II

1
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IHIIJHIHHI

NH

123
261
THS

 

A 5.2mm at? THE LEPEDS OF amp-3.3203?
TREFOEL (LQTUS gORN’ECQLAWS)

W

“mesh: {or Hm Dogma 0% M. S.
MECHIGAN STATE UNIVERSWY

Morris D. S. Richarés
£965

1145515 I.) LIBRARY ‘1
Michigan Stan
U . .

ABSTRACT

A STUDY OF THE LIPID CONSTITUENTS OF
BIRDS-FOOT TBEFOIL (LOTUS CORNICULAIUS)

by Morris D. S. Richards

The invention of gas chromatography:mu1the improved
techniques in column and thin-layer chromatography have
greatly facilitated the isolation and characterization of
lipids. This is eSpecially true with plant lipids where
isolations are hampered by the presence of pigments which
have similar chemical and physical properties. These methods
of chromatography have been employed in studying the lipids
of L. corniculatus.

The purpose of this study was to find a method by which
the lipids could be isolated and used individually to study
their in_1i§£g rumen metabolism. Lipids were extracted com-
pletely with any 'of the following three solvent systems: chloroform-
methanol, chloroform-isoprOpanol, and ethyl ether. The com-
plete separation of non-acidic lipids was accomplished by
using both column and thin-layer chromatography. With column
chromatography three fractions were obtained consisting of:
.sterol esters and fatty acid methyl esters; triglycerides and
free fatty acids; and free fatty acids and diglycerides. The
individual separation of the lipids was accomplished by thin-
layer chromatography.

After the fatty acids were released from the lipids by

saponification they were methylated and analyzed by gas-liquid

Morris D. S. Richards
chromatography. The fatty acids identified were lauric, myristic,
myristoleic, palmitic, palmitoleic, stearic, oleic, linoleic,
linolenic, arachidic, behenic and lignoceric acids. Palmitic,
linoleic and linolenic acids were the predominant acids. From
a comparison made with a first and second harvest there were
38% saturated acids and 62% unsaturated acids in the first
harvest and for the second these values were 35 and 65%,
reSpectively.

The individual lipid fractions varied in their fatty
acid composition. In the sterol esters, triglycerides and
free fatty acid fractions, palmitic and linolenic were the
major acids. Palmitic acid was the predominant acid in the
fatty acid methyl ester fraction but the concentrations of
linoleic and linolenic were also high. The diglyceride frac-
tion was high in myristic acid content. Palmitic and linolenic

acids concentrations were 20% of the myristic acid.

A STUDY OF THE LIPIDS 0F BIRDS-FOOT
TREFOIL (LOTUS CORNICULATUS)

 

By

Morris D. S. Richards

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry

1965

Approved _ RWY“. \QJLQ.

ACKNOWLEDGMENTS

The author expresses his sincere appreciation to
Professor Harold M. Sell for his encouragement and direction
throughout the course of this work. He is also indebted to
Professor J. William Thomas for his valuable suggestions and
added encouragement in the research and in preparation of the
manuscript. The assistance obtained from my colleagues was
immeasurable but Special thanks must be given to Amir
Fathipour whose cooperation in the research was very benefi-
cial; Keith K. Schlender for his help in the preparation of
both the thesis and manuscript; and Roger W. Ritzert and Ben
Homer Yeh who were always available for discussions in the
preparation of the manuscript. His thanks goes to the
Regional Project NC-63 of the U. S. D. A. for financial

support.

11

DEDICATION

This thesis is affectionately dedicated to my parents,
Mr. and Mrs. Lister Seymour Richards, who have contributed

immeasurably to my scholarship.

iii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . .
EXPERIMENTAL . . . . . . . . . . . . . .
Preparation of samples . . . . . .
Extraction of Lipids. . . . . . . .
Column Chromatography of Lipids . .

Thin-Layer Chromatography of Lipids

Saponification and Recovery of Acids.

Test for Sterols. . . . . . . . . .
Test for Glycerol . . . . . . . . .

