I4 -—

- .2: r1"

 

:1: STUDY G? SOME SDLVENTS

TSCSR “5‘3?! EX?RACT§ON £3? STEROLS

ROM AREALFA LEAF MEAL

‘fémséis faze- am Bums oé' M S.
MZCRSGAN S‘E'ATfi CGLLSGE
Jamséfiques Rcsiar

E3948

This is to certifg that the

thesis entitled

’4 W7 (3" Jam Jb/ymlzr fuv on
Ex flax/I'm {if ”I ra/J frlm Ayn/fa [turf ”N /

presented by

x/Pd/I— \jficfln’x “004/?”

has been accepted towards fulfillment
of the requirements for

i£mdegree in 8/‘oCéwnlwf7

(W390:

Major professor

 

Date div”? 4; ”4f

M496

A STUDY OF SOME SOLVENTS FOR THE EXTRACTION
OF STEROLS FROM ALFALFA LEAF MEAL

by

Jean-Jacques Rosier

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of
MASTER OF SCIENCE

MICHIGAN STATE COLLEGE

Department of Chemistry

l9h8

cmuamyuun
7‘4, / a,

61.5

RS”?

ill tilil It IIIIlIlIll'Ii Ilrgil'l’i'l‘l‘lllill

 

ACKNOWLEDGEMENT

I wish to express my sincere appreciation
to Professor C. D. Ball for the encouragement
and assistance he has given.me during the course
of this work and in the preparation of this
thesis.

203178

I.
II.

III.

V.
VI.

TABLE OF CONTENTS

INTRODUCTION

HISTORICAL

A. Separation or Isolation of Lipids

B. Isolation of the Sterols after
Saponification

EXPERIMENTAL

Saponification and Obtention of the
Unsaponifiable Material

Purification of the Sterols
Acetylation and Identification
Determination of Hydroxyl Groups
DISCUSSION

SUMMARY AND CONCLUSION
BIBLIOGRAPHY

Page NO 0

12
14
I8
20
25
30
31

INTRODUCTION

Different solvents have been used for the extraction
of sterols. They have been: ethanol, ethyl ether, petrole-
um ether, chloroform, benzene, acetone, methanol. They have
been used separately or in combination. Almost all of them
have a great disadvantage because of their inflammability,
their lack of specificity and their ability to extract
substances other than sterol. However, ethanol and ethyl
ether have been commonly used for this extraction.

In this work it was decided to try to use a nonin-
flammable solvent such as ethylene dichloride and to come
pare the yield and properties of the preparations with those
obtained by ethanol and ethyl ether.

The chief advantage of a chlorinated hydrocarbon is its
noninflammability; a disadvantage is its higher cost. Its
'specificity is not too good for it tends to dissolve non-
glyceride material thus interfering in the refining process
of the oil and, therefore, of the sterol.

The purpose of this study is to compare the different
amounts of sterols extracted from alfalfa leaf meal with
two different solvents, namely the pair (a) ethyl alcohol-
ethyl ether and (b) ethylene dichloride, and in addition to
study the specificity of each solvent.

ll\.\.[l\ Illll.,I\||I [‘{L/IL‘II [,(Illlllll 'lllll (Ill [I'll

 

 

{III

 

-2...

HISTORICAL

The sterols occur in three main forms: free sterols,
steryl esters, steryl glycosides. The last ones are nearly
insoluble in almost all of the solvents, little is known
about their solubility and, therefore, will not be con-
sidered. On the contrary, the other two are very soluble
in several solvents and are the object of this study.

In the past few years, different methods for the sepa-
ration of sterols from biological material have involved
distinct operations; (I) the separation and isolation of the
lipids and, (2) the separation of the sterols from the lipid
materials. A survey of these extractions, before and after

saponification, is listed below.

A. Separation or Isolation of Lipids.

Beneke (l) in Germany in 1872 was one of the first who
definitely studied the extraction of sterols, substances at
that time called "cholesterin". He extracted egg yolk, brain
material and calves eyes with ethanol in the cold.

The next one working with ethanol was Tanret (2) in
1879 in France. He used 95% ethanol to extract the lipids
from pulverized ergot.

Around 1910, Power and his co-workers (3,h,5,6,7,8,9)

\rll l.llllll\«"|k.[[\l(ll.[,fl|ll.[l‘|lll ll

 

 

[I {ll-Ill-

extracted the oil of numerous plants by percolation with
hot ethanol.

In 1932, Zeichmeister and Cholnoky (10) extracted
intermittently the oil of the dry blossoms of the plant,
Calendula officinalis, with 96% ethanol.

