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ABSTRACT

A STUDY OF THE LIPID FRACTIONS
IN THE POLLEN OF ZEA MAYS

by Amir Fathipour

A considerable number of papers have been published
on the chemical constituents of pollens. Analyses have
been made largely for minerals, amino acids, pigments, car-
bohydrates, vitamins and lipids. In the study of the latter
substances many of the fatty acids and lipids were not
isolated and characterized due to the lack of prOper methods.
However, the development of gas-liquid chromatography and
improved techniques in column and thin-layer chromatography
have greatly facilitated the isolation and identification
of lipids. The methods previously mentioned, were employed
to study the lipids in the pollen of ZSE.E§X§°

Complete extraction of the lipid material was accom-
plished by extracting the corn pollen with a boiling mixture
of chloroform-isopropanol (1:1) v/v solution. After extrac-
tion, the solution was evaporated to a small volume in vagug
and the concentrated extract was diluted with chloroform
and water. The chloroform layer was removed, washed with
water and evaporated to dryness. Further resolution was

made by dissolving the residue in chloroform and separating

Amir Fathipour

the extract by column chromatography into phosphatides and

non phosphatides. Fractionation of the nonphosphatides by
thin—layer chromatography yielded sterol esters, triglycerides,
free fatty acids, free sterols, methyl esters of fatty acids
and other glycerides. Identification of the individual fatty
acids in each fraction and their concentration were obtained
after transmethylation by gas-liquid chromatography. Lauric,
myristic, myristoleic,arachidic and behenic acids were charac-
terized for the first time. Other acids,also identified and
previously reported,were palmitic, palmitoleic, stearic,

oleic, linoleic and linolenic acids.

A STUDY OF THE LIPID FRACTIONS IN

THE POLLEN OF ZEA MAYS
By

Amir Fathipour

A THESIS

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

MASTER OF SCIENCE

Department of Biochemistry

1966

ACKNOWLEDGMENTS

The author is grateful to Professor Harold M. Sell
for his encouragement and supervision throughout the
course of this work. His thanks go to Mr. Roger W. Ritzert
and Mr. Keith K. Schlender for their wonderful discussions
and suggestions. The author is also thankful to Miss
Katherine Liang and Mr. Morris D. S. Richards for their
help in the preparation of the manuscript. He is indebted
to Mrs. Gerda Schrubkowski for her_help throughout the

preparation of the thesis.

ii

ACKNOWLEDGMENTS.
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION.

HISTORICAL .

TABLE OF CONTENTS

Storage and Viability . . .
Culture. . . . . .
Chemistry of Pollen. . . .
Nutritive Value of Pollen. .
Harmones and Related Substances
Enzymes. . . . .
Carbohydrates and Lipids . .

Pigments . . . . . .
EXPERIMENTAL. . . . . . .

Materials . . . . . . .

Extraction of Lipids . . . .

Column Chromatography Fractionation . .

Preparation of Silicic Acid—Celite

Column Packing. . . . .
Application of the Sample . .

Thin—layer Chromtography and Fractionation of Non-

Phosphatides . . .

Mixture

Preparation of Thin—Layer Chromatography Plates.

Application of the Sample . .

Purification of Methyl Esters by Thin-Layer

Chromatography. . . . . .
Sterol Esters . . . . .
Purification of Methyl Esters by
graphy .
Preparation of the Adsorbent .
Column Chromatography . . .
Separation of Triglycerides .

iii

Column Chromato—

0 O O O O O O O

Page

11

vi

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HFJF‘
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HFJF’
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U'II'UKOKO

Free Fatty Acids. .

Mono—and/or Diglycerides .

Phospholipids. . . .
Total Fatty Acids .
Gas—Liquid Chromatography.
Results and Discussions

SUMMARY . . . . . .

BIBLIOGRAPHY. . . . .

iv

LIST OF TABLES

Table

Page

1. Comparison of Various Methods of Extraction of

Lipids from Corn Pollen. . . . . ll
2. Fractionation of Column Chromatography of the

Lipid Extract of Corn Pollen . . . . lu
3. Distribution of the Methyl Esters of Fatty Acids

in Corn Pollen. . . . . . . . . . . . 19
4. Distribution of the Methylated Fatty Acids From

the Sterol Esters in Corn Pollen. . . . . 22
5. Distribution of the Methylated Fatty Acids from

the Triglycerides in Corn Pollen. . . . . . 25
6. Distribution of the Methylated Free Fatty Acids

in Corn Pollen. . . . . . . 26
7. Distribution of Methylated Fatty Acids from the

Mono- and/or Diglycerides in Corn Pollen . . . 29
8. Distribution of Methylated Fatty Acids from the

Phospholipids in Corn Pollen . . . . . . . 32
9. Distribution of the Total Methylated Fatty Acids

in Corn Pollen. . . . . . . . . 33

LIST OF FIGURES

Figure

l.

