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WH”M STATE UNE‘ﬁ'ERSH‘Y
Charies Richard Barr

1957

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THE HIGHER FATTY ACIDS OF POLLEN OF ZEA MAYS

By

Charles Richard Barr

AN ABSTRACT

Submitted to the College of Science and Arts
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Chemistry
1957

Approved L 3' ﬂ /‘, Ac“: (/4

Charles Richard Barr

Marv articles have been published on the chemical composition of
pollens. Analyses of corn pollen for minerals, amino acids, carbo-
hydrates, and vitamins have been reported in the literature, but there
has been no systematic study of the fatty acids.

An investigation was made of the higher fatty acids in the pollen
of _z__e__a mg. The lipids were separated from corn pollen by extraction
with eth ether. Saponification, dilution with water, and extraction
with Skellysolve "B" followed by acidification of the aqueous solution
yielded the free fatty acids. The fatty acids were esterified, and
the methyl esters distilled under reduced pressure using a Stedman
column. The methyl esters of the fatty acids were collected in five
fractions, and several plvsical constants were determined for each
fraction.

The fatty acids in each fraction were identified by making the
appropriate derivatives . The saturated fatty acids were identified
by the melting points of the p-bromOphenacyl esters, the mono-unsat-
urated fatty acids by the melting points of the dihydrom acid, and
the more unsaturated fatty acids by the melting points of the bromine
addition products. A quantitative estimtion of each fatty acid was
made by substituting the determined iodine numbers, thiocyanogen
numbers, and percentage of saturated fatty acids into empirical
equations.

The following higher fatty acids were identified in corn pollen:
palmitic, 21.6%; stearic, 1.15; oleic, 7.5%; linoleic, 22.9%; and

Charles Richard Barr

linolenic, 23.5%. The percentages listed indicate the relative abund-
ance of each fatty acid of the total fatty acids obtained.

Palmitic and linolenic acids were found to be present in larger
quantities in corn pollen than reported for other corn products; to
the contrary, stearic, oleic, and linoleic acids were found in smaller

quantities in corn pollen.

THE HIGHHI FATTY ACIDS 0F POLLEN 0F Z_;_EA HAYS

By
Charles Richard Barr

A THESIS

Submitted to the College of Science and Arts
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SHENCE

\

Department of Chemistry
1957

TABLE OF CONTENTS

INIRODUC TION O 0 O 0 I O O O O O O

I-IISWRICAL . C O O O O O O O O O O

EXPmImNTAL O O O O O O O O O O 0
Preparation of Fatty Acids. .
Preparation and Fractionation
Analytical Methods. . . . . .
Preparation of Derivatives. .

RESULTS AND DISCUSSION . . . . . .

SUMMARY. . .

O O O O O O O O O

BIBLIWRAPIIY O O O O O O O O O O O

of Methyl

10
ll
11

12

18

23

25

LIST OF TABLES

TABLE PAGE

I. Mineral Content of Corn Pollen . . . . . . . . . . . . . . . . h

II. Amino Acid Content of Corn Pollen. . . . . . . . . . . . . . . 6

III. Carbohydrate Content of Corn Pollen. . . . . . . . . . . . . . 7

IV. Vitamin Content of Corn Pollen . . . . . . . . . . . . . . . . 7

V. Fatty Acid Content of Some Corn Products . . . . . . . . . . . 9

VI. Fractionation of Methyl Esters of Fatty Acids

In Corn Pollen. . . . . . . . . . . . . . . . . . . . . . 20

VII. Quantitative Distribution of Higher Fatty Acids

In Corn Pollen. . . . . . . . . . . . . . . . . . . . . . 21

ACKNOWLEDGMENTS

The author wishes to express his sincere thanks to Professors
H. M. Sell and C. D. Ball for their interest and guidance throughout
the course of this work. The writer deeply appreciates the financial
support from.the National Science Foundation. Grateful acknowledgment
is also due Mrs. Jean Lagowski, Mrs. Audrey Anderson, and.Mrs. Jean

Brehmer for their assistance in the preparation of the manuscript.

INTRODUCTION

INTRODUCTION

The important role played by pollen in the life cycle of plants
has attracted the attention of scientists for many years in an attempt
to learn the chemistry through which pollen manifests its pm'siological
effects, culminating in fertilisation. Due to its importance in the
Production of foodstuffs, for consumption by man, pollen is among the
first of the natural products studied by modern chemists. Pollen re-
search in recent years has been influenced largely by the fact that
P011011 may be responsible for certain allergies. Much of the recent
literature in this field is devoted to studies of the causes of these
allergies, particularly the component responsible for that allerg
assumed to be produced by the pollen of ragweed.