The oil of the algae, Fucus Vesiculosus, was ex-
tracted with ethanol in the cold by Phipers and Wright
in 193A (11).

Simpson in 1937 (12) worked on dried Sanega root
with boiling ethanol and extracted the oil.

Dicholesteryl ether was obtained from.the spinal
cord of oxen by Silbermann (13) in 19h5 by continuous
extraction of the dried cord with hot ethanol.

The second solvent used was petroleum ether. In
1878, Hesse (1h) in Germany extracted the lipid material
from powdered Calabar beans with this solvent.

More recently, Clenshaw and Smedley mac Lean (15)
extracted green leaves with light petroleum ether (BP:
400-6o°).

Another solvent which has been used was ethyl ether
to extract the oil and, subsequently, the sterols.

Chibnall and Channon (16) in 1927 obtained the oil
from cabbage leaf cytoplasm with ethyl ether.

Anderson (17) also extracted wheat and corn germ

with ethyl ether.

Cholesterdl was extracted from blood by drying the
serum over sodium sulfate and then extracting it with
chloroform (22)(Myers-Wordell method, 1918).

In 1932, Hart and Heyl (18) extracted dried spinach'
leaves with acetone.

The total steroidal content of steers' urine was
determined by Marker in 1939 (19) after a treatment with
hydrochloric acid and an extraction with butanol.

Fernholz and Moore (20) in 1939 extracted dehydrated
alfalfa meal with hexane.

In l9hh, the oil of ground dried seeds of the plant,
Calycanthus floridus, was obtained with benzene by Cook
and Page (21).

Schoenheimer and Sperry (22) used acetone-ethanol to
precipitate the proteins and extract the cholesterol from

whole blood.

l[[n[[ [III
[.(I

 

-5-

B. Isolation of the Sterols after Saponification.

The foregoing extractions were carried out on various
biological tissues involving the isolation of total lipids
along with the sterols.

The most used procedure for the extraction of the
sterols involved a preliminary saponification by ethanolic
or methanolic potassium or sodium hydroxide, either in the
cold or at a warm temperature. water was added to the un-
saponifiable material and it was then extracted by a solvent,
usually ethyl ether. The sterol was extracted from the ether
phase either by formation of a derivative, such as, oxalate,
acetate, digitonide, or by a fractionated crystallization
in ethanol. However, there are a few exceptions to this
general rule, for sterols can be extracted from a biological
source without carrying out a saponification. These cases
will be listed at the end of this section.

Beneke (1) used a lead salt, PbO, to precipitate the
impurities. He dried the precipitate and extracted it with
ethanol. He recovered the sterols after saponification with
barium hydroxide and then made subsequent extractions of the
unsaponifiable material with ethanol.

Schulze (23) working with wool fat, Hesse (14), Tanret
(2), Power (3,h,5,6,7,8,9), Clenshaw and Smedley Mac Lean
(15), Simpson (12), all previously mentioned, used the usual

procedure mentioned above for the extraction of sterols.

 

 

'llll‘lllll’l‘rll

Chibnall and Channon in 1927 (16) treated the ethyl
ether extract with acetone to remove the phosphorus con-
taining lipids. A double saponification, first with sodium
hydroxide and then with sodium ethylate, was followed by
the usual procedure.

Fernholz and Moore (20) used the usual procedure
but evaporated the ethyl ether extract to dryness and
dissolved it in acetone. The acetone soluble material,
evaporated to dryness, was dissolved in hexane which was
extracted with methanol. The methanol extraction yielded
crystals which were purified by recrystallization in
ethanol. '

Zeichmeister and Cholnoky (10) after addition of water
transferred the sterols to an ethyl ether - petroleum ether
phase, dried the epiphasic layer over sodium sulfate and
then applied the usual procedure.

Hardt and Heyl (l8) and Phipers and Wright (11) elimi-
nated the chlorophyll by petroleum ether before proceeding
in the ordinary way.

Anderson (17) extracted the unsaponifiable material
fraction.with ethyl ether, dried it in vacuum at 10000.,
washed it with ice cold pentane and obtained practically
colorless crystals. Then an additional saponification,
extraction with ethyl ether and recrystallization from
ethanol yielded crystals of high purity.

 

 

 

[I’ll’lii

 

Pernholz (24) holds a patent in which he claimed to
obtain the sterols from.soybean oil by saponification; the
resulting soapstock was decomposed by hydrochloric acid
and the fatty acid, steam distilled in vacuo. The sterols
were recovered from the residue by ethyl ether.

