基于藏药余甘子酚酸类成分体内动态变化的抗癌药效物质与质量控制研究

The follow-up study on the inhibit tumor of Tibetan medicine Phyllanthus emblica shows that the extract has a very good effect in inhibiting tumor growth finnishing the project of National Natural Science Foundation, hydrolyzable tannins and phenolic acids are the mainly effective substances. Hydrolyzable tannins can be hydrolyzed to gallic acid, ellagic acid and polyol with acid, alkali and enzyme (in vivo : gastric juice, intestinal juice and enzymes), The explore the efficacy compositions of hydrolyzed tannins and phenolic acids in normal and pathological animals is the key problem of clarifing the efficacy material basis and method for quality control. of emblica respectively.The project will use the hydrolyzable tannin and phenolic acids as the quantiative indexs and quality substance, the pharmacokinetical (PK) character of the composition in the normal and tumor-bearing mice was studied, conjugate PK with pharmacodynamics(PD) to study the relationship and inquire the effective substance of. Phyllanthus emblica , the sources of composition and metabolic transformation in gastric juice, intestinal juice and blood of animals are analysised by HPLC, HPLC-MS and other method to clarify the anti-cancer efficacy substances. the characteristic spectrum of Phyllanthus emblica based on the anticancer efficacy ingredients in vivo and quality evaluation methods are established to provide the basis for the clinical application and medicinal quality control of the Tibetan Phyllanthus emblica.

在国家自然科学基金项目后续研究中发现藏药余甘子具有很好的抑制肿瘤作用，药效物质主要为可水解鞣质等酚酸类成分。可水解鞣质在酸、碱、酶（体内胃液、肠液、酶）等作用下可水解为没食子酸、鞣花酸和多元醇，探讨余甘子可水解鞣质等酚酸类化学成分在正常动物和病理动物体内的动态变化过程和变化成分是深入研究余甘子药效物质基础和质量控制的关键。.本项目将分别给正常动物和实体瘤模型动物口服余甘子总鞣质/总酚酸，采用体外化学成分分离,HPLC-MS等方法检测动物胃液、肠液、血液的酚酸类等成分，分析其来源和代谢转化关系，通过药代动力学/药效动力学（PK/PD）关联性分析确定与药效相关的有效成分组，阐明抗癌药效物质基础。建立基于体内抗癌药效成分的余甘子特征图谱和质量评价方法，为藏药余甘子的临床应用和药材质量控制提供依据。

余甘子为大戟科叶下珠属植物余甘子Phyllanthus emblica L．的干燥果实，为常用藏药，在自然基金后续研究中发现余甘子具有很好的抑制肿瘤作用。本项目以余甘子总鞣质和单体成分含量为指标筛选出余甘子鞣质提取和大孔树脂富集纯化工艺，使鞣质含量稳定在55 %以上。并通过体外抗癌活性筛选出余甘子的主要抗癌物质为鞣质和酚酸。. 体内实验显示余甘子总鞣质对荷肉瘤S180荷瘤小鼠抑瘤率56.19 %，荷肝癌H22荷瘤小鼠抑瘤率51.02 %。体外实验对A549 （IC50为0.019 g•L-1）、NCI-H1703（IC50为0.033g•L-1）作用最显著。其抗肿瘤作用与调节MAPK信号通路诱导肿瘤细胞凋亡， MMP-2和MMP-9的表达抑制人肺鳞癌细胞的转移和抑制肿瘤细胞侵袭有关。. 余甘子鞣质部位在人工胃液中主要成分柯里拉京、没食子酸、鞣花酸等在12h内都比较稳定，在人工肠液中鞣花酸比较稳定。没食子酸、柯里拉京和鞣花酸在Caco-2细胞模型中透过较低，3种成分在单体、单体混合物、提取物状态下表观渗透系数呈现先下降后上升的趋势。.从余甘子鞣质部位40个成分入血，确定了其中26个成分结构。并得到没食子酸、柯里拉京、鞣花酸的血清药代动力学参数和药时曲线。在人肠内菌作用下检测出4个代谢成分4个原型成分，其中没食子酸、柯里拉京、鞣花酸3个指标性成分时效曲线呈先增加后减少的趋势。大鼠尿液、粪便代谢与排泄研究显示没食子酸在尿液和粪便中排泄量均较大，柯里拉京主要经粪便中排泄。余甘子鞣质部位在组织分布趋势与血清代谢较吻合，没食子酸、柯里拉京、鞣花酸均以在胃肠道中含量最高，其次是肝组织和肺组织。. 抗癌药效动力学（PD）研究，选取HepG-2、A549癌细胞，进行血清药理学试验,得到抑制率随时间变化E-T曲线。结果表明随着给药剂量和给药次数的增加，含药血清对两株癌细胞的抑制率增加，且对A549的抑制率更强，并得到其效应时间曲线下面积（AUCE）。基于没食子酸、柯里拉京、鞣花酸的PK数据，与鞣质部位含药血清对A549、Hepg-2癌细胞两个效应量之间的PD数据，初步分析柯里拉京对癌细胞的抑制作用强于鞣花酸和没食子酸。