A 5mm 5% mg me FaAcnous 5N ms mus-w as 2355:3433ng Thesis €91 the Deg?“ of M. S. MICHIGAN STATE“ OWNER-SIN Amér Fai‘hipaur 196.5 LIBRA“ 1’1] Michigan Static University «‘7‘»? k VIP“! Y 1-0: ’ f m\u Whu U 131‘] hwy-g 1 Fit 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 \ouo \o arq~aomh¢upuJ L» H HFJF‘ rongm l—J UL) HFJF’ JZ'UOU.) FJH arq mwww 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. O )- J. @1“;1.:’1:5 y“? (‘75; AW l6 .1: ‘ )- 0 0h: 5-. 37' A_.",.L!j u- .. . 3°. : - ‘NOI . ~ I a -.-K" . _‘ ' a U : c u ‘ v 1? 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 ——.—-~.—..—-—. -..- .— ~- ~“a— -i.....__..._..-._. ""“ *‘1 - ——~+ -: . w_»__._.__ _ . .AH._..-L_._._. __.._...._. _. i.__._ ._. .ia«_._ --.~.._.._.. -+Mf__Lm. . .. -_.—-_...- ._. _-_ __ . 1,14-_._.__' _’_ ____ _ ._.___.__. «._.. 'H. -_-- 1» -—~‘—7*-l A .. , a _..__._— +— - , ‘— _-___;_..__‘+ +- ._.. ---——-t-———. 4 _ -_‘___.. - --- ___._._ _- m- 4— -_4_,__:_-_44__ -,~ +~+ — .._. _.__~—.._.._ ._- .__._'.+-___.- __;.__.,_i;_.-.__ ._._ . ._._ ..-_.--- ~..- .— ._0 A+~~-—-—~-—~—-+-+-—— ewe—mew» ---——+ -1-.. _ + _._-4 . *7-“ .: . 1. ,1 .u L _ i . .1 , + 1F 1 4 .r e + 1 _ m 1 1 ._. . _ a a . o . . . q a w _ __ 1 , . u _ . 1 h M 1 u . ._ _. . _ _ . _ _ ‘ a u _ . _ . x I _ 1 _ W . _ _ 1 _ . _. .1. . . . . . _ _ a __ H nt -- ‘ H + 11 J _ . _ _ ~ . _ _ . . ........ r; . ., M ’ i . lmlf a H N H N L L L i _ _ w _ _ _ .. .Z_ -~——f ---~- -- —. -- i“ ---— ~-+—-—— ~: I ~-———— —— .- .4+. ___._+...1 + 4-.._._...—___. _‘__. _ -_- -_- -_--4--_j - _. -1- ..____..___._. ;_+-_ . 1 ‘ 1» _ . _ q. 1_._,_ 1 _ M 11 _ 1f . _;_:_ H. im__. .. flm___n 41:: _ __.mwm _ k ,_._hH .. 1.. {m _ _, v _ m.._u_ p 1 41;:1 0..”h .... “uh .. .1]. 7.::. .. _, ,._ 4 H . Luv—1.flfl * .x. %. ______”__« _ .3 My .4214». 42.4.9.4.34. --«.X4I. .. 4 4 _ _.,_W W _ in Z i , _+ . k #f ._ + ._. _. .- 1 if 1 1.. . _ 1 ‘_ _1+ _ ._. ._ 1:: . “A W P..,» H 2 m , «M14. ._r._u HM.» .w x 4 F“ »a #4..“ 1.. mm__ .fi _wi.~.u w .. . ,,. ;_I_1f:T: ._._—l. .—...-. _ i ._ _..._..§__- 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 ”I.” m...‘ ”fl-fl ._-..A . _ 9» .p _ , . A +..v» 9.. off. , “on "-O‘ (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 “In -v.‘ n A" -~| ”ml-ul- III W. ”DI- nun-um 1n. 3‘... v .‘l 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 - ”W...’ -——‘. __--_. ..._‘ ... ,'_' I -—_——4 1 1----__..