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. In reverse-phase HPLC, it is known that TGs are eluted in the order of the PN (partition number) calculated with the known equation PN = CN (carbon number) - 2 × DB (number of double bonds). The fatty acid combinations in TG molecular species detected were OOL, POL, POO and PPO, which were common to all species of ratites. Nev- Fig.1 Separation of Triacylglycerols from Ratites into Mole- ertheless, the molecular species of ratite TG differed by cular Species by Reverse-Phase HPLC. ratites species as shown by the fatty acid patterns. In the The analytical details have been described in Materials emu, TG containing oleic acid accounted for 89% of and Methods. Peak numbers refer to Table 3 - Table 5. the total and POO (20.5%) and the amount of OOO (16.6%) was the highest among the three species. In the ostrich, TG containing linolenic acid accounted for the amount of POL (16.1%) was the highest among the 23% of total and the amount of POLn (7.4%) was the three species. The composition of the saturated fatty highest among the three species. In the rhea, TG con- acids (S) and unsaturated fatty acids (U) found in TG taining linoleic acid accounted for 55% of the total and are shown in Table 6. Amounts of U2S (diunsaturated

59 J. Oleo Sci., Vol. 52, No. 2, 57-63 (2003) S. Shimizu and M. Nakano

Table 3 Fatty Acid Compositions in Triacylglycerol Peaks from the Back Fat of the Domestic Emu. Fatty acid (mole%) Peak Ratio Suggested Estimated C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 numbera (%) TGb PNc (n-7) (n-9) (n-6) (n-6) 1 0.5 10.1 24.3 41.9 23.7 Unidentified 2 34.1 65.9 OLL 44 3 1.9 18.1 48.4 20.1 13.5 Unidentified 4 1.2 26.7 6.5 11.6 54.5 0.8 PLL 44 5 1.7 30.0 13.7 16.6 13.4 15.7 10.6 Unidentified 6 9.3 66.1 33.9 OOL 46 7 2.8 30.1 67.7 2.1 PoOO 46 8 8.9 32.2 34.7 33.1 POL 46 9 2.8 31.4 30.3 3.2 35.1 PPoO 46 10 1.9 64.9 4.4 30.7 PPL 46 11 0.7 56.3 20.0 23.8 Unidentified 12 16.6 100.0 OOO 48 13 3.7 28.0 44.5 27.6 SOL 48 14 20.5 33.5 66.5 POO 48 15 1.4 36.7 27.3 8.4 24.6 PSL 48 16 7.8 67.2 32.8 PPO 48 17 0.6 86.8 13.2 PPP 48 18 7.8 1.0 33.5 65.5 SOO 50 19 5.8 33.6 34.1 32.3 PSO 50 20 0.7 60.0 37.4 2.6 PPS 50 21 1.2 2.2 64.4 33.4 SSO 52 22 0.3 32.9 67.1 PSS 52

aSee Fig. 1, b,cAbbrevations described in the Materials and Methods section.

TG) + U3 (triunsaturated TG), which are liquid TG that polyunsaturated fatty acids deteriorate more due to species at room temperature, were 75.5% for the emu, oxidation than other fatty acids (7). Accordingly, emu 63.0% for the ostrich, and 70.7% for the rhea. oil may not be as sensitive to oxidation as other ratite oils. The molecular species of TG from the three ratite species reflected the differences in fatty acid composi- 4Discussion tion. The emu had a large amount of TG species con- The fatty acid composition and molecular species of taining oleic acid, the ostrich had a large amount of TG TG isolated from depot fat of the emu, ostrich and rhea species containing linoleic acid and linolenic acid, and were investigated and it was found that the major fatty the rhea had a large amount of TG species containing acids in depot fat TG from the emu were oleic acid and linoleic acid. The fatty acid composition of the wild palmitic acid, and the major TG molecular species were emu was different from that of the domestic one. In POO and OOO. This differed with the ratite species. general, fatty acid patterns in domestic bovines and pigs The amount of oleic acid was less in depot fat TG from are influenced by the kind of feed used. In the wild, the ostrich and rhea than in that from the emu, but the emus feed on leaves, seeds and insects. But on most amount of linoleic acid was greater in the ostrich and farms, domestic emus are fed a specially formulated rhea than in the emu. The amount of linolenic acid was emu feed that provides a balanced nutritional diet for greater in depot fat TG from the ostrich than in the them. Accordingly, it may be that the chemical charac- other ratites. The amounts of polyunsaturated fatty ter of depot fat from the three species of ratites, includ- acids in TG from emu depot fat were much less than ing domestic and wild emus, was affected by the differ- those from the ostrich and rhea. It is generally accepted ent diet, in addition to genetic factors.

