基于分子量分布的黄芪多糖抗炎活性组分筛选及代谢组学调控机制研究  

Screening for anti-inflammatory components of Astragalus polysaccharide and metabolomics research based on molecular weight distribution

作　　者：范信晖 李科[1,2,3,4] 杨一丹 秦雪梅 李震宇[1,2,4] 李雪琴 FAN Xin-hui;LI Ke;YANG Yi-dan;QIN Xue-mei;LI Zhen-yu;LI Xue-qin(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;Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China;Key Laboratory of Effective Substances Research and Utilization in TCM of Shanxi Province,Taiyuan 030006,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]中国科学院过程工程研究所,北京100190 [4]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

出　　处：《药学学报》2022年第3期783-792,共10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)。

摘　　要：黄芪多糖分子量分布广,常规水提醇沉制备的多糖为大分子混合物,虽有研究证明黄芪多糖具有双向免疫调节功能,然而免疫抑制或抗炎活性组分的多糖分子量分布及调控机制并不明确。因此,课题组通过水提醇沉法制备黄芪总多糖,采用凝胶色谱法对其测定分子量分布,结合超滤膜截留法分离制备不同分子量多糖组分,通过脂多糖(lipopolysaccharide, LPS)诱导小鼠单核巨噬细胞RAW 264.7过度炎症模型进行筛选,并采用LC-MS/MS代谢组学技术研究其调控机制。结果表明,黄芪多糖主要由APS-Ⅰ(大于2 000 kDa)和APS-Ⅱ(10 kDa)组成;APS-Ⅰ由Man、Rha、Gal A、Glu、Gal和Ara 6种单糖组成,各单糖组成比例约为0.54∶0.26∶12.24∶17.24∶8.46∶1;APS-Ⅱ由Rha、Gal A、Glu、Gal和Ara 5种单糖组成,各单糖组成比例约为0.26∶0.14∶24.04∶0.62∶1。APS-Ⅰ和APS-Ⅱ在0~100μg·mL^(-1)对RAW 264.7无毒性;与模型组相比, APS-Ⅰ在0~100μg·mL^(-1)内能明显抑制RAW 264.7分泌NO和TNF-α;并能明显促进IL-10的分泌,体外抗炎活性优于APS-Ⅱ。代谢组学发现,与空白组比较,模型组发现32种差异代谢物;给药组可显著回调18种差异代谢物;主要涉及精氨酸生物合成、精氨酸和脯氨酸代谢、嘧啶代谢、三羧酸循环、半胱氨酸和甲硫氨酸代谢、色氨酸代谢。本研究发现APS-Ⅰ的体外抗炎活性优于APS-Ⅱ,其机制可能与氨基酸代谢及能量代谢密切相关,以期为进一步阐明黄芪多糖药效物质基础指明方向。Molecular mass distribution of Astragalus polysaccharides is wide. Astragalus polysaccharides prepared by conventional water extraction and alcohol precipitation are mostly mixture of macromolecules.Although studies have shown that Astragalus polysaccharides have two-sided immunomodulation, the relationship between anti-inflammatory components and molecular mass distribution of Astragalus polysaccharides is not clear.Therefore, Astragalus polysaccharides were extracted by water extraction and alcohol precipitation. The relative molecular weight of them was determined by high performance gel permeation chromatography(HPGPC).Astragalus polysaccharides with different molecular weights were separated and prepared by membrane separation.RAW 264.7 cells were induced by lipopolysaccharide(LPS) to establish an inflammatory cell model in vitro and the anti-inflammatory polysaccharide were screened. The anti-inflammatory regulation mechanism of Astragalus polysaccharides was analyzed by the LC-MS/MS metabonomics technology. The results showed that APS was composed of APS-Ⅰ( > 2 000 kDa) and APS-Ⅱ(10 kDa). APS-Ⅰ was composed of mannose, rhamnose, galacturonic acid, glucose, galactose, arabinose and the molar ratios of these monosaccharide of APS-Ⅰ were 0.54∶0.26∶12.24∶17.24∶8.46∶1. APS-Ⅱ was composed of rhamnose, galacturonic acid, glucose, galactose, arabinose and the molar ratios of these monosaccharide of APS-Ⅱ were 0.26∶0.14∶24.04∶0.62∶1. APS-Ⅰ and APS-Ⅱ had no cell toxicity to RAW 264.7 macrophage in the range of 0-100 μg·mL^(-1). Compared with the model group, APS-Ⅰ at a concentration of 0-100 μg·mL^(-1)could significantly inhibit the secretion of NO and TNF-α by RAW 264.7, and can significantly promote the secretion of IL-10. APS-Ⅰ had better anti-inflammatory activity than APS-Ⅱ in vitro. The metabolomics results showed that 32 different metabolites were found between the model group and blank group;APS-Ⅰ group can significantly callback 18 different metabolites;mainly

关 键 词：黄芪多糖 黄芪多糖Ⅰ 代谢组学 机制 抗炎 

分 类 号：R282.5[医药卫生—中药学]