基于TLC和iTRAQ技术进行地蚕中抗金黄色葡萄球菌环肽的发现和其作用机制研究

Plant cyclopeptides are a kind of cyclic nitrogen compounds formed by amino acid peptide bonds in plants and have antibacterial, anti-tumor and anti-HIV activities. Stachy geobombycs is a traditional Chinese medicine, which is used for the treatment of tuberculosis, bruises and sores related tobacterial infection. In order to explore the basis of the active substance of the drug and search for antibacterial lead compounds, We have initially isolated three novel cyclic peptide compounds that have a significant inhibitory effect on Staphylococcus aureus. Therefore, this project is intended to (1) obtain more anti-Staphylococcus aureus cyclic peptides directly and quickly by using TLC. (2) Determination of the parameters of inhibition zone, IC50, MBC, MIC, Growth curve, electrical conductivity, intracellular and extracellular protein content of inhibition of S. aureus inhibitied by cyclopeptides of Stachy geobombycs, to systematically evaluate the antibacterial activities of the cyclopeptides, to analyze the interaction between the cyclopeptides and S. aureus cell walls, cell membranes and bacterial intracellular macromolecules, and to elucidate the molecular mechanism of S. aureus inhibitied by the cyclopeptides. (3) .To demonstrate the molecular mechanism of the anti-S. aureus activities of the cyclopeptides from the overall protein level through the iTRAQ quantitative proteomics analysis technique.The completion of this study will further enrich the types of antibacterial peptides and know the molecular mechanism of antibacteria of cyclic peptides of traditional Chinese medicines, and provide the exact theory and experimental basis for the application of plant cyclic peptides in antibacterial.

植物环肽是植物中由氨基酸肽键形成的一类环状含氮化合物，具有抗菌、抗肿瘤和抗HIV等活性。地蚕为传统中药，用于治疗细菌感染相关的肺痨、跌打和疮毒等。为了探明该药活性物质基础，寻找抗菌先导化合物，我们已初步从地蚕中分离得到3个对金黄色葡萄球菌有明显抑制作用的新颖环肽化合物。因此本项目拟（1）通过活性追踪和利用TLC，定向快速地获取更多抗金葡菌环肽化合物。（2）进行地蚕环肽抗金葡菌的抑菌圈、IC50、MIC、生长曲线、电导率、胞内外蛋白含量变化等参数测定，系统地评价地蚕环肽抑菌活性，分析地蚕环肽与金葡菌细胞壁、细胞膜和细菌胞内大分子的相互作用，并阐明地蚕环肽抑制金葡菌的分子机制。（3）通过iTRAQ定量蛋白质组学分析技术，从蛋白质整体水平上系统雪阐明地蚕环肽抑制金葡菌的分子机制。本研究的完成，将进一步丰富抗菌肽种类和认识中药抗菌环肽的分子机制，为植物环肽在抗菌中的应用提供确切的理论与实验基础。

耐药菌的出现致使传统药物失效，开发新的具有抗菌活性的药物，挖掘新的抗菌机制则显得尤为重要。地蚕为传统中药，用于治疗细菌感染相关的肺痨、跌打和疮毒等。前期研究发现地蚕含有环肽类化合物，且对金黄色葡萄球菌有明显抑制作用。为全面了解地蚕的环肽和其他类型的次生代谢产物，以及这些产物在抑菌特别是在抑制金葡菌方面的活性及潜力，本项目对地蚕进行了深入的分离纯化和鉴定，并对部分化合物进行抑菌活性测试和作用机制研究。结果从地蚕分离得到了20多个环肽，另外还有30多个其它类型的化合物，如苯甲酸酯类、生物碱类、苯乙醇苷类和环烯醚萜类等。其中确定的新化合物有21个。找到1个对金葡菌有活性作用的环肽，且发现该环肽可能通过神经活性配体与受体相互作用信号通路以及FoxO信号通路等发生作用。另外还发现部分环肽化合物对α-葡萄糖苷酶具有很好的抑制活性。上述研究结果促进了我们对地蚕活性物质基础和功能的认识，为后续进一步开发利用该植物提供了实验依据。项目已经发表论文3篇，其中SCI收录期刊论文2篇，中文核心期刊论文1篇，同时也已经申请了相关专利1项。