Preparation of Methyl Esters . . .

Conditions for Gas-liquid Chromatography.

RESULTS AND DISCUSSION . o . . . . . . .
SUMMARY 0 O O 0 O O O O O O 0 0 O 0 O O
BIBLIOGRAPHY O O 0 O O O O O O O O 0 O 0

iv

Page

\30\O\O\\0

ll
12
13
13
13
15
16
21+

25

LIST OF TABLES
Table Page
1. Sterols and glycerol present in various lipid
fractions . . . . . . . . . . . . . . . . . . . . 18
2. Percent of fatty acid in each fraction. . . . . . l9
3. Fatty acid composition in various lipid fractions
from the first harvest of L. corniculatus . . . . 21

u. Fatty acid composition in various lipid fractions

from the second harvest of L. Cornicu;atgs. . . . 22

INTRODUCT ION

INTRODUCTION

The study of the lipids of birds-foot trefoil (Lotus
corniculatus variety viking) is not only an attempt to further
understand and investigate plant lipids but comprises one of
a series of investigations being conducted with this Species.
Prompted by the need for a perennial agricultural legume more
persistent than red clover or alfalfa and more drought-
resistant than white clover, birds-foot trefoil has been
receiving much attention not only in its development from
seed germination to maturity, but in its value as a possible
forage.

Over the past years great strides have been made in
understanding this legume. Many chemical analyses (1) have
been made, but information is insufficient for any real con-
clusion about its composition in comparison to alfalfa and
red clover. In zizg and in ELEEQ nutritional studies (2)‘
revealed that birds-foot trefoil was more nutritious than
many of the other legumes. Further information on its chemi-
cal composition has been reported in references 3-6.

The isolation and characterization of the lipids of
L. corniculatus and their fatty acids are essential steps in
studying its metabolism in rumen digestion. These lipids,

like many other plant lipids, have been difficult to separate

1

2
because of their similar prOperties. Application of improved
chromatographic methods of analysis have achieved the isola-
tion of several of these lipids from L. corniculatus, namely,
sterol esters, fatty acid methyl esters, triglycerides, free
fatty acids and diglycerides. A comparison of these lipids
in a first and second harvest of this forage was carried out
in this study and it represents the first attempt to analyze
its lipid content.

REVIEW OF LITERATURE

REVIEW OF LITERATURE

Lipids from numerous plants have been under investiga-
tion by many workers who attempted to isolate and characterize
the various plant lipids as well as to determine their biolo-
gical functions. The fatty acids associated with these
lipids are also being identified. Although the slow develOp-
ment of good techniques has; delayed progress in isolating
many of the lipids, recent advances in lipid research have
enabled much success.

Lepage gt al. (7) reported the isolation of the deacy-
lated product of plant sulfolipid extracted from alfalfa
leaves. On analysis it was found to be sulfo-deoxyhex0pyra-
nosyl glycerol. Further study of this lipid by O'Brien and
Benson (8) revealed that it contained approximately equal
amounts of palmitic and linolenic acids. Continued study by
the later investigators (8) showed the presence of two glyco-
lipids: galactosyl diglyceride and digalactosyl diglyceride.
The fatty acids of these two lipids were highly unsaturated
and contained 82-94% linolenic acid. Lepage (9) identified
phosphatidyl serine, phosphatidyl inositol, phosphatidyl
choline, phosphatidyl glycerol, digalactosyl glyceride,
phosphatidyl ethanolamine, monogalactosyl glyceride and

neutral lipids by thin-layer chromatography. The fatty acid

3

2+
analyses by Van Der Veen and Olcott (lO)wwre reported from
several lipid fractions. These fractions consisted largely
of linolenic acid.

