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THE CHEMICAL EXAMINATION OF
THALOSPERMA GRACILE

BY

CLARENCE BARBRE

A.B. Eureka College, 1913
B.S. University of Illinois, 1914

THESIS

Submitted in Partial Fulfillment of the Requirements for the

Degree of

MASTER OF SCIENCE

IN CHEMISTRY

THE GRADUATE SCHOOL

OF THE
UNIVERSITY OF ILLINOIS

1915

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UNIVERSITY OF ILLINOIS

THE GRADUATE SCHOOL |

1 HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY
Clarence Barbre

ENTITLED... THE CHEMICAL EXAMINATION OF THE CONSTITUENTS OF

THELESPERMA GRACILE

BE AC@EPTED MAS: FULFILLING THIS PART OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE IN CHEMISTRY

Be oat

In Charge of Major Work

WAN 20

Head of Department

Recommendation concurred in:

Committee

on

Final Examination

Misoy eta
ADAEEU

TABLE OF CONTENTS

Pages
INTRODUCTION *@ee seo see eoe ee? eoeeee@ 1

- EXPERIMENTAL @ee@*@steo eee eseoe ee 22882208 5

SUMMARY AND CONCLUSIONS ...... at

BIBLIOGRAPHY

ACKNOWLEDGMENT

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Digitized by the Internet Archive
in 2013

http://archive.org/details/chemicalexaminatOObarb

THE CHEMICAL EXAMINATION OF THE CONSTITUENTS OF
THELESPERMA GRACILE,.

INTRODUCTION

From the earliest recorded writings of man we have the

| report of materials extracted from plants which are valuable for

| their curative power or ability to set up some nervous sensation.

| Many of the active agents in these preparations from plants have

| been isolated and identified as chemical compounds or definite mix-
| tures of compounds with the result that some of them have been pro-
| duced by synthesis. The laborious and costly collection and the

| still more difficult extraction processes have been eliminated,

The recordsof extraction, identification and synthesis have not
occupied a very large part of Chemical Literature for the reasons
that the products were so valuable that the methods were not writte
and. also,because of the difficulty of the work there has not been
a large amount of reliable information to report. Methods for the

| preparation and estimation of single plant constituents, however,

| are voluminous compared with the methods for the accurate proximate

|analysis of plants.

The first article on the analysis of plants was published |

at the University of Prague by Rocheleder, It was later translated

iby Bastick and published in The British Journal of Pharmacy in 1860,'

| It was revised by Maisch, the first American plant analyst,and pub-
lished in the American Journal of Pharmacy in 1862. “&n Introduc-

tion to the Chemical Analysis of Plants" by Wittstein was translat-

o> ly

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— aoe i delat

: of wd, & Chee +I

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1.4

zg.
ed into English by Mueller and published at Melbourne in 1878,
| During the time that Henry D. Parsons was a student under
| Prescott at the University of Michigan;he worked out and published
"} Method for the Proximate Analysis of Plants” in the American
Chemical Journal.

In 1884 Dragendorff published "S Qualitative and Quanti-
tative Analysis of Plants" which was translated in 1886 by Green-
ish. The Wellcome Research Laboratory was established in London
| by H. D. Wellcome for the study of Tropical Medicine, Physiology,
and Pharmacy. From this laboratory has come the best of all the
| methods proposed and about the only one that approached the accur-
| ate proximate santyeis of plants. Until this time the best analyse
gave only the percentages of ash, volatile mtter, and the relative
amounts of material extracted by various solvents. The Parson's
scheme sought to separate the constituents into their respective
classes such as. alkaloids, glucosides, volatile oils, sugars, et-
cetera. By means of the Power's method some advance has been made,
The pure compounds are identified, isolated and derivatives are
|made to prove the composition, The plan in brief is as follows:-
| twenty-five kilos of the air-dry material are taken for analysis.
It is ground in a mill until it will pass a sixty mesh sieve and a
| preliminary study made. & small portion (10 grams) is subjected to
a test for alkaloids. The material is extracted with Prollius Mix-
|ture (Ethyl ether 325 parts by volume, ethyl alcohol 25 parts,
Smmonium Hydroxide (sp.gr. 0.9) 10 parts.) ‘The ethereal liquid is
Shaken with some dilute Hydrochloric acid, after which the aqueous

