目的 富集水牛角Bubali Cornu中含巯基肽类成分(thiols-rich peptide fractions,SHPF)并探讨其抗氧化、抗炎活性。方法 通过巯基亲和树脂Thiopropyl Sepharose 6B从水牛角提取液中富集得到SHPF,并采用Ellman法测定游离巯基(-SH)含量;应用单因素实验法优选富集工艺;基于Nano LC-MS/MS技术鉴定SHPF。体外通过H₂O₂诱导小鼠脑微血管内皮细胞bEnd3氧化损伤,采用细胞增殖检测试剂盒(CCK-8)和乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒检测SHPF抗氧化活性;通过脂多糖(lipopolysaccharide,LPS)诱导小鼠巨噬细胞RAW264.7炎症损伤,采用实时荧光定量PCR法测定炎症因子白细胞介素-6(interleukin-6,IL-6)、IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的分泌。结果 优化SHPF富集工艺为上样量10 μmol游离-SH、吸附时间1 h、洗脱剂为含β-巯基乙醇(β-mercaptoethanol,β-Me)洗脱剂。从水牛角富集液中共鉴定到704条肽段,其中154条含半胱氨酸(cysteine,Cys)肽段,占总肽段的21.9%,主要来源于角蛋白和角蛋白相关蛋白。体外细胞实验结果表明,SHPF能够提高氧化损伤bEnd3细胞的存活率,同时降低其LDH的泄露,并减少炎症损伤RAW264.7细胞炎症因子IL-6、IL-1β、TNF-α的分泌。结论 采用优选富集工艺能明显提高SHPF的富集效率,SHPF具有良好抗氧化和抗炎活性,为水牛角"清热解毒"功效物质基础的研究提供了研究思路和方向。

Objective To enrich the thiols-rich peptide fractions (SHPF) from Shuiniujiao (Bubali Cornu, water buffalo horn) and investigate its antioxidant and anti-inflammatory activities. Methods SHPF was enriched from Bubali Cornu extract by Thiopropyl Sepharose 6B, and the content of free -SH was determined by Ellman method. The enrichment process was optimized by single factor experiment. SHPF was identified based on Nano LC-MS/MS technology. Oxidative damage of bEnd3 cells were induced by H₂O₂ in vitro, and cell proliferation assay kit (CCK-8) and lactate dehydrogenase (LDH) kit were used to determine the antioxidant activity of SHPF. The secretions of cytokines interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) were determined by qPCR after LPS induced inflammatory damage of RAW264.7. ResultsThe optimized SHPF enrichment process was as follows:loading volume of 10 μmol free -SH, adsorption time of 1 h, and eluent containing β-mercaptoethanol (β-Me). A total of 704 peptides were identified from the Bubali Cornu enrichment solution, among which 154 thiols-contained peptides, accounting for 21.9% of the total peptides, mainly derived from keratin and keratin-related proteins. SHPF can improve the survival rate of oxidatively damaged bEnd3 cells, reduce the leakage of LDH, and reduce the secretion of inflammatory factors IL-6, IL-1β and TNF-α in RAW264.7 cells. Conclusion The optimized enrichment process can significantly improve the enrichment efficiency of SHPF, and SHPF has good antioxidant and anti-inflammatory activities, which provides a research idea and direction for the study of the material basis of Bubali Cornu efficacy.

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国家自然科学基金面上项目（81973450）；国家重点研发计划资助（2018YFC1706100）；江苏省高校“青蓝工程”中青年学术带头人；江苏省农业科技自主创新项目［CX(22)3173］；南京中医药大学中药学一流学科开放课题（2020YLXK009）