基于高分辨质谱技术的黄芪多糖APS-Ⅱ酶解活性寡糖特征分支位点的研究  

Study on the characteristic branch sites of oligosaccharides of Astragalus polysaccharide APS-Ⅱenzymolysis based on high resolution mass spectrometry

作　　者：刘宇冲 李虎峰 李科[1,2,3,4] 秦雪梅 杜昱光[4] 李震宇 LIU Yu-chong;LI Hu-feng;LI Ke;QIN Xue-mei;DU Yu-guang;LI Zhen-yu(Modern Research Center for Traditional Chinese Medicine of Shanxi University,Taiyuan 030006,China;Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education of Shanxi University,Taiyuan 030006,China;Key Laboratory of Effective Substances Research and Utilization in TCM of Shanxi Province,Taiyuan 030006,China;Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]山西大学中医药现代研究中心,山西太原030006 [2]山西大学化学,生物学与分子工程教育部重点实验室,山西太原030006 [3]地产中药功效物质研究与利用山西省重点实验室,山西太原030006 [4]中国科学院过程工程研究所,北京100190

出　　处：《药学学报》2024年第7期2108-2116,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81872962);国家博士后科学基金资助项目(2019M650851);国家重点研发计划(2019YFC1710800);山西省重点研发计划重点项目(201603D311101);山西省优秀人才科技创新项目(201605D211030,201705D211020);山西省科技创新人才团队专项基金。

摘　　要：黄芪多糖是黄芪中免疫调节活性最强,含量最为丰富的物质,具有抗肿瘤、抗病毒、免疫促进等多种生物活性,临床应用广泛。前期研究发现黄芪多糖主要由2种不同相对分子质量多糖APS-Ⅰ(>2000 kDa)、APS-Ⅱ(10 kDa)组成,APS-Ⅱ(10 kDa)为黄芪多糖中活性最强组分,并采用α-1,4-葡聚糖内切酶将APS-Ⅱ降解为寡糖,通过体外免疫活性筛选发现2~9糖整体免疫活性较低,而10~14糖具有较强的免疫活性,且活性优于未降解的APS-Ⅱ。为了探究APS-Ⅱ降解寡糖发挥免疫活性的关键结构,本文采用MALDI-TOF-MS生物质谱以及高分辨质谱仪器ESIQ Exactive-MS对APS酶解寡糖进行解析,通过对比发现10~14糖中存在1→4和1→6两种连接方式共存的支链结构,推测1→4和1→6两种连接方式共存的支链结构为APS-Ⅱ发挥免疫活性的关键结构,为黄芪多糖和寡糖的构效关系研究奠定理论基础。Astragalus polysaccharides are the most immunoregulatory active and abundant substances in Astragalus,with anti-tumor,anti-viral,and immune-promoting biological activities.They have been widely used in clinical practice.Previous studies have found that Astragalus polysaccharides are mainly composed of two different polysaccharides,APS-I(>2000 kDa)and APS-Ⅱ(10 kDa),with APS-Ⅱ(10 kDa)being the most active component of Astragalus polysaccharides.We usedα-1,4-glucan endo-hydrolysis enzyme to degrade APS-Ⅱinto oligosaccharides,and screened the immune activity of oligosaccharides in vitro.We found that the overall immune activity of 2−9 oligosaccharides was low,while the immune activity of 10−14 oligosaccharides was strong,and the activity was better than that of untreated APS-Ⅱ.To investigate the key structural features of APS-Ⅱoligosaccharides that play a role in immune activity,we used MALDI-TOF-MS biochemical mass spectrometry and high-resolution mass spectrometry instrument ESI-Q Exactive-MS to analyze the APS enzymatic oligosaccharides.By comparing,we found that 10−14 oligosaccharides contain 1→4 and 1→6 branched structures with coexisting linkages,suggesting that 1→4 and 1→6 linkages in branched structures are key structural features that play a role in the immune activity of APS-Ⅱ,laying a theoretical foundation for the structure-activity relationship of Astragalus polysaccharides and oligosaccharides.

关 键 词：APS-Ⅱ 酶解 连接方式 结构解析 高分辨质谱 

分 类 号：R917[医药卫生—药物分析学]