In another patent, sterols having one reactive double
bond in the hydrophenanthrene nucleus were isolated by
Yoder in l9hh (25). The separation consisted of saponifi-
cation, solution of the unsaponifiable material in an
anhydrous solvent, such as, ethylene dichloride or acetone,
and treatment of this solution with anhydrous oxalic acid.
The sterols which were precipitated as oxalates, were
liberated by hydrolysis.

Mitchell (26) in a patent extracted the dried saponi-
fied product with liquid sulfur dioxide in order to obtain

sterols.

As mentioned above, methods have been reported which
have eliminated the saponification phase of the sterol
preparation.

In 1942, Oliver and Palmer (27) passed "talloel",
extracted by a solvent such as petroleum naphta, through
an adsorbent clay in order to recover the sterols.

Kruse and his co-workers (28) claimed to have

neutralized the fatty acids and precipitated the

 

 

 

.I’l‘lll .I”:
I'll fl Ill

 

phosphatides of soy-bean oil by addition of a base, with
insufficient alkali to saponify the oil itself. The solid
portion was then extracted with an organic solvent such as
acetone.

Kraybill, Brewer and Thornton (29) reported in a patent
a process for preparing an adsorbent suitable for removing
phosphatides, mucilages, sterols and pigments from vegetable
oils. The adorbent was prepared by mixing a water solution
of sodium silicate with a water solution of an acid reacting
aluminum salt .

Asquew (30), Almquist (31), Drummond (32), Embree (33),
Fawcett (3A), Hickman (35), and some others worked on the
molecular distillation of fats and oils. In such a method,
phospholipids and mucilaginous matters were eliminated by a
preliminary treatment. During the distillation, the fatty
acids passed off first, then the hydrocarbons and then the

sterols, which came off just before the triglycerides.

EXPERIMENTAL

The material used was dehydrated alfalfa leaf meal,
the so-called Leafy Irish Brand. The meal used had the
following analysis according to the report of its manu-
facturers, Ireland Alfalfa Mills Inc.

Crude Protein, not less than 17%

Crude Fat, not less than 1.75%

Crude Fibre, not more than 25.0%

Carbohydrates nitrogen free extract

not less than 60%

For comparison, the alfalfa leaf oil was extracted
by two different methods: the first preparation by the
use of ethanol and ethyl ether whereas, in the second
preparation, ethylene dichloride was used. Both ex-
tractions were performed at room temperature in order
to lessen the effect of heat.

The solvents used were:

1. 95% Ethanol from Commercial Solvents, Inc.

2. Ethyl Ether, USP 10, from Merck Co. Inc.

3. Ethylene Dichloride from the Dow Chemical Co.

Ethylene dichloride is a colorless liquid the boil-
ing point of which is 83.70. It is insoluble in water,
soluble in alcohol and ether and has a specific gravity

H H
of 1.257. This 1-2 dichloroethane, or Dutch oil, Cl-C-C-Cl

H H

-10..

is prepared industrially by direct chlorination of ethylene
in the presence of a catalyst, such as a ferric ion or a

peroxide.

Extraction No. l.

1 kg. of alfalfa leaf meal was weighed and put into
a cloth bag. Thgtgggawasplaced in a 12 liter cylinder.
Five liters of 95%kwere poured into the cylinder, the latter
then covered with a glass top. After twenty-four hours of
standing, the liquid was siphoned off and the process re-
peated until the extracting solvent remained colorless.
This condition was obtained after ten extractions.

’The accumulated solvent containing the oil was concen-
trated on the steam bath under reduced pressure. The re-
ceiving flask was packed in ice. This is shown in Figure
l.

 

 

 

 

-11-

Despite all these precautions a large part of the
ethanol did not condense but was lost through the water
pump.

The concentration was stopped when the liquid began
to boil too violently. At this stage, about 1.5 I.
remained in the flask.

The residue, after the ethanol extractions, was
treated with ethyl ether, USP. The ethyl ether was poured
into the cylinder containing the alfalfa leaf meal; and
the same procedure as described for the ethanol extractions
was used. The ethyl ether extract was condensed to about
1 1.in the same manner as described for the ethanol ex-
tractions and then combined with the ethanol extract. The

preparation was now ready for saponification.

Extraction No. 2.

As in the.case of the ethanol and ethyl ether ex-
tractions, 1 kg. of alfalfa leaf meal was put in a bag and
extracted with ethylene dichloride until the solvent re-
mained colorless. For this purpose l8 extractions were
necessary.