solution is made alkaline with Ammonium Hydroxide, and the liberat-

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ed alkaloid is extracted with ether. The ethereal liquid is evap-
orated leaving the alkaloid as a resinous or crystalline mass,
which may be further purified by reformation and extraction of the
Hydrochloride and subsequent treatment with Smmonia and ether.
The material is next examined for the presence of ensymes!
| bout two hundred grams of the ground plant is macerated with
| water at ordinary temperature for several days, the liquid is ex-
“i pressed and filtered and twice the volume of alcohol is added. The|
| precipitate produced in this way is tested with starch and other
| solutions for enzymatic change,
The moisture and ash are determined and the separation
| of the constituents into classes of organic compounds is begun,
While the method of analysis does not attempt to isolate the identi-
cal compounds as they occur in the plant in every case, it does |
try to get them out with as gentle treatment and as little change
| as possible.

First, the ground material is thoroughly extracted with
ninety-five per cent alcohol. This extract constitutes all of the
material to be analyzed. The solvent is removed and data on the

| weight of the extract and its per cent of the whole weight is taken.
| The essential oil is now removed by mixing with water and distillin
|with steam, The material is now divided into two parts, water solu-
| ble and insoluble, by extraction with water. From this point, each |
| part is extracted with various immiscible solvents in the order of

| their least solvent action. This is done so as to get as wide and

sharp a separation as possible. The order of the solvents is de-

termined on a twenty-five gram sample by extracting it successively

oy 4 f 5 “Or St
Tt o ry
7 5

in a Soxhlet apparatus with various solvents and the weight of

each extract taken, After the constituents are separated into
classes, the members of each class are separated by the usual meth- |
ods depending on their physical and chemical properties employed

| in the analysis of mixtures of organic compounds,

History of the Sample.

The material used in this investigation was collected in

Mexico through the courtesy of Mr. Geo. Findlay and sent to Dr. G.

D. Beal for analysis. With the plant came the statement that a
| water infusion of the plant was highly prized by the natives of faa
| locality of collection for its use as an antidote in cases of snake
| bite.

Professor William Trelease, of the Botany Department of
the University of Illinois, identified the plant as Thelesperma
| Gracile, Gray. No reference in the alkaloid literature could be
found. The only information obtained was that it grows in the state
| of Kansas and in the south west. Thinking that the plant might con-
tain an alkaloid,a preliminary study was made in 1913 by Mr. D. T.
|Englis. No alkaloid was found at this time nor subsequently.

The Object of the Investigation.

The purpose of the investigation was to test out the

lB, B. Power plan for the proximate analysis of plants and to deter-

r
\

imine,if possible, the chemical constituents of the plant Thelesperma

Gracile,.

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EXPERIMENTAL

The material used in this investigation consisted of the
| whole air dry plant, except the roots, ground until it would pass

| a sixty mesh sieve. & small portion of the sample (10 grams) was

subjected to the preliminary test for an alkaloid, when no indica-

| tions were obtained of the presence of such a substance.

For the purpose of complete examination 2.24 kilos of the
ground plant were completely extracted with hot alcohol when after
| the removal of the greater part of the solvent 120 grams (about

15.3%) of a viscid, dark green extract were obtained.

Distillation of the Extract with Steam and the Separation of the

| Essential Oil,

The entire amount of the above mentioned extract was mixe
|with water and distilled with steam for five hours, On extracting

| the distillate with ether and removing the solvent, a quantity of a
|pale yellow essential oil was obtained. There was about one cubic

centimeter and since it was not a normal product its constants were

not determined,

| Non-volatile Constituents of the Extract.

| &fter the removal of the essential oil as above described,
there remained in the distillation flask a dark aqueous liquid (A),
sik a dark green oily resin (B), The resin was separated and, in
order to wash it thoroughly, it was mixed with boiling water when

an emulsion formed. The latter was shaken with petroleum ether

6.
(B.P.40-50), after which the aqueous liquid which contained suspend-
ed matter was filtered, the filtrate being concentrated and added |
to the main portion of the aqueous liquid. The petroleum liquid
was then evaporated and the residue mixed with the solid which had
been collected on the filter, the total amount of the resin being

thus brought together.

