基于蛋白组学的三七叶稀有原人参二醇型皂苷抗肿瘤机制研究及资源综合利用

The roots of Panax notoginseng is one of the most widely used natural tonics in oriental countries. Due to distinct multi-bioactivities of saponins, the supply of roots of P. notoginseng can hardly meet the increasing market demands every year. For over a thousand years, P. notoginseng has been grown in Wenshan of Yunnan, where over 85% of the total production of about 2 million kg per year in China. Although the Good Agriculture Practice (GAP) program was established for the optimal utilizat However, their composition and contents vary greatly depending on the tissue. Our recent observation indicated that existence of abundant protopanaxtriol saponins in leaves is the most significant difference compared to the roots of P. notoginseng. Renewed interests in the cytotoxic activity of ginsenosides have come from the ?nding that protopanaxdiol ginsenosides have stronger anti-tumor activities than protopanaxtriol ginsenosides ion of the medicinal plant resources, yield of P. notoginseng was frequently affected by extremes of climate in the past decade. From the point of multipurpose and optimal utilization, it is urgent to explore new resource and usage from other parts of this plant. Compared to roots, exploitation and research of leaves are lack of attention. Contemporary structural-function relationship studies of ginsenosides have proven that their anti-tumor activities increase with the decrease of sugar number. Sugar molecules within a ginsenoside have an inverse impact on tumor cells. Our previous study has demonstrated that the anticancer effects of ginsenosides Rg3 (two sugars), Rh2 (one sugar), Pn-1 (one sugar) are more powerful than Rb1 (three sugars), Rb2 (four sugars) against different types of cancer cell lines. The results imply that the low polar saponins fraction may be enriched with stronger anti-carcinogenic PPD group. From the points of time consuming and accurate mass measurement capability, the LC-QTOF-MS is employed for screening and guiding target separation of new compounds in herbal extract..To identify the cellular targets of the active saponins, two-dimensional gel electrophoresis will be performed, and proteins altered in expressional level after saponins exposure of cells will be identified by MALDI-TOF MS/MS. The regulation of proteins will be also confirmed by Western blotting and other in vivo or in vitro experiments. The results of the present study will shed light on the anticancer mechanism of ginsenoside from a molecular perspective. Furthermore it is possible that we can obtain promising saponins for cancer therapy either by isolating them leaves of Panax notoginseng or by modifying the structure of natural saponins.

三七Panax notoginseng(Buck)FoHoChen为我国名贵中药材，由于独特疗效在临床上得到了广泛应用。近年频繁的自然灾害使三七产量锐减，在提供根及根茎时，地上部分除三七花外均被废弃。为综合利用三七资源，本研究结合新技术对三七叶的新活性化合物探寻。前期本实验室对三七叶和根茎做了定性分析和化学研究，结果表明在三七叶小极性部位富含小分子量的原人参二醇型皂苷，且许多人参皂苷的构效关系研究表明该类皂苷能抑制肿瘤细胞的生长，如：我们新分离到的新骨架皂苷Pn-1在多种肿瘤细胞株中抗肿瘤效果是Rg3的3-5倍。基于蛋白组学在发现药物靶点和阐明作用机制方面的优势，本课题组运用二维凝胶电泳，基质辅助激光解析飞行时间质谱，药物靶点预测软件等前沿技术并结合细胞水平实验以及体内抗肿瘤实验，多角度分析三七茎叶中稀有原人参二醇型化合物的作用靶点，为促进三七叶的资源综合利用提供理论依据

三七Panax notoginseng(Buck)F•H•Chen为我国名贵中药材，由于独特疗效在临床上得到了广泛应用。在提供根及根茎的同时，地上部分除三七花外均被废弃。现已证实三七中原人参二醇型皂苷的抗肿瘤活性强于原人参三醇，而本课题组发现三七叶中含有丰富的原人参二醇型皂苷。因此，为综合利用三七资源，寻找高效低毒的抗肿瘤天然活性成分，本项目运用靶向分离策略，结合分子生物学、药理学、蛋白质组学、生物信息学等多学科技术，发现三七叶中具有抗肿瘤活性的原人参二醇型皂苷并阐明其作用机制。..1.我们利用现代分析方法和药理学手段确定了大孔树脂85%乙醇洗脱部位为三七叶活性部位。且为了扩展原人参二醇型皂苷的植物资源，我们对三七叶以及同属植物—人参叶和西洋参叶进行了全成分表征，结果表明三七叶和西洋参叶中原人参二醇型皂苷资源丰富，可作为原人参二醇型皂苷开发的主要资源。.2.对活性部位85EF进行了系统的化学成分分离，鉴定了13个原人参二醇型皂苷，包括两个新原人参二醇型皂苷（Com12，Com13）。抗肿瘤活性筛选发现，Com12，Com13和人参皂苷F2（F2）在多种肿瘤细胞株上表现出较强的抗肿瘤活性。.3.我们选择F2为研究对象，采用人胃癌细胞株SGC7901为模型，结合体内动物和体外细胞实验对F2的抗肿瘤机制进行研究。F2可能通过ROS介导JNK途径激活线粒体通路，下调Bcl-2家族蛋白的表达，改变线粒体膜通透性，导致Cyto c从线粒体释放入胞浆，引发caspase联级反应，抑制PARP的表达，诱导了SGC7901细胞凋亡。.4.运用MALDI-TOF以及iTRAQ技术对F2作用后的差异蛋白进行了研究，结果显示F2引起SGC7901细胞205个差异蛋白丰度改变，对其中6个差异蛋白（PRR5，CISD2，Bcl-xl，NLRX1，RPS15和RPL26）进行了western blotting验证；其诱导凋亡的机制与ribosomal protein-p53 signaling pathway和Bcl-xl/Beclin-1 pathway有关。.本项目为三七叶资源的合理利用提供了理论依据，为小极性原人参二醇型皂苷的药用价值深度开发奠定了基础。本项目共发表论文7篇，其中SCI论文5篇；获授权专利2项；培养博士研究生1名，硕士研究生3名。按期完成了计划书的指标。