The ethylene dichloride extract was evaporated to
dryness under reduced pressure and then 600 m1. of ethanol
were added. Since ethylene dichloride is soluble in ethyl

ether, trouble would have occurred later in the separation

-12-

of the unsaponifiable portion from the bulk of material

if the ethylene dichloride had not been removed.

Saponification and Obtention of the Unsaponifiable

From now on, the instructions given by Fernholz and
Moore (20) were followed. Since they started with 2.8 kg.
of dehydrated alfalfa meal while, in these preparations,

1 kg. only of dehydrated material was used, proportional
amounts of the reagents were employed. The same method
was applied to both extractions.

The saponifications were done directly on the concen-
trated extracts. For that purpose 18 g. of potassium
hydroxide dissolved in 360 ml. of 95% ethanol were heated
with the extracts on the steam bath with a reflux condenser
for a period of two hours. After cooling at room tempera-
ture, 2 1. of water were added. About 1 l. of the solution
was placed in a 2 1. separatory funnel and 200 m1. of ethyl
ether were added. A gentle rotation permitted the upper
layer to absorb the unsaponifiable matter and thus to become
colored. The same Operation was repeated about seven times
for each liter of the solution. Some persistent partial
emulsions were encountered at this point but were ignored

for in later concentration, as described below, the two

-13-

phases separated completely. The combined ether extracts
were concentrated to 1.5 1. and washed thoroughly with
water using phenolphtalein to denote the removing of alkali
present. The three last washings were made with a solution
of water containing 10% of ethanol in order to remove the

water dissolved in the ether.

-1p-

PURIFICATION OF THE STEROLS

_The ether extract containing the unsaponifiable matter
was evaporated to dryness. In order to prevent the oxidation
of the product, the distillation was done under reduced
pressure and an atmosphere of dry carbon dioxide. A cylinder
furnished the carbon dioxide which was dried by passing through
concentrated sulphuric acid. The liquid to be evaporated was
contained in 100 cc. beaker which in turn was placed on a ground
glass plate. A truncated suction flask with the bottom cut off
was placed on the ground glass plate. Silicone grease was put
on the low rim.of the flask to effect a seal. When the pressure
equilibrium was obtained, the current of carbon dioxide was kept

at about one bubble every second through the sulfuric acid.

, fl to VQCWM

 

y",

4! ' 1 i

n,sag

Ftaf 2.

C0, «stiflin-

,Between evaporations, the carbon dioxide cylinder was
.disconnected in order to prevent the sulphuric acid from
backing up in the rubber tubing. The apparatus described

above is shown in Figure 2.

-15-

The evaporation of ethyl ether to complete dryness re-
quired about 20 hours. A dark brown waxylike material remained
at the bottom of the flask. This residue was dissolved in a
proportional amount (71 ml.) of hot acetone, and the material
coming out on cooling was removed by filtration and set aside.
This was probably phosphatides but a crystalline substance,
not further considered, was present.

The acetone filtrate was evaporated to dryness with the
same apparatus as described above. This evaporation required
about the same period of time as the ether evaporation.

At this step of the method, a modification.vas made from
that followed by Fernholz and Moore (20) because of the lack
of hexane. Instead of hexane, pentane was used. The residue
was dissolved in a proportional amount (71 m1.) of pentane)
and the solution was placed in a 500 m1. separatory funnel.
This dark solution was washed five times in succession with
71 ml. each time of 95% methanol saturated with pentane. The
methanol layer was hyponhasic. The first washing was brown;
subsequent washings change progressively from yellow to about
colorless.

After concentration on the steam bath, the combined
methanol extracts were evaporated under reduced pressure and
atmosphere of carbon dioxide with the apparatus described
above. The resulting product consisted of a brownish waxylike

material.

-16-

The results of the different extractions both with
alcohol-ethyl ether and with ethylene dichloride as the

starting materials are shown in Tables I and II.

-17-

TABLE I

Amount of material Obtained

by

Alcohol - Ethyl Ether Extractions

 

 

 

 

 

No. Crude material Partially Refinedi Methanol
of (after ethyl Material Extracted
Preparation ether evapora- (after acetone Material
tion) evaporation) (after treat-
ment with
1. - pentane)_
lst 5.1 8' 2.13 .6306
3rd 32.70 7.0 1.6
TABLE II
Amount of Material Obtained
by
Ethylene Dichloride Extractions
“No. ‘* Crude materiaITPartially Refined_ MEthanol
of (after ethyl Material . Extracted
Preparation ether evapora- (after acetone Material
tion) evaporation) (after treat-
ment‘with
pentane)_
lst 4.6 g 1.606 .1898
2nd 901 #02 chi?
3rd 16.1 5.21 1.18

 

-18-

ACETYLATION AND IDENTIFICATION

This waxylike material obtained from either the ethanol-
ethyl ether extraction or the ethylene dichloride extraction
was dissolved in acetic anhydride. (30 ml. per gram) and the
solution heated on the steam bath for 1 hour. The solution
was let stand overnight and then filtered.

