| Objective: HPLC fingerprint and grey correlation analysis(GRA)were used to predict the effective toxic components of Bupleuri Radix decoction extract in reducing lipid activity and hepatotoxicity.The biotransformation of effective toxic substances in artificial gastric juice,artificial intestinal juice,intestinal flora and liver microsomes was preliminarily analyzed in order to provide pharmacological evidence for the clinical safety of Bupleuri Radix decoction.Methods: HPLC-MS fingerprint was used to characterize the material basis of the Bupleuri Radix decoction;TG content in Hep G2 cells induced by oleic acid,ALT and AST content in L-02 cells were determined to study the lipid-lowering activity and hepatotoxicity of the Bupleuri Radix decoction;correlation analysis of spectrum effect toxicity was carried out by GRA to predict the effective toxic components corresponding to the lipid-lowering activity and hepatotoxicity respectively;L-02 cytotoxicity experiment,to explore the influence of Bupleuri Radix decoction serum group and water extract group on L-02 cells;to study the influence of artificial gastric juice,artificial intestinal juice,intestinal flora and liver microsomes on Bupleuri Radix decoction extract by HPLC-MS fingerprint,to analyze the in vitro biotransformation of effective components of Bupleuri Radix decoction,and to summarize its transformation characteristics.Result: The HPLC-MS fingerprint of Bupleuri Radix decoction was established and 29 common peaks were identified.The GRA results showed that the common peaks of 8,12,27,20,14,10 and 26 had a high correlation with the lipid-lowering activity of Bupleuri Radix;the common peaks of 13,12,8,26,10,14,27 and 11 had a high correlation with the liver toxicity of Bupleuri Radix,among which the common peaks of 8,10,12,14,26 and 27 had a higher correlation with the effective toxicity of water extract The common peaks of Gao,11 and 13 were only related to the liver toxicity of Bupleuri Radix.The results of L-02 cytotoxicity experiment in vitro showed that the survival rate of L-02 cells in the same dose of Bupleuri Radix serum group was significantly higher than that in the Bupleuri Radix decoction group(P < 0.001).The results of biotransformation in vitro showed that the representative components of No.8,10,12 and 14 related to Bupleurum toxin were relatively stable in the incubation system of artificial gastric juice and liver microsomes,and the change rate of each component in samples at different time points was less than 10%;After 24 hours of transformation in the artificial intestinal fluid,the content of the specific components of Bupleuri Radix liver toxicity decreased by 31%,15%,37% and 31%,among which the peaks 11 and 13 decreased by 33% and 18%;the effective components of Bupleuri Radix decoction were unstable in the intestinal flora,among which the peak 8 increased by 107% in 24 hours,and the representative components of the peaks 10,12 and 14 decreased by 32%,32%,77,respectively %The content of 11 and 13 peaks of Bupleurum was reduced by 61% and 42%.The results of L-02 cytotoxicity experiment in vitro showed that there was no significant difference in the effects of the samples transformed at different time on the activity of L-02 cells;compared with the group of 0 h transformation,the samples transformed at 24 h with the water extract of Bupleuri Radix decoction had a significant difference in the activity of L-02 cells(P < 0.001).Conclusion: The results of biotransformation in vitro showed that the effective components of Bupleuri Radix decoction were relatively stable in the artificial gastric juice and liver microsomes,and transformed in the artificial intestinal juice and intestinal flora;the content of toxic components in the artificial intestinal juice and intestinal flora decreased and changed L-02 fine Cellular vitality. |
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