On studying the acetone-soluble lipids of red clover,
Weenink (11) reported that they consisted largely of the
galactolipids: galactosyl-l-glycerol and digalactosyl-l-
glycerol linolenate. Like the galactolipids of alfalfa, they
contained 95% linolenic acid. Further work by Weenink (12)
indicated that these acetone-soluble lipids also contained
triglycerides, diglycerides, and sterol esters. Unlike the
investigations by Van Der Veen and Olcott (10) on alfalfa
linoleic acid was the major unsaturated acid in all the above
fractions. Over 50% of the fatty acids of the sterol esters
was palmitic acid.

Many other plant lipids have been studied for their
lipid constituents. Among them is the work on the fatty acid
composition of pasture grass lipids by Carton (13). The
latter forage was a mixture of rye, cocksfoot and timothy
grasses and their total fatty acids contained 61% linolenic,
13% linoleic and 15% palmitic acids. Shorland (14) found
marked differences between the fatty acids of the dialysable
lipids and the non-dialysable lipids in which linolenic acid
was largely concentrated and comprised 88% of the total fatty
acids. The lipids in this study were the acetone-soluble
lipids from rye, cocksfoot, white clover and rape grasses.
From lettuce and cabbage, Nichols (15) reported the separation
and identification of the leaf phOSpholipids in addition to

5

mono- and di-galactolipids, sterol esters and triglycerides.
The glycolipids in runner-bean leaves, namely mono- and
di-galactosyl diglycerides and lecithin were isolated and
characterized by Sastry and Kates (16). Analysis of their
fatty acids demonstrated that the glycolipids contained 96%
and 93% linolenic acid respectively, and the lecithin contained
27% palmitic, 36% linoleic and 24% linolenic acids. Further
work by the same investigators (1?) established that the
structures of the two glycolipids were 2, 3-di-O-linolenoyl-
l—O-fl «D-galactopyranosyl-D-glycerol and 2,3-di-O-linolenoyl-
l-O-fi (6-O-°<-D-galact0pyranosyl-D-galactopyranosyl)-D-
glycerol.

EXPERIMENTAL

EXPERIMENTAL

Preparation of Sample

Two samples of birds—foot trefoil (L. corniculatus),
were from the first harvest gathered on July A, 1962 and the
other from the second harvest reaped on July 16, 1962 at the
experimental farm of Michigan State University. After the
first cutting, the hay got wet on the field from rain. Both
samples were air dried in the field and baled after which it
was further dried in a barn dryer with heated air (38-650).
In the laboratory the samples were ground in a Wiley mill to
pass a 20 mesh seive and finally dried over phOSphoric acid
in a vacuum oven at #00. Samples were studied separately for

their lipid and fatty acid content.

Extraction of Lipids
The extraction of the lipids was done with two different
combinations of solvents and also with a single solvent; (1)
chloroform-methanol (2:1), (2) chloroform-iSOprOpanol (2:1)
and (3) methanol-free ethyl ether. L
Chloroform-Methanol: Chloroform-methanol was added to
the tissue in the ratio of 17 ml‘per gram of tissue and stirred
with a magnetic stirrer for 2h hrs. The mixture was filtered
through a fat-free filter paper and the extraction process was

repeated twice with the same solvent mixture. Extraction by

6

7
this process removed 95% of the lipids (18) and the other 5%
was finally extracted in a Soxhlet apparatus for 8 hrs, with
chloroform-methanol. All four extracts were combined and
taken to dryness in vagug. The inorganic salts that might
have been extracted with the lipids along with any other water
soluble materials were washed from the extract by dissolving
it in chloroform-methanol (2:1) and adding water to make up
20% of the total volume (19). The mixture was shaken in a
separatory funnel and allowed to settle in two layers. The
non-lipid materials were taken up in the aqueous layer.
Chloroformjlsopropanol and Ethyl Ether: One extraction
with chloroform-isopropanol followed the same procedure as
that described for the extraction with chloroform-methanol.
Another extraction followed the procedure used for the extrac-
tion with ethyl ether. Extraction with ethyl ether was done
in a Soxhlet apparatus for 2# hrs. No specific amount of
solvent was used in this method and the amount varied accord-

ing to the size of Soxhlet apparatus used.

