J. Jpn. Oil Chem. Soc. Vol.47, No.2 (1998) 187

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Compositions of Phytosterols in the Seeds of Echinochloa crus-galli and Setaria italica Beauv.

Suguru TAKATSUTO and Takahiro KAWASHIMA Department of Chemistry, Joetsu University of Education (1, Yamayashiki-machi, Joetsu-shi, Niigata-ken 943-0815)

Abstract: Determination was made of phytosterol composition in the seeds of Echinochloa crus-galli and Setaria italica Beauv. The phytosterol fractions were extracted, purified and analyzed by GC-MS following trimethylsilylation. In the seeds of E. crus-galli, cholesterol, 24-methylenecholesterol, campe- sterol, campestanol, stigmasterol, sitosterol, sitostanol and isofucosterol were identified. In the seeds of

S. italica, campesterol, campestanol, stigmasterol, Ģ22-stigmasterol, sitosterol, sitostanol, and Ģ7-stig- mastenol were found present. Phytosterol compositions in the two species of seeds of E. crus-galli and S. italica were clarified. It is interesting that in the seeds of S. italica, sitostanol was found to account for

26% of the total sterols.

Key words: seed, Echinochloa crus-galli, Setaria italica Beauv., phytosterol, sitostanol

1 Introduction

There have been many reports of phytosterols in major Gramineae plants such as rice, wheat, corn, and so on, and it is well known that campesterol, stigmasterol and sitosterol occur as major phytosterol components, along with small amount of cholesterol and isofu- costerol1). However, with respect to 5 ƒ¿-stanols in Gramineae plants, sitostanol was identi- fied in Triticum spp2) and cholestanol, 24-methylcholestanol (campestanol), 24-ethylcholest- anol (sitostanol) in Zea mays3). These 5ƒ¿-stanols are very minor sterol components in these

plants. However, from the nutritional points of view, it has been clarified that sitostanol is superior to sitosterol in inhibiting cholesterol absorption and lowering the level of serum cholesterol4). Thus, we have now undertaken a new program searching for edible seeds, in which sitostanol is contained as relatively major sterol components. At first, we have investi-

gated some seeds of the Gramineae plants. During the course of our investigation, we have now found that a considerable amount of 5ƒ¿-stanols was contained in the seeds of Echino- chloa crus-galli and Setaria italica Beauv. Perusal of literature revealed that Phytosterols in S. sphacelata5) and the fatty acid compositions of the seeds of E. crus-galli, S. glauca (L.) Beauv. and S. faberii Herrm. have been investigated6). In the seeds of S. italica and S. viri-

dis, Iwama and Hirose reported the compositions of fatty acids, hydrocarbons and phytos- terols, and, with respect to phytosterols in the two seeds, they identified sitosterol, stigmas- terol, and campesterol as major sterol components, along with small amounts of cholesterol, brassicasterol, isofucosterol and avenasterol7). However, the phytosterol composition of the seeds of E. crus-galli has not been investigated and 5ƒ¿-stanols contained in the seeds of S. italica have also not been clarified until now. Therefore, in this paper we report the phytos- terol compositions, including some 5ƒ¿-stanols, of both seeds of E. crus-galli and S. italica, which are obtained by GC-MS analysis using a fused silica capillary column.

Corresponding author: Suguru TAKATSUTO

53 188 J. Jpn. Oil Chem. Soc. Vol.47, No.2 (1998)

2 Experimental

2.1 Plant materials The mature seeds of S. italica and E. crus-galli are commercially available as foods for birds and were purchased at Musashi home center, Joetsu-shi, Niigata, Japan. 2.2 Chemicals All reagents and solvents were of reagent grade quality or better and were purchased from