The work done on pollen chemistry has been somewhat limited be-
cause of the difficulties experienced in collecting sufficient pollen
samples for chemical analyses. Analyses of a large number of different
P011ens for specific materials have been reported in the literature,
but only thirteen pollens have had relatively complete analysis (1).

The present study was made on the pollen of Egg mg, a‘highly
important agricultural crop in this country. The pollen of this plant
a180 lends itself more readily to hand collection than that from most
other species of plants. The flower of £93 £52.! is of the incomplete
type with a single, easily accessible stamen located at the top of the
Plant. The plant depends upon the wind to shake the pollen onto the

”time, thus the pollen must be produced in large quantities and easily

shaken from the pollen sacs.

HISTORICAL

HISTORICAL

VA review of the literature suggests that a great deal of chemical
analysis and research remains to be done on corn pollen. Lundén (2)
has reviewed the recent literature on pollen chemistry and summarizes
his major findings regarding the pollen chemistry of many plant species.

The ash content of corn pollen varies from 2.55 to 3.83i. The
mineral composition of the pollen ash has been determined quantita-

tively (l, 3). The results of these analyses are shown in Table I.

TABLE I

MINERAL CONTENT OF CORN POLLEN

(Percentage of ash)

 

r v , ——__._. -"ﬁ-v» w_._‘__.'.o." m .— _'..—.,..~ W

 

 

 

A ﬁeferences ’1

Mineral *‘ (l) (3)
x 26.31 35.58
Na - 0.69
P 10.19 18.20
Ca 3.92 1.02
Mg 8.23 b.60
A1 - 0.22
Fe 0.05 0.25
s .. 0.69

C]. "‘ 0.80

'1’

5

The amino acids in Egg may! have been identified by chromatographic
techniques (h), and several have been estimated quantitatively (h, 5).
These results are tabulated in Table II. In the table, column 2 indi-
cates the presence (+) or absence (-) of amino acids as determined by
chromatographic techniques; columns 3 and b show the percentage of
each amino acid of the crude protein in freshly collected pollen and_ E
pollen that had been stored for one year, respectively. Column 5 shows
the percentage of amino acid of the crude protein in sweet corn pollen,
and colunn 6 gives the average amino acid content of crude protein of
pollen for a number of plant species. Vinson (6) isolated from.air-
dried pollen a protein behaving like a glutelin which contained 214.98%
of the total nitrogen in the pollen. iAlso present were adenine, cho-
line, and the free amino acids arginine, lysine, tyrosine, and.p-
hydroxyglutamic acid. Hlyake (7) has reported the presence of adenine
and choline in corn pollen.

The carbohydrates of corn pollen have been reported to consist of
starch, dextrin, sucrose, glucose, fructose, and hemicellulose, the
latter hydrolyzing into glucose and.xylose; free pentoses are probably
absent (7). Some quantitative data on.carbohydrates in corn pollen
are given in Table III.

Redemann gt 5}. (10) have identified quercetin as the major yellow
pigment of the pollen of Egg &.

Sagromeky (ll) determined the thiamine content and Nielsen gt El.
(h) have determined a number of other deitamins present in Egg 55:3.

Table IV sunrises the findings of these authors.

THE HIGHER mm ACIDS 0F POLLEN OF ye} nus

By
Charles Richard Barr

A THESIS

Submitted to the College of Science and Arts
Michigan State University of Agriculture and
Applied Science in partial Mfillment of
the requirements for the degree or

MASTER OF $IENCE

Department of Chemistry
1957

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . .

I-IISTURICAL O O O O O O O O O O 0 O

EXPDTIMENTAL O O O O O O O O 0 O 0
Preparation of Fatty Acids. .
Preparation and Fractionation
Analytical Methods. . . . . .
Preparation of Derivatives. .

REQULTS AND DISCUSSION . . . . . .

SUMMARY. . .

BIBLIWHJ‘PTIY O O O O O O I O O O C

of Methyl

Esters.

10
ll

11

23

25

LIST OF TABLES

TABLE PAGE

I. Mineral Content of Corn Pollen . . . . . . . . . . . . . . . . D

II. Amino Acid Content of Corn Pollen. . . . . . . . . . . . . . . 6

III. Carbohydrate Content of Corn Pollen. . . . . . . . . . . . . . 7

IV. Vitamin Content of Corn Pollen . . . . . . . . . . . . . . . . 7

V. Fatty Acid Content of Some Corn Products . . . . . . . . . . . 9

VI. Fractionation of Methyl Esters of Fatty Acids

In Corn Pollen. . . . . . . . . . . . . . . . . . . . . . 20

VII. Quantitative Distribution of Higher Fatty Acids

In Corn Pollen. O O O O O O O O O O O O O O O O O O O O O 21

ACKNOWLEDGMENTS

The author wishes to express his sincere thanks to Professors
H. M. Sell and C. D. Ball for their interest and guidance throughout
the course of this work. The writer deeply appreciates the financial
support from the National Science Foundation. Grateful acknowledgment
is also due Mrs. Jean Lagowski, Mrs. Audrey Anderson, and Mrs. Jean

Brehmer fer their assistance in the preparation of the manuscript.

