JOURNAL OF OLEO SCIENCE Copyright ©2003 by Japan Oil Chemists’ Society J. Oleo Sci., Vol. 52, No. 2, 57-63 (2003) JOS Molecular Species of Triacylglycerol Isolated from Depot Fats of Ratites 1, 2 3＊ Satoru SHIMIZU and Masuo NAKANO 1 The United Graduate School of Agriculture Sciences, Iwate University, Morioka (Iwate 020-8550, JAPAN) 2 Total Science Institute, Zukohsha Co. Ltd. (Obihiro, Hokkaido 080-0048, JAPAN) 3 Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine (Obihiro, Hokkaido 080-8555, JAPAN) Edited by K. Miyashita, Hokkaido Univ., and accepted October 11, 2002 (received for review September 6, 2002) Abstract: The depot fat of the emu, ratite native to Australia, has recently been found to be a source for cosmetics and pharmaceuticals. In this study, the chemical characteristics of the depot fat from the emu have been clarified and compared with those from the ostrich and rhea, other ratites. The fatty acid combination in triacylglycerol (TG) molecular species detected were dioleoyllinolein, palmitoyloleoyllinolein, palmitoyldiolein and dipalmitoylolein, which were common to all major species of ratite. In the emu, TG containing oleic acid accounted for 89% of the total and palmitoyldiolein (20.5%) and the amount of triolein (16.6%) was the highest in the three species. In the ostrich, TG containing linolenic acid accounted for 23% of the total and the amount of palmitoyloleoyllinolenin (7.4%) was the highest in the three species. In the rhea, TG containing linoleic acid accounted for 55% of the total and the amount of palmitoyloleoyllinolein (16.1%) was the highest in the three species. Key words: emu oil, ratite, fatty acid, triacylglycerol latory properties. It had been shown that enteral admin- 1 Introduction istration of a mixed fuel containing a physical mix of The depot fat of the emu, a ratite native to Australia, long-chain triacylglycerol and medium-chain triacyl- has recently been found to be a source for cosmetics glycerol improves protein anabolism and attenuates net and pharmaceuticals. It has been reported that emu oil protein catabolism in burned rats (5). It had also been derived from emu fat has skin-penetrating, moisturizing reported that feeding an enteral diet containing a fish and anti-inflammatory properties (1, 2), but the chemi- oil/medium-chain triacylglycerol structured lipid was cal characteristics of the oil have not been reported. associated with a reduction in eicosaniod production The major lipid class of animal depot fats is triacyl- from peripheral blood mononuclear cells (5). Therefore, glycerol (TG). The actions of TG molecular species and the physiologic functions of emu oil may be associated fatty acids in the fats on nutritional and physical charac- with the fatty acid composition and molecular species teristics have been reported in detail (3, 4). On the other of TG. hand, it is known that some TG molecular species and In this study, the fatty acid composition and molecu- fatty acids have anti-inflammatory and immunomodu- lar species of TG from emu depot fat were determined ＊ Correspondence to: Masuo NAKANO, Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, JAPAN E-mail: [email protected] Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online 57 http://jos.jstage.jst.go.jp/en/ S. Shimizu and M. Nakano by reverse-phase high-performance liquid chromatogra- 2mg/5mL. phy (HPLC) and gas-liquid chromatography analyses. They were then compared with those of the ostrich, a 2･4 Gas-liquid Chromatography ratite native to Africa and the rhea, one native to South Fatty acid methyl ester derivatives of TG separated America. by TLC and HPLC were prepared with methanolic HCl (50mL/L) for 2 hours at 125℃, and analyzed by gas- liquid chromatography (GC-14B, Shimadzu Co., 2Materials and Methods Kyoto, Japan) with a flame ionization detector and an 2･1Materials HR-SS-10 capillary column (25mm I.D.