Fedoreyev S.A., Bulgakov V.P.
В журнале Journal of Biotechnology
Год: 2008 Том: 136 Приложение: S1 Страницы: 140
Isoflavonoids, an interesting and restricted group of secondary metabolites of legumes, exhibit estrogenic, antiangiogenic and anticancer activities, and are now popular as dietary supplements [1,2]. Amur maackia (Maackia amurensis Rupr. et Maxim.) is the only woody plant representative of Leguminosae family in the flora of the Russian Far East. This species is a relict tree of the tertiary flora [3]. Both polyphenolyc complexes extracted from the core wood (PHW) and the callus cultures (PHC) of M. amurensis decreased the acute toxicity of tetratrachloromethane on liver with similar efficiency, showing a prominent favorable effect on animal survival. This effect was due to the suppression of the dystrophy and necrosis of hepatocytes, the normalization of aminotransferase and ?-glutamyltransferase activities in the blood, and the stimulation of bilirubin conjugation [4-6]. The HPLC and NMR analyses of PHW yielded isoflavones, pterocarpans, monomeric and dimeric stilbenes [3]. We initiated the research of plant cell cultures possessing increased ability to synthesize these polyphenolic compounds. Cell cultures of M. amurensis were established during the investigation. A selection of seed-derived cell aggregates yielded a callus line designated A-18. This culture produces 20 isoflavonoids, namely isoflavones: genistein, daidzein, formononetin, calycosin, derrone, pseudobaptigenin; their glycosilated conjugates genistin, 6''-O-malonylgenistin, ononin, 6''-O-malonylononin, daidzin, 3'-metoxydaidzin, 4'-O-?-D-glucopyranosyldaidzin, 4'-O-?-D-glucopyranosylgenistin, 7-O-?-D-glucopyranosylcalycosin; pterocarpans maackiain and medicarpin; their glycosilated conjugates: 6''-O-malonyl-3-O-?-D-glucopyranosylmaackiain, 6''-O-malonyl-3-O-?-D-glucopyranosylmedicarpin; and a new pterocarpan glucoside 6''-O-malonyl-3-O-?-D-glucopyranosyl-6,6a-dehydromaackiain. HPLC chromatography and 1H and 13C NMR spectroscopy of the isolated compounds were used for isolation, identification and structural elucidation of isoflavonoids. The A-18 calli, which have been established in 2001, were analyzed for two years (2006-2007) by HPLC. During this time, the calli produced in total 1.437±0.178% DW of isoflavonoids. The analysis of the results showed that the A-18 callus culture produced free and glycosilated isoflavones in the ratio ?1:7 and free and glycosilated pterocarpans in the ratio ?1:5. This result indicates stable production of isoflavonoids during the prolonged periods of the cultivation. The dynamics of polyphenol accumulation during a subculture was characterized by continuously increasing levels of polyphenols up to 45-50 days (stationary phase of the growth); after that their levels declined. Interestingly, despite M. amurensis plants produced large amounts of stilbenes, we could not detect any substantial quantities of these substances in our callus cultures. On the other hand, we had not found in PHW, isolated from the core wood M. amurensis, glycosilated conjugates of the isoflavones and pterocarpans. Therefore, the possibility to create a renewable source of Maackia isoflavonoids had been demonstrated.
