Agric. Biol. Chem., 54 (4), 1035-1042, 1990 1035

Chemical Composition of , Especially Triacylglycerol, in Grape Seeds Masao Ohnishi, Shuji Hirose,* Masayuki Kawaguchi,* Seisuke Ito and Yasuhiko Fujino1 Department of Agricultural Chemistry, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan * Tokachi-Ikeda Research Institute for Viticulture and Enology, Ikeda, Hokkaido 083, Japan Received November 13, 1989

Total lipids were extracted from five varieties of grape seeds and systematically analyzed for their chemical compositions. The yields of the total lipids were 10-16 %, and triacylglycerol (TG) usually amounted to c. 90 %of the whole. From a reversed-phase high-performance liquid chromatographic analysis, the major molecular species of TGwere shown to be trilinolein (40 %), oleoyldilinolein (21 %) and palmitoyldilinolein (18 %). The component fatty acids were asymmetrically distributed at C-l and C-3 of the TGmolecule. was exclusively located at the C-l position, although unsaturated fatty acids, especially , were predominant at the C-l position, as at the C-2 and C-3 positions. Compared with TG, higher proportions of palmitic and linolenic acids were generally observed in thirteen other classes isolated from grape seeds, although the compositions of the diacylglycerol and free fatty acids were roughly identical with that of TG. As component sterols, sitosterol, campesterol and stigmasterol, especially the former, were predominant. Their relative proportions were somewhatdifferent from each other between the neutral and polar sterol lipids.

In the process of wine making, large quan- Europe. It has been reported that grape seed tities of the pomace are produced as by- contain large amounts of unsaturated fatty products, which are utilized after fermentation acids such as linoleic and oleic acid, and mainly as a soil conditioner. The effective use sitosterol is the predominant sterol constituent of food processing by-products is essential not in the oils.2~4) However, little is known about only to minimize the production cost, but also the classification of the lipids and their com- to augment limited resources. Grape seeds ponent fatty acids. Especially, there have been which remain unchanged in the pomacerepre- no comprehensive studies on the molecular sent a high protein content. Therefore, the level of TG. A determination of the lipid seeds are considered to be a potential source of composition in grape seeds is an essential edible protein, and a procedure for preparing a foundation on which further examination of protein concentrate has been developed.1'2) such subjects as production on an industrial Moreover, grape seeds have been extracted on scale, utilization and nutritional quality can be a small scale to get edible vegetable oils in based. f Present address: Department ofFood Science, Sonoda CampusCollege, Amagasaki, Hyogo 660, Japan. Abbreviations: TG, triacylglycerol; AS, acylsterol; DeMS, desmethylsterol; FFA, free fatty acid; DG, diacylglyc- erol; ASG, acylsterylglycoside; CE, cerebroside; SG, sterylglycoside; DGDG, diglycosyldiacylglycerol; MGDG, monoglycosyldiacylglycerol; PC, phosphatidylcholine; PE, ; PI, ; APE, acylphosphatidylethanolamine; LAPE, lysoacylphosphatidylethanolamine; LPC, lysophosphatidylcholine; PG, phos- phatidylglycerol; PA, phosphatidic acid; LLL, trilinolein; OLL, oleoyldilinolein; PLL, palmitoyldilinolein; POL, palmitoyloleoyllinolein; SLL, stearoyldilinolein; OOO, ; OOL, dioleoyllinolein; POO, palmitoyldiolein; TLC, thin-layer chromatography; GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography. 1036 M. Ohnishi et al.

