt. 157. General reactions of alkaloids. Make a 05 ^/q solution of quinine
sulphate in warm water and add a few drops of each of the following reagents :

{a) Tannic acid solution. A white precipitate is formed.

(6) Mercuric iodide in potassium iodide solution [Briicke's reagent : 50 gms. of
potassium iodide in 500 c.c. water are saturated with mercuric iodide (120 gms.) and
made up to 1 litre]. A white precipitate is formed.

(c) Phosphotungstic acid (50 gms. of phosphotungstic acid and 30 c.c. of cone, sul-
phuric acid are dissolved in water and made up to a litre). A white precipitate is formed.

{d) Iodine in potassium iodide solution. A brown precipitate is formed.

(e) Picric acid solution. A yellow precipitate is formed.

ExpL 158. Extraction of the free base from quinine sulphate. Add strong sodium
carbonate solution drop by drop to some of the quinine sulphate solution until a
white precipitate of quinine is formed. Then add ether and shake up in a separating
funnel. The precipitate will disappear as the quinine passes into solution in the ether.
Separate off the ethereal solution and let it evaporate in a shallow dish. The quinine
is deposited. Take up the quinine again in dilute sulphuric acid and test the solution
with the alkaloidal reagents.

The alkaloids are classified into five groups according to the nucleus
which constitutes the main structure of the molecule. These five groups
are:

1. The pyridine group.

2. The pyrrolidine group.

3. The tropane group.

4. The quinoline group.

5. The isoquinoline group.

174

PLANT BASES

[CH.

Pyridine

Nn^

Pyrrole

\^

Tropane

n

V

Iminazole

Quinoline

./N

L^«>

Isoquinoline Pyriinidine Iminazole Purine

1. The pyridine alkaloids.

These are, as the name implies, derivatives of pyridine. (Pyridine

is a colourless liquid which boils at 115° C. It is a strong base and forms

salts with acids.)

CH

/\
CH CH

II I

CH CH

\^
N

Pyridine

The more important members of this group are: arecoline, coniine,
nicotine, piperine and trigonelline.

Arecoline occurs in the "Betel Nut" which is the fruit of the Areca
Palm (Arecha Catechu).

Coniine occurs in all parts of the Hemlock (Conium maculatum), but
more especially in the seed.

Nicotine occurs in the leaves of the Tobacco plant {Nicotiana
Tahacum). It is a colourless oily liquid which is intensely poisonous.
Its constitution may be represented as:

CH

CHo— CH2

^\

i ' 1

CH C-

-CH CHa

1 11

\/

CH CH

N

\/^

1

N

CH,

It is readily soluble in water and organic solvents.

XI] PLANT BASES 175

Expt. 159. Extraction and reactions of nicotine. Weigh out 100 gms. of plug
tobacco and boil up the compressed leaves with water in an evaporating dish or in a
saucepan. Filter off' the extract and concentrate on a water-bath. The concentrated
solution is made alkaline with lime and distilled from a round -bottomed flask fitted
with a condenser, the flask being heated on a sand-bath. The distillate has an un-
pleasant smell and contains nicotine in solution. Test the solution with the alkaloidal
reagents employed in Expt. 157. A precipitate will be obtained in each case.

The nicotine can be obtained from solution in the following way. Acidify the
•aqueous distillate with oxalic acid and concentrate on a water-bath. Make the con-
•centrated solution alkaline with caustic soda, pour into a separating funnel and shake
up with ether. Separate the ethereal extract and distil off" the ether. The nicotine
is left behind as an oily liquid which oxidizes in air and turns brown. The alkaloidal
tests should be made again with the extracted nicotine.

Piperine occurs in various species of Pepper {Piper nigrum). The
fruit, which is gathered before it is ripe and dried, yields a black pepper,
but if the cuticle is first removed by maceration, a white pepper. Piperine
is a white solid which is almost insoluble in water but soluble in ether
a,nd alcohol.

Expt. 160. Extraction and reactions of piperine. Weigh out 100 gms. of black
pepper. Put it into an evaporating dish, cover well with lime-water and heat with
■constant stirring for 15-20 minutes. Then evaporate the mixture completely to
<iryness on a water-bath. Grind up the residue in a mortar, put it into a thimble
•and extract with ether in a Soxhlet. Distil off" the ether and take up the residue in
hot alcohol from which the piperine will crystallize out. With an alcoholic solution
make the following tests :

{a) Add the alkaloidal reagents mentioned in Expt. 157 and note that a pre-
■cipitate is formed in each case.

{h) Pour a little of the solution into water and note that the piperine is pre-
■cipitated as a white precipitate.

(c) To a little solid piperine in a white dish add some concentrated sulphuric
•acid. It dissolves to form a deep red solution.

Trigonelline occurs in the seeds of the Fenugreek (Trigonella
Foenum-graecum), Pea(Piswm sativum), Kidney Bean {Phaseolus vulgaris),
Strophanthus hispidus, Hemp (Cannabis sativa) and Oat (Avena sativa).
It is also found in the Coifee Bean (Coffea arabica); in tubers of Stachys
* tuberifera, Potato and Dahlia and in roots of Scorzonera hispanica. It
is really a betaine (see p. 172).

2. The pyrrolidine alkaloids.

These are derivatives of pyrrolidine, of which the mother substance
is pyrrole. (Pyrrolidine is a liquid boiling at 91° C. It is a strong base
•and forms stable salts with acids.)

176

PLANT BASES

[CH.

CH— CH

I I

CHo CHij

CH CH

\/ \/

NH NH

Pyrrole Pyrrolidine

These alkaloids form a small group containing:

Hygrine and cuskhygrine which occur in Coca leaves {Erythroxylon
Coca).

Stachydrine which occurs in tubers of Stachys taherifera and leaves
of the Orange Tree (Citrus Aurantium) and in various other plants
(Betonica). The formula is :

CH2 — CHq

I I

CO— CH CH2

I \/
0 N(CH3)2

from which it is seen that it is really a betaine (see p. 172).
3. The tropane alkaloids.

These are derivatives of tropane, which may be regarded as formed

from condensed piperidine and pyrrolidine groupings. (Tropane is a

liquid boiling at 167° C.)

CH2

CH CH

NCH3

CH2 — CHo
Tropane
The alkaloids in this group are limited to four natural orders and are
as follows:

Solanaceae: Atropine occurs in the root and other parts of the
Deadly Nightshade (Atropa Belladonna), the Thorn Apple (Datura
Stramonium) and Scopolia japonica. Atropine may be represented as :

CH— O— CO— CH • CH2OH
/\ I

CH2 CHa

I I

CH CH

\/
NCH.

C«H,

XI] PLANT BASES 177

Hyoscy amine occurs in the Henbane {Hyoscyamus niger), H.
muticus and also in the Mandrake (Mandragora),

Erythroxylaceae: Cocaine and tropacocaine occur in Coca leaves

{Ej'ythroxylon Coca) together with smaller quantities of allied alkaloids.

Cocaine has the formula:

H OCOCgHs

\/
C

/\
CHo CHCOOCH3

i " I
CH CH

\/
NCH<

Punicaceae : Pelletierine and other allied alkaloids occur in the root
and stem of the Pomegranate Tree (Punica Granatum).

Leguminosae: Sparteine occurs in the Broom (Spartium scoparium):
lupinine in the yellow and black Lupins {Lupinus luteus and L. niger)
and cytisine in the Laburnum (Cytisus Laburnum).

4. The quinoline alkaloids.

These are derivatives of quinoline. (Quinoline is a colourless liquid
which boils at 239° C.) Its constitution is:

CH CH

/'\/\
CH C CH

I II I

CH C CH

\/\^
CH N

Quinoline

These alkaloids form two natural groups, (a) the cinchona alkaloids,
i.e. quinine, cinchonine and allied forms, and (6) the strychnine alkaloids,
i.e. strychnine and brucine.

Quinine occurs in the bark of various species of the genus Cinchona
(Rubiaceae) which are trees, originally natives of S. America, but now
cultivated on a large scale in Ceylon, Java and India. The species
employed are C. Calisaya, Ledgeriana, officinalis, succirubra. The yellow
bark of Calisaya has the highest percentage, i.e. 12 7o. of alkaloid.