Column Chromatography of Lipids

Two different size columns were employed for the partial
separation of the lipids and both were packed with a minus
325 mesh silicic acid (20). The larger column was 2.5 cm
inside diameter and packed with 36 gms of the silicic acid.
The extract from 10 gms. of tissue, after taken to dryness
in laggg, was dissolved in petroleum ether (B. R. 50-650)
and placed on top of the adsorbent in the large column. Since

the lipid extract was not too soluble in petroleum ether great

8
care was necessary to facilitate a quantitative transfer of the
contents to the column. This transfer of the extract was
accomplished by not drying the lipid extract completely
in gaggg. The addition of small quantities of petroleum ether
to the partially evaporated extract prevent the lipid material
from adhering to the walls of the flask while the first sol-
vent is removed. The total lipid was separated into three
fractions by eluting the column adsorbent containing the sam-
ple with 750 ml. of 1% ethyl ether in petroleum ether followed
by 130 ml.of 4% ethyl ether in petroleum ether, fraction 1;
650 ml. of ethyl ether, fraction 2; and 850 ml.of methanol,
fraction 3 (20). After the three fractions were taken to
dryness and analyzed by thin-layer chromatography, fractions
1 and 2 were further separated on a smaller column.

The smaller column was 1 cm inside diameter and was
packed with 18 gms of silicic acid. Fraction 1 in petroleum
ether was separated into two fractions by eluting the packing
plus sample with 350 ml.of 1% ethyl ether in petroleum ether,
fraction la; and #00 ml.of ethyl ether, fraction lb. If
fraction la and lb were not completely resolved after checking
it on thinulayer chromatography, complete resolution was
obtained by rechromatographing each fraction with the same
set of solvents on the same washed column. Fraction 2 in
petroleum ether was also separated into fractions by eluting
with 350 ml. #% ethyl ether in petroleum ether, fraction 2a;
and 350 ml. of ethyl ether, fraction 2b.

The larger columns with packingsIW£fi3reused by washing

them with 100 ml. of absolute methanol followed by 100 m1 of

ABBREVIATIONS FOR FLOW SHEET

SE ----- Sterol esters
ME ----- Fatty acid methyl esters
TG ----- Triglycerides
FA ----- Free fatty acids
F.A ----- Fatty acids
DG ----- Diglycerides
PL ----- PhOSpholipids
S ----- Sterols
P ----- Pigments
UK ----- Unknown

TLC ----- Thinelayer chromatography

EXTRACTION AND SEPARATION

TISSUE (L. Corn/bylaws)

EXTRACTION

I) CHLOROFORM-METHANOL (2zl)
2) CHLOROFORM-ISOPROPANOL (2zl)
3) ETHYL ETHER

 

I
LIPID EXTRACT

COLUMN CHROMATOGRAPHY
TLC ANALYSIS

 

 

I I I
FRACTION l FRACTION 2 FRACTION 3
(SE, ME,) (FA, 06,) (PL, P,)

TC, FA S, P, UK UK

COLUMN CHROMATOGRAPHY

TLC ANALYISIS

 

I 1
FRACTION 2b FRACTION 20
(S, P, UK) (FA, 06, S)

 

 

I I
FRACTION lo FRACTION lb
(SE, ME) (TO, FA)

TLC TLC TLC
I T I l I . I

SE ME TO FA FA 06 S

 

 

 

 

11
dried acetone, 100 m1 of anhydrous peroxide-free ethyl ether
and 100 ml.of petroleum ether (21). Smaller columns were
washed with proportional amount of the same solvents used

for the larger columns.

Thin-Layer Chromatography ofLiEids

Thin-layer plates were made of silica gel G and silica
gel H. Plates with silica gel G were used for qualitative
work, primarily for comparing Rf values of the lipids from
the extract with Rf values of known lipids to determine what
lipids were present in the lipid extract. The former plates
were made 0.25 mm thick and those with silica gel H were
0.5 mm thick. The silica gel H plates were used to quantita-
tively isolate and recover the individual lipids. A 10% solu-
tion of phosphomolybdic acid in ethanol was used as the indi-
catorjxlthequalitative work. When the plates were sprayed with
this indicator they were heated at 110°F for 20 to 30 minutes
or until all the spots become visible. In the quantitative
work the lipids could not be heated and the indicator used
was a 0.2% solution of 2',7'dichlorofluorescein in ethanol.
The spots were detected with the aid of a short-wave ultra-
violet light.

