ИСТИНА

In this work the structural diversity and the features of the triterpene glycosides accumulation in cell cultures of members of Araliaceae (Panax ginseng, Panax japonicus var. repens, Polyscias filicifolia, and Polyscias fruticosa) were studied. The systematic phytochemical investigation of the two Polyscias species suspension cell cultures has been carried out for the first time. The presence of triterpene glycosides with the oleanolic acid as an aglycon in P.filicifolia and P.fruticosa plant cell cultures was proved and the structure of four of them was identified on the basis of spectroscopic evidence (1H-, 13C-NMR and HRESI MS/MS data). These compounds are ladiginoside A, polyscioside A, polyscioside E and glucopyranosyl-(1-2)-glucuronopyranosyl-3-oleanolic acid-28-glucopyranosyl ester. It was shown the difference of qualitative and quantitative triterpene glycosides composition in P.filicifolia and P.fruticosa suspension cell cultures. It was established that the P.filicifolia cell culture exposed higher level of triterpene glycosides accumulation. The presence of large amounts of ginsenosides malonyl-Rb1, -Rc, -Rb2, and -Rd in a suspension culture of Panax japonicus var. repens cells was demonstrated for the first time. Identification of ginsenoside malonyl-Rb1 was based on chromatographic, chemical, and spectroscopic evidence. Ginsenosides malonyl-Rc, -Rb2, and -Rd were identified on the basis of chromatographic and chemical data. Content and composition of the individual ginsenosides (Rg1, R0, malonyl-Rb1, Rb1, Rc, Rb2, and Rd) were monitored in the suspension culture over four years. The RP-HPLC-UV analysis showed that Rg1, R0, and malonyl-Rb1 accounted for more than 75% of the total pool of ginsenosides. In accordance with this result, and data analysis reported in the literature, we propose that ginsenoside formation in the cells of P. japonicus var. repens in vitro is closely related to the cellular compartmentation of these substances. In particular, the accumulation of the 20(S)-protopanaxadiol ginsenosides (especially Rb1) is strongly dependent on their pattern of malonylation, which likely targets them for transport into the vacuole. As opposite to the 20(S)-protopanaxadiol-type ginsenosides, the esterification (particularly malonylation) is not typical of 20(S)-protopanaxatriol ginsenosides. Therefore, we suggest that the unique pattern of glycosylation of the 20(S)-protopanaxatriol-type ginsenosides (attachment of one of the sugar chains to the α-hydroxyl group at the C-6 position of the dammarane-type aglycone) makes them resistant to non-specific glycosyl hydrolases. Accordingly, the accumulation of ginsenoside Rg1 is possible without significant disturbance of the metabolic activity of P. japonicus var. repens cells in vitro. This finding is supported by the results of the ginsenoside profiling during growth of Panax ginseng cell culture in flasks. Here, the total amount of the ginsenosides varied over a wide range but the ratios of the major groups remained nearly constant: Rg1+Re and malonyl-Rb1 accounted for more than 60% of the total ginsenoside pool. These findings provide a new approach for understanding of the ginsenoside accumulation machinery, and may be helpful for the rational and efficient optimisation of their production in ginseng cell cultures and intact plants.