× 25m, Shin- Back fats of domestic emus (n=20) were supplied wa Chemical Industries, Kyoto, Japan). The initial col- from Double LL Emu Ranch (n=12), Montana, U.S.A., umn temperature was 150℃, the final temperature and Pyramid Hill Products Pty. Ltd. (n=8), Victria, Aus- 220℃ and the column program rate was 2℃/min. tralia. The back fat of the wild emu was supplied from Helium carrier gas was employed at a pressure of Pyramid Hill Products Pty. Ltd., Victria, Australia. The 2.0kg/cm2. Fatty acids in the samples were identified by back fat is a layer of fat that surrounds its body much comparison with retention times of external standards. like a saddle blanket. The back fat, peritoneal cavity fat The acyl groups examined in TG were palmitic acid and kidney leaf fat of the domestic emu were supplied (P), palmitoleic acid (Po), stearic acid (S), oleic acid from Double LL Emu Ranch, Montana, U.S.A.. The (O), linoleic acid (L) and linolenic acid (Ln). TG was back fat, peritoneal cavity fat and kidney leaf fat of the not distinguished by the position of the acyl group domestic ostrich were supplied from Big Bird Farms, bond; for example, it is shown as POL. Inc. Pres., Oregon, U.S.A.. The back fat of the domestic rhea produced in Paraguay, was supplied from Emu 3 Results Marketing International Pres., Oregon, U.S.A.. These depot fats was stored at －40℃ prior to preparation. 3･1 Fatty Acid Compositions of Triacyl- glycerols from Ratites 2･2 Extraction of Lipid and Preparation of The fatty acid compositions of depot fat TG from Triacylglycerol domestic and wild emus are shown in Table 1. Major The extraction of total lipids from the samples fol- fatty acids of TG from the domestic emu were oleic lowed the procedure described by Folch et al. (6). Total acid (49.9 ± 2.5%) and palmitic acid (24.6 ± 2.0%). lipids were dissolved in chloroform/methanol (2:1, by Major fatty acids of TG from the wild emu were oleic vol.) and applied on silica gel TLC plates (Silica gel 60, acid (38.6%), palmitic acid (22.4%) and linolenic acid Merck, Germany). The plates were developed in n-hex- (19.8%). The amount of linolenic acid in the depot fat ane/diethylether/acetic acid (80:30:1, by vol.) to 4/5 of TG of the wild emu was much greater than in the the plate height. The silica gel corresponding to TG of domestic emu. the zone was scraped off separately and extracted with The fatty acid compositions of TG from the three C/M (2:1, by vol.). ratite species depot fat are shown in Table 2. TG was 2･3 Reverse-phase HPLC Table 1 Fatty Acid Compositions in Triacylglycerol from TG were dissolved in chloroform and subjected to the Back Fats of the Wild and Domestic Emus. HPLC (LC-10A, Shimadzu Co., Kyoto, Japan) for sep- Fatty acid (mole%) aration of TG into molecular species. The mobile phase Domestic (n=20) Wild consisted of acetone/acetonitrile (64:36, by vol.) at a C16:0 24.6 ± 2.0 22.4 flow rate of 1.5mL/min. Peaks were monitored with a C16:1(n-7) 5.2 ± 1.2 3.4 refractive index detector (RID-10A, Shimadzu Co., C18:0 9.4 ± 1.4 7.1 Kyoto, Japan). The column used for the separation was C18:1(n-9) 49.9 ± 2.5 38.6 a Mightysil RP-18 GP (5mm, 6mm I.D.× 250mm, C18:2(n-6) 10.0 ± 3.4 8.7 Kanto Co., Japan), with two connected in tandem. The C18:3(n-3) 0.9 ± 0.3 19.8 column temperature was 30℃. The sample size was 58 J. Oleo Sci., Vol. 52, No. 2, 57-63 (2003) Molecular Species of Triacylglycerol Isolated from Depot Fats of Ratites Table 2 Fatty Acids in Triacylglycerols from the Depot Fats of the Domestic Ratites. Fatty acid (mole%) Emu Ostrich Rhea Back Peritoneal Kidney Back Peritoneal Kidney Back fat cavity fat leaf fat fat cavity fat leaf fat fat C16:0 22.5 22.8 21.1 29.9 28.9 29.7 31.5 C16:1(n-7) 3.5 4.1 3.4 5.5 7.3 6.9 6.0 C18:0 8.3 8.7 8.7 7.7 6.5 6.8 5.4 C18:1(n-9) 54.4 53.0 55.4 32.2 33.1 33.7 34.2 C18:2(n-6) 10.4 10.3 10.3 17.1 16.6 16.4 21.3 C18:3(n-3) 0.9 1.0 1.0 7.6 7.5 6.5 1.6 composed of fatty acids with carbon numbers of 16 and 18, compositions characteristic of ratites species. The major fatty acids of emu TG were palmitic acid (22.5%) and oleic acid (54.4%), and those of the ostrich and rhea were oleic acid (ostrich, 32.2% and rhea, 34.2%), palmitic acid (ostrich, 29.9% and rhea, 31.5%) and linoleic acid (ostrich, 17.1% and rhea, 21.3%). Ostrich TG contained more linolenic acid than other ratite TG. In the emu and ostrich, the fatty acid composition of the back fat was homologous with that of the peritoneal cavity fat and the kidney leaf fat. 3･2HPLC of Triacylglycerols from Ratites The results of reverse-phase high-performance liquid chromatography of ratite depot fat (back fat) are shown in Fig. 1. There were 22 TG peaks for the emu, 34 TG peaks for the ostrich and 25 TG peaks for the rhea detected by HPLC separation. The relative ratios of the TG peaks are shown in Tables 3-5.