Examination of the Squeous Liquid (s).
The whole of the aqueous liquid was first thoroughly ex-

tracted with ether (extract £1), then with petroleum ether (42),
| then with chloroform (A3), and finally with iso-amyl alcohol (4&4).
| These solvents removed some brown viscid material which gave a
| Slight coloration with ferric chloride, Their respective solutions |
(f1-2-3-4) were each shaken successively with ammonium carbonate,
sodium carbonate, potassium hydroxide, hydrochloric acid and chloro
form; after which the alkaline solutions were acidified whereupon
a dark green resinous material was thrown down which was filtered
|| out and reserved. The acid solutions were concentrated and set
aside. The acidified filtratewith the green resinous material were
extracted with ether and various attempts were made to detect phe-
nols; to separate them from the green coloring matter by the addi-
| tion of various solvents to the ethereal solution. No satisfactory
separations were obtained and the lightest colored precipitates
could not be crystallized from alcohol of various concentrations in
|water. No green coloration with ferric chloride was observed and
nothing definite could be obtained by this treatment.

After extracting the aqueous liquid with iso-amyl alcohol

as above described, it was concentrated somewhat under diminished

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. ‘ t i
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7.

pressure. (It was necessary to set this portion aside for some

time, and although chloroform and xylol were added to prevent fer-

mentation by the action of bacteria or moulds, some decomposition

aid occur which may have caused the following negative tests for

sugars.) The concentrated solution was then treated with a slight

excess of basic lead acetate solution, when a brown precipitate
was produced. The latter was collected, washed, suspended in water
decomposed by hydrogen sulphide and the liquid filtered. The light |
reddish brown filtrate, on concentration under diminished pressure, |
| yielded a dark colored product which was examined for tannin.
The liquid was heated with potassium hydroxide, acidified,|
extracted with ether and the ethereal liquid shaken out with aqueous
/ammonium carbonate. On acidifying amiextracting with ether and the
| subsequent evaporation of the solvent, no evidences of tannin were
| obtained.
The filtrate from the precipitate produced by basic lead
acetate was deprived of the lead by means of sodium sulphate and
the lead sulphate filtered out. Slumina cream was added and the
solution filtered and concentrated under diminished pressure. The
|concentrated solution failed to give any of the tests for sugars
| even after a portion had been hydrolyzed with dilute hydrochloric
acid, The osazone test was run for a period of two hours with negea-'|
| tive result. |
{
|

Examination of the Resin (B).

The crude resinous material which had been separated from

the aqueous liquid (&) was extracted successively in a flask under

i@ reflux condenser with various solvents in the following order:

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petroleum ether (Bl), ethyl ether (B2), alcohol (B3).

Petroleum Extract of the Resin.

This extract, after the complete removal of the solvent
formed a dark green, viscid mass. The entire extract was then in-
troduced into a distillation flask, some water added and steam
passed through the mixture for five hours. By this means a small
amount of essential oil was eliminated which had not been removed
by the distillation of the original alcoholic extract with steam,
The contents of the distillation flask were saponified with alco-
| holic potassium hydroxide after the removal of a large part of the
| water, The resulting mixture was extracted with ether whereupon
| three layers separated; namely, the aqueous alkaline layer (Blal),
the ethereal layer (Bla3), and a relatively small amount of a spar-

ingly soluble solid in suspension (Bla2).

Examination of the Volatile Acids.
The aqueous alkaline layer (Blal) was acidified with ten

| per cent sulphuric acid, after removing the alcohol, and steam
| distilled. The distillate thus obtained was extracted with ether,
the ether evaporated in a tared dish and the average molecular
‘ij weight of the volatile acids determined, The average molecular

| weight was found to be 243.9. This corresponds to that of Pentade- |

cylic acid which has a molecular weight of 240, This material was |
probably impure lauric acid. Since only 0.0387 grams were obtained,
it was impossible to separate the mixture and determine the indivi-

dual acids as was originally intended.

j a ! «as - 4 mee ar
‘ wus, ato oF 9

Examination of the Fixed Fatty Acids.
The residue (Blala) left after the distillation of the

volatile acids was neutralized with sodium hydroxide and extracted
with ether to remove any unsaponifiable matter. The aqueous liquid |
was then acidified and extracted with ether. During the evapora-

| tion the acids were lost by fire.

| Examination of the Ethereal Layer (Bla3).
The ethereal layer obtained by extracting the hydrolyzed

petroleum extract of the resin with ether as above described was
| dried and the solvent removed. & viscid dark yellow product was

removed. It was found to consist for the most part of the solid
hydrocarbon described below and a light yellow oil of indefinite
| composition. The weight of this product was about 0.5 grams.

