黑果枸杞多酚对H2O2致PC12细胞损伤的功效物质基础研究

Lycium ruthenicum Murr.(LRM) is a newly discovered wild plant, and riched in polyphenols such as rare acrylated petunidin anthocyanins. Our previous research showed that the polyphenols from LRM such as anthocyanins has a protective effect on PC12 cells, and it's antioxidant activity was much higher than common plant resources, but the compositions, structure-activity relationship and the related mechanisms are not yet clear. In the project, 17 different germplasm materials of LRM as the research object, the polyphenols from LRM were separated, identified and the chemicalomics library were established by HPLC-MSn, pHPLC and NMR. The HPLC-MSn chemical fingerprint of polyphenol compounds from 17 different LRM were established by fingerprint similarity method, and analyzed by cluster analysis with principal component analysis to completely represent the chemical characteristics of the polyphenols. We also studied the protective effects of polyphenols extracts and monomers from different germplasm materials of LRM against oxidative damage of PC12 cell. The Partial Least Squares Regression analysis were adopted to establish spectrum-activity relationships of polyphenols extracts from LRM acting on PC12 cell. The data of chemomics and spectrum -activity relationships were integrated to analyze the relationship of polyphenols and activity. The RT-PCR and Western blot were used to study the protective mechanism of polyphenols on oxidative damage of PC12 cells, and the material basis of the polyphenol from LRM as an inhibition of PC12 cell apoptosis were finally elucidated.

黑果枸杞（LRM）是新挖掘的植物资源，富含鲜见酰化矮牵牛素花色苷等多酚。项目组前期发现，LRM花色苷等多酚具有保护PC12细胞活性，且抗氧化活性远高于常见植物资源，但其组成、构效关系及相关机制尚不清楚。本项目选用17个不同种质LRM，利用HPLC-MS/MS、pHPLC、NMR等技术，分离、鉴定LRM多酚，建立其化学组学库；采用指纹图谱相似度法建立LRM多酚HPLC-MS/MS指纹图谱，采用聚类和主成分分析法分析所建图谱，完整表征LRM多酚的化学特征；观察不同种质的LRM多酚提取物及单体对PC12细胞损伤作用，采用PLSR法构建LRM多酚对PC12细胞损伤的“谱-效”关系，整合“化学组学”和“谱效关系”数据，分析各多酚组分与活性的相关性，采用RT-PCR和WesternBlot等技术手段，探讨其对PC12细胞氧化损伤保护分子作用机制，阐明LRM多酚对PC12细胞凋亡抑制的功效物质基础。

黑果枸杞(LRM)是新挖掘的植物资源，富含鲜见酰化矮牵牛素花色苷等多酚｡本项目选用17个不同种质LRM，利用HPLC-MS/MS､制备色谱､核磁共振等技术，分离､鉴定LRM多酚，建立其化学组学库；采用指纹图谱相似度法建立LRM多酚HPLC-MS/MS指纹图谱；研究不同种质的LRM多酚提取物及单体对PC12细胞损伤作用及其“谱-效”关系；探讨并揭示黑果枸杞花色苷对PC12细胞氧化损伤保护分子作用机制｡.（1）采用大孔树脂-中压色谱、凝胶树脂-中压色谱分离获得了2个花色苷单体，采用UPLC-MS、1H NMR对其结构进行了鉴定，所得黑果枸杞花色苷化合物分别为矮牵牛素-3-O-芸香糖(反式-对香豆酰基)-葡萄糖苷-5-O-葡萄糖苷（Pt3G）和矮牵牛素3-O-芸香糖（反式-对香豆酰基）-5-O-葡萄糖苷。.（2）采用UPLC-MS鉴定了17个不同种质黑果枸杞的27个花色苷组分，分子量介于1095-433Da之间，整体具有分子量较大、芳香酸酰化、芸香糖和葡萄糖糖苷化，其中，最主要花色苷成分是分子量为933Da的Pt3G。并建立了UPLC指纹图谱。.（3）17种黑果枸杞多酚提取物以浓度依赖性的方式保护PC12免于氧化损伤，其中Pt3G与保护作用相关性最强，Pt3G能够显著降低H2O2诱导PC12细胞ROS产生、降低细胞凋亡，且其保护作用与降低ROS/L0X-1//NF-kB/TNF通路相关。