An attempt was made to recover from.the mother liquors
some of the acetylated product dissolved in the excess of
acetic anhydride. For that purpose the solution was concen-
trated and water added, but only a negligible quantity of
steryl acetate appeared. ‘

The solid acetate, crystallized after acetylation, was
separated from the acetic anhydride by filtration and saponi-
fied with an excess of 5% alcoholic potassium hydroxide
during two hours on the steam bath with a reflux condenser.
The alkalinity of the solution was checked in order to be
sure to obtain a complete saponification. Then water was
added to the solution and the solution was extracted with
ethyl ether seven times. The ethyl ether solution was
washed with water until free from base and finally washed
with 10% ethanol solution to remove the water. Then the
ethyl ether solution was evaporated to dryness on the steam
bath and the crude sterols obtained were dissolved in Just
sufficient boiling 85% ethanol to effect a complete solution.
This solution was let stand overnight and the material which

crystallized out of it was separated. The crystals were then

-19-

redissolved in the minimum amount of 85% ethanol and let
stand over night. This procedure was repeated eight times.
For the last time the solvent was methanol instead of
ethanol.

The sterols obtained with the ethanol - ethyl ether
extraction melted at 156° after the first crystallization
from ethanol and 1680 after the last crystallization. The
sterols obtained with the ethylene dichloride extraction
melted at 160-1620 after the first crystallization from
ethanol and 168-168.5° after the last crystallization. The
identity of the sterols obtained with these two different
solvents was furthermore proved by the melting point of
their acetate. The acetates were prepared by the usual
method. The melting points were respectively 179-1810 for
the sterols obtained from the ethanol-ethyl ether extraction
after one recrystallization from ethanol, and 180-1820 for
the sterols obtained from the ethylene dichloride extraction

after one recrystallization from ethanol.

DETERMINATION OF HYDROXYL GROUPS

In order to see whether there was some other substances
containing an hydroxyl group in the mother liquors of the
crystalline material, a determination of the hydroxyl group
was attempted.

P. J. Ehdng and B. Warshowsky (36) used the phthafiization
method with phthalic anhydride in hot pyridine solution for the
determination of the alcoholic hydroxyl content of complex
mixtures.

An attempt to apply this method for the determination of
the hydroxyl containing substances was tried.

A preliminary experiment applying this method was per-
formed on cholesterol (m.P. : 137-1380) from.Eastman.Kodak
Company.) A quantity of cholesterol (1.7lh' g) was placed in a
dry volumetric flask and dissolved in 50 m1. of dry redistilled
-pyridine (b.p. llh-llSo). The phthaflization mixture was pre-
pared by dissolving 20 g. of phthallic anhydride in 200 ml. of
redistilled pyridine. water must be avoided, otherwise low
values may be obtained, because of the hydrolysis of phthaInc
anhydride. Twenty-five ml. of the phthaflic anhydride solutions
were pipetted into a 2h/A0 standard taper flask. To this were
added 10 m1. of the solution containing the sample. A con-
denser was attached to the flask and the later was heated in a
boiling water bath for one hour. A blank determination was

made in the same manner on the reagents employed. In each case

-21-

50 m1. of distilled water were then added and the solution
was cooled and titrated immediately with 0.2027 N. sodium
hydroxide. The difference in amount of alkali consumed
between the blank and the sample represented the amount of

esterifiable hydroxyl present in the solution.

 

% hydroxyl = V x N x .017 x 100

W = weight in grams 0? the sample in the aliquot taken.

V 8 difference in volumes required by the blank and
sample titrations.

N = normality of the standard sodium hydroxide.

.017 = the milliequivalent weight of the hydroxyl group.

The solution containing the sample was yellow after
phthaflization. When the water was added, a white cloudiness
appeared due to the presence of phthallic anhydride,very in-
soluble in water. It persisted almost until the end of the
titration; about 5 m1. before the end point the solution
became transparent but still yellowish.

