HPLC法测定补阳还五汤中主要成分及其代谢研究

目的:探索补阳还五汤的主要成分在体内的代谢途径。方法:采用HPLC法对补阳还五汤中主要成分(毛蕊异黄酮苷、刺芒柄花苷、芍药苷、毛蕊异黄酮和刺芒柄花素)的含量进行测定,通过体外孵育方法考察补阳还五汤中主要成分在肝匀浆、肠道菌液、人工胃液和人工肠液中不同时间点的含量变化,以相对浓度的改变来反映各化合物量的变化。硝苯地平用来验证孵育体系中CYP450酶的活性。采用Shimadzu VP-ODS分析柱(250 mm×4.6 mm,5 μm) 色谱柱,柱温30 ℃,以0.1%甲酸水溶液(A)-乙腈(B)为流动相,梯度洗脱,流速0.8 mL·min^-1,UV检测器波长254 nm,二极管阵列检测器检测波长范围200~500 nm,进样量10 μL。结果:补阳还五汤中的5个主要成分在肝匀浆、肠道菌液、人工胃液和人工肠液中相对浓度均发生不同程度的改变,其中肝匀浆(毛蕊异黄酮含量增加超过500%)和肠道菌液(毛蕊异黄酮含量增加超过200%)孵育体系中相对浓度变化最为明显。结论:肠道菌群、消化道酶和肝药酶是补阳还五汤中主要成分的主要代谢因素,不同活性成分的主要代谢途径存在明显差异。

Objective:To explore the in vivo metabolic pathway of main ingredients in Buyang Huanwu decoction.Methods:The content of five main ingredients (calycosin-glucoside,ononin,paeoniflorin,calycosin and formononetin) in Buyang Huanwu decoction was determined by an HPLC method.The changes of the main ingredients in Buyang Huanwu decoction incubated with liver homogenate,bacteria liquid,artificial gastric juice or artificial intestinal juice at different time nodes were investigated.Nifedipine was used to verify the CYP450 enzyme activity in the incubation system.The separation column was Shimadzu VP-ODS(250 mm× 4.6 mm,5 μm),column temperature was 30 centigrade degree,and the mobile phase was a mixture of 0.1% formic acid aqueous solution (A) and acetonitrile(B).Gradient elution was utilized with a flow rate at 0.8 mL·min^-1,UV detector was set at 254 nm,the photodiode detector array (PDA) detector was set from 200 to 500 nm,and the injection volume was 10 μL.Results:The concentration changes of five main ingredients were observed in the liver homogenate,bacterium fluid and artificial intestinal fluid incubation systems to different degrees. Among the investigated incubation systems,liver and bacteria exhibited the most obvious changes of the relative concentration of the five ingredients.The calycosin changed more than 500% in the liver homogenate relative concentration and more than 200% in the bacteria solution.Conclusion:Intestinal flora,digestive enzymes and liver drug enzymes were the main metabolic factors for the main ingredients of Buyang Huanwu decoction.There were obvious differences on the main metabolic pathway for different bioactive ingredients.