Quinine is a white solid which crystallizes in long needles containing
water of crystallization. It is very slightly soluble in cold water, more

12

178 PLANT BASES [ch.

so in hot but readily soluble in alcohol, ether and chloroform. With
acids it forms salts, which are soluble in water, the sulphate being
commonly employed in medicine. Quinine is said to have the following
constitution:

CioHi5(OH)N

OCH,

Expt. 161. Extraction and reactions of quinine. Mix 20 gras. of quicklime with
200 c.c. of water in a basin and then add 100 gms. of powdered Cinchona bark. Stir
together well and then dry the mixture thoroughly on a water-bath, taking care to
powder the lumps. The dried mixture is then extracted in a Soxhlet apparatus with
chloroform. The chloroform extract is then shaken up in a separating funnel with
25 c.c. of dilute sulphuric acid. The chloroform layer is run off and again extracted
with water. The sulphuric acid and water extracts are mixed together and neutralized
with ammonia. The liquid is evaporated on a water-bath until crystals of quinine
sulphate begin to separate out. With the quinine sulphate the following tests should
be made. (It is better to use a solution of the hydrochloride prepared by adding a
few drops of hydrochloric acid to the sulphate solution) :

{a) Test with the alkaloidal reagents of Expt. 157.

(6) Add to a little of the solution some bromine water and then some ammonia.
A green precipitate is formed which gives a green solution with excess of ammonia.

(c) Dissolve a little of the solid quinine sulphate in acetic acid and pour into a
large volume of water. A blue opalescence is produced which is characteristic of
quinine.

Cinchonine occurs together with quinine in Cinchona bark. It is
very similar in constitution to quinine, the latter being methoxy-
cinchonine.

Strychnine and brucine occur in the seeds of Nux Vomica (Strych-
nos Nux-vomica) and St Ignatius' Bean {S. Ignatii).

Expt. 162. Tests for strychnine. Add a little concentrated sulphuric acid to a
small quantity of strychnine in an evaporating dish and then add a small amount of
powdered potassium bichromate. A violet coloration is produced which changes to
red and finally yellow.

Curarine, the South American Indian Arrow poison, occurs in
several species of Strychnos (S. toxifera and others).

5. The isoquinoline alkaloids.

These can be divided into two groups: {a) the opium alkaloids and
(6) the berberine alkaloids.

XI] PLANT BASES 179

The opium alkaloids again fall into two classes: (1) the papaverine
group which includes papaverine, laudanosine, narceine, narcotine
and others, and (2) the morphine group including morphine, apomor-
phine, codeine, thebaine and others.

Opium is the dried latex obtained by making incisions in the cap-
sules of the Opium Poppy (Papaver somniferum).

Allied to the papaverine group is hydrastine which occurs in the
root of Hydrastis canadensis (Ranunculaceae).

The constitution of all these alkaloids is very complex.

^^^^.163. Tests for morphine.

(a) Add a little ferric chloride solution to a solution of a morphine salt. A deep
blue coloration is formed.

(6) Dissolve some morphine in concentrated sulphuric acid and then after
standing about 15 hrs. add concentrated nitric acid. A deep blue-violet colour is
produced which afterwards changes to red.

Berberine occurs in the root of the Barberry (Berberis vidgaris) and
is also found in isolated genera in Anonaceae, Menispermaceae, Papa-
veraceae, Ranunculaceae and Rutaceae.

Corydaline occurs in Gorydalis cava (Fumariaceae).

Many other alkaloid substances have been isolated from a large
number of different plants, but since the constitution of most of them
is unknown, they have not been classified.

Purine and Pyrimidine Bases.

These substances, as indicated (p. 3), have a hecterocyclic ring
structure and are derivatives of purine and pyrimidine: the atoms of the
ring are numbered in the order indicated below:

N=:CH

I I
HC C— NH IN— «C N==CH

CH 2q 5Q_7f^ HQ Q„

^ i II V II II

N— C— N 3N_4c_9N^ N — CH

Purine Pyrimidine

Purine itself is a crystalline basic compound (m. p. 211-212° C.)
which forms salts with acids. It is composed of two rings, the pyrimi-
dine and the iminazole: the latter grouping also occurs in histidine
(see p. 135).

The chief purine bases which occur in plants are xanthine, guanine,
hypoxanthine, adenine, caffeine and theobromine.

12—2

180

PLANT BASES

[CH.

Xanthine may be regarded as 2, 6-dioxypurine :

HN— C=:0

I I

0=0 C— NH

\
CH

HN— C— N

It is widely distributed in plants and has been found in leaves of the
Tea plant {Thea sinensis), in the sap of the Beetroot (Beta) and in various
seedlings.

Guanine and hypoxanthine can be represented respectively as
2-amino, 6-oxypurine and 6- nionoxy purine:

H^

1 —

Z=0

1

Hh

i— C=0

1

HoN-

— c

c

) <

— <

jua

1

D— NH

\
CH

D— N

[line

1
HC C— NH

\
CH

N— C— N

Hypoxanthine

They usually occur together and have been found in the germinating
seeds of the Sycamore (Acer pseudoplatanus), Pumpkin (Cucurhita
Pepo), Common Vetch (Vicia sativa), Meadow Clover (Trifolium
pratense), yellow Lupin (Lupinus luteus) and Barley (Hordeum vulgare) :
also in the juice of the Beet (Beta).

Adenine is 6-aminopurine. It is represented as:

N=C— NHo

HC C-

N— C-

-NH
\

CH

N

It has been found in Beet (Beta), Tea leaves (Thea sinensis) and in
leaves of the Dutch Clover (Trifolium repens).

Guanine and adenine are obtained by the hydrolysis of plant nucleo-
proteins.

Caffeine or theine is 1, 3, 7-trimethylxanthine:

CHgN— C=0

II

0=C C— N • CH,

\

CH

CH. • N— C-

-N

XI] PLANT BASES 181

It occurs in the leaves and beans of the Coffee plant (Coffea arabica),
in leaves of the Tea plant {Thea sinensis), in leaves of Ilex paragueiisis
("Paraguay Tea"), in the fruit of Paullinia Cupana and in Kola nuts
(Cola acuminata).

Expt. 164. Preparation of caffeine from tea i. Digest 100 gms. of tea with 500 c.c.
of boiling water for a quarter of an hour. Then filter through thin cloth or fine
muslin using a hot-water filter in order to keep the liquid hot. Wash the residue
with a further 250 c.c. of boiling water. Add to the filtrate a solution of basic lead
acetate until no more precipitate is formed. This removes proteins and tannins.
Filter hot and to the boiling filtrate add dilute sulphuric acid until the lead is pre-
cipitated as sulphate. Filter from the lead sulphate, and concentrate the solution,
with the addition of animal charcoal, to 250-300 c.c. Filter and extract the filtrate
three times with small quantities (50 c.c.) of chloroform. Distil off the chloroform
on a water-bath, and dissolve the residue in a small quantity of hot water. On
allowing the solution to evaporate very slowly, long silky needles of caifeine separate,
which may have a slightly yellow tint, in which case they should be drained, re-
dissolved in water, and boiled with the addition of animal charcoal. The yield
should be about 1*5 gm.

Evaporate a little of the caffeine on a water-bath with bromine water. A reddish-
brown residue is left which becomes purple when treated with ammonia.

Theobromine is 3, 7-dimethylxanthine:

HN— c=0

I I
0=C C— N • CH^

\
CH

CHg-N— C— N

It occurs in the fruit of the Cocoa plant ( Theohroma Cacao), in leaves
of the Tea plant {Thea sinensis) and in the Kola nut {Cola acuminata).

The chief pyrimidine bases found in the plant are uracil (2, 6-dioxy-
pyrimidine) and cytosine (6-amino-2-oxy-pyrimidine). They are con-
stituents of the molecule of nucleic acid (see p. 141).

It seems appropriate at this point to mention the fact that urea is
said to have been detected in small quantity in the Spinach {Spinacia
oleracea), Cabbage (Brassica oleracea), Carrot (Daucus Carota), Potato
(Solanum tuberosum), Chicory {Cichorium Intybus) and other plants.
A point of considerable interest is the occurrence in the seeds of the Soja
Bean {Glycine hispida) and other Leguminosae of an enzyme, urease,
which decomposes urea into ammonia and carbon dioxide:

/NH2
o=c(^ + HoO = 2NH3 4- CO.,.