Fraction 1a was resolved by developing the spotted plate
in a solvent mixture of petroleum ether-ethyl ether-acetic
acid (9031031) (22). For the recovery of the lipids, one end
of each plate was spotted with the reference sample and the
other portion streaked with the lipid extract. After the-

plates were deveIOped only the end with the reference compound

12
was Sprayed with the indicator and the spots located. Indi-
vidual lipids from the extract were located according to
known Rf values from the references and they were scraped off
and extracted from the gel with chloroform. Fractions lb
and 2a did not give good resolutions in the above solvent
system or any other single solvent system tried. However,
good resolutions were obtained with two separate solvent
systems. This method is called two step thin-layer chromato-
graphy (23). First the plates were placed in isopropyl ether-
acetic acid (94:6) and the solventvas allowed to move half
way up the plate before ittwusremoved and air dried. The
plate was now placed in a different solvent system consisting
of petroleum ether-ethyl ether-acetic acid (9031031) and the
solvent permitted to move the full length of the plate. Iden-
tification and recovery of the individual lipids followed the
same procedure as described for fraction la. After each lipid
fraction was recovered it was rechromatographed on thinmlayer
plates with the same solvent systems that they were resolved
in as a means of purification.

Saponification and Recovery of Acids; The individual
lipids were saponified by refluxing with 100 m1 of 1.5 N
methanolic KOH for 6 to 8 hrs. The soaps were extracted
from the saponified mixture with water in a separatory funnel
and the water insoluble portions of the lipids were tested
for sterols and glycerol. In recovering the acids from the
soaps, the soaps were first acidified with RC1 in the water

and the fatty acids were extracted with petroleum ether,

13

(00»600). The petroleum ether extract was dried with anhydrous
sodium sulphate. After evaporation of the petroleum ether
under nitrogen the acids from the different fractions were
weighed.

Test for Sterols: (Liebermann-Burchard Reaction) (2“)
The alcohol portion of the lipid was dissolved in approximately
2 ml. of chloroform and a few drops of acetic anhydride were
added. The contents were mixed followed by the addition of a
few drOps of concentrated sulfuric acid. The final green
color“wm3a.positive test for the presence of sterols.

Test for Glycerol: To the alcohol portion of the lipid
in about 1 ml. of 50% ethanol was added 1 ml. of 95% ethanol.
Several dr0ps of 0.5 M_copper sulphate solution were than
added until there was approximately a 2:1 ratio of copper ions
to the glycerol. The addition of a few drops of 0.1 N_sodium
hydroxide forms a green precipitate whichwas a chelate of

glycerol and COpper (2Q).

Preparation of Methyl Esters

The fatty acids were methylated by adding 3 ml. of boron
trifluoride-methanol reagent to 100 to 200 mg. of the acids
and boiling the mixture on a steam bath for 2 minutes (25).
After the addition of approximately 15 ml. of water to the
boiled mixture itTmnstransferred to a separatory funnel. The
fatty acid methyl esters were recovered by extracting the
mixture with petroleum ether (40-600) and drying this fraction
with anhydrous sodium sulphate. The petroleum ether was.

evaporated under nitrogen and the fatty acid methyl esters

SAPONIFICATION AND F.A. ANALYSIS
LIPID FRACTIONS
(SE, TG, FA, 06)

SAPONIFIED INDIVIDUALLY
WITH LSN KOH (METHANOL)

 

