九味通窍汤及其不同萃取部位的体外药效学考察

In vitro Pharmacodynamic Studies of Jiuwei Tongqiao Decoctions and Its Different Extracting Parts

摘要:

目的: 研究九味通窍汤醇提物经有机溶剂梯度萃取后不同萃取部位的化学成分变化,通过体外药效学筛选有效萃取部位,探索抗脑胶质瘤细胞增殖作用的最有效部位,为阐明该复方作用机制提供实验依据。方法: 九味通窍汤经乙醇回流提取浓缩,采用石油醚、三氯甲烷、乙酸乙酯和水饱和正丁醇进行梯度萃取,得石油醚萃取物(部位Ⅰ)、三氯甲烷萃取物(部位Ⅱ)、乙酸乙酯萃取物(部位Ⅲ)、水饱和正丁醇萃取物(部位Ⅳ)、残留水层液(部位Ⅴ),选择毛蕊异黄酮葡萄糖苷(毛蕊异黄酮-7-O-β-D葡萄糖苷)、芒柄花苷(芒柄花素-7-O-β-D葡萄糖苷)、毛蕊异黄酮和芒柄花素指标成分,采用HPLC确定各萃取物中4种成分的含量,流动相(乙腈+0.05%甲酸)(A)-(水+0.05%甲酸)(B)梯度洗脱(0~50 min,5%~55%A;50~70 min,55%~60%A;70~90 min,60%~85%A;90~100 min,85%~100%A),检测波长254 nm。选用人源脑神经胶质瘤U251细胞活性进行跟踪筛选,应用MTT比色法测定各萃取部位对细胞生存的影响。结果: 部位Ⅲ中毛蕊异黄酮葡萄糖苷-芒柄花苷-毛蕊异黄酮的含量比1.0:10.8:8.2,部位Ⅲ的半抑制率(IC₅₀)46.89 g·L^-1,与其他部位比较,含各指标性成分较多且含量较高,对U251细胞增殖抑制作用较为明显,且呈一定的浓度依赖性。结论: 部位Ⅲ为九味通窍汤抗脑胶质瘤U251细胞增殖作用的最有效部位。

Abstract:

Objective: To study chemical compositions changes of different extracting parts of Jiuwei Tongqiao decoctions by gradient extraction with four kinds of organic solvents,screen effective extracting parts by in vitro pharmacodynamic,select the best effective parts on proliferation effect of anti-glioma cells,and provide experimental basis for further study on mechanism of Jiuwei Tongqiao decoctions. Method: After alcohol extract enrichment of Jiuwei Tongqiao decoctions,gradient extraction with petroleum ether,chloroform,ethyl acetate and water saturated n-butyl alcohol was to get petroleum ether extract(part Ⅰ),chloroform extract(part Ⅱ),ethyl acetate extract(part Ⅲ),water saturated n-butyl alcohol extract(part Ⅳ) and the residual liquid water extract(part Ⅴ).HPLC was employed to determined four index components,including calycosin-7-O-β-D-glucoside,formononetin-7-O-β-D-glucoside,calycosin and formononetin,mobile phase was consisted of (acetonitrile+0.05% formic acid)(A)-(water+0.05% formic acid)(B) for gradient elution (0-50 min,5%-55%A;50-70 min,55%-60%A;70-90 min, 60%-85%A;90-100 min,85%-100%A) and detection wavelength was 254 nm.U251 cells of anthropogenic brain glioma was selected for tracking,MTT was applied to determine effect of extraction parts for cell survival. Result: The content ratio of calycosin glucoside-ononin-calycosin in part Ⅲ was 1.0: 10.8: 8.2,its half inhibition rate(IC₅₀) was 46.89 g·L^-1.In comparison with other parts,index ingredients in part Ⅲ were more comprehensive and concentration of them were higher,inhibitory effect on U251 cells was more obvious and appeared a certain concentration-dependent manner. Conclusion: Part Ⅲ is the most effective parts in Jiuwei Tongqiao decoctions for anti-proliferative effect of U251 glioma cells.