Thin-layer Chromatographic Separation of
Different Classes of NonphOSphatides
on Silica Gel G. . . . . .

o o o o p

Gas——1iquid Chromatography of Naturally Occurring
Fatty Acid Methyl Esters. . . .

Gas— liquid Chromatography of Fatty Acids from
the Sterol Esters .

Gas— —liquid Chromatography of Methyl Esters from
the Free Fatty Acids . . . .
Gas— —1iquid Chromatographic Analysis of the Fatty
Acids from the Mono- and/or Diglycerides and
the Phospholipids . . . . . .
Gas- liquid Chromatographic Analysis of the Total
Fatty Acids. . . .

IR Spectrum of Naturally Occurring Methyl Esters

vi

Page

15

2O

23

27

30

3A
39

INTRODUCTION

Pollen plays an important role in the life cycle of
plants and has interested scientists for many years to learn
more about the physiology and biochemistry of pollen as it
relates to fertilization. The importance of pollen in the
production of food products has aroused many physiologists
and became one of the first of the natural products to be
investigated.

Horticulturist and plant breeders have cross-bred many
varieties, Species and genera to produce new and improved
types of plants better suited for food. Entomologists.have
been interested in pollen since it is the chief source of
all nutrients required by bees. Tsitin (H8) claimed that
the longevity of most of the centenarians in Russia depended
on their diet of honey.

In recent years pollen research has been influenced
largely by the fact that it is responsible for certain
allergies. Consequently, much of the recent literature is
devoted to the studies of these allergies.

Analysis of a large number of different pollen for
specific materials have been reported, but only thirteen
pollens have had relatively complete analysis (49). The
fatty acid content of the pollen of £33 gays was studied by
Barr gt al. (A) but the analyses were incomplete because

1

modern techniques were unavailable. The development of
thin-layer and gas—liquid chromatography and the improved
techniques in column chromatography have enabled scientists
to determine and fractionate the lipids and their fatty
acids in biological materials with a considerable degree
of accuracy. This present work is an extension of the
work by Barr et 91, (A) for the determination of the
lipids and associated fatty acids in the §2a_May§ of

pollen.

HISTORICAL

Storage and Viability

 

Systematic studies of the storage of pollen were
started in 1886 by Mangin (26). Johri and Vasil (1?),
reviewed the literature on the effect of temperature,
relative humidity, gases, and pressures on the viability
of stored pollen. They concluded that low temperatures
and a relative humidity of 25 to 35 percent are necessary
to retain viability for a prolonged period. The viability

of stored corn pollen has been reviewed by Andronescu (2).

Culture

The early history of pollen tube physiology was
reviewed in 1961 by Johri and Vasil (17) who described
the effects of pH, sugars, hormones, vitamins, carotenoids,
antibiotics, gibberellic acid and inorganic salts on the
culture of pollen. These substances are known to improve
germination in many cases.

Boron is the most effective inorganic salt. The effect
of boron on pollen germination and on elongation of tubes is
more marked than that of any known hormone, vitamin or
chemical. It increases absorption of sugars, aids in their

metabolism by forming a sugar-borate complex, increases the

3

oxygen uptake, and is involved in the synthesis of pectic
materials required for the wall of actively elongating
pollen tubes. Pollen of most species seems to be naturally

deficient in boron.

Chemistry of the Pollen

 

In comparison with the number of studies on the storage
and culture of pollen, very little work has been done on the
chemistry of this substance. The major limitation in this
field is the difficulty in obtaining sufficient quantities
of uniform samples. Consequently, studies have been re—
stricted to plants producing relatively large amounts of
pollen: e.g. Pinu§_and gag Téli- Nevertheless, the chem?
istry of pollen is gaining attention, partly, because of
its importance in physiological studies and also due to its
nutritional and medicinal value. Lunden (2A), and Johri
and Vasil (17) have written short review articles on the
chemistry and amino acids of pollen.

The protein content of pollen is of interest to medical
workers as an allergenic factor and to beekeepers as a
source of food for better honey production. Chromato—
graphic techniques have provided very helpful quantitative
and qualitative methods for the determination of amino acids.
These analytical techniques indicate that the content of
amino acids of pollen proteins varies from Species to

species and may be as high as 35%, as reported by Todd and

Bretherick (49). Almost all the common amino acids were from
pollen grains, either in the free state or bound to protein.
The presence of phenylalanine, tryptophan, hydroxyproline,
tyrosine and aminobutyric acid is not very common, but a
high proline content is reported by Bellartz (6) and
Bathurst (5). Weaver and Kuiken (51) studied the amino acid
composition of pollen protein and that of soybean flour,
casein and whole egg powder. They did not show any marked
differences. The histidine-proline quotient in pollen (50)
probably represents an important factor in pollen fertility.
Ray Sarkar gt a1. (39) determined the amino acid composition
of sweet corn pollen.