This paper provides such a background, and with a 163 gas chromatograph (Hitachi Seisakusho Co.) describes the isolation, quantification and equipped with a hydrogen-flame ionization detector. The characterization of the lipid classes, com- column was packed with 5% DEGS on Chromosorb WAW-DMCS,and the column temperature was held at ponent fatty acids and sterols from grape 175C. seeds. The experimental procedures for degradation of AS, ASG and SG were identical to these reported pre- viously.1n On the other hand, the neutral and polar lipid Materials and Methods fractions were, respectively, saponified with I n KOHU) Materials. Grape seeds were collected from the pomace and methanolyzed as already described to obtain the of five varieties of grapes (Campbell Early, Kiyomi, component sterols, originating from AS and DeMSin the Zweigeltrebe, Kanzler and Wild grape), which were har- case of the former fraction and from ASGand SGin that vested in Hokkaido during 1985 and 1986. The pomace of the latter one. The sterols thus liberated were chro- of the Kanzler variety used for white wine production matographed on a column packed with 1.5% OV-17 was immediately separated from the must after pressing, on Chromosorb WAW-DMCS,the column temperature whereas those of the other grapes were obtained after being 250 C. pressing and subsequent fermentation. For a comparison, the seeds were directly collected from fresh grapes (Camp- Analysis of TG. The stereospecific distribution of the bell Early variety). The grape seeds thus obtained were all component fatty acids of TGwas examined according to subjected to air-drying. Brockerhoff. 1 2) To separate the molecular species, TGwas dissolved in Extraction andfractionation of the total lipids. The grape chloroform and then subjected to reversed-phase HPLC seeds were ground to powder (30mesh) and extracted by with a Shimadzu Model 5A instrument. Reversed-phase shaking three times with five volumes of a chloroform- HPLC was carried out on an ERC-ODS-1282 column methanol (2: 1, v/v) solution and with three volumes of (6x250mm) at 30°C, acetone-acetonitrile (64: 36, v/v) water-saturated butanol. The combined extracts were being used as the mobile phase at a flow rate of 1.5 ml/min. washed in water,5} and the chloroform layer was evapo- The chromatograms were monitored by an SPD-24 re- rated to dryness to yield the total lipids. Part of the lip- fractive index detector (Erma Optical Co.) and an SPD-2A ids was applied to a silicic acid column for fractionat- ultraviolet detector (Shimadzu Seisakusho Co.). The TG ing into neutral and polar lipids.6' The total lipids from species fractionated by HPLCwere analyzed by GLCfor the Kiyomivariety were also fractionated into neutral lip- the quantification anddetermination of total carbon num- id, glycolipid and fractions by silicic acid bers.13' GLC analysis was carried out with an internal column chromatography, using the successive elution of standard (), using Diasolid ZT as the column chloroform, acetone and methanol.7) packing at 320°C. Simultaneously, part of the TG species was methanolyzed as already described to identify the Analysis and isolation of the lipid classes. The com- component fatty acids. ponent lipid classes in each fraction were analyzed by TLC on silica gel G with hexane-diethyl ether-acetic acid (80:20: 1, v/v/v), chloroform-methanol (95 : 12, v/v) and Results chloroform-methanol-water (65 : 16 : 2 and 65 : 25 :4, v/v/ Lipid content and composition v), and their relative proportions were estimated by den- sitometric determination.8) Two-dimensional TLC on As shown in Table I, the yield of total lipids silica gel G was carried out, using chloroform-methanol- from grape seeds ranged from 10 to 16% of the 28% ammonia water (65:36:7, v/v/v) in the first dimen- fresh weight. This distinct difference of lipid sion and chloroform-acetone-methanol-acetic acid-water content amongthe varieties maybe due to the (10:4:2:2: 1, v/v/v/v/v) in the second dimension. The major lipid constituents were separated and purified degree of seed maturity. The lipid content was from the grape seeds by a combination of silicic acid col- highest in the Kiyomi variety and lowest in the umnchromatography, DEAE-Sephadex CL-6B column Campbell Early variety. However, there was chromatography and preparative TLCon silica gel G, as no significant difference in the lipid content described previously.9 10' between seeds collected from the fresh grapes and the pomace. Of the total lipids, the neutral Analysis of component fatty acids and sterols. To de- termine the fatty acid composition, the lipids were heat- lipids usually amountedto morethan 90%. ed under reflux for 2hr with methanolic 3% HC1. Fatty The ratio between the glycolipid and phospho- acid methyl were extracted from the methanolyzates lipid fractions was approximately 2 : 1 in the with hexane and analyzed by GLC. GLCwas carried out case of the Kiyomi variety. Lipids in Grape Seeds 1037 The composition of lipid classes from aric acids. Campbell Early seeds is shown in Table II. The (2) Component fatty acids of the lipid classes. major neutral lipid was TG (90% of the neutral The composition of fatty acids in the neutral lipid fraction), with small amounts of AS (4%), lipid classes isolated from Campbell Early free DeMS (3%) and FFA (1%). In addition, seeds is shown in Table IV. The fatty acid DGand methylsterols were found to be pres- profiles of TG and FFA were quite similar to ent as minor components. In the glycolipid that of the total lipids already described. In fraction, ASG (31%), CE (28%), SG (24%) DG, linoleic acid was slightly less and oleic and DGDG (10%) were predominant, some and palmitic acids were richer, compared with minor glycolipids such as MGDG and sul- TG. The proportions of saturated fatty acids foquinovosyldiacylglycerol also being recog- (palmitic and stearic acids) were specifically nized. Two-dimensional TLC of the phospho- high in AS, although its major fatty acid lipid fraction revealed the presence of at least eight components, which were characterized as Table II. ApproximateComposition of the APE, LAPE, PE, PG, PC, PI, LPC and PA. Lipid Classes in Grape Seeds (%) Amongthese, the major lipids were PC (32%), Lipid class Relative proportion PE (28%) and PI (17%). Neutral lipids 9T TG 90" Fatty acid composition AS 4 (1) Component fatty acids of the total lipids. DeMS 3 Table III shows the composition of fatty acids FFA 1 Others in the total lipids from grape seeds. Six fatty 7 acid components were found, among which Glycolipids ASG linoleic acid was usually predominant; their CE proportions amounted to 69-81% of the SG whole. The other predominant fatty acids in DGDG MGDG decreasing order were oleic, palmitic and ste- Others Table I. Contents of Lipids in Grape Seeds (%) PC PE Ratio of neutral PI Variety Total lipids lipids to polar APE lipids LPC PG Campbell Early 9.9 94:6 Others Kiyomi 16.2 97:3 Zweigeltrebe 15.4 92:8 a Relative proportion of the neutral lipid, glycolipid Kanzler ll.7 94:6 and phospholipid fractions separated by silicic acid Wild grape 13.3 95:5 column chromatography. b Measured by quantitative TLCof each fraction.