INTRODUCTION

INTRODUCTION

The important role played by pollen in the life cycle of plants
has attracted the attention of scientists for new years in an attempt
to learn the chemistry through which pollen manifests its pkvsiological
effects, culminating in fertilisation. Due to its importance in the
production of foodstuffs, for consumption by man, pollen is among the
first of the natural products studied by modern chemists. Pollen re-
search in recent years has been influenced largely by the fact that
pollen may be responsible for certain allergies. Much of the recent
literature in this field is devoted to studies of the causes of these
allergies, particularly the component responsible for that allergr
assmed to be produced by the pollen of ragweed.

The work done on pollen chemistry has been somewhat limited be-
cause of the difficulties experienced in collecting sufficient pollen
samples for chemical analyses. Analyses of a large number of different
pollens for specific materials have been reported in the literature,
but only thirteen pollens have had relatively complete analysis (1).

The present stucb' was made on the pollen of £35 m, a‘highly
important agricultural crop in this country. The pollen of this plant
also lends itself more readily to hand collection than that from most
other species of plants. The flower of _Z_e_a HE is of the incomplete
type with a single, easily accessible stamen located at the top of the
plant. The plant depends upon the wind to shake the pollen onto the
at18ml, thus the pollen must be produced in large quantities and easily

shaken from the pollen sacs.

HISTORICAL

HISTORICAL

.A review of the literature suggests that a great deal of chemical
analysis and research remains to be done on corn pollen. Lunde’n (2)
has reviewed the recent literature on pollen chemistry and summarizes
his major findings regarding the pollen chemistry of many plant species.

The ash content of corn pollen varies from 2.55 to 3.83$. The
mineral composition of the pollen ash has been determined quantita-

tively (l, 3). The results of these analyses are shown in Table I.

TABLE I

MINERAL CONTENT OF CORN POLLEN

(Percentage of ash)

   

,. - "A... e — v - »._-.-_~ -*W«r_W~—.e.._—,. ”i W W...- w...»

 

 

References *‘
k Mineral # (1) (3) A
x 26.31 35 .58
Na -- 0.69
P 10.19 18.20
Ca 3.92 1.02
Mg 8.23 h.60
Al -— 0.22
Fe 0.05 0.25
5 .- 0.69

S

The amino acids in $33 me have been identified by chromatographic
techniques (h), and several have been estimated quantitatively (h, 5).
These results are tabulated in Table II. In the table, colunn 2 indi-
cates the presence (+) or absence (-) of amino acids as determined‘hy
chromatographic techniques; columns 3 and b show the percentage of
each amino acid of the crude protein in freshly collected pollen and,
pollen that had been stored for one year, respectively. Column 5 shows
the percentage of amino acid of the crude protein in sweet corn pollen,
and column 6 gives the average amino acid content of crude protein of
pollen for a number of plant species. Vinson (6) isolated from.air-
dried pollen a protein behaving like a glutelin which contained 21:38:!
of the total nitrogen in the pollen. .Also present were adenine, cho-
line, and the free amino acids arginine, lysine, tyrosine, and.B-
hydroxyglutamic acid. Miyake (7) has reported the presence of adenine
and choline in corn pollen.

The carbohydrates of corn pollen have been reported to consist of
starch, dextrin, sucrose, glucose, fructose, and hemicellulose, the
latter hydrolyzing into glucose and xylese; free pentoses are probably
absent (7). Some quantitative data on carbohydrates in corn.pollen
are given in Table III.

Redemiann .9}. g]... (10) have identified quercetin as the najor yellow
piencnt of the pollen of E2 E2.-

Sagromsky (ll) determined the thiamine content and Nielsen gt a}.
(h) have determined a number of other B-vitamins present in 23 m.

T‘ble IV smrises the findings of these authors.