___ _. -.__.. v ...._ ._._—._._ 1.. ._ _- '_-+--_ -____._ _;— 1 .— - ..- -.—._-‘._..- _.'. 1_.____._._: : __:._.+.4.-_.._.._' _;_.|_._ _ 5___.__...___._ __ _.»~.__ --.—._._A‘. . ...—4. ._ .4:— . .__..__+._ . . . . . . 1-;— L.~__.__: 1+:__. . + . . ._-.._ - _._- ._ A. ..___.._+_-.—~.______i ...——..__.' ' -_._-..-~_._—_‘. .. -....- _ _ 1 _ ,_. h. z .. H .1 _ . 1 _ _ 1 _ 1 n w 1 1 m m n M m. .. _ _ _ 4 1 . 1. . _ _ . a y... 14 . .w.* r L. 4 14. 4 w ._ .4. _ . 7 _ _ _ _ fl 1 _ m __ I... 4..-.qibt4ila l-_».,.43.oi+.qatilt.w..1 .1714 . . . . . u . _4 1 1 4 _ 4 _ .1 . v . f f A: A r it h. 1 H 1 A W 11.411 via . _ _. " --§_ _ c-w s—un . 111-..”... .. c c I s --- ----.“111 —+———- —~-—— — +-~——-J§«~——i—~s - ._ w A 4 H. i . 4;. 1 _ 1 _ w 4H _ t , . _ w .. ,_ _. U _ _ _ _ _ .4._:_:44..4.JL.. 1 . _ . . . . _ 1 _ _ _ . H .. .1 1 _ _ fl _ 4 _ . 4 _ 1 .4. .. 4. a . 1. _ L 1. . . : .....t. 1 _ 1 _ . H 1 . _ _ _ _ _ a 1 III. 1 ._.: 4 .1. 1....._.i-.-.1i #‘ .1 , ._ .1. Vi. . 11 t ... 1 11 ,_ . u _ 4.4;»: - _ --- .—_- _., ~ -_._ 1 4+- -14.-- —.~ + 1_ % ._._. n ._11” 1 ._.1_. 1: I: _ _HL.it|4 w” ”M. ,1 ~_ .1“." u . . V w. u h 4. iiuHiL+ ... 1.1:. flags 4 _ ._.. J“ _ __4 _ _. _._ ,.__ 1 u _. .- -..——.——~«--....._i- -_.. .. . . . 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 lgghog‘hUflz OUR¢IL2. CIExOOm 1‘34 :33. I a—lzv .1 stats! II I.‘ — — — — “ZOIUzg Z. frUZHJU>(; — fl ‘ — — I o. m. z 2 N. : 9 o o n o n v n u . 1 _ _ . . . o _ .fl 0 o. ..... o. 8 ................................................ 2 R ...... R 9 o. 8 on 8 8 2 2 8 8 o. . o. 8. 4|.9 .«IW.-- 0'0 . a. . . V . v4. 7-1 .1. v . . Yo Vii 10‘ of -u-% . :17. 010.10 8. Th 7.... Z. I.» .7: ....1... ._.... ...... t... .....r .2. .... :1: I... 2. a... “I. .21. ...... ...; TE. ._._: 2:: .1... :1: :2 8. R: 8. 8o 8. 8: 89 82 8: 8n. o8~ 82 88 88 88 180.8223. 1 o 3 ...-snicu apt-a4). VI. volt-.30»... lullv‘ ...!" tut-83.... ”...!o.’ 0.... Idol). ..qlo v.5! ’3? I! 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. BIBLIOGRAPHY Anderson, R. J. Composition of corn pollen, J. Biol. Chem. 55, 611 (1923). Andronescu, D. I. The physiology of the pollen of Zea mays with special regard to viability, Ph.D. Thesis, University of Illinois (1915). Association of Official Agricultural Chemists, Official methods of analysis, 9th ed., 371 (1960). 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