广西产拳卷地钱遗传差异与药材品质相关性的研究

Marchantia convoluta is abundant and widely distributed in Guangxi. Through previous studies of screening and evaluation of crude drugs for anti-HBV, our research group found that the total flavones from Marchantia convoluta have a strong anti-HBV activity in vitro, suggesting that the crude drug is potentially deserved to be developed. At present, our research group is trying to develop a new anti-HBV drug using Marchantia convoluta as the raw material. During the research, we found that there exist significant differences in the active ingredients content of Marchantia convoluta among different populations. The content and composition of active ingredients, which are material basis of the efficacy, determine the quality of crude drugs. Meanwhile, they are affected by genetic differences. Based on our early work, we plan to identify the material base of anti-HBV of total flavones from Marchantia convoluta; we also use SAM method to develop suitable SSR primers for Marchantia convoluta, through which to study the genetic differences of the crude drug; at last we analyze the relationship between the SSR molecular marker and the content and composition of active ingredients of Marchantia convoluta at the population and individual levels. This project may provide technical support for the identification of high-quality varieties, the screening of germplasms and the research of genetic diversity of Marchantia convoluta in future.

拳卷地钱在广西分布广泛，资源丰富。本课题组在前期进行的抗乙型肝炎病毒药材筛选评价研究工作中发现，拳卷地钱总黄酮具有较强的体外抗乙肝病毒活性，开发潜力巨大。项目组目前正在研究开发以拳卷地钱为原料的抗乙肝新药。在研究过程中发现不同居群的拳卷地钱成分含量差异较大。药材的有效成分含量及组成决定药材的品质，是疗效的物质基础。而遗传差异又影响着有效成分的高低及组成差异。因此项目组在前期研究工作的基础上，进一步确定拳卷地钱总黄酮抗乙肝作用的物质基础，依据SAM法开发拳卷地钱的SSR引物，用SSR分子标记研究拳卷地钱的遗传差异，分别从居群和个体的水平分析拳卷地钱的SSR分子标记与拳卷地钱有效成分含量及组成的关系，寻找与之相关的SSR分子标记。为今后拳卷地钱优质品种的鉴别、种质资源筛选、遗传多样性研究提供技术支持。

以广西产拳卷地钱为研究对象，1. 资源调查；2.扫描电镜SEM技术显微观察和RAPD分子鉴定；3.AAS、AFS对其重金属安全限量检测；4.GS对其农药残留安全限量检测；5.UV-Vis测定其总黄酮含量；6.HPLC-MS分析其化学成分；7.HPLC测定其芹菜素含量；8.HPLC指纹图谱研究；9.其总黄酮对 CCl4诱导急性肝损伤大鼠保肝效用及作用机制研究；10. SAM 法设计开发其SSR引物分子生药学鉴别；11.建立并优化其DNA 提取及 ISSR- PCR 反应体系；12.SCoT-PCR引物筛选及反应体系优化；13.基因组测序法对其遗传差异SSR引物筛选等。结果：广西产地钱属主要有3种（地钱、拳卷地钱和粗裂地钱）；SEM技术及分子鉴别方法可有效进行品种鉴别；提供6种重金属安全限量检查及9种有机氯农药残留检测结果；总黄酮含量4.0009~56.3107mg/g，差异较大，贺州昭平的含量较高；发现其含有芹菜素、木犀草素等黄酮类成分5个，非黄酮类成分有18个；测定芹菜素含量评价药材品质，贺州昭平的含量较高。其HPLC 指纹图谱显示，不同药材共有峰的相对峰面积存在较大差异；其总黄酮能降血清ALT和AST，病理切片示对急性肝损伤有保护；升高血清SOD活性，减少MDA，可能抑制脂质过氧化反应发挥作用；降肝组织TNF-α、IL-1β和IL-6水平，抑制炎症发挥护肝作用。SAM法开发其SSR引物100对并筛选，获30对SSR标记引物分子生药学鉴别方法。改进 CTAB 法提取DNA 质量高，适用于 ISSR-PCR 扩增，建立并优化了ISSR-PCR反应体系。正交设计方法对其SCoT-PCR反应体系进行优化，获最佳反应体系；采用基因组测序法对其SSR引物筛选得到29对引物，三条引物有特异性条带，对其扩增，观察特异性条带与样品组分关联及遗传多样性分析，遗传亲缘关系具有一定差异，高含量样品遗传亲缘性更近，易聚为一类，在 60个样品间多态性数据计算遗传相似系数， UPGMA聚类分析为9类，第1类为12个产地其总黄酮芹菜素含量高(如贺州昭平）。本研究达到预期目标，进一步完善了广西产拳卷地钱遗传差异与药材品质相关性评价体系及关键技术，提供新的思路与方法，将促进广西产拳卷地钱的开发利用。（经科技查新证实符合预期内容及未见相同报道，见附件）