1 From Cohen, Practical Organic Cliemistry.

182 PLANT BASES [ch. xi

Urease is quite specific in its action on urea, and the latter has been
detected in a few tissues which also yield the enzyme (grain of the
Wheat and seeds of the Bean) (Fosse, 4).

Expt. 165. Action of urease on urea. To 100 c.c. of water in a small flask add
1 gm. of urea and 3 gms. of Soja Bean meal. Connect the flask by glass tubing to a
second flask contaning 0*5 c.c. of strong sulphuric acid in 50 c.c. of water and a piece
of litmus paper. Place the flask containing the urea and enzyme in a beaker of
water kept at 37-40° C. and run a rapid current of air through the two flasks. After
two or three hours, the litmus paper will turn blue. Add sodium carbonate to the
second flask and heat. Ammonia will be evolved and can be detected by its smell
and by giving white fumes with a drop of strong hydrochloric acid on a glass rod.

EEFERENCES

Books

1. Abderhalden, E. Biochemisches Handlexikon, v. Berlin, 1911.

2. Allen's Commercial Organic Analysis. Vegetable Alkaloids (G. Barger),
Vol. 7, 1913, pp. 1-94.

3. Barger, G. The simpler Natural Bases. London, 1914.

4. Fosse, R. Presence simultanee de Puree et de I'urease dans le meme vegetal
C. B. Acad. Sci., 1914, Vol. 158, pp. 1374-1376.

5. Henry, T. A. The Plant Alkaloids. London, 1913.

6. Jones, W. Nucleic Acids. London, 1920. 2nd. ed.

7. Winterstein, E., und Trier, G. Die Alkaloide. Berlin, 1910.

INDEX

Figures in heavy type denote main references.

Abderhalden, 10, 26, 79, 99, 155, 168, 182
Abies, 108

pectinata, 151

Acacia, 63
Acacia Senegal, 63
A cer pseudoplatanus, 180
Acetaldehyde, 21, 24, 129
Acetic acid, 1, 81, 82, 89
Acetone, 158, 163
Achroodextrin, 59
Aconitic acid, 88
Aconitum, 88
Acorus Calamus, 170
Acrolein, 93
Acrylic acid, 3

aldehyde (see Acrolein)

series, 90

Adenine, 142, 180
Adipic acid, 83, 85
Adipo-celluloses, 67, 70
Adzuki-bean, 140
Aegopodium Podagraria, 111
Aesculetin, 159, 165
Aesculin, 159, 165
Aesculus, 127, 159, 166

Hippocastanum, 98, 106, 108, 149,

165

Aetiophyllin, 31

Aetioporphyrin, 33

Agar, 14, 51

Alanine, 81, 134

Albumins, 132, 138

Albiimoses, 132, 133, 143

Alcoholic fermentation, 22, 129

Alder Buckthorn, 161

Aleurone, 132

Alkaloidal reagents, 138, 173

Alkaloids, 3, 169, 172

Alkanet, 92

Allen, 10, 99, 168, 182

Allium, 64, 71

Cepa, 62, 76

Allocyanidin, 121, 122

Allocyanin, 122

AUose, 48

Almond, 63, 91, 140, 160, 162

Aloe, 64, 9t)

Althaea rosea, 64, 118

Althaein, 118

Altrose, 48

Alyssum, 126, 127

Amandin, 140

Amarantaceae, 116, 171

Amaranthus, 116

Amines, 3, 169, 170

Amino-acids, 21, 81, 133, 149, 171

Amino-acetic acid, 81
Amino-glutaric acid, 1, 83
Amino-iso-caproic acid, 81
Araino-iso-valeric acid, 81
Amino-propionic acid, 81
Amino-succinic acid, 1, 83, 84
Ampelopsidin, 118
Ampelopsis quinquefolia, 118

hederacea, 82

Amphoteric electrolytes, 16, 134
Amygdalase, 160

Amygdalin, 21, 26, 157, 158, 160, 162
Amyl acetate, 82

caproate, 82

formate, 82

Amylodextrin, 59
Amyloid, 68

Ananas sativus, 86, 154

Anchusa officinalis, 92

Anemone, 123

Aniline acetate (test for pentoses), 45

Anonaceae, 179

Anthocyan pigments, 2, 101, 114

artificial, 121

isomerization of, 115

reactions of, 115

Anthocyanidins, 114
Anthocyanins, 114

Antirrhinum, 110

majus. 111, 112, 116

Antiseptics, 19

Apigenin, 111, 159

Apiin, 112, 159

Apiose, 159

Apium graveolens, 171

Apocynaceae, 172

Apomorphine, 179

Apple, 65, 82, 84, 85, 123, 127, 160

Apricot, 140

Aquilegia, 114, 116

vulgaris, 161, 162

Araban, 45, 47, 65, 62, 63
Arabic acid, 63

Arabin (see Gum Arabic)

Arabinose, 42, 45, 46, 55, 62, 63, 64, 65, 74

Arabis, 125, 127

Araceae, 160

Arachidic acid, 89

Arachis hypogaea, 141

Araliaceae, 165

Arbutin, 102, 159, 166

Archichlamydeae, 123

Arctostaphylos Uva-ursi, 166

Areca Catechu, 174

Areca Palm, 174

Arecoline, 174

184

INDEX

Arginine, 135, 151

Armstrong, 20, 26, 79, 94, 99, 160, 161, 168

Aromatic acids, 2, 101, 103

alcohols, 2, 101, 103

aldehydes, 2, 101, 103

compounds, 2, 101

Arsenic trisulphide sol, 13, 17
Artichoke, 61, 151, 171
Arum maculatum, 161, 162
Asclepiadaceae, 160

Ash, 91

Asparagine, 150
Asparagus, 61, 62, 158, 166

officinalis, 150

Aspartic acid, 1, 83, 84, 134, 150
Aster, 118
Asterin, 118
Astragalus, 63

gummifer, 63

Atkins, 79

Atriplex, 116

Atropa Belladonna, 176

Atropine, 176

Aubrietia, 125

Aucuba, 159

Aucubigenin, 159

Aucubin, 159

Auld, 26

Autolysis, 20, 153, 161

Autoxidation, 123

Avena sativa, 139, 140, 171, 175

Avenalin, 140

Bach, 123, 129, 130

Balsams, 109

Bamboo, 151, 171

Bambusa, 151, 171 ^

Banana, 82, 84, 97, 128, 153

Barberry, 179

Barger, ^82

Barley, 60, 138, 141, 146, 170, 171, 180

Bassett, 111, 112, 131

Bayliss, 15, 17, 20, 26

Bearberry, 166

Beech, 171

Copper, 116

wood, 69

Beet, 54, 65, 73, 74, 78, 84, 85, 116, 128,

129, 151, 15H, 171, 180
Behenic acid, 89

Benzaldehyde, 21, 26, 158, 159, 160
Benzidine (test lor peroxidase), 124
Benzoic acid, 103, 158
Berberidaceae, 160
Berberine, 179
Berberis vulgaris, 179
Bertholletia excelsa, 91, 93, 139, 140, 149
Bertrand, 10, 142
Beta, 54, 75, 77, 116, 158, 180
vulgaris, 72, 73, 78, 84, 85, 128, 151,

153, 171
Betaines, 169, 171
Betonica, 176

oMcinalis, 111,^12

Betonicine, 172
Betony, 171, 172
Betulaceae, 90
Bignoniaceae, 160
Bilberry, 118
Bird Cherry, 46, 63, 160
Bird's-foot Trefoil, 161, 162
Biuret reaction, 136
Blackman, 57, 79
Blau, 168
Blood, 153, 155
Bodnar, 130
Bolton, 131
Boraginaceae, 123
Borneol, 108
Bornyl acetate, 108
Bourquelot, 162, 166, 168
Bran, 47, 56, 57, 70, 99
Brassica, 77, 91, 153, 164

alba, 141, 149, 153, 159

campestris, 141, 151

Napus, 91, 171

nigra, 159, 164

oleracea, 151, 181

rapa var. oleifera, 91

Brassidic acid, 90

Brazil nut, 91, 93, 139, 140, 149

Broad bean, 62, 138, 140, 147, 152, 153, 154,

171
Bromelin, 154
Broom, 177
Brown, 72, 76, 79
Brownian movement, 15
Brucine, 178
Briicke's reagent, 173
Buchner, 23
Buckthorn, 113
Burton, 17
Butter-nut, 140
Butyric acid, 81, 82, 89