Examination of the Suspended Solid (Bla2).

This solid was a neutral wax-like substance, and was

| easily obtained as a pure white solid free from chlorophyll by

| erystallization from alcohol diluted three to one with water. For
the final purification, it was crystallized from acetic anhydride

| and washed with ether, and allowed to stand in a vacuum dessicator
over potassium hydroxide. When prepared in this way, the solid

melted sharply at 70.5 degrees, was friable and tasteless. mn tet
| tion on platinum foil it burned with a non-sooty flame and had a |
|

paraffin odor, leaving no residue. The solid contained no Hitrogen,

| Sulphur or Halogens. It was insoluble in the following liquids:-
Water both hot and cold; Hydrochloric acid dilute and concentrated

both cold and hot; Potassium hydroxide dilute and concentrated both

10.
hot and cold. It was unaffected by hot concentrated sulphuric acid
and was insoluble in the cold reagent. It was but slightly soluble}
in ether. The analysis of a 0.0940 gram sample gave the following

results:-

Per cent Per cent Total
Cc H

Per cent found 86.13 13.15 99.68

Per cent calculated for
85.72 14.38 100.00

Ether Extract of the Resin (BZ).

This extract, when free from the solvent, formed a dark
green crisp mass. The ethereal solution was extracted with ammon-
ium carbonate, sodium carbonate and potassium hydroxide, The alka-
| line liquids in each case were acidified with ten per cent sulphur-
ic acid, when a very small quantity of green tarry material was pre-
cipitated. Since the combined precipitates amounted to less than
one gram,further attempts to separate any acid substances were

found impracticable.

The Alcoholic Extract of the Resin,
The residue from the above extractions was dissolved in

| alcohol and reserved for future investigation.

SUMMARY AND CONCLUSIONS

The material used in this investigation consisted of the
dry stem and leaves of Thelesperma Gracile collected in Mexico
' about three years ago (1911 or 1912).
For the purpose of a complete examination 2.24 kilograms
| of the plant were used. This material was ground completely, ex-
| tracted with hot alcohol, and the resulting extract distilled ina
current of steam in order to remove the essential oil.

From the portion of the extract which was soluble in water
| no definite compounds were obtained, although effort was made to

| identify sugars and any tannin that might have been present. The

sugar was probably small in amount and of this some was undoubtedly
destroyed by fermentation, The tannin was not found for the reason
that such a small sample was originally taken.

The portion of the alcoholic extract which was insoluble
in water consisted of a green oily resin. From this resin which
contained some occluded essential oil, the following substances
were isolated:- (i) a hydrocarbon which has the formula (CyHy4),.
| The molecular weight was not determined because of the small amount |
of material available; (ii) a mixture of volatile fatty acids which
| were not separated; (iii) a mixture of fixed fatty acids which were
| lost by fire.
In consideration of the results of the foregoing investi
the plant, as taken for analysis, contained no alkaloid, and ther

gation, the following conclusions are given. In the first place, |
e-
fore it is doubtful that the plant has any specific medicinal value.

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12.

In the second place, it seems highly important that at least 20
kilograms of a plant should be available before a complete analysis

is undertaken for there is no doubt in the writer's mind that the

method employed separates the constituents in such a way as to make |

| their identification possible. Although the method is long, it has

accomplished more in this direction than any other method,

a

BIBLIOGRAPHY

Rocheleder: British Journal of Pharmacy, Vol.I., p. 562 (1860) |

Maisch: American Journal of Pharmacy, Vol.XXXIII, p.81 (1860)

Wittstein; Introduction to the Chemical Analysis of Plants,
translated by Mueller (1878) Melbourne.

H, D. Parson's: American Chemical Journal, Vol.I., p.377.

Dragendorff: Qualitative and Quantitative Analysis of Plants,
(1884), translated by Greenish (1886)

F, B. Power et al: Publications of the Wellcome Research
Laboratories.

Prescott: Textbook of Organic Analysis.

Allen: Commercial Organic Analysis.

The United States Pharmacopeia, Eighth Revision,

AQKNOWLEDGMENT

This investigation was suggested by
and carried out under the direction of Dr.G.D.Beal.
I wish to take this opportunity of thanking him for
de patience and kind assistance.
I wish also to express my appreciation to

Mr. Geo. Findlay for his courtesy in collecting the

sample for this work.

|
|
|

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