Two different solutions of cholesterol in pyridine were
used. The percentage of hydroxyl group are given in Table
III. ‘

In order to verify further this phthallization method,
and the purity of the cholesterol used of which neither the
origin, nor the properties appeared on the label, the following

experiment was performed. A saponification of the cholesteryl

-22-

 

 

.TABLE III
Weight of Cholesterol Ml. 0.2027 N. % Hydroxyl
NaOH
Blank Sample
1st Exp. 0.3433 154.91 150.81. b.01
2nd Exp. 0.3h3 15A.60 150.15 b.15

3rd Exp. 0.209 153.08 l5l.hh h.07

 

-23-

acetate prepared from thesample of cholesterol used above
was carried out in order to know the number of milligrams

_of sodium hydroxyde necessary for the hydrolysis of the
acetate and consequently the OH content. A saponification
with .2027 N. sodium hydroxyde was performed and the solution
back titrated with .1015 N. hydrochloric acid. A similar
experiment was performed using stigmasterol acetate, which
has been recrystallized 1A times.

The results of both saponifications are given in Table
IV.

To check the presence of water in cholesterol, an
attempt to dehydrate the material at 100° under atmospheric
pressure failed because of decomposition before a sensible
loss of water.

The phthalflzation method however was applied to charac-
terize the hydroxyl containing group in the mother liquors
of crystallization after purification of the sterols.

The mother liquors were evaporated to eliminate the
ethanol and methanol. The respective weights of dry material
were .021 g. for the ethanol-ethyl ether extractions and
.016 g. for the ethylene dichloride extractions (only the
third extractions were considered in both cases).

The results are given in Table V.

TABLE IV

 

Sterol Weight of Steryl

%OH in Acetate

%OH in Sterol

 

 

Used Acetate calculated found calculated found
Cholesterol .456 8 3.97 4.21 4.40 4.63
.093 4.18 4.60
.235 4.36 4.80
Stigmasterol .105 3.74 4.06 t.10 4.47
.113 4.23 4.65
TABLE V

 

Ethanol-Ethyl Ether

Ethylene Dichloride

 

Extractions Extractions
wgt. of Material Wgt. of Material
in 8. in g.
.004 .0032
.008 .0064

 

a-.-

l~

no».

-25-

DISCUSSION

The appearance of the two different extracts is not
materially different until the last phase of the purifica-
tion. Whereas, the extract coming from the ethanol-ethyl
ether extraction is dark brown, that coming from the
ethylene dichloride extraction is light brown, almost orange
in color.

The acetylation products are also different. The
dark brown material from the ethanol-ethyl ether extraction
gives crystals which are small, flat, and seem to have a
tendency to be agglomerated one with the other. The light
brown material on the contrary gives large, flat, trans-
parent crystals.

If the best preparations in each of the methods used
in this study are compared with the method used by Fernholz
andeoore (20), the yields are still low. In Table‘VI is
given a comparison of the results published by Fernholz
and Moore with those obtained in the present study showing
the yields at the different steps of the purification, all
on the basis of g. per kg. of alfalfa leaf meal.

The extraction of crude material by diffusion, used
in the present work, gave a better yield then the extraction
with the percolation method as described by Fernholz and
Moore (20). At the subsequent step the results are about

the same although those of the present work are a little

-‘U’—

TABLE VI
(g. per kg. of alfalfa leaf meal)

 

 

Present Present
Fernholz and Work Work
Moore Ethanol- Ethylene
Hexane Method Ether method Dichloride
Method
Crude Material
(after ethyl ether 10 32.70 16.1

evaporation)

Partially refined
material (after 7.14 7.0 5.21
acetone evaporation)

Methanol extracted
Material (after 3.92 1.6 1.18
treatment‘with

pentane)

 

lower, particularly for the ethylene dichloride extraction.
At the last phase of this purification, the yield of the
Fernholz and Moore's percolation method is far ahead of the
yields obtained in this study. From the comparison of
these results, we can conclude that the extraction in the
stationary diffusion method isolates something else than
the sterols in an amount different from.that obtained by
percolation. Beside the phOSphatides which are extracted
in all the cases, it might consist of pigments, probably
not quite destroyed by the saponification. This might be
ameliorated by a second saponification recommended by
different authors such as, Chibnall and Channon (16),
Anderson (17) and others, after the extraction of the un-
saponifiable matter with ethyl ether. In fact, in the ethyl
ether extract, there was some greenish material standing at
the interphase and overlapping therefore into the ethyl
ether phase.

The final yields in this work are effectively much
smaller than the one obtained by Fernholz and Moore (20),
but there is to be noted that the cited authors used an
extraction by percolation and do not mention the duration
of their extraction, or the amount of solvent used. In
this study, the renewing of solvents was stopped when an
extraction remained colorless after twenty-four hours; but

nothing proves that the extraction of sterols stops when

the extraction of pigments ceases. Besides, if the solvent
is left long enough, for example seventy-two hours, it still
becomes colored.