Cabbage, 65, 66, 151, 153, 154, 171, 181

Cactaceae, 44

Caemlpinia, 105

Caffeic acid, 103, 104, 123

Caffeine, 180

Calabar Bean, 97

Calanthe, 167

Caldwell, 26

Callistephin, 118

Callistephus chinensis, 118

Calluna erica, 118

Campanulaceae, 60

Campbell, 147, 156

Camphene, 109

Camphor, ^09

' Tree, 109

Cane-sugar (see Sucrose)

Cannabis sativa, 69, 90, 139, 140, 148, 154,

171, 175
Gapparis, 159
Capric acid, 89, 90
Caprifoliaceae, 160, 165
Caproic acid, 1, 81, 82, 89, 90

INDEX

185

Caprylic acid, 89, 90
Carbohydrates, 1, 42

in leaves, 71

Carbon assimilation, 6, 27
Carboxylase, 21, 22, 24, 129
Carica Papaya, 154
Carnaiiba wax, 96
Carnaiibic acid, 89, 97
Carnaiibyl alcohol, 96

Carrot, 40, 67, 98, 153, 154, 170, 171, 181
Carum, 159

Petroselinum, 111

Carvacrol, 108, 109
Caryophyllene, 108, 109
Castanea, 105, 107

vulgaris, 106, 140

Castanin, 140
Castor oil, 91

Plant, 91, 93, 94, 98, 99, 129,

139, 150

seed, 138, 141, 148, 154

Catalase, 21, 22, 25, 129
Catalysts, 18

Catechol, 21, 70, 101, 122, 123, 126, 127
Celastraceae, 160
Celery, 151, 171
Celluloses, 42, 67

reserve, 67

tests for, 68

Centaurea, 159

Gyanus, 114, 116, 117, 118, 120

Centrospermae, 165

Cerasin (see Cherry gum)
Cerasus, 159

Padus, 160

Cerotic acid, 89, 97
Ceroxylon andicolumy 97
Ceryl alcohol, 96
Cetyl alcohol, 96
Chaerophyllum sylvestre, 74
Gheiranthus, 122

Gheiri, 113, 116, 122, 125

Ghelidonium majus, 84
Chenopodiaceae, 116, 171
Ghenopodium, 149

Vulvaria, 170

Cherry, 46, 63, 65, 85, 91, 118

Gum, 45, 46, 55

Laurel, 160, 161

Chervil, 74

Chibnall, 155

Chicory, 60, 62, 151, 171, 181

Chlorophyll, 21, 28

a, 28, 30, 33

b, 28, 30, 33

allomerized, 37

colloidal, 36, 38

crystalline, 33

Chlorophyllase, 21, 34
Chlorophy Hides, 34
Chlorophyllins, 30
Chodat, 122, 123, 130
Choline, 21, 98, 99, 170
Christmas Eose, 125

Chrysanthemin, 118
Chrysanthemum, 116, 118

indicum, 118

Chrysin, 112

Gichorium Intybus, 60, 151, 171, 181

Ginchona, 177, 178

Galisaya, 177

Ledgeriana, 177

officinalis, 177

succirubra, 177

Cinchonine, 178
Cinnamic acid, 103

aldehyde, 108, 109

Cinnamon oil, 109
Ginnamomum Gamphora, 109

zeylanicum, 109

Citral, 109
Citric acid, 87, 88
Citronellal, 108
Citronellol, 108, 109
Gitrus, 87, 159

Aurantium, 176

Limonum, 109

Clark, 78, 80

Clay son, 66, 79

Clove oil, 109

Clover, 61, 62, 67, 76, 77, 151

Dutch, 180

Meadow, 180

Clupanodonic acid, 90
Coca, 176, 177
Cocaine, 177
Cochlearia, 125

Annoracia, 124, 164

Cocoa, 91

plant, 181

Coconut, 56, 90, 93, 141, 149
Gocos, 91

nucifera, 90, 141, 149

Codeine, 179
Co-enzyme, 23

Gofea arabica, 61, 62, 91, 175, 181
Coffee Bean, 61, 62, 91, 175, 181
Gola acuminata, 181
Cole, 10, 45, 136, 150
Collodion dialyser, 137
Colloidal state, 5, 11

precipitation of, 16, 17

Columbine, 114, 116, 161, 162
Colza, 91

oil, 91

Combes, 121, 130

Combretaceae, 165

Compositae, 60, 91, 123, 160, 165

Conglutin, 140, 147

Coniferae, 56, 62, 151, 158, 166

Coniferin, 69, 158, 166

Conifers, 29

Coniferyl alcohol, 104, 158, 166

Coniine, 174

Gonium maculatum, 174

Continuous phase, 12, 15

Convolvulaceae, 160

Copal, 109

12—5

186

INDEX

Copernicia cerifera, 96

Corchorus, 69

Cork, 70

Cornflower, 114, 116, 117, 118, 119, 121

Corydaline, 179

Corydalis, 88

cava, 179

Corylin, 140

Corylus Avellana, 90, 140

var. rubra, 116

Cotton Plant, 54, 56, 68, 91

seed, 141, 149, 171

oil, 91

Cotyledon Umbilicus, 76
Coumaric acid, 103
Coumarin, 103, 165
Courtauld, 26

Cow Parsnip, 33

Pea, 140

Cradein, 154
Cranberry, 102, 118
Crassulaceae, 85, 123
Crataegus, 125

Oxyacantha, 111, 113, 161, 170

Cresol, 128

Cruciferae, 91, 123, 150, 160, 164
Cucumber, 125, 153, 154
Cucumis Melo, 153

sativus, 153

Cucurbita, 149, 150, 151

maxima, 141

Pepo, 91, 180

Cucurbitaceae, 91, 165
Curarine, 178
Currants, 65, 84, 87

Black, 161

Bed, 65, 66, 161

Cuskhygrine, 176
Cuticle, 70

Cutin, 67, 70
Cuto-celluloses, 67, 70
Cyanidin, 117, U8, 120,159
Cyanin, 118, 169
Cynips, 106
Cystine, 135, 137
Cytase, 21, 71
Cytisine, 177
Cytisus Laburnum,, 177
Cytopectic acid, 21, 66
Cytosine, 142, 181
Czapek, 10, 69, 70, 79

Daffodil, 113

Dahlia, 60, 77, 118, 128, 160, 151, 163, 171,

176
Dahlia variabilis, 60, 118, 128, 153
Daish, 57, 72, 73, 74, 77, 78, 79
Dammar, 109

Dandelion, 60, 62, 111, 125
Date-palm, 61, 62
Datura, 170

Stramonium, 176

Daturic acid, 89

Daucus Carota, 67, 98, 153, 170, 171, 181

Davis, 44, 57, 72, 73, 74, 77/78, 79, 8()

Dead Nettle, 125

Deadly Nightshade, 176

Dean, 153, 155, 156

Delphinidin, 117, 118, 120, 121, 159

Delphinin, 118, 159

Delphinium, 116, 159

consolida, 113, 118, 121

Dextrin, 42, 58, 69, 71, 72, 75

tests for, 60

Dhurrin, 158, 163

Dialysis, 11, 14

Diastase, 21, 58, 59, 71, 75

Digallic acid, 107

Digitaligenin, 159

Digitalin, 159

Digitalis, 159

Digitalose, 159

Dihydroxyphenylalanine, 104, 152

Dimethyl-jj-phenylenediamine (test for oxi-
dases), 125

Disaccharides, 42, 53

Dispersed phase, 12, 15

Dobbin, 81, 88

Dobson, 73, 78, 80

Dock, 111

Dog's Mercury, Annual, 170

Perennial, 170

Douglas Fir, 55

Dunstan, 168

Dyer's Green weed (Broom), 112

Weld (Kocket), 111, 112

Edestin, 139, 140, 143, 148

of Barley, 146

Elaeis guinensis, 90, 91
Elaidic acid, 90

Elder, 29, 70, 111, 151, 161

Ellagic acid, 105

Ellis, 100

Emulsin, 21, 157, 160, 162, 166, 167

Emulsions, 12, 13

Emulsoids, 12

Enolic form, 74

Enzymes, 8, 18

classification of, 21

hydrolysis by, 8, 18

synthesis by, 8, 20

Erepsin, 21, 152
Ergothioneine, 172

Erica cinerea, 111
Ericaceae, 102, 159, 166
Erucic acid, 90
Ervum Lens, 188, 140, 147
Erythrina Hypaphorus, 172
Erythrodextrin, 59
Erythroxylaceae, 177
Erythroxylon Coca, 176, 177
Esbach's solution, 138
Essential oils, 2, 82, 101, 108
Ethyl alcohol, 22, 23
Ethylene series, 81, 88, 92
Eucalyptus, 105
Eugenia caryophyllata, 109

