“桑黄”类药用菌的次生代谢产物资源及其部分化合物的抗肿瘤活性机理研究

In the project, 13 core species and relative species of "Sanghuang" fungi will be used as materials to systematicly isolate and identify the secondary metabolites of their fruiting bodies and fermentation products based on the completed National Natural Science Foundation Project. According to the chemical structure features, the regularity of distribution of different secondary metabolites will be found in Phellinus, Inonotus, Fomitiporia, Fomitopsis and Fomes genus, and the detailed secondary metabolites resources library of the 13 fungi will be established. The antitumor active mechanism and structure-activity relationship of the screened 8 antitumor active components and antitumor active extracts from the fungi of Phellinus, Inonotus and Fomitopsis genus will be studied with the methods of inhibited activity of microtubule protein, inhibited protein tyrosine kinase activity and others. The project will give a comprehensive scientific explanation to antitumor effects of Traditional Chinese Medicine "Sanghuang" fungi with the multidisciplinary research methods, such as resources classification, secondary metabolites isolation and identification, antitumor activity mechanism research and others. The project will provide exactly basic research results for the further development and utilization of "Sanghuang" fungi, and this has extremely important significance in the medicinal fungi area.

选择13个"桑黄"类真菌的核心种类及相近种类，在已结题基金项目基础上，进一步对其子实体和发酵产物进行系统的次生代谢产物的分离鉴定，按着结构特点进行分类，寻找不同类型的次生代谢产物在Phellinus、Inonotus、Fomitiporia、Fomitopsis和 Fomes属中的分布规律，建立这13个"桑黄"类真菌及其相关种的较详细的次生代谢产物资源库。对已筛选出的8种抗肿瘤活性成分以及筛选出的Phellinus、Inonotus和Fomitopsis属真菌的具有抗肿瘤活性的提取物采用抑制微管蛋白活性、抑制蛋白酪氨酸激酶活性等多种方法进行活性机理和构效关系的研究。利用资源分类、次生代谢产物分离鉴定、抗肿瘤活性机理实验等多学科的研究方法对传统药用真菌"桑黄"的抗肿瘤作用给予较全面的科学解释。本项目对"桑黄"类真菌的进一步开发利用有着极其重要意义，并提供确切的基础研究成果。

经本草学研究和野外及市场调查，生物活性成分与药理学相关研究证实，粗毛纤孔菌与古代本草记载吻合度最高，提供新观点。课题组已成功驯化栽培粗毛纤孔菌。.对几种桑黄类真菌的石油醚提取物、水提取物、粗多糖进行了抗肿瘤活性对比，结果均具显著活性。粗毛纤孔菌和红缘拟层孔菌的固体发酵产物具有显著的抗肿瘤活性，主要机理与其抗氧化、提高机体免疫力及抑制肿瘤组织血管生成有关。药用层孔菌中的3-酮基-去氢硫色多孔菌酸和去氢齿孔酮酸通过有效促进细胞的凋亡体现出较显著的体内抗肿瘤活性。粗毛纤孔菌中分离的多酚类成分对HepG2的抑制效果明显。开展了3-酮基-去氢硫色多孔菌酸、3-乙酰氧基-8，24-羊毛甾二烯-21-酸和麦角甾醇的实验动物体内药代动力学研究。.麦角甾醇、麦角甾醇过氧化物、3-乙酰氧基-8，24-羊毛甾二烯-21-酸、麦角甾-7,22-二烯-3β-醇-棕榈酸酯和麦角甾-4, 6, 8(14), 22(23)-四烯-3-酮对人肝癌细胞株HepG2和人乳腺癌细胞株MCF-7的细胞毒活性均较高，前三者具显著的细胞毒活性。3-乙酰氧基-8,24-羊毛甾二烯-21-酸的抗肿瘤活性机制与细胞凋亡相关，与BAX、BCL-2、Cytochrome C和PARP等凋亡信号通路蛋白表达有关。红缘层孔菌和药用拟层孔菌分离的3-酮基-去氢硫色多孔菌酸等7种羊毛甾烷型三萜(LTT)类化合物抗肿瘤活性构效关系研究表明在3位羟基被酮基或乙酰基取代和C环中15位连有羟基时，此类化合物抗肿瘤作用增强。甾类抗肿瘤活性成分的构效关系为甾核的C‐3位被羟基或羰基取代后，其抗肿瘤活性较显著。.建立了次生代谢产物资源库，包括本项目中得到的27个化合物，有1个新化合物、1个自然界中首次发现的化合物。多糖类、甾醇类、三萜类、多酚类化合物在不同属中的分布与抗肿瘤活性有一定规律，Inonotus和Phellinus属真菌中甾类和多酚类化合物较丰富；Fomitopsis和Inonotus中三萜类成分丰富，前者的氯仿和乙酸乙酯提取物的抗肿瘤活性显著；黄酮类成分主要在Phellinus中发现；甾类化合物和多糖类普遍存在。桑黄类真菌的这些有效物质共同发挥了“多靶点”效应，体现出了显著的抗肿瘤活性。