Table III. Composition of Fatty Acids in the Total Lipids from Grape Seeds (%) Fatty acid Campbell Early Kiyomi Zweigeltrebe Kanzler Wild grape

16:0 8.9 6.8 7.2 7.2 6.7 16:1 <0.1 <0.1 <0.1 <0.1 <0.1 18:0 4.4 5.3 4.2 1.1 3.4 18:1 17.5 12.0 10.9 ll.2 9.7 18:2 69.2 75.9 77.7 80.5 80.2 18:3 <0.1 <0.1 <0.1 <0.1 <0.1 1038 M. Ohnishi et al.

Table IV. Composition of Fatty Acids in the Lipid Classes from Grape Seeds (%) Fattyacid TG FFA DG AS ASG MGDG DGDG PE PC PI LPC APE PG

16:0 8.6 10.3 12.9 31.0 38.6 22.1 18.9 34.2 31.1 36.9 40.0 40.6 61.8 22.7 18:0 3.9 3.8 4.9 14.7 4.7 2.9 1.5 2.9 4.5 5.6 2.0 2.2 3.5 2.5 18:1 20.0 19.0 24.6 14.9 4.8 4.3 4.4 5.5 10.3 4.2 4.7 9.5 5.6 8.5 18:2 67.5 64.2 54.2 31.9 41.0 23.2 28.7 53.6 50.5 51.9 50.2 42.4 26.0 60.1 18:3 <0.1 1.5 1.7 2.9 9.8 43.4 45.0 2.8 2.5 1.1 2.0 4.4 <0.1 5.2 Others <0.1 1.2 1.7 4.6 1.1 4.1 1.5 1.0 1.1 0.3 1.1 0.9 3.1 1.0 S.I.a 1.6 1.5 1.4 0.9 1.2 1.9 2.0 1.2 1.2 1.1 1.1 1.1 0.6 1.5

S.I. =saturation index (average number of double bonds per acyl chain).