Wako Pure Chemical Industries Ltd., Osaka, Japan, and they were used as such without further purifications. Cholesterol, cholestanol, brassicasterol, campesterol, campestanol, stigmasterol, sitosterol, and sitostanol were kindly supplied by Tama Biochemical Co. Ltd., Tokyo, Japan. ƒ¢22-Stigmastenol was synthesized according to the methods previously de- scribed9). 24-Methylenecholesterol and isofucosterol were obtained from the pollen of orange, as previously described10). 2.3 Extraction and solvent partitioning The seeds of E. crus-galli (100g) were powdered and extracted with methanol (300mL) for two weeks. The methanol extract was filtered and the solvent was evaporated to dryness in vacuo. The residue was partitioned between water (50mL) and chloroform (50mL•~3). The combined organic phases were concentrated and the residue was partitioned between hexane (50mL) and 90% methanol (50 mL•~3). The hexane fraction was concentrated to give an oil E-a (0.37g). In the same method, the methanol extract of the seeds (100g) of S. italica was subjected to solvent partitioning to give the hexane fraction as an oil S-a (0.50g). 2.4 Purification of a phytosterol fraction The hexane-soluble oil E-a (0.37g) in 80% ethanol (20mL) was treated with sodium hy- droxide (0.5g) at 90•Ž for 1h. The cooled reaction mixture was extracted with toluene (50 ml) and the separated organic phase was washed with brine, dried over anhydrous magne- sium sulfate, filtered and concentrated in vacuo. The residue was applied to a column of sili- ca gel (1.5cm i.d. •~20cm). Elution with toluene-ethyl acetate (50:1 to 25:1, vol/vol) gave a phytosterol fraction (18mg). This was further chromatographed on silica gel (1.5cm i.d. •~25cm) eluting with hexane-ethyl acetate (20:1 to 3:1, vol/vol) to provide a purified phy- tosterol fraction E-b (15mg), which showed a single spot with the same Rf value as that of authentic stigmasterol. In the same method, the hexane-soluble oil S-a (0.50g) was saponified and the unsaponifi- able lipid was repeatedly chromatographed on silica gel to provide a purified phytosterol fraction S-b (30mg). 2.5 GC-MS analysis of a phytosterol fraction A Shimadzu GC-MS QP-5000 equipped with a fused silica ULBON HR-1 capillary column

(50m•~0.25mm i.d., 0.25ƒÊm film thickness, Shimadzu) was used under the following con- ditions: ionization, electron impact mode (70eV); mass range, 100•`550; scan interval, 0.5 sec.; injection temp., 300•Ž; interface temp., 250•Ž; carrier gas, helium; carrier gas pres- sure, 170kPa; total flow rate, 50mL/min; split ratio, 61:1; flow rate, 0.7mL/min; col- umn temp., 300•Ž. A small portion (0.5mg) of each phytosterol fraction E-b and S-b was converted to a trimethylsilyl (TMS) ether by treatment with trimethylsilylimidazole (100ƒÊL) and pyridine

(50ƒÊL) at 60•Ž for 30min. The characteristic MS data of the identified phytosterols as TMS ethers were as follows: cholesterol TMS ether MSm/z: 458 (M+, 14%), 368 (28), 353 (15), 329 (28), 129 (100). 24-Meth- ylenecholesterol TMS ether MSm/z: 470 (M+, 0.3%), 386 (18), 343 (19), 296 (18), 281 (8), 257 (8), 129 (100). Campesterol TMS ether MSm/z: 472 (M+, 12%), 457 (3), 382 (29), 367 (11), 343 (29), 275 (2), 255 (6), 129 (100). Campestanol TMS ether MSm/z: 474 (M+, 37%), 459 (32),

54 J. Jpn. Oil Chem. Soc. Vol.47, No.2 (1998) 189

417 (14), 384 (14), 369 (28), 306 (31), 257 (10), 230 (21), 230 (21), 215 (100), 201 (19), 147 (34), 121