INBEX

187

Eugenol, 108, 109
Euler, 26
Euphorbia, 64
Euphorbiaceae, 91, 160
Everest, 115, 121, 130, 131
Excelsin, 140, 149

Fagus sylvatica, 171

var. purpurea, 116

Fats, 1, 89

— — tests for, 91
Fatty acids, 89

synthesis of, 96

Fehling's test, 50
Fenugreek, 171, 175
Ferric hydroxide sol, 13, 17
Fibrin, 154

■ carmine, 155

Fichtenholz, 166, 168

Ficus, 154

Fig, 154

Fischer, 107, 130

Fisetin, 114, 120

Fisher, 156

Flavone pigments, 2, 101, 110, 120

Flavonol pigments, 2, 101, 110, 120

Flax, 64, 69, 91, 97, 139, 141, 162, 163

Flowering Currant, 108

Forget-me-not, 125

Formaldehyde, 27, 28, 37, 38, 39, 81, 82

Formic acid, 1, 81, 82, 89

Fosse, 182

Fraxetin, 159

Fraxin, 159

Fraxinus, 159

excelsior, 91

Omus, 55

Freudenberg, 107, 130
Fructomannans, 61, 71
Fructose (see Laevulose)
Fumaria, 88
Fum^riaceae, 88, 179
Fumaric acid, 88
Fungi, 21, 170, 172
Funkia sinensis, 76
Furfural, 44, 46, 70

phloroglucide, 57, 66

Gaillardia, 116

Galactans, 42, 51, 62, 63, 71

Galactoaraban, 62, 71

Galactomannan, 61, 62, 71

Galactose, 42, 48, 51, 52, 62, 63, 64, 65, 74,

159
Galactoxylan, 62, 71
Galanthus, 57

nivalis, 72, 111

Galeopsis, 35

Tetrahit, 34

Gallic acid, 2, 104, 105
Gall-nuts, 104
Gallotannic acid, 107
Galls, 105

oak, 106

Garden cress, 91

Gaultheria, 159

Gaultherin, 159

Gelatine, 14

Gels, 14, 65

Genista tinctoria, 112

Gentian, 55

Gentiana, 55

Gentianose, 54

Geraniol, 108, 109

Geranyl acetate, 108

Glaucium, 88

Gliadin, 132, 141, 146

Globulins, 132, 138

Glucomannans, 61, 71

Glucose, 42, 48, 49, 58, 64, 72, 75, 78, 167

a and /3, 49

tests for, 50

Glucosides, 50, 74, 101, 105, 167

a and /3, 49, 158

coumarin, 165

cyanophoric, 160

mustard-oil, 164

Glucotropaeolin, 159
Glutamine, 150

Glutaminic acid, 83, 85, 134, 149, 160

Glutaric acid, 1, 83, 86

Glutelins, 132, 141, 145

Gluten, 146, 150

Glutenin, 146

Glyceria aquatica, 161

Glycerol, 21, 89, 92, 93, 99

Glycerophosphatase, 21, 99

Glycerophosphoric acid, 21, 98, 99

Glycine, 1, 81, 134, 171

Glycine hispida, 140, 181

Glycinin, 140

Glycogen, 22

Glycogenase, 22, 25

Gly collie acid, 82

Glyoxylic reaction, 136, 151

Gold sol, 13, 17

Goodeniaceae, 60

Gooseberry, 65, 67, 84, 87, 161

Goosefoot, 149

stinking, 170

Gosney, 99

Gossypium, 54, 56, 91, 159

herbaceum, 68, 91, 141, 149, 171

Gout weed, 111

Graham, 14

Gramineae, 90, 123, 145, 149, 160, 165

Grape, 82, 84, 86, 87, 96, 118

sugar (see Glucose)

Great Millet, 163
Greater Celandine, 84
Greengage, 127
Greshoff, 160, 168
Guaiaconic acid, 124
Guaiacum gum, 124
Guaiacum officinale, 124

sanctum, 124

Guanine, 142, 180
Guggenheim, 152, 156

188

INDEX

Guignard, 164, 168

Gulose, 48

Gum Arabic, 13, 45, 55, 63

Tragacanth, 63

Gums, 42, 51, 62
Gun-cotton, 68
Guttiferae, 165

Haas, 10

Harden, 23, 24, 25, 26

Harris, 147, 156

Hatschek, 17

Hawthorn, 111, 113, 125, 161, 170

Haynes, 65, 80

Hazel, 90

Nut, 140

Hazel, Eed-leaved, 116
Hedge Woundwort, 33
Helianthm, 62, 77, 150

annuus, 57, 76,91, 141, 149, 171

tuberosus, 60, 61, 76, 151, 171

Helleborus niger, 125

Hemerocallis fiilva, 76
Hemi-cellulose, 21, 62, 71
Hemlock, 174
Hemp, 69, 90, 139, 140, 171, 175

seed, 148, 155

Hemp-nettle, 34
Henbane, 170, 177
Henry, 26, 168, 182
Heracleum, 34, 35

Sphondylium, 33

Hesperidin, 159
Hesperitin, 159
Hexosephosphatase, 21, 22
Hexosephosphate, 21, 22
Hexoses, 42, 47

Hill, 10

Histidine, 3, 135, 161

trimethyl, 172

Hollyhock, 64, 118

Hop, 76

Hopkins, 136

Hordein, 141, 146

Hordenine, 170

Hordeum vulgare, 60, 76, 138, 141, 146, 180

Horse Chestnut, 98, 106, 108, 127, 149, 165

Horse-radish, 124, 125, 126, 164

Horsfall, 112, 130

Horton, 160, 168

Hummel, 113, 130

Humulus Lupulus, 76

Hyacinth, 153, 154

Hyacinthm, 57, 60

orientalis, 153

Hyaenic acid, 89
Hydrastine, 179
Hydrastis canadensis, 179
Hydrocaffeic acid, 123
Hydrocharis Morsus-ranae^ 76
Hygrine, 176

Hymenophyllum demissum, 76
Hyoscyamine, 177
Hyoscyamus muticus, 170, 177

niger, VJl

Hypaphorine, 172
Hypoxanthine, 180

Idaein, 118

Idose, 48

Ilex paraguensis, 181

Iminazole, 3, 174, 179

Indican, 159, 167

Indigo, 167

plants, 167

Indigofera, 159

Anil, 167

erecta, 167

sumatrana, 167

tinctoria, 167

Indole, 129

Indoxyl, 159, 167
Inositol, 21, 101, 102
Inulase, 21, 60
Inulin, 21, 42, 52, 60

tests for, 61

Invertase, 21, 22, 25, 52, 78
Invert sugar, 52
Iodoform, 23

Iris, 57, 60
Irvine, 73, 78, 80
Isatis tinctoria, 167
Isobutyric acid, 82
Isocetic acid, 89
Isochlorophyllins, 31
Isoleucine, 134, 150
Isolinolenic acid, 90
Iso-oleic acid, 90
Isoquercitrin, 159
Isoquinoline, 174
Isorhamnose, 42
laothiocyanate, acrinyl, 159

allyl, 21, 159, 164

benzyl, 159

j9-hydroxybenzyl, 164

Isovaleric acid, 82

Jasininum, 55, 158 *^

Jerusalem Artichoke, 60

Jones, 182

Jorgensen, 28, 37, 41

Juglandaceae, 90

Juglans cinerea, 140

nigra, 140

regia, 90, 102, 106, 140

Juglansin, 140

Jute, 69

Kaempferol, 113, 120, 121, 159

Kastle, 78, 80

Keracyaniu, 118

Kidd, 28, 37, 41

Kidney Bean, 62, 138, 140, 147, 149, 175

Kishida, 110, 131

Kola nut, 181

Labiatae, 109, 123
Laburnum, 177
Laccases, 122, 128
Lacquer, 124

INDEX

189

Lacquer Tree, 124, 128

•y-Lactone, 49

Laevulose, 42, 62, 60, 72, 74

tests for, 53

Lamium album, 125
Larch, 55, 106
Larix, 55, 108

europaea, 106

Larkspur, 113, 116, 118
Latex, 12, 124
Lathyrus odoratus, 76

pratensis, 76

Laudanosine, 179
Lauraceae, 109
Laurie acid, 89, 90
Laurocerasin (see Prulaurasin)
Lavandula vera, 109
Lavender oil, 109