Table V. Composition of 4-Desmethylsterols in the Sterol Lipids from Grape Seeds (%) Kiyomi Zweigeltrebe Campbell Early

NSLfl PSL" NSL PSL AS DeMS ASG

1.3 0.3 0.9 <0.1 1.1 1.2 0.7 0.4 ll.6 4.2 12.1 5.5 ll.1 9.8 4.0 4.1 7.6 2.0 7.9 1.9 6.4 8.6 5.0 5.8 75.6 93.4 72.7 92.6 81.4 80.2 90.3 89.7 0.1 <0.1 1.4 <0.1 <0.1 0.2 <0.1 <0.1

a Neutral sterol lipids (AS and DeMS). Componentsterols in the neutral lipid fraction were analyzed. b Polar sterol lipids (ASGand SG). Component sterols in the polar lipid fraction were analyzed. components were linoleic and palmitic acids. and oleic acid were slightly richer in APEthan The compositions offatty acids in the major in PE. On the other hand, the fatty acid glycolipid and phospholipid classes from the composition of PG was significantly different same variety are also shown in Table IV. from those of the other phospholipids already Linoleic and palmitic acid were rich in ASG, as described, palmitic acid amounting to more in AS, but its content was lower than 60%. Among the phospholipids, the than that in AS. In MGDGand DGDG, lowest palmitic acid content was found in linolenic acid as well as linoleic acid were PA, whereas its proportion of linoleic acid predominant, which largely differed from the was conversely the highest. fatty acid profiles of the other lipids. The major component fatty acids in the phospho- Sterol composition lipid classes were usually linoleic and palmitic As component 4-desmethylsterols of non- acids, the proportions of the former acid being polar sterol lipids (AS and DeMS)and polar significantly lower in the phospholipids than ones (ASG and SG) from Zweigeltrebe and TG. The fatty acid compositions of PE, PC Kiyomi seeds, at least five compounds were and PI were similar to one another, although a found by GLC analyses (Table V). Among slightly higher content of oleic acid was found these the principal sterol was usually sitosterol; in PC. In LPC, palmitic acid amounted to 40% this sterol amounted to 93%in the polar sterol of the whole, from which the lipid was pre- lipids. The next most abundant ones were sumed to be largely of the 1-isomer, since campesterol and stigmasterol. Although minor saturated fatty acids hardly ever located at the amounts of and Zl5-avenasterol C-2 position in PC.10) APEshowed a fatty acid were also detected, the sterols are knownto be composition similar to that of PE, but palmitic widespread in higher plants.14) The sterol com- Lipids in Grape Seeds 1039

Table VI. Positional Distribution of the Fatty Acids in Triacylglycerols from Grape Seeds (%) Campbell Early Kiyomi Zweigeltrebe Fatty acid C-l C-2 C-3 C-l C-2 C-3 C-l C-2 C-3

16:0 17 3 <1 12 4 <1 12 4 2 18:0 7 1 2 9 2 1 ll 1 1 18:1 16 28 17 ll 23 7 15 28 10 18 : 2 60 68 81 68 71 92 62 67 87

