| Trillium kamtschaticum Pall.is a perennial medicinal plant of Trillium genus in Liliaceae,which medicinal parts are ripe fruit and dried rhizome.Trillium kamtschaticum Pall.is kind of traditional and rare Chinese herbs which has complicated chemical constituents and multipled structure.It shows strong pharmacological effects when it comes to immunological regulation,antitumor antioxidation,analgesic coagulation and anticaner and so forth.The main steroidal saponins were extracted and purified in this study,and their structures were identified;The extraction process of steroidal saponin extracted from Trillium kamtschaticum Pall.was optimized by orthogonal test;At the same time,the immune activity of the extract was studied.The results are as follows:1.Chemical constituents study of steroidal saponins of the rhizome of Trillium kamtschaticum Pall.7 steroidal saponins were identified through qualitative analysis of steroidal saponins from Trillium kamtschaticum Pall.: pennogenin-3-O-?-D-glucopyranside-rhamnopyraosyl-O-(?-L-rhamnopyraosyl)-?-D-glucopyranside or its isomer(1),protodioscin-26-O-?-D-glucopyranside-3-O-?-potato triglycoside(2),(25R)-24?-hydroxypenogenin-3-O-?-L-rhamnopyranosyl-(1?2)-?-D-glucopyranoside(3),pennogenin-3-O-?-D-glucopyranside-(1?6)-[O-?-L-rhamnopyraosyl-(1?2)]-O-?-D-glucopyranside(4),pennogenin-3?-O-?-L-rhamnopyraosyl-(1?4)-O-?-L-rhamnopyraosyl-(1?4)-[O-?-L-rhamnopyraosyl-(1?2)]-O-?-D-glucopyranside(5),pennogenin-3-O-?-L-rhamnopyraosyl-(1?4)-[?-L-rhamnopyraosyl-(1?2)]-?-D-glucopyranside(6),pennogenin-3-?-O-?-L-rhamnopyraosyl-(1?2)-O-?-D-glucopyranside(7).Four steroidal saponins were obtained through the extraction,separation and purification of steroidal saponins from Trillium kamtschaticum Pall.,4 compouds are identified as follows: compoud 1:(25R)-24?-hydroxypenogenin-3-O-?-L-rhamnopyranosyl-(1?2)-?-D-glucopyranoside;compoud 2:pennogenin-3?-O-?-L-rhamnopyraosyl-(1?4)-O-?-L-rhamnopyraosyl-(1?4)-[O-?-L-rhamnopyraosyl-(1?2)]-O-?-D-glucopyranside;compoud 3: pennogenin-3-O-?-L-rhamnopyraosyl-(1?4)-[?-L-rhamno pyraosyl-(1?2)]-?-D-glucopyranside;compoud 4: pennogenin-3-?-O-?-L-rhamnopyraosyl-(1?2)-O-?-D-glucopyranside.The contents of the 4 steroidal saponins extracted from Trillium kamtschaticum Pall.were by HPLC method,and the methods of determination were investigated.The results show: The average contents of 4 compouds are 0.463mg/g,0.922mg/g,3.135mg/g,3.899mg/g.2.Optimize Extraction Process of steroidal saponins extracted from Trillium kamtschaticum Pall.with orthogonal test methodThe influence of solid-liquid ratio,time and temperature on extraction contents of the 4 steroidal saponins extracted from dry rhizomes of Trillium kamtschaticum Pall.were discussed by orthogonal test method,with a HPLC method for the determination of its contents.The optimum extraction process of 4 steroidal saponins obtained from the variance analysis of the experiment are as follows:compoud 1: solid-liquid ratio of 1: 8,extraction time of 4h,and extraction temperature of 80?;compoud 2: solid-liquid ratio of 1: 9,extraction time of 4h,and extraction temperature of 90?;compoud 3: solid-liquid ratio of 1: 10,extraction time of 4h,and extraction temperature of 90?;compoud 4: solid-liquid ratio of 1: 12,extraction time of 4h,and extraction temperature of 90?.3.Study on the immune activity of extract from Trillium kamtschaticum Pall.The 8 extracts of Trillium kamtschaticum Pall.are as follows: Component 1: ethanol crude extract,Component 2: n-butanol extract,Component 3: Ethyl acetate extract,Component 4: aqueous phase,Component 5: pennogenin-3?-O-?-L-rhamnopyraosyl-(1?4)-O-?-L-rhamnopyraosyl-(1?4)-[O-?-L-rhamnopyraosyl-(1?2)]-O-?-D-glucopyranside,Component 6: chonglouoside ?,Component 7: pennogenin-3-O-?-L-rhamnopyraosyl-(1?4)-[?-L-rhamnopyraosyl-(1?2)]-?-D-glucopyranside,Component8: chonglouoside ?.Study on the effect of extracts on mouse peritoneal macrophages show that: The extraction phase activity of n-butanol was strongest,then component 8,5,6,7 in order after 2,there was no significant difference in the activity of the three components.A lot of saponins are contained in n-butanol phase,so the saponins of Trillium kamtschaticum Pall.has the function of enhancing immunomodulatory is inferred.Study on the effect of extracts on mouse spleen lymphocytes shows that: The extraction phase activity of n-butanol was strongest,the telative grouth rate of SPL is 75.2%,then component 6,8,5 in order after 2,the telative grouth rate of SPL of them are 61.1%,60.8% and 56.8%;The extraction phase activity of ethyl acetate was weakest,the telative grouth rate of SPL of it is 3.6%.Study on cytotoxicity of extracts by MTT shows that: Only ethyl acetate extract showed weak cytotoxicity among the 8 extracts from rhizome of Trillium kamtschaticum Pall..Therefore,it is proved that the development of Trillium kamtschaticum Pall.has relative safety and stability in improving the immune function of the body. |
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