日 蚕 雑 66(2),136-137(1997) Short report J.Seric.Sci.Jpn. Identification of isofucosterol in mulberry leaves, Molus alba MASAYOSHI YAMAZAKI, NAOKO NAKAMURA and AKIRA KURIOKA Silk Science Research Institute, Dainippon Raw Silk Foundation (Received September 9, 1996) Key words: mulberry leaves, phytosterol, isofucosterol, silkworm As silkworm larvae are known to have no Scientific) at 280•Ž, and each sterol was deter- ability to synthesize sterols de novo, they require mined with GC/MS by a selected ion monitoring dietary sterols for their normal growth and devel- method (SIM). opment (ITO,1961). In mulberry leaves, the sole ƒÀ-sitosterol , campesterol, stigmastrol and food for silkworm larvae, the presences of /3- fucosterol were purchased from Funakoshi Co.. sitosterol (NAITO and HAMAMURA, 1961) campes- Isofucosterol was isolated from the sterol mix- terol (MIYAUCHI et al., 1964) and stigmasterol ture of honey bees. (SUZUKI and IKEKAWA, 1966) have been reported. Results and Discussion In the present study, isofucosterol was discovered in the leaves, in addition to sterols. Total ion chromatogram (TIC) of the Isofucosterol had been believed to be an interme- mulberry leaf sterol mixture is shown in Fig. 1. diate in the conversion of sitosterol to cholesterol Mulberry leaf sterols showed 4 peaks on TIC. in silkworm larvae (MORISAKI et al., 1981). Peaks at retention times of 20.2, 21.0 and 22.8min (-OH) and 17.1, 17.6 and 18.8min (-TMS) were Materials and Methods assigned by comparison with authentic samples to Dried mulberry leaf powder (50g) was campesterol, stigmasterol and ƒÀ-sitosterol, extracted for 24 hours with diethylether in a respectively, for which sterols have been soxhlet apparatus. The ether extract was identified. The fourth retention peak time of 24. saponified with 6% ethanolic potassium hydrox- ide for 4 hours. Extraction with ether produced Table 1. The sterol composition of mulberry leaves an unsaponifiable fraction. in various leaf oder (%) The fraction was applied to a Sep-Pak silica cartridge(Waters Associates, Milford, MA.) and the sterol mixture (90mg) was obtained from the eluate with ethylacetate-hexane (15:85). This was analyzed as free and trimethylsilyl (TMS) ether with Shimazu QP 5000 gas chromatograph- mass spectrometer (GC/MS) using a fused silica l: Leaves collected from largest glossy leaf to eighth capillary column (DB-17, 30m •~ 0.32mm, J&W in leaf order 2: Leaves from nineth to fourteenth 3-25-1, Hyakunincho, Shinjuku-ku Tokyo, 169, Japan 3: Leaves from fifteenth to twentieth -136- MASAYOSHI YAMAZAKI et al.: Identification of isofucosterol in mulberry leaves, Molus alba 137 The sterol composition of the leaves for early autumn rearing, which was determined by SIM with GC/MS, is shown in Table 1. More isofucosterol was found in younger leaves than in older ones. In contrast, there was no difference in other sterol contents among leaf orders. The nutritional experiment revealed that isofucosterol satisfied the sterol requirements for silkworm larvae (MORISAKI et al., 1974). Fig. 1. Total ion chromatogram of sterols of mul- Isofucosterol was identified in silkworm larvae berry leaves. and reported to be an intermediate in the conver- sion of sitosterol to cholesterol (MORISAKI et al., lmin (-OH) and 20.0min (-TMS) was derived from 1981). The present study suggests that this sterol an unknown sterol. The mass spectrum of un- is not a metabolite of sitosterol in silkworm lar- known sterol(Fig. 2) coincided with that of fucos- vae. terol or isofucosterol. The retention times of fucosterol and isofucosterol were 23.5 and 24.Imin Acknowledgements: The authors wish to (-OH), and 19.8 and 20.0min (-TMS), respectively. thank Dr. H. Ooi of the Institute of Silkworm From the results, the unknown sterol was Genetics and Breeding for supplying the mulberry identified as isofucosterol. This was the first leaves. such identification from mulberry leaves. Fig. 2. Mass spectrum of unknown sterol. MORISAKI, M., Ying, B. and IKEKAWA, N. (1981): References Identification of both fucosterol and isofucos- ITO T. (1961) Sterol requirements of the silkworm, terol in the silkworm, Bornbyx mori. Exper- Bombyx mori. Nature, Lond. 191, 882-883 ientia, 37, 336-337. MIYAUCHI K., M. SAITO and N. IKEKAWA (1964): NAITO, K. and HAMAMURA, Y. (1961): Studies on Identification of ƒÀ-sitosterol and campesterol in the micro constituent in mulberry leaves-‡V mulberry leaf sterol by gas chromatography. Isolation of ƒÀ-sitosterol from mulberry leaves. Bull. Sericul. Exp. Sta. 18 (6) 431-439. (In J. Agric. Chem. Soc. Japan 35, 848-850 (In Japanese) Japanese) MORISAKA, M., OHTAKA, H., AWATA, N., IKEK SUZUKI, M. and IKEKAWA, N. (1966): Studies on AWA,N., HORIE, Y. and NAKASONE, N. (1974): the sterol of Bombyx mori. V. Lupeol in the Nutritional effect of possible intermediates of silkworm blood, Chem. Pham. Bull. 14. 1049 -1051 phytosterol dealkylation in the silkworm, Bom- . byx mori, Steroids 24 (2), 165-176..