基于特征肽类成分的动物药整体质量评价体系研究：以地龙为例

The main reason for the irregularity and confused animal drugs is the weak quality standard which remains deficiency of specific and active index. In this study, Pheretima, usually mixed with counterfeit in the market, is used as a model. Previous works proved that the protein fraction of Pheretima exhibited anti-stroke activity. Thus, the anti-stroke active peptides and the species-specific peptides will be used as signature peptides, which would be applied for developing the overall quality evaluation system for Pheretima with multiple sources. The study could be divided into four steps: Firstly, the fingerprint-efficacy relationship will be established based on the activity against stroke, and then it will be used to screen for active peptides. Secondly, Protease-catalyzed 18O-labeling combined with peptidomics will be used for screening for specific peptides of Pheretima from four species in Chinese pharmacopoeia. Thirdly, both the active peptides and the specific peptides would be prepared by solid phase synthesis. All those peptides would be used as signature peptides for the quality control method of Pheretima. At last, based on signature peptides, the overall quality evaluation system for characteristic fingerprint analysis and multi-component quantitative analysis would be developed. It would be used for the species discrimination and the quality evaluation of Pheretima from market. Besides, the established method would also be used an example for improving the quality control system of animal drugs.

动物药市场混乱、质量参差不齐，究其原因主要是其质量标准薄弱，缺乏专属性指标和活性指标。本课题以市场正伪品混杂的动物药地龙为研究对象。前期实验表明，地龙蛋白部位具有抗脑卒中活性。基于此本研究提出将抗脑卒中活性肽类和种属特异肽类成分作为特征肽类成分，建立动物药地龙的整体质量评价体系。该研究主要包含以下四个方面：首先基于抗脑卒中活性建立地龙肽类部位的谱效关系，进而寻找地龙抗脑卒中的活性肽类成分；其次利用酶催化的氧18同位素标记结合多肽组学技术，对药典四个种属来源的地龙进行差异性分析，寻找种属特异性肽类成分；第三，通过固相合成，分别得到活性肽类成分和种属特异肽类成分，作为特征肽类指标性成分用于地龙质量评价体系的建立；最后基于特征肽类成分，建立特征图谱和多成分定量分析相结合的整体质量评价体系，用于药典多基原动物药地龙的种属区分和质量评价，进而为动物药质量评价体系的提升提供借鉴。

动物药市场混乱、质量参差不齐，究其原因主要是其质量标准薄弱，缺乏专属性指标。本课题以市场正伪品混杂的动物药地龙为研究对象，采用转录组结合蛋白组学的方法，筛选酶解特征多肽，用于多基原地龙药材的种属区分。具体而言，首先基于转录组构建地龙理论蛋白质数据库，其次，基于蛋白组学，深入探讨了不同品种地龙的蛋白质类成分，明确了蚓激酶在药典品种地龙中的分布情况；第三，筛选了市场三个主要品种地龙的特征酶解多肽，并构建质量评价方法用于市售药材的种属区分。通过本项目的开展，完善了地龙理论蛋白质数据库，明晰了地龙蛋白类成分的分布特别是蚓激酶类成分，构建了基于特征酶解多肽的MRM方法体系，实现了市售地龙品种的种属区分，在地龙的质量标准方面显示出了较好的应用前景，该方法的建立为动物药特征多肽的筛选及其种属鉴别的应用提供参考。