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Table 4 Fatty Acid Compositions in Triacylglycerol Peaks from the Back Fat of the Domestic Ostrich. Fatty acid (mole%) Peak Ratio Suggested Estimated C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 numbera (%) TGb PNc (n-7) (n-9) (n-6) (n-6) 1 0.3 32.8 67.2 LLnLn 38 2 0.6 65.8 34.2 LLLn 40 3 1.0 10.2 22.0 12.7 55.1 OLnLn 40 4 1.1 30.7 69.3 PLnLn 40 5 0.5 100.0 LLL 42 6 2.8 6.6 27.9 37.5 28.0 OLLn 42 7 0.5 24.8 33.4 12.4 29.4 PoOLn 42 8 2.3 31.6 2.8 1.6 32.9 31.1 PLLn 42 9 0.4 24.4 19.5 6.3 6.1 6.2 37.6 Unidentified 10 3.2 34.4 65.6 OLL 44 11 3.6 2.3 14.5 49.6 15.5 18.1 OOLn? 44 12 3.1 32.4 15.8 49.6 2.2 PLL? 44 13 7.4 29.0 9.6 3.4 22.9 12.2 22.9 POLn 44 14 2.4 59.7 4.8 2.3 5.7 27.5 PPLn 44 15 0.9 58.0 6.1 1.6 8.6 25.6 PPLn? 44 16 6.3 66.6 33.4 OOL 46 17 1.7 30.2 69.8 PoOO 46 18 11.9 31.9 34.3 33.7 POL 46 19 3.5 31.2 29.2 3.4 36.2 PPoO 46 20 4.6 66.3 33.7 PPL 46 21 1.1 51.3 13.2 22.3 13.2 Unidentified 22 2.0 57.6 22.0 6.0 8.1 1.9 4.5 PPPo 46 23 4.8 3.4 95.4 1.2 OOO 48 24 3.1 7.5 27.8 36.6 27.5 SOL 48 25 9.6 31.1 2.7 66.2 POO 48 26 1.6 33.1 0.9 31.9 4.6 29.5 PSL 48 27 8.5 61.1 3.3 5.1 30.5 PPO 48 28 2.0 94.2 3.4 2.4 PPP 48 29 0.3 36.0 5.0 28.1 30.9 Unidentified 30 2.4 1.4 33.4 65.1 SOO 50 31 4.1 32.2 34.8 32.9 PSO 50 32 1.4 60.2 1.3 37.3 1.1 PPS 50 33 0.5 66.0 34.0 SSO 52 34 0.4 31.5 5.4 63.1 PSS 52

aSee Fig. 1, b,cAbbrevations described in the Materials and Methods section.

In the emu and ostrich, the fatty acid compositions of It has been reported that emu oil derived from emu back fat were homologous to those of the peritoneal fat has skin penetrating and moisturizing properties (1). cavity fat and the kidney leaf fat. In depot fats from the Generally, the melting point is affected by the content cow and pig, fatty acid compositions differ with differ- of U2S+U3, liquid TG species at room temperature (9). ent kinds of adipose tissue (8,9). The above results sug- In this work, ratite depot fat contain large amonts of the gest that the peritoneal cavity fat and the kidney leaf fat above TG species, but content did not differ among the from the emu are available as sources for cosmetics and three species. In TG from human sebum, the main fatty pharmaceuticals, like the back fat. acids are palmitic acid, palmitoleic acid and oleic acid

61 J. Oleo Sci., Vol. 52, No. 2, 57-63 (2003) S. Shimizu and M. Nakano

Table 5 Fatty Acid Compositions in Triacylglycerol Peaks from the Back Fat of the Domestic Rhea. Fatty acid (mole%) Peak Ratio Suggested Estimated C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 numbera (%) TGb PNc (n-7) (n-9) (n-6) (n-6) 1 0.9 100.0 LLL 42 2 1.9 19.2 3.2 14.5 48.2 14.9 Unidentified 3 1.1 27.0 4.9 4.5 35.4 28.2 PLLn 42 4 4.5 33.8 66.2 OLL 44 5 2.7 27.5 38.3 29.8 4.5 PoOL 44 6 5.3 30.0 3.9 4.5 61.6 PLL 44 7 4.8 30.1 20.0 5.4 12.9 22.3 9.4 PPoL 44 8 7.8 66.9 33.1 OOL 46 9 1.8 4.2 26.9 5.9 58.9 4.1 PoOO 46 10 16.1 32.1 1.1 33.0 33.8 POL 46 11 4.1 31.4 29.6 1.2 36.0 1.7 PPoO 46 12 5.7 63.0 3.9 2.3 30.8 PPL 46 13 1.1 67.3 32.7 PPL? 46 14 2.0 71.3 28.7 PPPo 46 15 4.7 99.5 0.5 OOO 48 16 3.1 10.4 25.8 38.2 26.0 SOL 48 17 11.9 33.4 66.6 POO 48 18 1.4 36.5 32.4 31.1 PSL 48 19 10.2 64.6 3.3 32.1 PPO 48 20 1.9 95.0 2.3 2.7 PPP 48 21 1.9 4.3 35.5 60.2 SOO 50 22 3.4 32.7 34.2 33.1 PSO 50 23 1.0 64.8 35.2 PPS 50 24 0.3 6.6 3.2 58.0 32.3 SSO 52 25 0.2 28.0 4.2 61.3 6.4 PSS 52 aSee Fig. 1, b,cAbbrevations described in the Materials and Methods section.

Table 6 Existence Rate and Compositon of Saturated Fatty ing properties. Acids and Unsaturated Fatty Acid in Ratite Triacy- Politis and Dmytrowich reported that emu oil lotion lglycerol. was potentially a useful agent in the treatment of full- Triacylglycerol (%) thickness wounds if applied after the inflammatory Emu Ostrich Rhea stage of wound healing (2). Human granulocyte elastase a S3 1.6 3.8 3.1 has been implicated in a number of inflammatory dis- b US2 18.1 23.7 23.0 eases, both in the production of mediators of inflamma- c U2S 44.9 41.3 48.3 tion and in the destruction of connective tissue (11). It d U3 30.6 21.7 22.4 is known that unsaturated fatty acids inhibit human a b granulocyte elastase. The most potent is oleic acid (12). S3: Trisaturated TG, US2: Monounsaturated TG, c d U2S: Diunsaturated TG, U3: Triunsaturated TG. The main fatty acid in depot fat from the ratites was oleic acid. The amount of oleic acid in depot fat TG from the emu was the greatest among the three ratite (10). Emu depot fat contained more of the above fatty species, and its TG containing oleic acid accounted for acids than the other ratites. Accordingly, emu depot fat 89% of the total. These physiological properties may be might be most appropriate for use in cosmetics, though attributed to the above chemical characteristics of emu depot fats from all three ratite species had skin penetrat- oil.

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