The proteins of pollen have fairly low molecular weights
(5000). Some are combined with sugar. Free-proteins and
those combined with sugars or pigments (artefolin, trifidin,
and pratensin) have been isolated and purified. Nucleopro—
teins have also been reported, however, more work appears
necessary (24).

Stanley (45) recorded an interesting observation con-
cerning deoxyribonucleic acid turnover in pine pollen.
Deoxyribonucleic acid is known to be synthesized when cells
are ready to divide; however, Stanley presents data to con—
firm the presence of deoxyribonucleic acid in vegetative

nuclei of pollen nuclei which do not divide.

Nutritive Value of Pollen

Pollen grains appear to be highly nutritive materials
as far as water soluble vitamins are concerned. The pollens
of gymnosperms have a low vitamin content, whereas, those of
angiosperms are rich in vitamins of the B complex but poor
in fat-soluble vitamins (36). Biotin, folic acid, inositol,
nicotinic acid, pantothenic acid, pyridoxine, riboflavin,
thiamine, ascorbic acid, and vitamins A, D, E, and K have
been reported in the pollen of maize, date, plum and many
other plants (42).

Folic Acid has been reported in the pollen of Egg mgyg
and many other plants by Nielsen and Homstrom (34). This
acid appears to be a very important component of the so—

called Vitamin T a growth factor for insects. Kakhidze

l’
and Medvedyeva (18) observed that the pollen of tobacco
excretes considerable quantities of vitamins into artificial
germinating media. Nielsen (35) indicated that while most
of the vitamins from the pollen of several species including
Egg mgyg did not show any appreciable change after a year

of storage (in a cool, dry place) the panthothenic acid con—

tent had substantially decreased in all of the samples.
Hormones and Related Substances

Estrone and estrogenic substances have been reported

in the pollen of Salix and date palm by Lunden (24) and also

by Hassan and Aboul Wafa (15). Anderson (1) observed a
mixture of phytosterols in corn pollen.

Mitchell and Whitehead (30), Wittwer (52), and
Redemann (40), reported growth promoting substances from
corn pollen which gave a positive Azggg response. Others
have reported similar substances from many other pollens.
Fukui g3 g1. (13) have reported 3—indoleacetic acid and two
other unidentified growth promoting substances from the
acidic fraction of ether extract of corn pollen.

Tanaka (46) believes that the slow growth of the pollen

tube of Pinus densiflora may be due to the presence of

 

inhibitors in its own pollen.

Enzymes

Pollen tubes metabolize externally supplied sugars and
digest the food reserve while passing through the tissues of
the style which indicate that they secrete enzymes (37). As
expected, the presence of many enzymes and coenzymes have
been reported from the pollen of different sources including

maize (24, 14, 40, 34).

 

Carbohydrates and Lipids

Miyake (31, 32) determined the carbohydrate content of
corn pollen. Nielsen _g _l. (36) evaluated the reducing

sugars from the pollen of several species including Zea mays.

Mameli (25) reports that generally anemophilous plants

(wind pollinated) have starchy pollen. Those of entomo-
philous plants are rich in fat and sugar which serve as a
nutrient for insects, but this hypothesis has not yet been
completely confirmed. Heyl (16) showed the presence of
formic, acetic, valeric, a C10 unsaturated, myristic,
palmitic and oleic acids in corn pollen. A high content
of heptacosane is indicated in sugar beet pollen (20). In

hazel pollen, a C acid, palmitic acid, tricosane, hexa-

l2
decanol, and two unsaturated sterols have been found (44).
Tappi and Monzani (47) isolated and identified heptacosane,

sitosterol and phytofluene from the pollen of Lilum candidum.

 

Mariella g2 gl. (28) have separated seven different com—
pounds from the unsaponifiable fraction of ragweed pollen.
Barr _g _i. (4) obtained the following fatty acids from the
saponifiable fraction of the ether extract of corn pollen:
palmitic, stearic, oleic, linoleic and linolenic. Phytos-
terol palmitate, a saturated hydrocarbon, a saturated

alcohol and lecithin have been shown to be present in corn

pollen (l, 31).

Pigments

Most of the pigments are located in the holes of
pollen in the form of oil and fats. These pigments serve
as a filter to protect the germinating power of the pollen
from ultraviolet light. The pollens of wind pollinated
flowers rarely contain carotenoids. Redemann (40) reported

quercetin in the pollen of ea mays.

“—

EXPERIMENTAL

Materials

 

Pollen was obtained by surrounding the tassels of
sweet corn with paper bags and collecting the pollen from
them after two days. The fresh pollen was screened through
a 100~mesh sieve and immediately stored in plastic bags

at -20°C.

