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。 OBJECTIVE: To study the chemical constituents in different extraction sites of alcohol extract of Jiuwei Tongqiao Decoction by organic solvent gradient, and to screen the effective extraction site through pharmacodynamics in vitro to explore the most effective site of anti-glioma cell proliferation. The compound mechanism of action to provide experimental evidence. Methods: Nine-flavored Tongqiao Decoction by ethanol reflux extraction and concentration, using petroleum ether, chloroform, ethyl acetate and water saturated n-butanol gradient extraction, petroleum ether extract (part Ⅰ), chloroform extract Ⅱ), ethyl acetate extract (part Ⅲ), water-saturated n-butanol extract (part Ⅳ), residual aqueous layer (part Ⅴ) and calycium isoflavone glucoside Glucoside), montmorillonoid-7-O-β-D-glucosidase, the constituents of calyciflorin and formononetin, and the contents of four components in each extract were determined by HPLC. The mobile phase (0-50 min, 5% -55% A; 50-70 min, 55-60% A; 70 (acetonitrile + 0.05% formic acid) ~ 90 min, 60% ~ 85% A; 90 ~ 100 min, 85% ~ 100% A). The detection wavelength was 254 nm. Human glioma U251 cells were selected for follow-up screening. MTT assay was used to determine the effect of each extract site on cell survival. Results: The content of calycosin-venonin-calyciflorin in part Ⅲ was 1.0: 10.8: 8.2, and the half-inhibitory ratio (IC50) of part Ⅲ was 46.89 g · L-1. Compared with other parts, More sexual ingredients and higher levels of U251 cell proliferation was more obvious, and a certain concentration-dependent. Conclusion: Part Ⅲ is the most effective site of anti-glioma U251 cell proliferation effect of Jiuwei Tongqiao Decoction.