TABLE II
AMINO ACID CONTENT OF CORN POLLEN

(Percentage of crude protein)

 

 

 

 

 

Amino Acid £22 we (5) - 5:? Aggie
Present Fresh Aged Pollen (h) Pollen SQ;
Crude Protein (ll-6.25) 25.63 26.25 26.88 26.31.
Arginine + 6.3 5.7 h.7 5.3
Histidine + - - 1.5 2.5
Lysine + 5.9 5.0 5.7 6.):
Tryptophan + 0.6 0.6 1.6 l.h
Phenylalanine + 2.9 2.3 3.5 h.1
Cystine + - - 0.6 ..
Methionine + 1.6 1.6 1.7 1.9
Threonine + - - h.6 h.1
Leucine + 7.5 5.6 5.5 7.1
Ieolcucine + -— -- 13.7 5.1
Valine + - -§ 6.0 5.8
Glutamic Acid + -— .. 9,1 ..
.GIYcine +
Alanine +
Sarina +
Aspartic Acid +
Proljxu, +
Hydrﬁxypreline +
TWOSi-ne + 1.9 1.9 -- --

““mino Butyric Acid +

4

TABLE III
CARBOHYDRATE CONTENT OF CORN POLLEN
(Percentage of pollen)

W

 

 

4_ References .4
WWW“ (1; g (3)‘1 (9)
Starch 22.h0 16.0h 16.19
Dextrin - - 0.80
Reducing Sugar (as Glucose) 6.88 b.61 0.59
Non-reducing Sugar (as Sucrose) 7.31 8.714 7.80
Pentosans - - 5.73

 

1'Average of three varieties.

TABLE IV
VITAMIN CONTENT OF CORN POLLEN

(use/s. of pollen)

 

 

 

 

Vitamin References
0:) (ll)

Thiamin - 1.l:--7.91
Rihoflavin 6.2 ..
Nicotinic Acid 71.8 --
Pyridoxine 12.7 '-
Pantothenic Acid 5.5 -
Biotin 0.55 -
Inositol (mg./8e) 30 --

 

1Variation for seven different pollens.

Paton (12) has determined the enzymes present in a number of
plants and has reported the following to be present in corn pollen:
amylase, invertase, catalase, reductase, pectinase, trypsin, and pepsin.

A pmrtosterol and an inositol were isolated from corn pollen by
Hiyake (9). Anderson (13) isolated a phytoeterol palmitate, which
upon twdrolysis gave two different phytosterol fractions, a saturated
twdrocarbon (apparently g-nonacosane) , a Gag-saturated alcohol, and a
phosphatide. ~

The lipid fraction of pollens other than that of _Z_e_a M has been
studied; Heyl (11:) found the following acids in ragweed pollen: formic,
acetic, valeric, lauric, an unsaturated acid 010111.03, oleic, linoleic,
palmitic, and mistic. Kiesel and Rubin (15) reported a high content
of heptacosane in the crude lipid from sugar beet pollen. In hazel
pollen, Sosa and Sosa-Bourdouil (16) noted the presence of palmitic
acid, one Ola-acid, tricosane, hexadecanol, and two unsaturated sterols.
Mariella g_t_ £1. (17) reported analyses and approximate empirical formu-
lae for seven compounds isolated from the nonsaponifiable fraction of
ragweed pollen. From the pollen cement of M candida, Tappi and
Monsani (18) have isolated heptacosane, ptvtofluene, and 7-sitosterol.

Redemann (19) has indicated the presence of a growth regulator in
the ethyl ether soluble fraction of corn pollen. Eyen though the fatty
acids of several corn products have been reported, as shown in Table V,
those of corn pollen have received little attention.

The work described in this thesis consists of the analysis and
identification of the higher fatty acids in the lipid fraction of corn

Pollen.

TABLE V
FATTY ACID CONTENT OF SOME CORN PRODUCTS

(Percentage of product)

 

 

 

 

I'm—WWW
Fatty Acid References Germ Oil Starch Grain
w (20)1 (21)1 (22)1L (23)” (2h) (25) (26)
Myristic 0.1 - -- 1.7 0.2
Palmitic 8.1 7.8 7.8 11.0 13.0 21.2 8.0-8.7
Stearic 2.5 3.6 3.5 2.9 0.9 7.8 b.1-5el
Arachidic 0.0 0.1: 0.1.. - 1.5
Behenic - - - - 0.2
Eicosenoic - - - - 1.5
Lignoceric 0.0 0.2 0.2 - -
Palmitoleic 1.2 -— - 1.6 0.2
Oleic 30.1 h6.3 h6.3 h8.8 h1.9 37.7 31.0-31.9
Linoleic 56.3 111.? 1.1.8 3h.o no.6 31.1 53.8-56.7
Linolenic - - -— - - 1.2

 

l
Expressed as weight percent.

IExpressed as mole percent.

IAcids above C1.-1.7 percent.

EXPER TENTH.

