葛根芩连汤氯仿部位含量测定及对羧酸酯酶的抑制作用研究

Determination of chloroform fraction in Gegen Qinlian decoction and its inhibition on carboxylesterase

目的:建立葛根芩连汤氯仿部位的多成分含量测定方法,并考察其抑制羧酸酯酶(CES2)的作用。方法:采用溶剂萃取法提取葛根芩连汤氯仿部位,以葛根素、小檗碱、黄芩苷、甘草苷4种指标性成分,采用超高效液相色谱(UPLC)对其进行含量测定;采用荧光素二乙酸酯(FD)为CES2的特异性荧光探针底物,在人肝微粒体(HLM)孵育体系中考察葛根芩连汤氯仿部位对CES2的抑制作用。结果:葛根芩连汤氯仿部位中指标性成分含量测定结果为:葛根素347 mg·g^-1、小檗碱1.65 mg·g^-1、黄芩苷14.9 mg·g^-1、甘草苷1.04 mg·g^-1,该部位抑制CES2活性的IC50为0.5 mg.mL-1。结论:葛根芩连氯仿部位及其单体均对CES2具有较强的抑制作用,揭示葛根芩连汤有效部位可通过抑制羧酸酯酶以缓解伊立替康所致迟发型腹泻,为中药缓解其毒副作用的机制提供科学依据。

Objective: To establish a method for the determination of multiple components in the chloroform faction of Gegen Qinlian decoction and to investigate its inhibitory effect on carboxylesterase(CES2). Method: The chloroform faction of Gegen Qinlian decoction was obtained by solvent extraction method. The contents of four representative components, puerarin, berberine, baicalin and glycyrrhizin, were simultaneously detected by ultra-high performance liquid chromatography(UPLC). Fluorescein diacetate(FD) was used as the specific fluorescent probe substrate to investigate the inhibitory effect of chloroform faction of Gegen Qinlian decoction on CES2 in the human liver microsome(HLM) incubation system. Result: The contents of these components in the chloroform faction of Gegen Qinlian decoction were as following: puerarin 347 mg·g^-1, berberine 1.65 mg·g^-1, baicalin 14.9 mg·g^-1, and glycyrrhizin 1.04 mg·g^-1, respectively. The value of IC50 chloroform faction of Gegen Qinlian decoction on CES2 activity was 0.5 mg.m L-1 Conclusion: The chloroform faction of Gegen Qinlian decoction and chemical components all exhibit a strong inhibitory effect on CES2 through which it relieves delayed diarrhea caused by irinotecan in clinics. The results provide a scientific basis for the combination of Chinese and Western medicine with Chinese medicine alleviating the toxic and side effects of Western medicine.