Extraction of Lipids

 

Several methods have been described for extracting
lipids from biological materials (10). A standard method
of fat extraction with ethyl ether is described (3), but
in the case of corn pollen complete extraction of lipid was
not accomplished with this procedure, as was shown by a
quantitative determination of the extract. Anderson (1)
used ohloroform and ethanol to extract lipids from corn
pollen and he reported 13.88% total fat obtained from corn
pollen (dry weight basis). Barr g3 gl. (4) using diethyl
ether as the extracting solvent reported only about 4% as
the amount of fat present in corn pollen. This contradic—
tory result indicated that diethyl ether does not extract
the lipid completely from the tissues. Although tissue
extract obtained by using chloroform-methanol as the

9

IO

extracting solvent is known to give complete extraction of
lipid materials (7), prolonged standing of tissue in
methanol is known to cause transmethylation because of the
presence of sodium carbonate in the tissue (23). Diethyl
ether being a solvent of low polarity does not permeate the
tissue sufficiently to carry polar lipids through the cell
membrane quantitatively. The same is true of chloroform

or petroleum ether as extractants. Extraction with
chloroform-is0propanol gives quantitatively the same amount
of extract as chloroform-methanol as shown by gas—liquid
chromatographic analysis. The use of isoprOpanol eliminates
the possibility of transmethylation. Table 1 summarizes
the data obtained by different extraction methods.

The extraction procedure finally adopted was as
follows: twenty gm. of lyOphillized polled was extracted
with 200 ml. boiling iSOprOpanol (freshly redistilled) for
three minutes on a steam-bath. The suspension was filtered
through a fat—free filter paper and the pollen re—extracted
twice in the same manner (19). The pollen residue was ex-
tracted with boiling chloroform-isoprOpanol (lzl) v/v. three
times. Each time the solution was filtered through a fat—
free filter paper and the pollen re-extracted. The final
residue on the filter paper was then washed with 200 m1. of

chloroform.* The combined filtrates were concentrated

 

*Chloroform was redistilled in a 6 ft. Stedman Column,
washed with one-half its volume of distilled water 5 times
in a separatory funnel, dried over CaCl2 over-night, and

redistilled over P205 (41).

ll

TABLE 1

COMPARISON OF VARIOUS METHODS OF EXTRACTION

 

 

OF LIPIDS FROM CORN POLLEN
Weight Weight
Extraction of Extract of Extract
Solvent Method (e) g. %
Carbontetra— Soxhlet
chloride 30 hrs. 0.802 1.61
Chloroform Soxhlet
(Alcohol—Free) 30 hrs. 2.015 4.03
Ethyl Ether Soxhlet
(Alcohol-Free) 30 hrs. 1.912 3.82
Ethyl Ether Macerated
(2% Alcohol) twice in
24 hrs. 2.179 4.36
Chloroform— Maceration (a)
Methanol (2:1) and Soxhlet 7.108 13 85 (b)
Chloroform-
Isopropanol (c) 6.820 13'54 (d)

 

(a) Method of Bottcher gt 1. (7)

(b) Weight percent after washing according to Folch g3

(12).

(c) Extracted according to Kates and Eberhardt (19).

(d) After being washed according to Kates and Eberhardt

(19).

(e) From 50 g. of pollen.

12

under vacuum in a flash evaporator to 40 ml., diluted with
200 ml. of chloroform and added dropwise in a separatory
funnel to 200 ml. of water. The chloroform layer was
separated and the water layer was washed four times with
50 ml. of chloroform. The combined chloroform layer was
evaporated to dryness under vacuo in a flash evaporator.
The residue was dissolved in 20 ml. of chloroform (each ml.

equivalent to 1 g. original material).

Column Chromatggraphy Fractionation

Preparation of Silicic Acid-Celite Mixture

200 gm. silicic acid (Mallinckrodt) and 50 g. celite
(lOO—mesh) (John Manville Corporation) were mixed and
washed in a Buchner funnel with 100 ml. of chloroform, then
with 1000 ml. of methanol, and finally with 1000 m1. of
distilled water (9). The silicic acid-celite mixture was
dried and heated at llO-l20° C. for four hrs. (22) and

then stored in.a vacuum desiccator.

Qolumn Packing

A slurry was made by adding 75 g. of silicic acid-
celite mixture to 100 ml. of chloroform. A 2.8 cm. (i.d.)
glass column fitted with a Kimax stOpcock and sintered
glass disk was used. The slurry was poured slowly into the
column by means of a long stem glass funnel with constant
stirring by use of an electric motor connected to a long

glass rod. The column was left to settle for one hr. and

13

was washed with 200 ml. of chloroform at the rate of
2 mL/min. (under low nitrogen pressure) with prOper manipu-

lation of the stOpcock.

