中药蛇胆的资源与品质评价研究

The Chinese medicine "Shedan" is a valuable but scarce medicinal resource, lots of preparations used Shedan as the crude material are collected in the "Chinese Pharmacopoeia". However, there is still no national standard for this herb and the resource quality of these preparations also can not be guaranteed. The underlying reason of this situation is that the resource quality control of Shedan have not been solved. In this study, on the premise of medicine market and origin investigation, along with the establishment of breeding base, we will identify the Shedan species by animal morphological classification and CO I primer amplification-based DNA barcode, and then, we will isolate and identify its effective substances by chromatographic and spectroscopic techniques, combied with high sensitive LC/MS technology. Furthermore, we will illustrate the relationship between snake liver transporter protein expression and bile secrection by proteomics technology, and also use in vivo model to evaluate Snakedan effecicacy of anti-cough and eliminating phlegm. Based on this work, we will clarify the relationship among germplasm resources-genetic material-effective substances, screen for the high quality Shedan resource and establish the evaluation database for Shedan resource and quality to make the foundation for quality control of Shedan herb and its preparactions, even for development of alternatives, rational resource conservation and sustainalble usage.

蛇胆是常用名贵资源稀缺的中药材，大量以中药蛇胆为原料的制剂收载于《中国药典》，但是蛇胆药材还没有国家标准，制剂源头的质量无法得到保证，究其原因是中药蛇胆的资源和品质问题没有真正解决。本课题在药材市场和产地调查并建立养殖基地的前提下，采用动物形态分类和以COⅠ序列引物扩增为基础的DNA条形码技术鉴定其物种，高灵敏高选择性的LC/MS/MS联用技术分析其药效物质，色谱和波谱学技术分离鉴定化合物的结构，蛋白组学技术探讨蛇肝转运蛋白表达与胆汁酸分泌的联系，动物体内药理模型评价其止咳祛痰的药效，综合阐明中药蛇胆种质资源-遗传物质-药效物质之间的关系，筛选出优质的蛇胆资源，建立中药蛇胆资源与品质评价数据库，为中药蛇胆药材和制剂的质量控制，甚至为替代品开发、资源的合理保护和持续利用奠定基础。

蛇胆是常用名贵稀缺中药材，来源复杂，鉴定困难，市场常出现掺伪现象，其资源和品质的问题没有得到解决，而且缺乏科学合理的质量控制手段。本项目对中药蛇胆的原动物资源及市场商品进行了较全面的调查、标本的收集及形态学鉴定，基本澄清了目前中药蛇胆的资源现状。通过DNA条形码技术对来源于3科25种290个蛇胆样品进行了准确的基源鉴定，并首次向GenBank提交了10种蛇的COⅠ条形码序列，建立了蛇胆的DNA条形码鉴定方法，建议将自行设计SNCOI2引物作为蛇胆DNA条形码鉴定通用引物，并对目前主要市场的蛇胆商品进行了基源鉴定；同时本项目运用UPLC-MS/MS技术，鉴定出15种蛇胆胆汁酸成分，并发现3种新的胆汁酸类化合物，对中药蛇胆及其掺伪猪牛羊鸡鸭胆的胆汁酸成分进行了全面的定性和定量分析，为蛇胆药材的质量控制及混伪品的鉴定提供了重要依据；本项目还通过聚合酶链式反应（PCR）和基因克隆的方法，得到了BSEP编码基因ABCB11的全序列信息，并进行了同源性分析；通过蛋白组学技术，得到了可靠的BSEP肽段，从基因水平和蛋白组学水平分别对BSEP进行了初步鉴定，首次证实了蛇肝脏中胆汁酸转运体BSEP蛋白的存在，为蛇胆汁代谢调控的进一步研究奠定基础。本项目的实施建立了中药蛇胆资源与品质评价数据库，对中药蛇胆的资源与品质评价具有重要意义。