新疆维药多伞阿魏抗胃癌作用物质基础及其机制研究

At present, plant resources of Ferula sinkiangensis K.M.Shen and Ferula fukangensis K.M.Shen, recorded in “Chinese Pharmacopoeia” were extremely scarce. Herbs of Ferula ferulaeoides (Steud.) Korov., which is widely used as national medicine by Uighur native people, is mainly circulated on Chinese herbal medicine market. It is rich in resources, unique distributed in Xinjiang drought regions, China. In recent years, researching of Ferula ferulaeoides was focused on anti-tumor activity, but research on its material basis and mechanism of action have not been systematically reported by scientists. On the basis of anti- gastric cancer cell activity study on extracts of Ferula ferulaeoides in vitro, the first purpose of this project is to screen the active parts of anti- gastric cancer cell activity from Ferula ferulaeoides, anti-gastric cancer cell activity screening assay in vitro were made on different polar parts of Ferula ferulaeoides by MTT and CCK 8. The second purpose of this project is to explore anti-gastric cancer activity in vivo and its safety from Ferula ferulaeoides, by means of detecting indexes of inhibitory rate and more pathology inspection on building baby mice model of gastric cancer. And also purpose of this project is to preliminary study molecular mechanism of anti-tumor of Ferula ferulaeoides by flow cytometry and Western blot detection, separating of gastric cancer cell apoptosis and Caspase, Bax and Bcl-2 protein factors. On the basis of biological activities study on Ferula ferulaeoides in vitro, several purification and separation methods (column chromatography on silical gel, polyamide, Sephadex LH-20) and many kinds of structure elucidation and identification technologies (UV, IR, NMR) were used, and to carry out the structure-activity relationship study between them. Through this project research, clarifying the material basis and mechanism of action from Ferula ferulaeoides fighting anti- gastric cancer cell activity, and then to rich for the anti-tumor natural products, promoting development and utilization of Ferula resources in Xinjiang region, and further accelerate the development of national pharmaceutical industry, driveing the local economic growth.

目前，《中国药典》收载的新疆阿魏和阜康阿魏植物资源已极度稀缺，市场流通的主要为多伞阿魏，资源丰富，是新疆干旱地区特有的民族药资源。近年已有学者关注多伞阿魏的抗肿瘤活性，但其物质基础和作用机制尚未见系统研究报道。在我们前期初步验证多伞阿魏能抑制体外胃癌细胞增殖活性的基础上，本课题首先采用MTT和CCK-8法对多伞阿魏不同部位体外抗胃癌细胞活性进行系统筛选；其次，构建胃癌幼鼠模型，通过检测抑瘤率、病理学等指标考察多伞阿魏体内抗胃癌活性与安全性；Western-blot等检测分离胃癌细胞Caspase、Bax、Bcl-2等蛋白因子，初步探索抗胃癌的分子机制。在此基础上，对活性部位进行分离纯化，IR、MS、NMR进行结构解析，并开展构效关系研究。通过本课题研究，阐明多伞阿魏抗胃癌物质基础和作用机制，为丰富抗肿瘤天然产物与促进新疆地区阿魏资源开发利用，加快民族医药产业发展，带动当地经济增长奠定基础。

多伞阿魏是新疆地区特有民族药，资源丰富，目前代替药材阿魏使用。我们前期初步验证多伞阿魏能抑制体外胃癌细胞增殖活性，为了系统研究多伞阿魏抗胃癌活性部位、作用机制及其物质基础，本课题组开展了下列研究： .1.体外实验结果表明多伞阿魏不同提取部位对5种人胃癌细胞均具有不同程度的增殖抑制作用，其中多伞阿魏挥发油、氯仿部位对AGS、SGC-7901、MGC-803的增殖抑制作用最强（IC50＜10μg/mL）；Hoechst33258荧光染色法和流式细胞凋亡检测结果验证挥发油和氯仿部位为多伞阿魏体外抗胃癌活性部位。.2.本项目先后成功建立了SGC-7901、MGC-803裸鼠皮下移植性胃癌肿瘤模型，结果表明多伞阿魏氯仿部位能显著抑制SGC-7901、MGC-803皮下异位移植瘤的生长，最大抑制率大于63%。HE及TUNEL结果显示，多伞阿魏氯仿部位可显著诱导SGC-7901、MGC-803皮下移植性胃癌肿瘤细胞凋亡，实验结果明确验证了多伞阿魏氯仿部位有显著的体内抗胃癌作用。通过RT-PCR、Western-blot等研究结果表明，多伞阿魏氯仿部位抗胃癌作用分子机制可能与下调裸鼠血液中Survivin mRNA表达水平，上调肿瘤细胞中凋亡蛋白Bax、Caspase-3和Caspase-9表达水平有关。.3.多伞阿魏挥发油可能的物质基础为D-柠檬烯、崁烯、愈创木醇等，其中D-柠檬烯相对百分含量最高（52.84%）；多伞阿魏氯仿部位可能的物质基础为香豆素类化合物，分析推断出4个香豆素、1个苯乙酮类化合物。急性毒性实验结果表明多伞阿魏氯仿部位属于低毒，较为安全。.4.构效关系研究表明，D-柠檬烯对AGS、SGC-7901均具有增殖抑制作用，特别对AGS细胞毒活性最强。.5.通过大鼠单次口服多伞阿魏氯仿部位后，愈创木醇吸收迅速，Tmax约为0.5h，半衰期为9.18±3.75h，建立的基于SIM的GC-MS法可很好应用于多伞阿魏有效成分血清药代动力学研究。.6.除胃癌细胞外，多伞阿魏挥发油和氯仿部位对5种人肿瘤细胞（Caco-2、PC-3、HeLa、HepG2、MDA-MB-175-VII）具有不同程度的增殖抑制作用，抑制率最高达90%，表明这两个部位具有广谱抗肿瘤活性。.7.通过本项目的实施，发表论文4篇，其中SCI论文1篇；申请专利受理1项。培养青年教师1人、硕士研究生2人。