Application of the Sample

Ten ml. of the chloroform solution of the pollen
extract was applied to the top of the adsorbent in the
column by means of a long stem funnel (avoiding contamination
of the side of the column). After the sample was adsorbed
into packing, the side of the column was washed with 20 ml.
of chloroform. Then the adsorbent was eluted 18 times the
column volume with chloroform (1800 ml.) The adsorbent was
further washed with 50 ml. of chloroform and the latter
fraction concentrated to 0.5 ml. The completeness of the
elution of nonphOSphatides was checked in this fraction by
means of thin-layer chromatography and the Liebermann-
Burchard reaction for sterols. The result of the separation

of the lipid material into two fractions by column chroma-

tography is given in Table 2.

Thin-Layer Chromatography and Fractionation
of Nonphosphatides
Preparation of Thin-layer Chromatography Plates
Thin—layer plates having a dimension of 20 x 20 cm.
were employed in this experiment. Before analysis the
plates were scoured with Ajax detergent under running warm

water, soaked in cleaning solution over night, rinsed under

14

TABLE 2

FRACTIONATION BY COLUMN CHROMATOGRAPHY
OF THE LIPID EXTRACT OF
CORN POLLEN

 

 

 

Volume
of
Eluent Total
Fraction Elution Collected Weight Extract
# Solvent ml. mg. %
l Chloroform 1800 395 28.95
2 Methanol 1000 908 66.56
TOTAL 95.51

 

tap water and distilled water and finally dried in the

oven (38).
Thirty-two g. of silica—gel G was added to 65 ml. of

distilled water to make a slurry and then shaken for 30 sec.
The plates were coated to a thickness of 250—300 microns
with an applicator, dried in air, and then activated in a

preheated oven at 1100 C. for 20 min. prior to use (33).

Application of the Sample
The sample, 138.25 mg. of lipid in chloroform, was

applied as streaks to the plates. Figure 1 indicates the
separation of different classes of lipid extract obtained

by different extraction methods from corn pollen.

FIGURE 1
THIN—LAYER CHROMATOGRAPHIC SEPARATION OF
DIFFERENT CLASSES OF NONPHOSPHATIDES
ON SILICA GEL G

The sample was resolved in a solvent system of petro—
1eum ether—ethyl ether-acetic acid (84:16:l v/v) for 65 min.
for a distance of 15 cm.

The columns represent from left to right: extract
from chloroform—isopropanol applied as streaks; extract from
carbon tetrachloride; standards; extract from chloroform-
isopropanol; purified fatty acid methyl esters; and extract
from chloroform-methanol.

The standards (third column from left) are, from bottom
to top, sterol esters, methyl esters of fatty acids,

triglycerides, and free fatty acids.

 

 

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Chromatograms were developed in freshly redistilled
light petroleum ether (40-60° C.), ethyl ether* and acetic
acid. The ratio of the mixture differed according to the
compounds to be separated. For resolution of sterol esters
from the methyl ester of fatty acids, ethylene-dichloride
was used as the developing solvent (38).

Separation of different classes of nonphospholipids
was accomplished by streaking 100 ml. of extract on each
plate and developing the plate in petroleum ether (40-60° C.)
diethyl ether and acetic acid (90:lO:l) (27). The plates
were air dried for 15 minutes and sprayed with 0.2% solution
of 2'7'-dichloro-fluoresceine in 95% ethanol. The spots
were observed under short wave length U.V. light (27).

Under the above conditions the separation of the
fatty acid methyl esters and the sterol esters were not
completely resolved. The methyl esters after being scraped
off the plates were highly contaminated with the sterol
esters and further fractionation was necessary.

Purification of Methyl Esters by
Thin—layer Chromatography

 

 

For further purification of methyl ester fraction
from the sterol esters, the adsorbent was washed with

chloroform on a Buchner funnel; the filtrate was evaporated

 

*To avoid formation of any ester in the presence of
methanol and/or ethanol in ethyl ether, ethyl ether (Mallin-
krodt) was washed in a separatory funnel with 1/3 of its
volume of a 10% solution of NaCl (21), dried over CaC12
overnight, and redistilled.

18

under vacuo to a very small volume, and rechromatographed
by thin—layer chromatography using ethylenedichloride as the

developing solvent. Streaks showing correSponding R values

f
to known methyl esters were scraped off and extracted with
chloroform and filtered on a Buchner funnel. The filtrate
was taken to dryness and adjusted to a known volume before
chromatographed on a gas-liquid chromatograph for analysis
of its fatty acids content. The various acids are listed

in Table 3 and are also represented on the chromatogram,

Figure 2.