(49), 107 (98). Stigmasterol TMS ether MSm/z: 484 (M+, 20%), 469 (3), 394 (25), 379 (8), 355 (8), 351 (7), 343 (5), 282 (8), 255 (31), 129 (100). Ģ22-Stigmastenol TMS ether MSm/z: 486 (M+, 74%), 374 (61), 353 (77), 345 (46), 285 (47), 257 (87), 215 (16), 161 (73), 147 (75), 135 (99), 123 (53), 121 (95), 107 (100). Sitosterol TMS ether MSm/z: 486 (M+, 10%), 471 (3), 396 (24), 381 (9), 357 (23), 275 (5), 255 (9), 129 (100). Sitostanol TMS ether MSm/z: 488(M+, 27%), 473 (28), 431 (11), 398 (18), 383 (26), 306 (24), 290 (9), 257 (9), 230 (15), 215 (100), 201 (17), 147 (43), 121 (46), 107 (90). Isofucosterol TMS ether MSm/z: 484 (M+, 0.8%), 386 (43), 371 (8), 355 (3), 296 (38), 281 (32), 257 (17), 253 (10), 129 (100). Ģ7-Stigmastenol TMS ether MSm/z: 486(M+, 62%), 471

(20), 396 (10), 381 (15), 345 (11), 255 (100), 229 (22), 213 (31), 173 (17), 145 (49), 131 (42), 119 (49), 107 (79).

3 Results and Discussion

The phytosterol compositions in both seeds of S. italica and E. crus-galli were investigat- ed. Each methanol extract of the powdered seeds was subjected to a conventional solvent partitioning to give a hexane-soluble fraction. This fraction was saponified and the un- saponifiable lipid was purified, as described in Experimental, to provide each phytosterol fraction. The fractions were respectively analyzed by GC-MS using a fused silica capillary column, after trimethylsilylation. Phytosterol TMS ether was identified by direct compari- son with authentic samples, in terms of retention time in GC and of mass spectrum. The phytosterol compositions in both seeds were obtained by total ion chromatogram and the re- sults are summarized in Table 1. Since separation of the C-24 stereoisomers of the 24-alkyl- sterols was found to be difficult under our analytical condition, it is unknown whether or not the identified 24-alkylsterols are mixed with each C-24 stereoisomer. In this paper, we tenta- tively used the common names. Under our analytical condition, the TMS ethers of sitostanol and isofucosterol have the same retention times in GC. Thus, in the seeds of E. crus-galli, it has been found that sito- stanol was contaminated with ca. 20% of isofucosterol, because characteristic fragment ions of isofucosterol TMS ether were clearly observed in the mass spectrum of sitostanol TMS ether. However, in the seeds of S. italica, the characteristic fragment ions of isofucosterol TMS ether have not been found in the mass spectrum of sitostanol TMS ether, showing that sitostanol identified in S. italica seeds does not seem to be contaminated with isofucosterol and that, if any, the content of isofucosterol seems to be very low. Therefore, we conclude that sitostanol was detected as a major sterol component (26%). As reported previously in other Gramineae plants1), campesterol, stigmasterol and sitos- terol have been identified as major phytosterol components in the two species seeds of E. crus-galli and S. italica. Sitosterol was reported to contain in the leaves of S. italica8) and the occurrence of sitosterol, stigmasterol, campesterol, isof ucosteriol and cholesterol, in the seeds of S. sphacelata have been reported5). In the seeds of S. italica and S. viridis, Iwama and Hirose previously identified sitosterol, stigmasterol, and campesterol as major sterol components, along with small amounts of cholesterol, brassicasterol, isofucosterol and ave- nasterol, by GC analysis7). In the present study of the seeds of S. italica, we have identified sitosterol, campesterol and stigmasterol, as major sterol components, along with small amounts of campestanol and Ģ22-stigmastenol and a large amount (26%) of sitostanol. We could not detect cholesterol, brassicasterol and avenasterol. Furthermore, it is found that the seeds of E. crus-galli contain campestanol and sitostanol in substantial amounts. Previ- ously, in Gramineae plants, sitostanol were identified in Triticum spp2) and cholestanol, campestanol and sitostanol in Zea mays3). From the nutritional points of view, the seeds of S. italica, in which sitostanol accounts for 26% of the total sterols, are worth mentioning, because it has been clarified that sitostanol is superior to sitosterol in inhibiting cholesterol

