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

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.