EXPERIMENTAL

Preparation of Fatty Acids

The lipid fraction used in this study was extracted from corn pollen
(variety Golden Cross Eggtam) with ethyl ether in l9h5 (19) and had been
stored at temperatures below 00 C. The lipids (21h.1 g.) were saponified
with 31.6 g. of potassium hydroxide in hOO ml. of 95% ethanol by refluxing
fer 8 hours on a steam bath. After diluting the solution with 1600 ml. of
distilled water, the unsaponifiable material was removed by extraction with
Skellysolve "B". The Skellysolve "B" solution was in turn extracted several
times with distilled water, and the water extracts combined with the ethanol-
water phase. The ethanoldwater solution, which contained the potassium salts
of the fatty acids, was acidified with to m1. of concentrated hydrochloric
acid, and the liberated fatty acids were extracted with ethyl ether. The
ether was removed in 33232 (15 mm.) at 35° C. in an atmosphere of carbon
dioxide. A residue of ll2.h g. of fatty acid remained in the flask. The
saponifiable fraction represented h9% of the original lipid material, while

51% consisted of unsaponifiable materials.
Preparation and Fractionation of Methyl Esters

Preparation of Methyl Esters of Fatty Acids. A solution of 112.1; g.
01‘ the fatty acids in 250 m1. of absolute methanol containing 5% sul-
furic acid (by weight) was refluxed for 6 hours on a steam'bath. The
methanol was removed from the methylated fatty acids _i__n m (15 m.)
at 110° C. The residual oil was diluted with water and neutralized with a

10% sodium carbonate solution. The methyl esters were extracted with ethyl

6"their, and the ether solution was washed with water to remove traces of

12

sodium carbonate. After removal of the ether in 32229 (15 mm.) at 35° 0.,
105 g. of crude methyl esters was recovered.

Fractionation of’Methyl Esters of Fatty Acids. AA Stedman packed
column constructed of Pyrex glass which was 0.762 inch in diameter and
2h inches long, and contained Stedman No. 10h packing, was used in this
study. The column was centered in a h2 mm. (i.d.) Pyrex tube by wrapping
both ends of the column with asbestos tape. A nichrome wire helix was
placed around the tube to permit the column to be heated electrically.
The heating jacket was insulated, and protected by another Pyrex tube
of 6h mm. (i.d.). The 250 m1. flask containing the methyl esters was
heated by a Glas-Col heating mantle. The temperatures of the flask and
column was regulated by means of A. C. variable transfermers. A still
head with an enclosed thermometer was used at the top of the column and
the reflux ratio was regulated by a stopcock located below the condenser.
The crude mixture of the methyl esters of the fatty acids was fractionated
in the column previously described. Five fractions of the methyl esters

were collected between 155-181;0 C./2-3 mm. at.a reflux ratio of 10:1.
Analytical Methods

Iodine Absorption Number (Wijs Method) (27). A sample of 0.2 g.
of'metbyl ester or fatty acid was weighed and transferred to a 500 ml.
glass-stoppered iodine flask containing 20 ml. of carbon tetrachloride
(C.P.). Twenty-five milliliters of iodine-chlorine solution was added
fronla.pipette and the reaction flask allowed to stand in the dark for
30 minutes. At the end of this period 20 ml. of 15% potassium iodide

solution and 100 ml. of recently boiled and cooled water were added.

13
The liberated excess iodine was titrated with a 0.1 N sodium thiosulphate
solution using starch indicator. Two blank determinations were made.
Preparation of.Reagent. Washed and dried chlorine gas was passed into
a solution of 13 g. of iodine in 1 l. of glacial acetic acid (99.5%)

until thiosulphate titration of the solution was approximately doubled.

Iodine No. - [ML of 8303(blankﬂ-Egl. of 8203(sampleﬂ (N)(126.91)(100)
G. of sample 1000

Refractive Index (27). The refractive indices were determined with

 

an Abbe refractometer at 250 C. using the D line of sodium.
Saponification Egijalent (28). A mixture of 0.14 g. of the methyl
ester and 15 ml. of 3 N alcoholic sodium hydroxide solution was refluxed
in a 150 ml. Erlenmeyer flask for 1 1/2 hours. After cooling, the excess
alkali was titrated with 0.1 N hydrochloric acid solution to a phenol-

phthalein end point.

Saponification Eq. - (G. of Is )(1000)
(NI. of Nam)(N)-(NI. of ROTH!)