I e I
STEROLS SOAPS OF
(;_YCEROL FATTY ACIDS
STEROL TEST CONC. HCI
LIEBERMAN - BURCHARD
REACTION 1

GLYCEROL TEST

COPPER-GLYCEROL FATTY ACIDS
COMPLEX I

BORON TRIFLUORIDE-
METHANOL REAGENT

I

FATTY ACID
METHYL ESTE RS

GAS-LIQUID
CHROMATOGRAPHY
ANALYSIS

15
dissolved in chloroform for gas-liquid chromatography analysis.
Conditions for Gas-liquid Chromatography.
Length of column-~6 ft.
Inside diameter of column--4 mm.
Packing:
Solid support--diat0port S.
Stationary phase--diethylene glycol succinate coated 15%
Mesh--80 to 100
Carrier gas~-helium
Flow rate of carrier gas--M5 ml/min
Gas pressure at outlets--40 psig
Temperature of oven~~l95o
Temperature of detector--220o
Temperature of injection port--2h0o
Sensitivity of Recorder
Range--10
Attenuation 32

Dector~~flame ionization dector

The gas~1iquid chromatograph was a F & M Biomedical
model #00. Reference samples were obtained from Applied

Science Laboratories, State College, Pa.

RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

The extraction of the second harvest of ngug corniculatus
with chloroform-methanol gave 7.7 mg.total fatty acids per
gram of tissue, chloroform-isopropanol gave 7.5 mg, and ethyl
ether gave 7.8 mg. The values of the first two solvent mix-
tures were the averages from three sets of extractions each
and the value from the ethyl ether extract was the average
from five extractions. All three solvents were effective for
extracting the lipids from the tissue and further proof was
given when a thin-layer chromatographic assay showed that the
lipid classes from the extracts were the same both in amount
and prOportion. Chloroform-methanol extracts were used in
this work because most of the lipid was extracted before heat
was applied. This included extractions from both the first
and second harvest.

On partial separation of the total lipids into three
fractions, 1, 2 and 3, thin-layer chromatographic analysis
showed that fraction 1 consisted of sterol esters, fatty acid
methyl esters, triglycerides and free fatty acids. Fraction
2 consisted of free fatty acids, diglycerides, sterols and
other unknown Spots along with pigments; and fraction 3 con-
sisted of phOSpholipids and pigments. Fraction 1 and 2 were
further separated on column to yield four fractions, 1a,.1b,

2a and 2b. Analysis of these fractions showed that, fraction
16

17
la contained sterol esters and fatty acid methyl esters;
fraction 1b contained triglycerides and free fatty acids;
fraction 2a contained free fatty acids, diglycerides and traces
of sterols; and fraction 2b contained sterols and other
unknown spots along with pigments. The latter fractions
proved very difficult to separate on the column even when
many different polarities of solvent were used. Thin-layer
chromatography, however, gave good resolutions and all the
non~acidic lipids identified were separated and collected
individually by this method.

The fatty acid methyl ester fraction was verified by
gas~liquid chromatography, then purified by thin-layer chroma-
tography and weighed. The fractions of sterol esters, trigly-
cerides, diglycerides and free fatty acids were saponified
and after the soaps were extracted the other portions of the
lipids were tested for sterols and glycerol. Table 1 showed
that only one fraction contained sterols and two fractions
contained glycerol. The result of these tests confirmed the
content of each fraction. The phospholipid fraction was also
saponified and after the soaps from all the fractions were
acidified they were weighed. The weight of these fatty acids,
including the extracted methyl esters, was used to represent
the amount of lipid classes present. Their percentages and
the weight of the total fatty acids are given in Table 2 for
both the first and second harvest. The sterol esters, fatty
acid methyl esters, triglycerides and diglycerides decreased

in the second harvest as compared to the first. In like manner

the total phospholipids and free fatty acids increased in the

18

TABLE I

Sterols and Glycerol Present in the

Various Lipid Fractions

 

 

Fractions Sterol Triglyceride Free Diglyceride
Ester Fatty
Acid
Test for + - - -
Sterol
Test for - + - +

Glycerol

 

19

TABLE 2

PERCENT OF FATTY ACIDs IN EACH LIPID FRACTION“

 

lst harvest 2nd harvest
Sterol esters 16.2 8.0
Methyl esters 9.7 4.8
Triglycerides 12.5 8.8
Free fatty acids 19.2 22.h
Diglycerides 11.8 7.2
Phospholipids 30.6 #8.?