Fig. 1. Separation of TGMolecular Species from Grape Seeds by Reversed-phase HPLC. HPLCanalysis was carried out on an ERC-ODS-1282column. The column temperature was 30°C, and the mobile phase was acetone-acetonitrile (64: 36, v/v) at a flow rate of 1.5ml/min. The peaks were monitored with a refractive index detector. position in each sterol lipid class from the C-3 position. Campbell Early variety is also shown in Table (2) High-performance liquid chromatogram. V. The sterol composition was similar between Reversed-phase HPLC of TG from the AS and FS, and between ASGand SG. Campbell Early variety revealed at least seven- teen peaks (Fig. 1). Table VII shows the Characterization of triacylglycerol relative proportions as well as the fatty acid (1) Stereospecific distribution offatty acids. combinations of each TG peak. The principal The composition of the fatty acids located at TG species were LLL (40%), OLL (21%) and the C-l, C-2 and C-3 positions of the TG PLL (18%), with minor amounts ofPOL (5%), molecule is shown in Table VI. Saturated fatty SLL (4%), OOO (3%), OOL (3%) and POO acids (palmitic and stearic acid) were distrib- (3%). The relative proportions of the other TG uted at the C-l position together with un- peaks were less than 1%of the whole. saturated fatty acids, but were scarcely de- (3) Fatty acid distribution in the triacylglyc- tected at the C-2 and C-3 positions, at which erol species. Table VIII shows the fatty acid linoleic and oleic acids were exclusively pres- composition at the C-2 position in the major ent. Thus, TG from grape seeds generally TG species that were separated by reversed- showed an asymmetric distribution of com- phase HPLC. In the OLLand POL species, the ponent fatty acids at the C-l and C-3 po- ratios of oleic acid to linoleic acid were found sitions. Although oleic acid was equally dis- to be roughly equal, whereas that in the OOL tributed between both the positions, the species was approximately 3 : 1. Saturated - proportion of linoleic acid was highest at the ty acids were hardly recognized at the C-2 1040 M. Ohnishi et al.

Table VII. Reversed-phase High-performance Liquid Chromatographic Data of TGfrom Grape Seeds Carbon number^ FAcomposition Peak number" Fatty acid c combination 16:0 18:0 18:1 18:2 18:3 Ad Be Cs. 1 100 75 LLLn 0.2 0.7 2 100 100 LLL 40.2 41.9 3 7 Not analyzed 54 PLLn? <0.1 0.1 4 100 36 64 OLL 21.3 19.9 3 5 99 1 3 65 PLL 17.9 12.5 6 100 69 31 OOL 3.2 4.6 7 3 5 95 9 59 SLL 4.3 6.4 8 86 15 24 26 41 POL 4.9 4.3 9 100 52 14 25 PPL 0.4 0.3 10 100 <1 98 2 OOO 3.3 2.6 ll 100 4 38 30 SOL 0.5 2.2 12 92 8 24 57 7 POO 2.9 2.7 13 Not analyzed 34 22 22 PSL? <0.1 <0.1 14 100 64 29 4 PPO 0.4 0.6 15 100 1 64 2 SOO 0.3 0.4 16 100 16 19 17 SSL 0.1 0.2 17 100 35 30 PSO 0.1 <0.1

SeeFig.1. Composition based on the carbon number of the acyl chains (%). Abbreviations indicate fatty acids that are esterified with in the molecule where positional isomerism is not regarded. P, palmitic acid; S, stearic acid; O, oleic acid; L, linoleic acid; Ln, linolenic acid. Determined by GCanalysis using an internal standard. Relative proportion of each peak recorded by a refractive index detector.

Table VIII. Distribution of Fatty Acids at the usually LLL (42-52%), OLL (17-22%), PLL C-2 Position in TG Species from Grape Seeds (14-18%) and POL, including roughly equal Fatty acid composition (%) amounts of SLL (9-11%). Thus, HPLC anal- yses of grape seed TG show no substantial 18:0 TGspecies* 16:0 18:1 18:2 difference in the molecular species composition 49 amongthe varieties. 5 95 25 <1 100 Discussion 49 In the literature,1~3) the lipid content in Each TG species by reversed-phase HPLC was grape seeds has been reported to be ll-16%, hydrolyzed by pancreatin, and its product (2- which is identical with the results obtained in monoacylglycerol) was analyzed for the constituent this study. Grape seed oil is considered to be a fatty acids. drying oil with high vitamin E content and to have a good effect on human health.16) Grape position in any TGspecies. seeds weigh no more than 2-3% of the fresh (4) TGcomposition. The relative proportions fruits, but amounted to 20-26% of the pom- of TGspecies from five grape varieties are ace.2) Therefore, the seeds could be utilized shown in Table IX. Although the percentages as a potential source of edible oil on an in the Table do not reflect the accurate com- industrial scale, although the lipid content is position of TG species because of peak de- not as high as oil-bearing seeds such as rape tection at 205 nm,15) the principal species were and sunflower.17) Lipids in Grape Seeds 1041