Leathes, 99

Lecithin, 2, 21, 98, 99

Lecythidaceae, 91, 98, 165

Legumelin, 138, 147

Legumin, 139, 140, 147

Leguminosae, 62, 76, 123, 139, 147, 'j 149,

160, 165, 172, 177, 181
Lemon, 87, 88

oil, 109

Lentil, 138, 140, 147
Lepidiuvi, 159

sativum, 91

Leucine, 134, 149
Leucosin, 25, 138, 145
Lewkowitsch, 99
Lignin, 67, 69, 70
Ligno-celluloses, 67, 69
Lignoceric acid, 89, 97
Lignon (see Lignin)
Ligustrum, 158
Lilac, 67, 111
Liliaceae, 123, 165
Lilium bulbifenim, 62

candidum, 62, 111

Martagon, 62

Lily, 62

White, 111

Lima bean, 140
Lime, 87

Limonene, 108, 109
Linaceae, 91, 160
Linalol, 108, 109
Linalyl acetate, 108, ^09
Linamarin, 158, 163
Ling, 111, 113
Linolenic acid, 90
Linolic acid, 90
Linseed, 64, 91, 139, 149
Linum, 64, 158, 163

perenne, 162

usitatissimum, 69, 91, 97, 139, 141,

149

Lipase, 21, 94, 99
Lobeliaceae, 60
Loganiaceae, 165
Lotase, 163
Lotoflavin, 159, 163

Lotus, 159

arabicus, 163

comiculatus, 76, 161, 162

uliginosun, 161

Lotusin, 159, 163
Lubrzynska, 100
Lucerne, 67, 82

Lupin, 62, 98, 140, 147, 149, 150, 153, 154,

171, 177, 180
Lupinine, 177
Lupinus, 62, 76, 98, 140, 149, 150, 151

alhus, 151

hirsutus, 153

luteus, 147, 151, 177, 180

niger, 177

Luteolin, 112, 120
Lycium barbarum, 171
Lycopersicum esculentum, 82, 86, 153
Lysine, 135, 161

Lyxose, 45

Mackenzie, 79

Maclean, 99

Magnoliaceae, 165

Maize, 90, 140, 141, 146, 153, 154

cobs, 56

Malic acid, 1, 86, 8G

Mallison, 131

Mallow, 118

Malonic acid, 83, 84

Malt, 77

Maltase, 20, 21, 22, 23, 24, 43, 77

Maltose, 20, 53, 58, 59, 60, 71, 72, 75, 77

tests for, 53

Malva, 159

sylvestns, 118

Malvaceae, 91
Malvidin, 118, 159
Malvin, 118, 159

Mandelonitrile glucoside (see Prunasin)

Mandragora', 177

Mandrake, 177

Mangold, 71, 72, 73, 74, 77

Mangrove, 105

Manna, 55

Ash, 55

Mannans, 42, 52, 61, 71
Mannitol, 55
Mannocelluloses, 61

Mannose, 42, 48, 52, 61, 64, 74, 159

Martin, 131

Matthiola, 111, 125

Maxwell, 62, 80

Maysin, 140

Meadow Eue, 161

Sage, 171

Sweet, 103, 167

Medicago sativa, 67, 82
Mekocyanin, 118
Melanin, 21, 128
Melicitose, 55
Melissic acid, 89, 97
Melissyl alcohol, 96
Melon, 125, 153, 154
Menispermaceae, 179

190

INDEX

Menthol, 108, 109
Menthyl acetate, 108, 109
Mercerized cotton, 68
Mercurialis annua, 170

perennis, 170

Mesembryanthemum, 85, 96
Metaproteins, 132, 143

reactions of, 143

Methyl pentoses, 42

salicylate, 108, 159

Methylamine, 170

Methylene blue, 25

Mieg, 131

Mignonette, 112

Milk, 12

Miller, 100

Millon's reaction, 136

Mirande, 161, 168

Mistletoe, 64, 102

Molisch's reaction, 137

Monkshood, 88

Monocotyledons, 57, 60, 71, 123

Monosaccharides, 42, 43

Moore's test, 50

Moraceae, 90

Morphine, 179

Morris, 72, 76, 79

Mountain Ash, 86, 88, 160, 170

Mucic acid, 51, 52, 64, 65

Mucilages, 42, 51, 52, 62, 64, 67

Musa Cera, 97

sapientum, 82, 128, 158

Muscari, 60

Mustard, Black, 91, 164, 165

White, 91, 141, 164

Seed, 149, 153

Myosotis, 125
Myrica, 159

Myricetin, 114, 120, 159
Myricitrin, 159

Myricyl alcohol (see Melissyl alcohol)

Myristic acid, 89, 90, 97

Myrosin, 21, 157, 164

Myrtaceae, 160, 165

Myrtillidin, 118, 119

Myrtillin, 118

Nagai, 110, 131

a-Naphthol tests, 44, 46, 124

Narceine, 179

Narcissus, 111, 113, 121, 122

incomparabilis, 113

poeticus. 111

Pseudo-Narcissus, 113

Tazetta, 113

Narcotine, 179
Narigenin, 159
Naringin, 159

Nasturtium, 57, 62, 77, 116, 153

Garden, 72, 74, 150

Nepenthes, 154

Nettle, 29, 81, 82
Neville, 64, 80
Newbury, 130
Nicotiana, 170

Nicotiana Tabacum, 171, 174
Nicotine, 174, 175
Nolan, 131
Nonadecylic acid, 89
Norris, K. V., 25, 26

F. W., 66, 79

Nucleic acid, 3, 44, 45,fl41, 181
Nucleoproteins, 3, 132,, 141, 180
Nucleotides, 142

Nux Vomica, 178

Oak, 105, 106, 107

wood, 69, 107

Oat, 139, 140, 171, 175
(Enanthylic acid, 89
Oenidin, 118, 119, 159
Oenin, 118, 159
Oil Palm, 90
Olea europaea, 91
Oleaceae, 91, 160, 165
Oleic acid, 90
Olive, 91

oil, 91

Onion, 62, 65, 66, 113
Onslow, 122, 130
Opium, 179

Poppy, 90, 179

Orache, 116
Orange, 65, 66, 87

Tree, 176

Orchid, 104, 124, 167
Orchidaceae, 62
Orchis Morio, 64
Orcinol, 44, 70

test for pentoses, 46

Ordonneau, 87, 88
Oriental Poppy, 128
Ormerod, 99

Oryza saliva, 141

Osazones, 50, 51

Osborne, 145, 146, 147, 148, 149, 155, 156

Osmic acid, 91

Ostwald, 14

Oxalic acid, 1, 83

Oxalis, 83

Oxidases, 122, 123, 125, 126, 127, 166, 167

Oxidizing enzymes, 9, 122

Oxybenzoic acid, 159

Oxygenase, 21, 122

Oxyproline, 172

Paeonia, 62, 96, 159

officinalis, 108, 116, 118

Paeony, 62, 108, 116, 118
Page, 131

Palladin, 10, 129, 130
Palm, 61, 62

oil, 91

wax, 97

Palmaceae, 90
Palmitic acid, 89, 90
Pansy, 113, 118
Papain, 154
Papaver orientate, 128