Sterol Esters

 

The combined adsorbent containing the sterol esters
was removed from the plates and transferred directly to a
50 ml. round bottom flask. Methylation was accomplished
by addition of 15 ml. of 10% solution of concentrated
sulfuric acid in dry methanol, and refluxing for one hr. in
the presence of hydroquinone (8). After methylation the
mixture was transferred to a separatory funnel with 15 ml.
of distilled water and the methyl esters were extracted
twice with 30 ml. of light petroleum ether (40—60° C.)

The combined petroleum ether layers were washed with
30 m1. of water, dried over sodium sulfate-sodium bicarbonate
(4:1), and the solvent evaporated. The methyl esters
obtained in this manner were further purified by column
chromatography (19) before being studied on gas-liquid

chromatography.

19

TABLE 3

DISTRIBUTION OF THE METHYL ESTERS OF FATTY
ACIDS IN CORN POLLEN

 

 

Methyl
Esters ug./g. %
Laurate 2.21 0.86
Myristate 5.12 2.01
Palmitate 162.62 63.82
Stearate 2.55 1.00
Oleate 5.20 2.04
Linoleate 9.25 3-63
Linolenate 65.47 25.69
Archidate 2.27 0.89
Behenate 0.20 0.07
TOTAL 254.80 100.01

Purification of Methyl Esters by Column
Chromatography

Preparation of the Adsorbent

Eight g. of silicic acid and 2 g. of celite were
mixed and activated at 110-120° C. for four hrs., then trans-
ferred to a crystallizing dish, and placed in a desiccator
containing 4 ml. of water. The adsorbent after being equi-

librated overnight was transferred to a sealed bottle (19).

20

FIGURE 2

GAS-LIQUID CHROMATOGRAPHY OF NATURALLY
OCCURRING FATTY ACID METHYL ESTERS

A, represent the sample and B, standards. The peaks
in the standards are, from left to right, methyl esters of
lauric, myristic, myristoleic, palmitic, palmitoleic,

stearic, oleic, linoleic, arachidic, cis-eicos-5-enoic,

linolenic, and behenic acids.

21

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22

Column Chromatography
A slurry was prepared by mixing 10 g. of silicic acid-

celite (4:1) and 30 ml. of light petroleum ether (40-609 C.).
The contents were poured into a 2 cm. (i.d.) column and
washed with 100 ml. of petroleum ether. The sample of
impure methyl esters was placed on the tOp of the adsorbent
in the column and the column packing was subsequently eluted
with 250 ml. of petroleum ether. The eluate was concentrated
to 2 ml. and subjected to gas-liquid chromatography. The

results are given in Table 4 and Figure 3.

TABLE 4

DISTRIBUTION OF THE METHYLATED FATTY
ACIDS FROM THE STEROL ESTERS
IN CORN POLLEN

 

 

Methyl Esters ug-/8- %
Palmitate 555-5 19-05
Stearate Trace ---
Oleate 125.0 4.29
Linoleate 546.8 18.74
Linolenate 1686.0 57-84
Arachidate Trace

 

TOTAL 2913.2 99-93

23

FIGURE 3

GAS—LIQUID CHROMATOGRAPHY OF FATTY ACIDS
FROM THE STEROL ESTERS

A, represent sample and B, standards. The peaks in
the standard represent, from left to right, methyl esters
of lauric, myristic, myristoleic, palmitic, palmitoleic,
stearic, oleic, linoleic, arachidic, eis-eicos-S—enoic,

linolenic, and behenic acids.

24

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. _
9»
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_ , . A

+..v» 9.. off.
,

 

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(no

 

 

 

 

 

25

Separation of Triglycerides

Triglycerides separated in the same manner as
mentioned for sterol esters were contaminated with free
fatty acids, and further purification was necessary.

The solvent system which gave the best resolution of
triglycerides and free fatty acids was petroleum ether,
ethyl ether and acetic acid (82:18:1) (43). The triglyceride
fraction obtained from first thin-layer chromatogram was re-
chromatographed in this solvent system. The triglycerides
separated in this manner were directly trans-methylated in
the presence of hydroquinone (8), as mentioned before, and
then the methyl esters were extracted and purified before
being analyzed with the use of the gas-liquid chromatograph.
The fatty acids present in the triglyceride fraction are

given in Table 5.