The solvents act by diffusion. This method does not
attain 100% extraction but rather approaches complete
extraction asyékotically due to the fact that there is always
an equilibrium of solute between the solvents and the plant
tissue immersed in the solvent.

If a comparison is made with the work of’King (37), it
can be seen that the sterols obtained in this work are
similar to the one obtained by King from.alfa1fa seed. The
melting points of the purified sterols are about the Same
and also the melting point of the acetates. The amount of
material obtained in the present work was too small to
attempt a separation of the different homologues ofaspina-
sterol as performed by King.

For the titration of the hydroxyl group, the phthalli-
zation method is not too satisfactory. A comparison between
the percentage of hydroxyl group of‘a known sterol shows a
fairly wide discrepancy as shown in Table IV.

This error can be explained partly by the small size
of the sample. In the method Ehing and warshowsky (36) a
sample of l g. of ethanol was advised.(MW : 46 g.) to obtain
reliable results. For cholesterol the same accuracy would be
obtained with 8.4 g. ( 86 in proportion to the MW. Consequent-
1y the results obtained for the hydroxyl content of the mother

liquors is not definite, due to extremely low
the sample. However, the result may indicate
of some 0H containing substances, probably of
weight.

The phthallization method was checked by
sisting of the sapononification of the sterol
here also the titration is not very accurate.
the acetate is dissolved in ethanol, a volume
sodium.hydroxyde is added, the flask refluxed
bath for two hours. The back titration, with
chloric acid and always gives a higher result

retical because of an insufficiency of acid.

weight of
the presence

lower molecular

a method con-
acetate, but

In this meulod
of standard

on the steam
standard hydro-
than the theo-
The phenol-

phthalein in ethanol has its PK raised, and besides that the

equilibrium seems to be very slowly obtained.

These methods of titration of the hydroxyl content can

give an idea but are not very reliable, especially when the

samples are small.

The results given in Table V show however that some

other OH.containing substances of lower molecular weight were

present in the mother liquors from.the crystals of spinasterol.

-30-

SUMMARY AND CONCLUSION

A comparison of the yield of extraction of ethanol-
ethyl ether and of ethylene dichloride has been determined.
The pair, ethanol-ethyl ether, extract more unsaponifiable
product than ethylene dichloride.

A qualitative comparison between the unsaponifiable
portion has been carried out. The ethanol-ethyl ether
extract does not yield as large an amount of sterols as
expected from.the weight of the unsaponifiable matter.

This pair of solvents extract a slightly larger amount of
sterols than does ethylene dichloride.

A derivative has been prepared from each of the solid
fractions separated.

The main crystalline substances in each of these
fractions has been identified ask“ spinasterol.

There is an indication of some uncrystallized material

containing hydroxyl groups.

l.

3.

h.

5.

9.

10.

11.

12.

13.

lb.

BIBLIOGRAPHY

Beneke F. W., Cholesterin im.Pflanzenreich aufgefunden,
Lieb. Ann. 122, 2A9, (1862).

Tanret G., De 1' ergotinine, Ann. Chim. Phys. ‘11,
A93, (1879).

Thorpe T. E., and Holmes J., The occurence of paraffins
in the leaves of tobacco, J. Chem. Soc.
.22. 982. (1901).

Power F. B. and Tutin F., The constitution of homo-
eriodictyol, J. Chem. Soc. 21, 887,
(1907).

Power F. B. and Mbore H., The constituents of the bark
of Prunus Serotina, J. Chem. Soc. 22,
2&3. (1909).

Tutin F. and Clewer H. W., The constituents of Cluytia
Similis, J. Chem. Soc. 101, 2221, (1912).

Power F. B. and Tutin F., The constituents of hops,
J. Chem. Soc. 103, 1267, (1913).

Power F. B. and Browning H., Constituents of the flowers
of Anthemis Nobilis, J. Chem. Soc. 105,
1865, (l9lh).

Power F. B. and Browning H., Constituents of the flowers
of Matricaria Chamomilla, J. Chem. Soc.
102. 2280, (1916).

Zeichmeister. L. and Cholnoky L. V., The pigment of
Marigold (Calendula Officinalis), Z. Phys.
Chem. 208, 26, (1932). (GOA-O)

Phipers R. F. and Wright H. R., The algae sterol:
fucosterol, J. Chem. Soc. 1572, (193h).

Simpson J. C., The constituents of Senega Root, J. Chem.
Soc. 730, (1937).