Rhoeas, 118

INDEX

191

Papaver somniferum, 90, 179

Papaveraceae, 88, 90, 172, 179

Papaverine, 179

Papaw Tree, 154

Parkin, 62, 72, 80

Parsley, 111

Passiiioraceae, 160

Paullinia Cupana, 181

Pea, 59, 62, 65, 75, 76, 77, 98, 125, 136, 138,

139, 140, 147, 149, 153, 154, 171, 175
Peach, 91, 140, 160
Pea-nut, 141
Pear, 85, 125, 127, 166
Pectase, 21, 67
Pectic substances, 42, 66, 70
Pectin, 21, 66, 67
Pectinogen, 65, 66, 67
Pectocelluloses, 67, 70
Pelargonic acid, 89
Pelargonidin, 117, 118, 120, 159
Pelargonin, 118, 119, 159
Pelargonium, 159

zonule, 108, 118, 119, 153

Pelletierine, 177

Pentosans, 44, 55, 56, 57, 63, 69, 71, 72

Pentoses, 42, 44, 56, 57, 65, 66, 72

Peonidin, 118, 159

Peonin, 118, 159

Pepper, 175

Peppermint oil, 109

Pepsin, 21, 144, 152

Peptones, 132, 133, 143, 144, 152

Periderm, 70

Perkin, 111, 112, 113, 130

Peroxidase, 21, 22, 24, 122, 123, 124, 125, 126

inhibitor, 24

Peroxides, 123, 126
Petunia violacea, 118
Petunidin, 118
Petunin, 118
Phaeophorbides, 35
Phaeophytin, 32, 39
Phajiis, 124, 167
Phaselin, 138, 147
Phaseolin, 140, 147
Phaseolunatin, 163
Phaseolus, 62, 149, 158

limatus, 140, 163

multijiorus, 76, 153, 154

radiatiis, 140

vulgaris, 138, 140, 147, 151, 175

Phellandrene, 108, 109

Phellouic acid, 70

Phenolase, 122

Phenols, 2, 70, 101, 108

Phenylalanine, 103, 135, 136, 161

^-Phenylenediamine(test for peroxidase), 124

Philip, 17

Phipps, 113, 130

Phloionic acid, 70

Phloretin, 159

Phloridzin, 159

Phloroglucinol, 2, 7, 44, 70, 102

(test for pentoses), 46

Phlox, 111

Phoenix, 62

Phosphotungstic acid, 138, 173
Phyllins, 31
Phytase, 21, 102
Phytelephas macrocarpa, 62
Phytin, 3, 21, 101, 102
Phytochlorins, 32
Phytol, 21, 31, 34, 39
Phytolaccaceae, 116
Phytolacca, 116
Phytorhodins, 32
Picea exceUa, 150, 151
Picramic acid, 161
Pine-apple, 86, 154
Pinene, 108
Pink, 111
Pinus, 108

sylvestris, 151

Piper nigrum, 175
Piperaceae, 165
Piperidine, 176
Piperine, 175
Pisang wax, 97
Pisum, 62, 136, 149, 151

sativum, 59, 75, 76, 77, 98, 125, 138,

140, 147, 153, 154, 171, 175
Pitcher-plant, 154
Pittosporaceae, 165
Plantago, 159

lanceolata. 111

Plastid pigments, 40, 116
Plimmer, 10, 99, 100, 129, 131
Plum, 63, 91, 96, 125, 140, 160
Polarization, 49
Polemoniaceae, 165
Polygalaceae, 165
Polygonum, 159

tinctorium, 167

Polypeptides, 132

Polysaccharides, 42, 56

Pomegranate Tree, 177

Poplar, 112, 167

Poppy, 118

Populin, 158

Populus, 76, 112, 158, 167

Porphyrins, 33

Portulaca, 116

Portulacaceae, 116

Potassium hydrogen sulphate, 21, 159

Potato, 72, 77, 125, 127, 128, 129, 140, 149,

150, 151, 171, 175, 181
Priestley, 37, 41, 70, 80
Primulaceae, 165
Prolamins, 132, 141, 145
Proline, 3, 135, 151, 172
Propionic acid, 81, 82, 89
Proteaceae, 165
Proteases, 22, 25, 152
Proteins, 2, 13, 17, 132

crystalline, 139, 141, 148

of cereals, 145

of fat-containing seeds, 148

of Leguminosae, 147

tests for, 136

Proteoses, 132, 143

192

INDEX

Protocatechuic acid, 2, 103, 105, 123

Protoplasm, 5, 9

Prulaurasin, 159, 161

Prunase, 160

Prunasin, 159, 160

Prunus, 113, 158, 159, 160

Amygdalus, 63, 91, 140, 160

Arvieniaca, 140

Cerasus, 46, 63, 91, 118

domestica, 63, 91, 140, 160

Laurocerasus, 160, 161

Padus, 46, 63

Persica, 91, 140, 160

Prussic acid, 21, 26, 158, 159, 160
Pseudotsiiga, 55

Pumpkin, 91, 180
Punica Granatum, 177
Punicaceae, 177
Purine, 3, 174, 179

bases, 141, 142, 169, 179

Purpurogallin, 126
Putrescine, 170

Pyridine, 174
Pyrimidine, 3, 174, 179
Pyrogallol, 70, 124, 126, 128
Pyrola, 166

Pyrrole, 3, 33, 174, 176
Pyrrolidine, 33, 170, 176
Pyrus, 158
Aucnparia, 86, 88, 160, 170

communis, 166

Malm, 82, 160

Pyruvic acid, 21, 22, 24, 96

Quercetin, 112, 113, 120, 121, 122, 159

Quercitrin, 159

Quercus, 105, 106, 113, 159

Rohur, 107

Quinine, 173, 177

Quinol, 101, 102, 128, 159, 166
Quinoline, 174, 177

Eacemic acid, 86

Kadish, 125, 141

Eaffinose, 42, 54

Eanunculaceae, 88, 123, 160, 165, 172, 179

Rartunculus aquatilis, 149

Eape, 91, 141

Raphanus sativum, 141

Raphia Rvffia, 97

Eaphia wax, 97

Easpberry, 67

Eed Seaweeds, 51

Eeductase, 21, 22, 25, 129

Eeed Poa, 161

Reseda luteola, 111, 112

Eeserve celluloses, 67

materials, 10, 57, 60, 73, 90, 132

Eesins, 101, 109

Eesorcinol, 70, 101
Eespiration, 6, 73, 129
Eeversible reactions, 19
Eeynolds Green, 94, 100
Ehamnaceae, 160, 165
Ehamninose, 54

Ehamnose, 42, 47, 54, 113, 159
Rhamnus, 113

Frangula, 161

infectoria, 54

Rheum Rhaponticum, 83, 84
Rhizophora, 105
Ehodophyceae, 14, 51
Ehubarb, 65, 66, 83, 84
Rhus, 105, 114

Conaria, 107

Cotinus, 107

vernicifera, 124, 128

Ribes Grossularia, 161

nigrum, 161

rubi'um, 161

sanguineum, 108

Eibose, 45, 142
Eibwort Plantain, 111
Eice, 141

Eicin, 138, 148
Eicinoleic acid, 90
Ricinus, 91, 93, 95, 150

communis, 91, 94, 98, 99, 129, 138,

139, 141, 148, 154

Eobertson, 73, 78, 80
Robinia, 159
Eobinin, 159
Rosa, 123, 159

centifolia, 109

gallica, 118, 120

Eosaceae, 63, 91, 109, 123, 159, 160, 165
Eose, 108, 116

oil, 109

Eubiaceae, 62, 91, 124, 160, 172, 177
Rumex Acetosa, 83

obtusifolius, 111

RuscuA, 61

Ruta, 159

Eutaceae, 109, 160, 165, 179

Eutin, 159

Eye, 97, 138, 141, 146

Saccharomyces, 21
Saccharum officinarum, 82
St Ignatius' Bean, 178
Salicin, 50, 103, 158, 163, 167
Salicylic acid, 103