Table IX. Relative Proportion of TG Species from Grape Seeds (%)fl TGspecies Campbell Early Kiyomi Zweigeltrebe Kanzler Wild grape LLLn 1.4 1.8 1.8 2.0 1.4 4 5 LLL 41.8 7.9 52.2 43.8 3.6 PLLn 1.2 1.1 1.7 1.3 1.2 2 1 OLL 22.0 1.3 17.0 19.9 8.3 1 1 PLL 15.2 5.6 14.1 18.1 4.8 OOL 4.1 2.1 1.7 2.5 1.5 SLL& POL 10.8 8.9 9.5 ll.1 8.5 OOO & PPL 0.9 1.2 1.6 1.3 0.7 < POO & SOL 2.6 0.1 0.4 <0.1 0.1 < Others <0.1 0.1 <0.1 <0.1 0.1

Calculated from the TGpeaks on a reversed-phase high-performance liquid chromatogram recorded by UV (205 nm) detection.

The lipid components in the grape seeds omers in TG species having saturated fatty consisted largely of TG, as in the cases of acids would be, therefore, considerably dif- other oil-rich seeds; the polar lipid classes ferent between grape and sunflower seeds. amounted to less than 5%. As reported previ- Namely, PLL from grape seeds was mostly ously,2 ~4) the fatty acid composition of grape of l-palmitoyl-2-linoleoyl-3-linolein, whereas seed oils was similar to that of sunflower in sunflower TG, the two enantiomers, 1- oy15-18) jn j)Otj1 tke ojis? linolenic acid, palmitoyl-2-linoleoyl-3-linolein and 1-linoleo- which could be a source of potent off-flavor yl-2-linoleoyl-3-palmitin, could be equally substances in the event of oxidative dete- present. From the viewpoints of nutritional rioration, is of extremely low content. The value and utilization, however, TGenantio- principal TGspecies in grape seeds were found mers may be regarded as the same molecu- to be LLL, OLL and PLL, which amounted to lar species, since there are probably no signif- approximately 80% in all. Although analyses icant differences among optical isomers con- of the TG compositions of seeds collected cerning their physiological properties and from fresh grapes and differing in harvest years reactivity, which will need further investi- were also carried out, there was no significant gation. difference in the molecular species among them As component 4-desmethylsterols in grape in either case. seeds, sitosterol, campesterol and stigmasterol Previously we have reported that LLL, OLL were present, the former being predominant, and PLLwere also predominant in sunflower which is identical with the results previously oil, their proportions being 36%, 23% and reported.34) Moreover, the compositions of 12%, respectively.15) From these results, it may sterols from AS and ASGwere, respectively, be likely that TGfrom grape and sunflower like those offree DeMSand SG, although the seeds is similar regarding the molecular species sterol pattern of neutral sterol lipids (ASand levels as well as the fatty acid composition. DeMS) from grape seeds was different from However, a stereospecific analysis of the com- that of polar sterol lipids (ASG and SG), as ponent fatty acids in grape seed TGshowedan was seen in the sterol lipids from rice bran.U) asymmetric distribution between the C-l and In this study, fourteen lipid classes were C-3 positions. The component fatty acids of isolated from grape seeds, and their com- TG from sunflower and safflower, which are ponents fatty acids were determined. Pre- also linoleic acid-rich oils, are known to be viously we reported that, of the 1,2-DG moi- almost symmetrically distributed at the C-l eties of glycerolipids from plant seeds, the and C-3 positions.18 19) The ratios of enanti- glycolipids tend to be highly unsaturated, the 1042 M. Ohnishi et al.

