鹿茸蛋白模拟胃肠消化物中活性成分肠吸收及免疫调节机理研究

Velvet antler, considered as important as ginseng, is believed to have body-strengthening, immunomodulatory and anti-aging effects. It has been used in Chinese commercial functional foods and nutraceuticals. According to the published literatures, there is a lot of weakness of the study on velvet antler. On one hand, functional study of velvet antler is limited to its crude extract for a long time and characteristic functional components are not clearly understood. On the other hand, protein is considered the major component of velvet antler with a content of more than 50% (w/w), but it has been underestimated in the literature. Proteins are broken down into small peptides during gastrointestinal digestion. To the best of our knowledge, systematic studies on identification of bioactive components in velvet antler protein digests, and clarification of their intestinal absorption mechanism are also lacking. Nowadays, the sub-health state is a great concern for people. A strong immune system could help people get rid of sub-health conditions. Therefore, proteins from velvet antler are selected as our research objects. First, simulated gastrointestinal digest of velvet antler proteins (SGD-VAP) will be prepared by two-step proteases hydrolysis. Second, the active components will be consecutively fractionated by molecular exclusion chromatography, ion exchange chromatography and HPLC. Then, the immunomodulatory effect of SGD-VAP and its fractions will be investigated. The main active components will be identified by spectroscopic and mass-spectrometric technique. Finally, intestinal absorption and immunomodulatory mechanism of the main active components will be intensively studied. This study can provide theoretical basis for the development of functional food of velvet antler proteins with immunomodulatory activities, which helps to improve physical fitness of sub-health population.

鹿茸与人参齐名，具有强壮机体、免疫调节和抗衰老等作用，常用于功能性食品和保健品。现有研究尚存在很多不足。一方面，长期以来对鹿茸功能活性的研究还局限于粗提取物，其功能成分尚未被系统揭示。另一方面，对鹿茸中含量占50%以上的蛋白质研究缺乏，其经胃肠消化后降解成何种小分子肽从而发挥其功能活性，肠吸收情况和转运方式等也未见报道。目前，亚健康已引起人们的关注，通过免疫调节改善自身免疫状况，是人们远离亚健康的最理想方法。本研究主要针对鹿茸蛋白，首先采用体外二步酶解法制备鹿茸蛋白模拟胃肠消化物。随后，依次采用凝胶层析、离子交换层析、HPLC等分离技术对模拟胃肠消化物进行分离，并检测其免疫调节活性。应用光谱、质谱等技术对主要活性成分进行鉴定。最终，对主要活性成分的肠吸收情况、转运机制及免疫调节机理进行深入研究。为开发具有免疫调节活性的鹿茸蛋白功能性食品，从而改善亚健康人群的身体状况，提供坚实的理论基础。

鹿茸是我国传统中药，与人参齐名，具有强壮机体、免疫调节和抗衰老等作用，常用于功能性食品和保健品。本项目以改善自身免疫情况并远离亚健康为目标，对占鹿茸含量50%以上的蛋白质经模拟胃肠消化后的免疫调节活性和肠吸收机制进行研究。研究选用100mmol/L Tris＋6mol/L盐酸胍＋0.02mol/L EDTA-2Na＋1%蛋白酶抑制剂为蛋白提取液，鹿茸蛋白得率为34.2%（以干鹿茸计）。在模拟胃肠消化过程中，模拟胃消化时间对鹿茸蛋白消化的影响不显著，模拟小肠消化时间以5 h为宜。模拟胃肠消化物的分子量主要集中在7,800 Da以下。采用超滤法从鹿茸蛋白模拟胃肠消化物中获得小分子量超滤组分（＜3kDa），在浓度为200μg/mL时可显著促进脾细胞增殖；在浓度为0.5~200μg/mL范围内，对ConA和LPS诱导的小鼠脾淋巴细胞增殖有显著促进作用。另外，小分子量超滤组分（＜3kDa）对LPS诱导的炎症巨噬细胞模型NO生成量有显著抑制作用。体内实验表明，小分子量超滤组分（＜3kDa）对二甲苯致小鼠耳肿胀、角叉菜胶致小鼠足肿胀的均有显著的抑制作用，其可能是通过提高机体抗氧化酶促体系的酶活，使机体清除过多的活性氧，从而达到减轻炎症的目的。依次采用大孔吸附树脂、凝胶层析、HPLC对小分子量超滤组分（＜3kDa）进行分离制备，获得组分H3对炎症巨噬细胞NO生成量抑制作用最强，在浓度为500μg/mL时抑制率高达51.6%。组分H3还能够抑制LPS诱导的炎症巨噬细胞中TNF-α、IL-6和IL-1β的释放。采用LC-Q-TOF-MS/MS分析组分H3，鉴定得出其主要成分是：AHG，AHWK，VH，Y，LAN，IA，AY，AL和TL。上述8种多肽在组分H3发挥抗炎活性时存在交互作用，其中AL和IA之间具有较好的协同抗炎作用。采用Caco-2单层细胞为小肠内皮细胞转运模型，对小分子量超滤组分（＜3kDa）的肠吸收情况进行研究，发现随着转运时间的延长和样品浓度的增加，透过物中肽氮的含量先增加随后趋于稳定。小分子量超滤组分（＜3kDa）主要通过PepT1载体运输和旁路转运两种方式被吸收进入肠细胞。至项目结题，已发表研究论文12篇，其中SCI收录3篇，EI收录2篇，CSCD收录6篇，申请国家发明专利1项，基本完成了项目计划的主要内容。