青龙衣抗肿瘤成分组合设计及其作用机理研究

A large number of clinical reports have been confirmed that traditional Chinese medicine green walnut husks had a notable inhibitory effect on many kinds of malignant tumors such as Esophagus, stomach, liver cancer. But the role of the anti-tumor efficacy material foundation of green walnut husks has not been consistent with the determinations yet. This study takes three types of components including naphthoquinones, diarylheptanoids and triterpenoids as the objects which have been isolated from green walnut husks and Initially proved the antitumor activity, and will carry out the research of establishment of "principal component" and combination of components by in vitro anti-tumor experiment.We'll carry on the further mechanism discussion and effect contrast to the principal components and the superiority combination which is for the purpose of illustrating the synergy between the components, the antagonistic relationship. The detailed contents are as follows: According to results of acute toxicity test and preliminary antitumor activity investigation of MTT, a horizontal comparison will be made to screen out the representative component possessing the characteristics of "high performance and low toxicity"from the above three groups separately; "principal component" will be determined by carrying out the longitudinal comparison and the quality synthetic evaluation in three anti-tumor represents; Based on human-computer fusion research, namely the uniform design experiment-efficacy trials-LARS regression-Fitting Correction, the superiority combination will be defined; Finally comparing the principal component against combination of components in the tumor cell influence, the mechanism research which induces apoptosis will be summarized.

经大量的临床报道证实，中药青龙衣对食道癌、胃癌、肝癌等多种恶性肿瘤有非常明显的抑制作用，但目前对青龙衣产生抗肿瘤作用药效物质基础的确定意见尚不一致。本项目以从青龙衣中分离获得并初步证实有抗肿瘤活性的萘醌类、二芳基庚烷类、三萜类成分为研究对象，通过体外抗肿瘤作用比较进行"主成分"的确立及成分间组合研究，对主成分、确定的最佳优势组合再进行深入的机理探讨及比照，旨在说明成分间的协同、拮抗关系。研究包括：进行急毒及MTT初步抗肿瘤活性考察，从上述三大类群中分别横向筛选出一个"高效低毒"的代表成分；对三个代表成分的抗肿瘤作用进行纵向比较和综合评价，确立"主成分"；基于均匀试验设计- - 药效试验- - LARS回归模型建立- - 拟合校正的人机融合研究思路，进行最佳优势组合研究；最后进行主成分、组合成分诱导肿瘤细胞凋亡的机制的比较研究。

目前从天然药物中寻求具有显著细胞毒活性的化学成分进而开发成为有效抗肿瘤药物越来越受到全世界的关注和重视。经大量的临床报道证实，中药青龙衣对食道癌、胃癌、肝癌等多种恶性肿瘤有非常明显的抑制作用，但研究者对青龙衣产生抗肿瘤作用药效物质基础的确定意见尚不一致。本项目以从青龙衣中分离获得并初步证实有抗肿瘤活性的萘醌类、二芳基庚烷类、三萜类成分为研究对象，通过体外抗肿瘤作用比较进行“主成分”的确立及成分间组合研究，对主成分、确定的最佳优势组合再进行深入的机理探讨及比照，旨在说明成分间的协同、拮抗关系。该研究从青龙衣中分离并鉴定了51种化学成分，其中新化合物5种，另有29种化合物在该植物中首次发现，尤其是系列三萜类成分此前未见报道。抗肿瘤谱研究结果表明有效成分对人源细胞株敏感性强弱顺序大致为胃癌BGC-823＞结直肠腺癌细胞HCT-15＞肝癌HepG-2＞宫颈癌Hela＞乳腺癌细胞MCF-7＞白血病细胞K562＞肺腺癌细胞A549＞结肠腺癌细胞HT-29。构效关系研究结果表明青龙衣抗肿瘤活性主要来源于1,4-萘醌，达玛烷型四环三萜以及大环环醚型二芳基庚烷类化合物，抑制活性的差异主要来源于立体异构、是否成环、取代基团的种类和连接位置的不同。通过比较筛选出对上述瘤株均有明显作用且收率较高的代表成分做为优势组合的药效物质基础，结合小鼠急毒实验和荷瘤裸鼠体内抗肿瘤作用研究结果选择胡桃醌、Juglanin A、20(S)-原人参二醇为最佳优势组合的组成成分，其中胡桃醌为主成分，基于均匀实验设计-药效试验-LARS回归模型拟合确定组成比例为胡桃醌：Juglanin A：20(S)-原人参二醇=11:2:6。研究结果发现最佳优势组合成分可有效调节Bcl-2呈低表达，Bax和Capase-3呈高表达，表明抑制肿瘤生长作用是通过促进肿瘤细胞凋亡而实现的。另外研究还发现最佳优势组合成分通过升高细胞内ROS水平，促进细胞内Ca2+浓度升高，降低线粒体膜电位诱导其严重损伤。青龙衣抗肿瘤成分组合是通过多个作用靶点抑制胃癌细胞BGC-823体内和体外的生长，从整体动物、细胞、蛋白水平等阐明了其抗肿瘤作用机理。定量研究该药有效成分组合与相关药效间的关联规律，对于揭示青龙衣成分间相互关系、抗肿瘤作用机理及今后对该药进行多指标质量控制都有现实意义。