Silbermann H., The occurrence of dicholesteryl ether in
the spinal cord of the ox, J. Biol. Chem.
159, 603, (19h5).

Hesse 0., Phytosterine und Cholesterine, Lieb. Ann.
192, 175, (1878).

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

-32-

Clenshaw C. H. and Mac Lean S., The nature of the
unsaponifiable matter extracted from
green leaves, Biochem. J. .22, 107.

Chibnall A. C. and Channon.H. J., The ether soluble
substances of cabbage leaf cytoplasm,
Biochem. J. .21, 225, (1927).

Anderson R. J., The phytosterols of the endosperm of
corn, J. Am. Chem. Soc. .56, 1&50,
(l92h).

Anderson, R. J., The phytosterols of the wheat endo-
sperm, J. Am. Chem. Soc. .59, 1717,
(1924).

Anderson R. J., Extraction of the grain with ether,
J. Am. Chem. Soc. .E§: 2987, (1926).

Hart M. C. and Heyl F. W., Spinasterol and some of
its esters, J. Biol. Chem. .22, 311,
1932 .

marker R. E., The Steroidal content of steer's urine,
J. Am. Chem. Soc. ‘61, 287, (1939).

Fernholz E. and Moore M., Isolation of Spinasterol
from alfalfa, J. Am. Chem. Soc. ‘éi,
2h67, (1939).

Cook J. W. and Page M. F. 0., The sterols of
Calycanthus floridus, J. Chem. Soc.
336. (1944).

Schoenheimer R. and Sperry W. M., A micromethod for
the determination of free and combined
cholesterol, J. Biol. Chem. 'lgé, 7h5,
(193h).

Schulze E., Ueber die Zusammensetzung des Wollfetts,
Ber. 1075, (1872).

Fernholz E., Obtaining sterols from.oils, Pat. US,
'3, 280, 815, (l9h2).

Yoder L., Isolation of sterols from.fats and oils,
Pat. US, '2, 322, 906, (1911).

Mitchell J. E., Extraction of vitamins and sterols from
compressed yeasts, various fish oils, etc.,
Pat. US, ‘3, 32A, 270, (19hh).

-33-

27. Oliver F. and Palmer R. C., Concentrating the sterol
content of "talloel", Pat. US, .2, 280,
8A3, (1942).

28. Kruse H. D., Concentrated sterol fractions from oils
such as soybean oil, Pat. US, .2, 296,
796, (19h3).

29. Kraybill H. R., Brewer P. H.,‘Thornton M., Adsorbent
material for treating oils and sludges,
Pat. US, ‘2, 17h, 177, (l9hl).

30. ASQUBW’F. A., Bourdillon R. B., Bruce H. M., Jenkins C.
and Webster T. A., Distillation of
Vitamin D. Proc. Roy. Soc. (London)
1073, 76, (1930).

31. Almquist H. J., Purification of the antihemorrhagic
vitamin by distillation, J. Biol. Chem.
117, 517, (1937).

32. Drummond J. C., The fat soluble vitamins, Ann. Rev.
Biochem. Z, 335, (1938).

33. Embree N. D., The separation of natural components of
fats and oils by molecular distillation,
Chem. Rev. .22, 317, (l9hl).

3h. Fawcett E. W., Refining crude fats and fatty oils,
Pat. US, _2_, 01.7, 196.

35. Hickman K. C., High vacuum distillation of the steroids,
Ind. Eng. Chem. ‘22, 1&51, (1910).

Hickman K. C., Extracting vitamin containing fish oils,
Pat. Brit. 539, 089, (l9hl).

36. Elving P. J. and Warshowsky B., Determination of the
alcoholic hydroxyl group in organic
compounds, Ind. Eng. Chem., Anal. Ed.
12, 1006, (191.7).

37. King L. C., A study of the sterols, sterolins and
certain alcohols of some legume seed oils,
PhD. Thesis, Michigan State College, (1912).

My} 3. If.“
JUN 2 7 .50

JUL 2 0 '55

 

 

 

 

6613361?“ 208178
3“ £819 Rosier

1

5412.”! “y m” .

, T612.015 203178

i 3819 Rosier

. .L study of some solvents
) for the extraction of

sterols from alfalfa leaf
meal 0

 

 

iNYB ’49

406%:6 .
.«Q ‘ ' .

_____'__ . 1_4’__ ._._.._ ____. ._.._¥

(4‘ {Iii}: ’~,._- H

 

 

 

M IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

MI\\\\\|\\\\\|\\\|\\\\\\NW)H\\|\\|\\\|\|\\\H|N\| .

3 1293 02446 7197