TG and neutral phospholipids to be mod- KaishU 52, 525 (1978). erately unsaturated, and acidic phospholipids 9) N. Murata, N. Sato, N. Takahashi and Y. and sulfolipid to be a little unsaturated.10'20) Hamazaki, Plant Cell Physiol., 23, 1071 (1982). 10) M. Ohnishi, Y. Yasui, Y. Mano, S. Ito and Y. Fromthe saturation index (the average num- Fujino, Agric. Biol. Chem., 53, 565 (1989). ber of double bonds per acyl chain) shown in ll) N. Kuroda, M. Ohnishi and Y. Fujino, Cereal Table IV, the glycerolipids from grape seeds Chem., 54, 997 (1977). 12) J. Brockerhoff, J. Lipid Res., 6, 10 (1965). are also presumedto have the same character- 13) T. Miyazawa, H. Tazawa and Y. Fujino, Cereal istics, although the fatty acid composition of Chem., 55, 138 (1978). PI in grape seeds (an acidic phospholipid) re- 14) G. A. Bean, Adv. Lipid Res., ll, 193 (1973); T. sembled those of neutral phospholipids (PE Akihisa, /. Jpn. Oil Chem. Soc. (Yukagaku), 35, 715 and PC) unlike maize.10) Compared with other (1986). plant seeds such as maize,10'21) rice,22) wheat23) 15) Y. Mano, M. Ohnishi, S. Sasaki, M. Kojima, S. Ito and Y. Fujino, Nippon Eiyd Shokuryo Gakkaishi, 42, and pea,24) however, oleic acid was lower and 251 (1989). linolenic acid was richer in the glyceroglyco- 16) G. W. Rohne, Fette Seifen. Anstrichmittei, 86, 172 lipids and ASGfrom grape seeds. Moreover, a (1984); I. Elmadfa and S.-W. Kim, ibid., 86, 606 higher proportion of palmitic acid was gen- (1984). erally observed in the grape seed phospho- 17) M. I. Gurr, in "The Biochemistry of Plants," Vol. 4, lipids, with less oleic acid. ed. by P. K. Stumpf, Academic Press, New York, 1980, pp. 205-248. 18) A. J. Sheppard, J. L. Iverson andJ. L. Weihrauch, in References "Handbook of Lipid Research," Vol. 1, ed. by A. Kuksis, Plenum Press, New York, 1978, pp. 341-379. P. Fantozzi, J. Am. Oil Chem. Soc, 58, 1027 (1981). 19) K. Ichihara and M. Noda, Phytochemistry, 19, 49 B. B. Kamel, H. Dawson and Y. Kakuda, J. Am. Oil (1980). Chem. Soc, 62, 881 (1985). 20) Y. Fujino, Nippon Nogeikagaku Kaishi, 56, 353 V. L. Puchinger, F. Wurst, T. Prey and G. Graefe, (1982). Fette Seifen. Anstrichmittel., 87, 1 (1985); Y. Hirose 21) H. Tanaka, M. Ohnishi and Y. Fujino, Nippon and F. Iwama, Jpn. Oil Chem. Soc. (Yukagaku), 35, Nogeikagaku Kaishi, 58, 17 (1984). 768 (1986). T. Miyazawa, Y. Yoshino and Y. Fujino, /. Sci. Fd. E. Fedeli and C. Mariani, Riv. It. Sost. Grasse, 51, Agric, 28, 889 (1977); T. Miyazawa and Y. Fujino, 129 (1974); A. Seher and H. Vogel, Fette Seifen. Nippon Nogeikagaku Kaishi, 52, 37 (1978). Anstrichmittel, 78, 301 (1976). S. Ito, K. Ohba and Y. Fujino, Nippon Nogeikagaku J. Folch, M. Lees and G. H. Sloane-Stanley, J. Biol. Kaishi, 57, 1231 (1983); S. Ito, H. J. Park and Y. Chem., 226, 497 (1957). Fujino, ibid., 58, 881 (1984). H. E. Carter and C. B. Hirschberg, Biochemistry, 7, T. Miyazawa, S. Ito and Y. Fujino, Cereal Chern., 51, 2296 (1968). 623 (1974); T. Miyazawa and Y. Fujino, Nippon G. Rouser, G. Kritchevsky, G. Simon and G. J. Nogeikagaku Kaishi, 50, 169 (1976); T. Miyazawa, S. Nelson, Lipids, 2, 37 (1968). Ito and Y. Fujino, Proc. Jpn. Conf. Biochem. Lipids, M. Ohnishi and Y. Fujino, Nippon Nogeikagaku 19, 223 (1977).