Thiogzanogen Number (27). A sample (0.1 to 0.5 g.) of fatty acid,
obtained by the saponification of the methyl ester, was dissolved in
25 ml. of thiocyanogen solution, and placed in the dark for 214 hours.
Ten milliliters of 20% potassium iodide was added; after diluting with
100 ml. of water, the excess iodine was titrated with a 0.1 N sodium
thiosulfate solution, using starch indicator. Blank determinations were
made.
Egeparation of Reagents. Thiocyanogen solution (0.2 N) was prepared by
dissolving 8.1.: g. of dry bromine in 100 ml. of anhydrous carbon tetra-
chloride, and diluting to 250 ml. with glacial acetic acid. Glacial

acetic acid (250 ml.) was added to 25 g. of anhydrous, reagent grade

it
lead thiocyanate in a l 1. glass stoppered bottle. The bromine solution
was added in small quantities and shaken after each addition until the
bromine was decolorized. When addition was complete, the suspension was
allowed to settle, the solution was filtered into a dry, brown, glass-

stoppered bottle and stored in the refrigerator.

son No. - [141. of sao§(blank)J-!'_r_11. of 8303(s%1e)] gN)(126.9)(100)
. o samp 6

Quantitative Determination of Fatty Acids (30). A quantitative

 

estimation of unsaturated fatty acids was made by substituting the
experimentally determined iodine number, thiocyanogen number, and per-
centage of saturated fatty acids in the fellowing equations.

In the absence of linolenic acid:

5 Oleic acid a 2.h21(T.V.)-1.293(I.V.)
% Linoleic acid - l.19h(I.V.)-l.202(T.V.)

In the presence of linolenic acid:
-O.6617(S)+66.l7

1.6h23(s)+16h.23
+1.30h0(s)-130.bo

% Oleic acid - 1.61h6(T.V.)-1.2275(I.V.)
% Linoleic acid = l.3565(l.V.)—3.20h8(T.V.)
% Linolenic acid: 1.5902(T.V.)-O.1290(I.V.)

T.V. - Thiocyanogen number
I.V. - Iodine number
5 s % Saturated fatty acid

Preparation of Derivatives

Lead Salt-Ether Method of Separating Saturated and Unsaturated

.Eatty Acids (27). To 5 g. of fatty acid in a 100 m1. Erlenmeyer flask

 

was added 15 ml. of ethanol and sufficient 15% potassium hydroxide
solution to produce a distinct pink color with phenolphthalein. The
soapisolution'was added cautiously to 60 m1. of 10% lead acetate sol-

ution (hot), and the flask rinsed with small portions of ethanol and hot

15

water. The solution was boiled gently for 5 minutes, shaken, cooled,
and the flask rotated to cause the precipitated lead salts to adhere to
the walls. After cooling, the supernatant liquid was decanted and the
precipitate was washed with cold water. The flask was drained for 10
minutes and moisture removed from the soaps with filter paper strips.
Sixty milliliters of ethyl ether was added and the lead salts disinte-
grated or dissolved by gently refluxing the ethereal suspension. The
walls of the flask were rinsed with sufficient ethyl ether to bring the

final volume to 75 ml. After cooling the ethereal solution in a refrig-
erator for 15 hours, the precipitate was collected quantitatively on a Buchner
funnel; the precipitate was washed with ethyl ether and dried thorough-

hr by suction. The precipitate was transferred immediately to a 100 ml.
separatory funnel containing 25 ml. of ethyl ether and the filter paper

was placed in a small flask. The lumps of lead salt were dispersed by
Shaking and allowed to settle. Ten milliliters of hydrochloric acid

( 2:1) was added, and the solution shaken for 2 minutes to decompose the
lead soaps. The precipitate adhering to the filter paper was decomposed

by the addition of 3 m1. of hydrochloric acid (2:1) and washed into the
separatory funnel with ethyl ether and water. After shaking, the con-
tents of the separatory funnel were allowed to stand for 10 minutes and

1file aqueous solution drained off. he ether layer was washed with 15 ml.
portions of water until free of hydrochloric acid. After drying with

l g. of anhydrous sodium sulfate, the ethereal solution was filtered

into a tared 100 ml. flask. The sodium sulfate was washed with several
Small portions of anhydrous ethyl ether and the ether washings combined

with the ether extract. The ether was removed at 35° C./l§ MI. and the

acids dried to constant weight at 110° C.

16

The ether solution containing the soluble lead salts was transferred

quantitatively from the filter flask to a 100 ml. separatory funnel. A

solution of 15 ml. of hydrochloric acid in I40 m1. of water was added and
the separatory funnel shaken for 2 minutes. After standing for 10 min-
utes, the aqueous suspension of lead chloride was drained into a beaker.

The lead chloride was allowed to settle, the supernatant liquid decanted,

and the lead chloride washed with a small portion of ethyl ether. The

ether washings were combined with the contents of the separatory funnel,

rotated, and allowed to stand for 10 minutes. The aqueous solution was

separated and the ether layer washed with 25 ml. portions of water until

free of hydrochloric acid. The ether layer was transferred to a tared

o
flask and the ether removed at 35 C./lS 1231.. The residue in the flask

was dried at 1100 C. in a carbon dioxide atmosphere for 1 hour and the

Weight of residue determined by difference.