55 190 J . Jpn. Oil Chem. Soc. Vol.47, No.2 (1998)

Table 1 The Compositions of Phytosterols in the Seeds of Echinochloa crus- galli and Setaria italica Beauv.

a) Relative retention time with respect to cholesterol TMS ether (13.75min).

b) Not detected.

c) Sitostanol was contaminated with isofucosterol.

d) Tentatively identified.

e) Several unidentified sterols are contained.

absorption and lowering the level of serum cholesterol4).

(Received Sept. 3, 1997; Accepted Nov. 10, 1997)

References

1) T. Akihisa, W.C.M.C. Kokke, T. Matsumoto, •gPhysiology and Biochemistry of Sterols•h ed. by

G.W. Patterson, W.D. Nes, American Oil Chemists' Society, Champaign, Illinois, USA, (1991) p. 172.

2) A.M.M. Berrie, B.A. Knights, Phytochemistry, 11, 2363 (1972).

3) N.L.A. Misso, L.J. Goad, Phytochemistry, 23, 73 (1984).

4) M. Sugano, Nippon Nogeikagaku Kaishi (review in Japanese), 71, 769 (1997).

5) B.N. Bowden, P.M. Williams, Phytochemistry, 10, 3135 (1971).

6) E.W. Stoller, E.J. Weber, J. Agr. Food Chem., 18, 361 (1970).

7) F. Iwama, Y. Hirose, Mem. Fac. Liberal Arts Educ., Part 2 (Yamanashi Univ.), 32, 43 (1981).

8) M. Kamil, N. Jain, M. Ilyas, Indian J. Pharm. Sci., 54, 83 (1992).

9) S. Takatsuto, N. Ikekawa, J. Chem. Soc., Perkin Trans. 1, 1986, 2269.

10) S. Takatsuto, K. Omote, C. Motegi, Biosci. Biotech. Biochem., 56, 670 (1992).

56 210 日本 油 化 学 会 誌 第47巻 第2号 (1998)

[ノー ト] ヒエ お よ び ア ワ種 子 中 に 含 ま れ る 植 物 ス テ ロ ー ル の 組 成

高 津 戸 秀 ・ 川 島 崇 弘 上 越 教 育 大 学 自然 系 化 学 教 室 (〒943-0815新 潟 県 上 越 市 山 屋 敷 町1)

ヒ エ(Echinochloa crus-galli)お よ び ア ワ(Setaria italica Beauv.)種 子 中 に 含 ま れ る 植 物 ス テ ロ ー ル の 組 成

に つ い て 調 べ た 。 両 種 子 か ら 植 物 ス テ ロ ー ル 分 画 を 抽 出 し,精 製 後 ト リ メ チ ル シ リル 化 してGC-MS分 析 を 行 っ た 。 ヒ エ 種 子 か ら8種 の ス テ ロ ー ル(cholesterol, 24-methylenecholesterol, campesterol, campestanol, stig- masterol, sitosterol, sitostanol, and isofucosterol)を 同 定 し,ア ワ 種 子 か ら7種 の ス テ ロ ー ル(campesterol, campestanol, stigmasterol, Δ22-stigmastenol, sitosterol, sitostanol, and Δ7-stigmastenol)を 同 定 し,両

種 子 の 植 物 ス テ ロ ー ル 組 成 を 解 明 し た 。 ア ワ種 子 に お い て,sitostanolは 総 ス テ ロ ー ル の26%を 占 め る こ と は 興 味

深 い 。 (連 絡 者:高 津 戸 秀)Vol.47, No.2, 187 (1998)

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