alcohol, 50, 103, 163, 167

aldehyde, 103

Saligenin, 103, 158, 163, 167
Salix, 103, 158, 167
Salkowski, 55, 80

Salvia pratensis, 171
Sambucus, 151, 159

nigra, 70, 111, 161

Sambunigrin, 159, 161

Saponaria, 165

Saponification, 92

Saponins, 12, 166

Sawdust, 47, 56

Sawyer, 44, 57, 72, 73, 74, 78, 79, 80

Saxifragaceae, 160, 165

Scarlet Geranium, 108, 118, 119, 153

Eunner, 153, 154

Schenckia blumenaviana, 124

INDEX

193

Schryver, 39, 40, 41, 65, 66, 79, 80, 82, 155

Schulze, 62, 80

Schweizer's reagent, 68

Scilla, 60, 64, 71

Scopolia japonica, 176

Scorzonera, 158

hispanica, 171, 175

Scotch Fir, 151

Secale cereale, 97, 138, 141, 146

Seliwanoff's test, 44, 53

Serine, 184

Shibata, 110, 131

Silicic acid, 14

Silver Fir, 151

sol, 13, 17

Sinalbin, 159, 164

Sinapin acid sulphate, 159, 164
Sinapu alba, 91, 164

nigra, 91

Sinigrin, 21, 157, 159, 164
Sitosterol, 97

Smedley, 100

Snapdragon, 111, 112, 116
Snowdrop, 57, 72, 111
Soap, 12, 13, 14, 17, 92, 93, 94

tests for, 94

wort, 165

Sodium picrate test, 161
Soja Bean (see Soy-bean)
Solanaceae, 123, 172, 176
Solanum, 77

tuberosum, 72, 128, 140, 181

Sorghum, 158

vulgare, 163

Sorrel, 83

Soy-bean, 140, 171, 181

Spanish (sweet) chestnut, 105, 106, 107, 140

Sparteine, 177

Spartium scoparium, 177

Spatzier, 164, 168

Spinach, 153, 181

Spinacia^ 153

oleracea, 181

Spiraea, 159

Ulmaria, 103, 167

Spoehr, 44, 80
Spruce Fir, 150
Squash, 141
Stachydrine, 172, 176
Stachyose, 42, 56
Stachys sylvatica, 33

tuberifera, 55, 175, 176

Staehelin, 83, 88

Starch, 13, 14, 17, 67, 71

soluble, 58, 76

tests for, 58

Stearic acid, 89
Steiger, 62, 80
Sterculiaceae, 91
Stereoisomerism, 45, 48, 158
Sterols, 97

Stiles, 28, 41
Stizolobium, 152
Stock, 111, 125
Stoll, 28, 41, 126, 131

Straw, 47, 56, 70
Strawberry, 65, 66, 67
Strophanthidin, 159
Strophanthin, 159
Strophanthus, 159, 171

kispiduSf 175

Strychnine, 178
Strychnos Igmitii, 178

Nux-vomica, 178

toxifera, 178

Suberiu, 70
Suberinic acid, 70
Suberogenic acids, 70
Substrate, 19

Succinic acid, 1, 83, 84, 86
Sucrose, 42, 64, 72, 78

tests for, 54

Sugar-cane, 82

Sulphur reaction (for proteins), 137

Sumac, 105, 107, 114

Sunflower, 57, 77, 91, 141, 149, 150, 171

Suspensions, 12, 13

Suspensoids, 12

Sweet Flag, 170

Sycamore, 180

Sympetalae, 123

Synthesis by condensation, 4

of aromatics, 7

of carbohydrates, 6, 27

of fats, 7, 96

of proteins, 6, 133

Syringa, 158

vulgaris, 67, 76, 111

Syringeuin, 158

Syringin, 158

Talose, 48

Tannic acid, 107, 137, 173

Tannin, 2, 76, 101, 103, 104, 106, 125

reactions of, 106

Taraxacum, 62

otJUcinale, 60, 111, 125

Tartaric acid, 86, 87
Tautomerism, 50

Taylor, 17

Tea, 104, 107, 181

plant, 180, 181

Terpeaes, 2, 108, 109
Tetrasaccharides, 42, 54
Thalictrum aquilegifolium, 161
lliea, 159

sinensis, 180, 181

Thebaine, 179
Theine (see Caffeine)
Theobroma Cacao, 91, 181
Theobromine, 181
Thomas, 10
Thorn Apple, 170, 176
Thyme oil, 109
Thymelaeaceae, 165
Thymol, 108, 109
Thymus vulgaris, 109
Tiglic acid, 90
Tihaceae, 160
Tobacco, 170, 171, 174, 175

194

INDEX

Tomato, 82, 86, 87, 153
Tragacanth, 63
Tridecylic acid, 89
Trier, 182
Trifoliim, 61, 151

ochroleucum, 76

pratense, 67, 76, 77, 180

repens, 180

Trigonella Foenum-graecum, 171, 175

Trigonelline, 172, 175

Trimethylamine, 170

Trisaccharides, 42, 54

Tristearin, 92

Triticum vulgare, 97, 98, 128, 138, 141, 145,

154
Trommer's test, 50
Tropacocaine, 177
Tropaeolum, 62, 77, 150, 151, 159

majus, 57, 72, 74, 76, 116, 153

Tropane, 174, 176

Trypsin, 154

Tryptophane, 25, 135, 136, 151, 153, 154, 167

trimethyl, 172

Tuberin, 140

Tulip, 153, 154

Tulipa, 158

Turnip, 65, 66, 67, 77, 125, 151, 153

Turpentine, 108

Tutin, 80

Tyrosinase, 21, 128

Tyrosine, 2, 21, 103, 128, 135, 136, 151

Ultramicroscope, 15
Umbelliferae, 62, 123
Undecylic acid, 89
Uracil, 142, 181
Urea, 3, 21, 181, 182
Urease, 21, 181, 182
Urtica, 29

dioica, 81

Urticaceae, 160
Usher, 37, 41

Vacciniurriy 166

Myrtillus, 118

VitiS'Idaea, 102, 118

Valerian, 82

Valeriana, 82
Valeric acid, 1, 81, 89
Valine, 1, 81, 134, 149
Van Rijn, 168
Vanilla planifolia, 104
Vanillin, 69, 104
Vegetable acids, 1, 81

ivory, 62

Marrow, 149

Velvet Bean, 152
Vernon, 26

Vetch, 138, 140, 147, 149, 163

Common, 161, 180

Hairy, 161

Vicia, 149, 150, 151, 159

angustifolia, 163

Faha, 138, 140, 147, 152, 153, 154, 171

Vicia hirsuta, 76, 161

sativa, 76, 138, 140, 147, 150, 151,

161, 180
Vicianin, 159, 163
Vicianose, 159, 163
Vicilin, 140, 147
Vigna sinensis, 140
Vignin, 140
Vine, 72

Vines, 153, 155, 156
Viola, 125

odorata, 57, 111, 116

tricolor, 113, 118, 121

Violaceae, 60

Violanin, 118
Violet, 57, 111, 116, 125
Virginian Creeper, 118
Viscum album, 64, 102
Vitis, 159

vinifera, 72, 86, 118

Voorhees, 145, 156

Waage, 102, 131

Wallflower, 113, 116, 122, 125

Walnut, 56, 90, 102, 106, 140

American, 140

Water Ranunculus, 149

Wax Palm, 97

Waxes, 96

Wehmer, 10

Weil, 131

Wester 10 109

Wheat,' 58,' 97, 98, 128, 129, 138 141, 145,

154, 171, 182
Wheldale, 110, 111, 112, 114, 120, 130, 131
White Jasmine, 55
Wig Tree, 107
Wilkinson, 113, 130
Willow, 50, 103, 167

Willstatter, 28, 41, 114, 117, 121, 126, 131
Wine, 104

Winterstein, 168, 182
Woad, 167
Wohlgemuth, 26
Wood Gum, 56

Xanthine, 180

Xanthone, 101

Xanthophyll, 28, 29, 30, 40

Xanthoproteic reaction, 136

Xylan, 47, 55, 66, 57, 62, 69

Xy Ionic acid, 47, 57

Xylose, 42, 45, 47, 56, 57, 62, 63, 64, 65, 74

Yeast, 20, 141

Zea Mays, 90, 140, 141, 146, 153
Zechmeister, 117, 131
Zein, 141, 146, 147
Zilva, 24, 25, 26
Zollinger, 131
Zymase, 21, 22, 23, 129
Zymin, 23, 24
Zymogen, 94

CAMBRIDGE : PRINTED BY

J. B. PEACE, M.A.,
AT THE UNIVERSITY PRESS

0

BINOIHG DEPT. JUN 11958

QK
865
057
1923

Onslow, Muriel (Wheldale)

Practical plant
biochemistry 2d ed.

UNIVERSITY OF TORONTO LIBRARY