基于人肝癌细胞SMMC-7721细胞周期调控的薏苡附子败酱散复方抗肿瘤物质基础研究

Yiyi-Fuzi-Baijiang San (YFBS) is a traditional Chinese medicinal formulation that is composed of three herbs, which include Coix lachrymal-jobi Linn, Patrinia Juss and Aconieum carmichacli Debx.. This herbal formulation, with the function of clearing heat, eliminating dampness and eliminating stagnation, has been widely used for over 1500 years to treat bacterial liver abscess and chronic pelvic inflammatory disease. Recently, YFBS has been used as an antitumor drug to treat liver cancer and cervical cancer clinically. Pharmaceutical research also indicated that the formulation and the individual herbs in the formulation showed antitumor activity significantly. But there was no research on the material basis and mechanism of antitumor activity of YFBS. In this study, a method of "formulation-effective chemical fraction-chemical constituent-material basis" for studying the antitumor material basis of YFBS was applied. Material chemical fractions of the herbs in the formulation were prepared firstly, and then the effective chemical fraction of YFBS was obtained when the optimum proportioning ratio of individual fractions was confirmed by taking antitumor effect on SMMC-7721 cell lines as the evaluating indicator. The chemical constituents in the effective chemical fraction of YFBS were identified by the method of HPLC/MS/MS, and the cell cycle arrest in human hepatoma SMMC-7721 cells induced by effective chemical fraction of YFBS was analyzed by flow cytometer in the further research to explore the antitumor mechanism of the effective chemical fraction of YFBS. This method can serve as a support for the delvlopment of material basis of traditional Chinese medicine.

薏苡附子败酱散始载于《金匮要略》，由薏苡仁、败酱草和熟附子三味中药组成，具有清热、利湿、化瘀、散结之功效，在临床上主要用于治疗细菌性肝脓肿和慢性盆腔炎等疾病。近年来，有关该复方用于肿瘤治疗的临床报道越来越多，现代药理研究也表明该方剂及其中各单味药材均具有明确的抗肿瘤作用，但有关薏苡附子败酱散抗肿瘤药效物质基础研究较少而作用机制研究更是空白。 本课题采用中药药效组分研究方法，从中药复方整体出发，按照"复方-有效物质组-单体化合物-物质基础"的研究模式对薏苡附子败酱散复方进行化学组分拆分、优化组分配伍，结合体内、外抗肿瘤药效评价，确定该复方抗肿瘤有效物质组，进一步采用质谱技术对有效物质组化学成分进行指认，并应用流式细胞仪评价该有效物质组对人肝癌细胞SMMC-7721肿瘤细胞周期特异性的调控作用，阐明薏苡附子败酱散复方抗肝癌药效物质基础及初步作用机制，为中药复方药效物质基础研究提供思路。

薏苡附子败酱散始载于《金匮要略》，由薏苡仁、败酱草和熟附子三味中药组成，具有清热、利湿、化痰、散结等功效，临床主要用于治疗细菌性肝脓肿及慢性盆腔炎等疾病。近年来，有关该复方及其中单味中药用于肿瘤治疗的报到越来越多，其中薏苡仁油脂已经开发为新药，并广泛用于临床。在此背景下，我们通过前期药效研究明确了该复方对SMMC-7721细胞的增殖抑制作用，在国家自然科学基金的资助下，我们按照课题的设计方法对该薏苡附子复方进行了较为深入的研究。取得了以下结果：1）采用中药化学提取分离方法从败酱草中分离并鉴定化学结构70个，其中新化学成分5个，首次分离化学成分51个。抗肿瘤活性评价结果表明新化合物3’-hydroxyl-impecylone对肿瘤细胞具有选择性抑制作用，对人肝癌细胞HepG2和人非小细胞肺癌A549抑制作用较强。该化合物可诱导两种细胞凋亡并呈剂量依赖性，可使两种细胞周期阻滞在G2/M期。2）首次应用UPLC-QTOF-MS对薏苡附子败酱散复方中主要化学成分进行定性鉴别，目前已确定其中26种化学成分，生物碱类成分为复方中的主要成分。3）制定了复方中各单味药材的主要有效物质组的提取、纯化工艺，如：薏苡仁油脂、败酱总黄酮、败酱总皂苷、附子总生物碱等。4）以体外药效为指标，分别采用均匀设计法及响应面法优化有效物质组配伍，最终得到配伍比例基本一致，说明响应面法可以用于中药有效物质组配伍设计。薏苡附子败酱散有效物质组的抑制SMMC-7721细胞的最佳配伍量为败酱草总皂苷0.4030 mg/mL、薏苡仁挥发油2.0550 mg/mL、败酱草总黄酮1.0595 mg/mL、附子总生物碱1.6950 mg/mL。5）应用流式细胞仪对复方有效物质组最佳配伍诱导SMMC-7721细胞凋亡作用进行评价，并明确了该有效物质组可将SMMC-7721细胞阻滞于G2/M期，从而抑制肿瘤细胞的增殖。通过本项基金的资助，我们阐明了薏苡附子败酱散有效物质组调控SMMC-7721细胞的物质基础及基于细胞周期调控的作用机制。这些成果为课题的后续申请及薏苡附子败酱散开发应用奠定了坚实的工作基础。