 

*Totai fatty acids for let and 2nd harvests were 715 and

788 mg. per 100 g. of dried sample, respectively.

20
second harvest. In the first harvest the phospholipids and
free fatty acids comprised 50% of the total lipids and in the
second harvest they comprised 70%.

The fatty acids from all fractions were analyzed after
being methylated with boron trifluoride-methanol reagent and
the results from the first and second harvest appear in
Tables 3 and A. The acids present in both harvest are lauric,
myristic, myristoleic, palmitic, palmitoleic, stearic, linoleic,
linolenic,arachidic, behenic and lignoceric. No attempt was
made to identify acids of chain length less than lauric acid.
In the first harvest there was 38% saturated acids and 62%
unsaturated acids. In the second harvest these values were
92% and 108% of the values of the first harvest, respectively.
Although the total unsaturated acids increased, the concentra-
tion of linoleic acid in the second harvest was 85% of the
value for the first harvest. Similar values for palmitic
and linolenic acids in the second harvest were 86 and 117%,
respectively. Palmitic, linoleic and linolenic acids were
the major acids of both samples.

Fatty acid composition of the individual lipid classes
were markedly different. In the sterol ester fraction palmitic,
linoleic and linolenic acids were the major components. For
the first harvest palmitic comprised 38% of the total fatty
acid of this lipid, linoleic 17% and linolenic 2#%, however,
the values for the second harvest as a percentage value of the
first were palmitic-6A, linoleic-87 and linolenic-18h. For!

the fatty acids in the methyl ester fraction myristic, palmitic,

21

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22

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23

linoleic and linolenic were predominant. In the second har-
vest palmitic acid decreased 41% and linolenic acid increased
31% while the other major acids remained approximately the
same in both cases. Fatty acid distribution in the trigly-
ceride fraction did not follow the pattern of the above two
lipids. Myristic, palmitic, linoleic and linolenic acids
were of greatest concentration in the first harvest while
palmitic, stearic and linoleic acid predominated in the second
harvest. Unlike the two latter fractions the saturated acids
in the triglyceride fraction were greater in concentration
than the unsaturated acids. The fatty acid composition of
the free fatty acid fraction was very similar for both the
first and second harvest of the forages. The principal acids
were palmitic, linoleic and linolenic and these acids made up
over 80% of the total acids. On the diglyceride fraction the
saturated acids again predominated with myristic acid com-
prising 50% of the total acids present in the first harvest.
In the second harvest there was a 30% increase in the propor-
tion of myristic acid. The phOSpholipid fraction of the
first harvest contained 44% myristic and 39% linolenic acids,
but for the second harvest it contained 30% palmitic and 49%
linolenic acids. There was no analysis for the individual
phospholipid in the phospholipid fraction.

Any nutritional implication of these findings must be
speculative at this time. However, these results indicate that
the biologically active fatty acids may vary in the different

harvests of this and probably other forages and that their

proportions varies considerably between different lipid fractions.

SUMMARY

til

SUMMARY

The neutral and non-acidic lipids of birds-foot trefoil
(L. corniculatus) and their associated fatty acids have been
studied with the use of column, thin-layer and gas-liquid
chromatography. Sterol esters, fatty acid methyl esters,‘
triglycerides, free fatty acids and diglycerides were the
lipids that were isolated and identified. A study of the
total fatty acids from the tissue showed the presence of
lauric, myristic, myristoleic, palmitic, palmitoleic, stearic,
oleic, linoleic, linolenic, arachidic, behenic and lignoceric
acids. The major acids were palmitic, linoleic and linolenic.

A comparison of the first and second harvest was made
to note the differences in the lipids and their fatty acid

composition in the different harvests.

24

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