TABLE 5

DISTRIBUTION OF THE METHYLATED FATTY ACIDS
FROM THE TRIGLYCERIDES IN CORN POLLEN

T

 

ug./g. of
Methyl Esters Pollen %
Myristate 5.85 0.58
Palmitate 862.96 86.05
Stearate 22.55 2.24
Oleate 23.52 2.34
Linoleate 23.30 '2.32
Linolenate 48.71 4.85
Arachidate 15.91 1.58

 

TOTAL 1002.80 99.96

26

Free Fatty Acids

The free fatty acid fraction was esterfied in dry
methanol with 10% H280“ at 60° C. for one hr. in the
presence of the adsorbent. A crystal of hydroquinone was
added to avoid oxidation. The methyl esters were extracted
twice with 30 ml. of petroleum ether after addition of
water. The combined petroleum ether extract was washed with
water, dried over a mixture of anhydrous sodium sulfate-
sodium bicarbonate (4:1), taken to dryness under vacuum
adjusted to a known volume, and injected in gas-liquid
chromatography column. The results shown in Table 6 and

Figure 4.

TABLE 6

DISTRIBUTION OF THE METHYLATED FREE FATTY
ACIDS IN CORN POLLEN

 

 

Methyl Esters ug./g. %
Laurate 2.48 .03
Myristate 27.04 .40
Myristoleate 4.48 .06
Palmitate 5988.31 91-59
Palmitoleate 45.41 -70
Stearate 116.36 1.80
Oleate 116.86 1.81
Linoleate 28.26 -“3
Linolenate 93.55 1.45
Arachidate 115.91 1.79
Behenate Trace

 

TOTAL 6537.66 100.06

27

FIGURE 4

GAS-LIQUID CHROMATOGRAPHY OF METHYL
ESTERS FROM THE FREE FATTY ACIDS

A, represents the standards and B, sample. The
standard represent, from left to right, methyl esters of
lauric, myristic, myristoleic, palmitic, palmitoleic,

stearic, oleic, linoleic, arachidic, cis-eicos—5—enoic and

linolenic acids.

28

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III W.

 

 

 

 

 

 

 

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29

Mono-and/or Diglycerides

 

This fraction, which was mixed with free sterols, was
directly transmethylated under the same conditions mentioned
under sterol esters. The methyl esters were purified (19)
before being analyzed by gas-liquid chromatography. Table 7

and Figure 5A indicate the results.

TABLE 7

DISTRIBUTION OF METHYLATED FATTY ACIDS
FROM THE MONO- AND/OR DIGLYCERIDES
IN CORN POLLEN

 

 

Methyl Esters ug./g. %

Myristate 13.60 0.28
Palmitate 3620.00 63.25
Stearate 58.46 1.02
Oleate 156.09 2.72
Linoleate 181.72 3-17
Linolenate 1634.63 28.56
Arachidate 44.84 0.78
Behenate 13.60 0-23

 

TOTAL 5722.94 99 96

LA)
0

FIGURE 5

GAS—LIQUID CHROMATOGRAPHIC ANALYSIS OF THE FATTY
ACIDS FROM THE MONO— AND/OR DIGLYCERIDES AND
THE PHOSPHOLIPIDS

A, represents the fatty acids from the mono- and/or
diglycerides and B, standards; and C, the fatty acids

from the phospholipids. The peaks of the standards from

left to right represent the methyl esters of lauric,

myristic, myristoleic, palmitic, palmitoleic, stearic,

oleic, linoleic, linolenic and arachidic acids.

 

 

 

 

32

Phospholipids

 

The separation of the phospholipids from the total
lipid extract was achieved by the use of column chromato-
graphy. Fraction 2 of Table 3 represents the fraction with
phospholipids. These phospholipids were not further separa-
ted and were transmethylated as the other lipids with
sulfuric acid and dry methanol. The methyl esters were
analyzed by gas—liquid chromatography and the associated
acids are listed in Table 8 and Figure 50 shows the chro—

matogram.

TABLE 8

DISTRIBUTION OF METHYLATED FATTY ACIDS FROM THE
PHOSPHOLIPIDS IN CORN POLLEN

 

 

Methyl Esters ug./g. %

Myristate 11.53 0.11
Palmitate 8809.25 85.56
Stearate 123.75 1.20
Oleate 191.85 1.86
Linoleate 143.33 1-39
Linolenate 922-43 8°95
Arachidate 93.05 0°90

 

TOTAL 10295.19 99.97

33
Total FattygAcids

One g. of pollen was extracted with chloroform-
isoprOpanol and the combined extract was evaporated to dry-
ness ig nggg. The methyl esters were prepared by re-
fluxing the extract in dry methanol with 10% sulfuric acid
and a crystal of hydroquinone for 16 hrs. The methyl esters
were then extracted twice with 30 m1. petroleum ether, dried
over sodium sulfate-sodium bicarbonate, and evaporated ip
Kgggg. Purification of the methyl esters by column chromato-
graphy was done before analysis by the gas-liquid chromato-
graph. The results of the analysis of the fatty acids are

summarized in Table 9 and Figure 6.