Preparation of p-Bromophenacyl Esters of the Saturated FattLAcids
( 28). A suspension of l g. of fatty acid in 5 ml. of water in a small

flask was neutralized with a 10% sodium hydroxide solution to a phenol-

Phthalein end point and then made slightly acid to litmus. one gram
01' 3—bromophenacyl bronide in 10 ml. of ethanol was added and the mixtlme
heated under reﬂux 1'01 ,‘5 hours. If a solid separated during reﬂuxing,
Sufficient ethanol was added to effect solution. After cooling, the

Precipitated ester was recrystallized from ethanol until a constant

mEi‘lting point was attained.
Preparation of Dihydroxy Derivatives of Mono-unsaturated Acids (23").
One gram of acid and l g. of sodium hydroxide were added to 100 ml. of

Imate):- and the mixture warmed on the water bath until solution was effected.

17

The solution of the sodium salt of the acid was cooled and diluted with

800 m1. of ice-cold water, and 80 ml. of a lie-'- potassium permanganate

solution was added quickly with stirring. After 5 minutes, the liquid

was decolorized with sulfur dioxide, and 30 ml. of concentrated hydro-
chloric acid was added. The white flocculent precipitate of crude

solid dimrdroxy saturated acid was collected on a Buchner funnel, washed

with 10 ml. of ethyl acetate, and air dried. The filter cake was mixed with

30 ml. of warm ethyl acetate, cooled, and filtered; then the acid was

washed with several small portions of cold ethyl acetate. The acid was

recrystallized to a constant melting point.

Preparationéof Bromo-derivatives of Unsaturated Fatty Acids (30).

One gram of fatty acid was dissolved in 25 ml. of ethyl ether and the

SOlution chilled to 00 C. in an ice bath. Bromine was added at such

0
a. rate that the temperature did not exceed 20 C. and until a deep red

COlor persisted. The reaction mixture was placed in the refrigerator

OVernight, and the solid white precipitate was collected on a Buchner

funnel .

The brominated acids were separated on the basis of their different

SOlubilities in organic solvents. The dibromo-acid is soluble in

petroleum ether, while the tetrabromo-acid, insoluble in petroleum ether,

is soluble in ethyl ether. The hexabromo-acid, insoluble in petroleum

ether and ethyl ether, is soluble in hot benzene. The melting points of

1‘vhe brominated acids were determined.

RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

The saponifiable material in corn pollen represented h9% of the total

ether extract, while the remaining 51% consisted of unsaponifiable mater—

ial.

The results of fractionation of the methyl esters formed by ester-
ification of the saponifiable material in corn pollen are summarized in
Table VI. Some physical and chemical characteristics of the 5 fractions
collected are given. The fatty acids identified and their derivatives
in each fraction are listed. The saturated fatty acids identified were

palmitic and stearic, while the unsaturated acids were oleic, linoleic,

and linolenic.

The quantitative values of the fatty acids in corn pollen are shown
in Table VII. Calculation of the percentage of each acid in the distillate

gave the fellowing results: palmitic, 21.6%; stearic, 1.1%; oleic, 7.5%;

linoleic, 22.9%; and linolenic, 23.5%.

A comparison of these values with those tabulated fbr corn oil,
corn germ oil, corn starch, and corn grain, in Table V, shows that stearic,
oleic, and linoleic acids were present in smaller quantities in corn
Joellen, whereas palmitic and linolenic acids were present in larger
(auantities. Of these differences, that of linolenic acid was outstand-
Zing. This acid was approximately 20 times more abundant in corn pollen
tﬂhan.in corn starch and the only corn product in which it has been re-

Fkirted. The unsaturated fatty acids in corn.pollen were found to represent

53 .9% of the fatty acids and were lower than those values found for the

20

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QUANTITATIVE DISTRIBUTION OF HIGHER FATTY ACIDS IN CORN POLLEN

TABLE VII

21

 

 

 

 

Fraction __
1 2 3 h 5 Total
weight in Grams 9.5 16.3 15.8 22.0 8.7 72.3
Iodine Number 21.1 8h.6 13h.h 227.9 220.5
Thiocyanogen NuMber 15.h hh.2 79.5 1hl.6 132.0
Percent Saturated Fatty Acid 89.3 51.7 21.6 2.6 5.0
Palmitic Acid
Percent 89.3 51.7 21.6 -- --
Grams 8.51 8.h3 3.b1 -- -- 20.35
Stearic Acid
Percent -- -- -- 2.6 5.0
Grams -- -- -- 0.57 0.hb 1.01
Oleic.Acid
Percent 3.1 0.h 1h.8 16.9 7.9
Grams 0.30 0.07 2.3h 3.72 0.69 7.12
Linoleic Acid
Percent 7.6 h7.9 56.6 8.2 27.0
Grams 0.72 7.80 8.95 1.81 2.3h 21.62
.Linolenic Acid
Percent -- -- 7.0 72.3 60.1
Grams -- -- 1.10 15.90 5.23 22.23

 

22
other corn products previously mentioned. The high percentage of lino-
lenic acid may be of considerable significance, since the unsaturated
fatty acids may be more easily oxidized and metabolized.by pollen during
growth of the pollen tube than are the other fatty acids. If these acids
are metabolized, the unsaturated fatty acids could serve as a better

source of nutrients for the growing part of the pollen tube.

SUMMARY

SUMMARY

A study was made of the saponifiable fatty acid fraction of pollen
OfL§E§.95X.'

The fatty acids extracted from corn pollen were methylated and
fractionated in a Stedman column. Some chemical and physical properties
were determined.

Palmitic and stearic acids were identified as the p-bromophenaqyl
derivatives, and oleic, linoleic, and linolenic acids as the hydroxy
and bromine addition compounds.

The percentage of each acid in the saponifiable fraction was:
palmitic, 21.5%; stearic, 1.1%; oleic, 7.5%; linoleic, 22.9%; and
linolenic, 23.5%.

A higher percentage of linolenic acid was found in corn pollen
than was reported for corn oil, corn germ oil, corn starch, and corn

grain.

BIBLIOGRAPHY

9.

10.

ll.

12.

13.

BIBLIOGRAPHY

F. E. Todd and 0. Bretherick, The Composition of Pollens, J. Econ.
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R. J. Anderson and W. L. Kulp, Analysis and Composition of Corn
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It
N. Nielsen, J. Grommer, and R. Lunden, Investigations on the
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B. 0. Ray Sarkar, S. H. Wittwer, R. W. Luecke, and H. M. Sell,
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C. T. Redemann, S. H. Wittwer, C. D. Ball, and H. M. Sell, The
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H. Sagromsky, Determination of the Vitamin B, Content of Pollen hy
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J. B. Paton, Enzymes of Pollen, Proc. Soc. Exptl. Biol. Med.,.ll,
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A. Kiesel and B. Rubin, Untersuchungen uber pflanzliche
Fortpflanzungszellen, Z. physiol. Chem. , 183, 2111 (1929).

A. Sosa and C. Sosa-Bourdouil, études de quelques constituents
chemiques du pollen de Corylus avellana E” Compt. rend., _2__3_§,
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R. P. Marielle, T. B. Bernstein, and A. L. Masher, Some Purification
Studies on Ragweed Pollen, B01 col. quim. Puerto Rico, 2, 15 (1952);
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G. Tappi and A. Monzani, Constituents of the Pollen Cement of
Lilium Candidum, Gazz. chim. ita1., 85., 725 (1955); C.A., 32, 11.1922

I39557.

C. T. Redemann, Biochemical Studies of Pollen from 233 m, Ph.D.
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F. J. Baur, Jr. and J. B. Brown, The Fatty Acids of Corn Oil, J. Am.
Chem. Soc., 61, 1899 (19145).

W. F. Baughman and G. S. Jamieson, The Chemical Composition of Com
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H
K. H. Kahler, The Preliminary Processing of Corn with Special
Consideration of Biochemical Factors, Getreide, Mehl u. Brot, 1,
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H. E. Longenecker, Deposition and Utilization of Fatty Acids. II.
The Non-pro ferential Utilization and Slow Replacement of Depot Fat
Consisting Mainly of Oleic, and Linoleic Acids; and a Fatty Acid
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A. D. Fortunato, Omical Composition of an Argentine Corn-Germ
Oil, Industria y'q ice, 1}, 132 (19h9; C.A., Q5, 370.

J. W. Evans and D. R. Briggs, The Lipids of Corn Starch, Cereal
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R. L. Shriner and.R. C. Fuson, The Systematic Identification of
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A. Lapworth and E. N. Mottram, Oxidation Products of Oleic Acid.
Part 1. Conversion of Oleic Acid into Dihydroxystearic Acid and the
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Natural Sources, J. Chem. Soc., 121, 1628 (1925).

H. K. Dean, Utilization of Fats, New York, Chemical Publishing Co.
of N. Y., Inc. (1938), p. 61.

J. W. McCutcheon, Linoleic Acid, Organic Syntheses, Coll. Vol. 3,
New York, John Wiley and Sons, Inc. (1955), p. 527.

 

-— ,Linolenic Acid, Organic Syntheses, Coll. Vol.3, New
York, John Wiley and Sons, Inc. (1955), p. 532.

G. S. Jamieson, Vegetable Fats and Oils, New York, Reinhold Publishing
Corp. (l9h3), P. 3h5.

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