TABLE 9

DISTRIBUTION OF THE TOTAL METHYLATED FATTY
ACIDS IN CORN POLLEN

 

 

 

Methyl Esters ug./g. %
Laurate Trace

Myristate 53.8 0.19
Myristoleate 16.9 0.06
“Palmitate 20789.0 75.03
Palmitoleate 94.5 0.34
Stearate 364.2 1.31
Oleate 660.0 2.38
Linoleate 720.0 2.59
Linolenate 4585.0 15.54
Arachidate 378.0 1.36
Behenate 44.0 0.16

TOTAL 27,705-Ii

 

34

FIGURE 6

GAS-LIQUID CHROMATOGRAPHIC ANALYSIS
OF THE TOTAL FATTY ACIDS

A, represent the sample and B, standards. The peaks
in the standards, from left to right, represent methyl
esters of lauric, myristic, myristoleic, palmitic, palmito-

leic, stearic, oleic linoleic, arachidic, linolenic and

behenic acids.

35

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36
Gas-Liquid Chromatography

The gas-liquid chromatography was accomplished on a
F & M #00 "Biomedical Gas—liquid Chromatograph" with
hydrogen flame ionization detector. Gas-liquid chromato-
graphy packing was done according to Desty (ll) and Miller
(29) on a l/U inch (i.d.) x 6 foot glass column with 15% E
diethylene glycolsuccinate polyester on diatOport S, 80-100

mesh* and conditioned overnight at 210° C. The conditions

 

for chromatography were as follows: oven temperature,
195° C. flash heater 235° C.; sensitivity--attenuation 32 fi
and range 10; and the flow of carrier gas (helium) MS ml./min.
Injection of the sample was done with a microliter syringe.

Thin-layer chromatography and gas—liquid chromato—
graphy standards were obtained from Applied Science
Laboratories.** The area under the peak was calculated by
multiplying 1/2 of the peak height by the perpendicular
midway between the peak and the base. The concentration of
the sample was determined by comparing the peak areas of

the sample with that of peak areas with known concentration.
Results and Discussions

Sweet corn pollen was found to contain 13.64% lipids

from a chloroform-isoprOpanol extract. On fractionation by

 

*Applied Science Laboratory, State College, Pennsyl—
vania 39-B LAC 3R—728 (Polyester).

**Applied Science Laboratories, State College, Pennsyl—
vania.

37

column chromatography the following two fractions were
obtained: neutral lipids, 3.95%, and acidic lipids, 9.68%.
The neutral lipids were further fractionated by thin-layer
chromatography and the following classes of lipids were
identified: sterol esters, methyl esters of fatty acids,
triglycerides, free fatty acids, free sterols, and other
glycerides.

Each fraction was transmethylated and the derived
fatty acid methyl ester was identified by gas-liquid chroma—
tography. The fatty acids of sterol esters comprised 10.5%
of the total fatty acids. These were linolenic, palmitic,
linoleic and oleic acid in decreasing order of concentration.
Six and one-half percent of the total fatty acids were in
the triglyceride form. Most of the triglycerides occur as
tripalmitin and trilinolenin. The other glycerides consist
of 20.65% of the total fatty acids. Twenty three and one-
half percent of all fatty acids are in the free state in
corn pollen. Palmitic acid was the most abundant acid in
this fraction.

A fraction of the total acids (0.7%) occurred
naturally in the form of methyl ester including mainly
methyl palmitate and methyl linolenate.

Further evidence for the presence of the naturally
occurring methyl esters of the fatty acids was demonstrated
by the preparation of chloroform (alcohol-free) or ether

(alcohol-free) extracts of pollen. The methyl esters were

38

obtained in a pure state by utilizing combined column and
thin—layer chromatography methods. The identity of the
methyl ester was established by infrared Spectroscopy and
the result is shown in Figure 7. Characteristic absorption
maxima were observed at 3.45, 5.75, 8.0, 8.38, and 8.5

microns.

39

FIGURE 7

IR SPECTRUM OF NATURALLY OCCURRING
METHYL ESTERS

40

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SUMMARY

The lipid extract of corn pollen was fractionated by
column chromatography into phosphatides and nonphOSphatides.
NonphOSphatides were further fractionated by thin-layer
chromatography. Individual fatty acids in each fraction
were identified and their concentration estimated by gas-
liquid chromatography. The following classes of lipids-were
found to be present in corn pollen: sterol esters,
triglycerides, free fatty acids, free sterols, and other
glycerides. One fraction contained methyl esters of longer~
chain fatty acids.

The total fatty acids (27 mg.) were obtained after
transmethylation of the lipid from one gram of dried corn
pollen. Lauric, myristic, myristoleic, arachidic, and
behenic acids were identified for the first time. Other

acids present were palmitic, palmitoleic, stearic, oleic,

linoleic, and linolenic.

Ml

BIBLIOGRAPHY

A2

 

IO.

11.

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