黄芪多糖中抗炎组分的结构及其活性的初步研究  

Preliminary study on structure and activity of anti-inflammatory components in Astragalus polysaccharides

作　　者：范信晖 李科[1,2,3] 杨一丹 秦雪梅 李震宇[1,2] 李雪琴[1,2] FAN Xinhui;LI Ke;YANG Yidan;QIN Xuemei;LI Zhenyu;LI Xueqin(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;Institute of Process Engineering,Chinese Academy of Sciences)

机构地区：[1]山西大学中医药现代研究中心,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 [3]中国科学院过程工程研究所

出　　处：《山西医科大学学报》2021年第10期1346-1356,共11页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(81872962);国家博士后科学基金项目(2019M650851);国家重点研发计划项目(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：目的从黄芪中分离得到两种多糖,分别对两种多糖的化学结构和抗炎活性进行分析。方法采用水提醇沉法提取黄芪总多糖(Astragalus polysaccharides,APS),膜分离法得到两种多糖APS-Ⅰ和APS-Ⅱ。高效凝胶色谱法(HPGPC)测定APS-Ⅰ和APS-Ⅱ的分子量,高效液相色谱法(HPLC-UV)测定两者的单糖组成,通过红外(IR)及甲基化分析对APS-Ⅰ和APS-Ⅱ的结构进行表征。将巨噬细胞株RAW264.7培养于含不同浓度APS、APS-Ⅰ和APS-Ⅱ的培养基,MTT法筛选合适的实验浓度,采用1μg/ml LPS诱导巨噬细胞株RAW264.7建立细胞炎症模型,应用不同浓度APS、APS-Ⅰ和APS-Ⅱ处理被LPS诱导活化的巨噬细胞株RAW264.7,利用ELISA试剂盒测定各组细胞培养上清液一氧化碳(NO)、白细胞介素-10(IL-10)和肿瘤坏死因子(TNF-α)含量。结果APS由APS-Ⅰ(大于2000 kD)和APS-Ⅱ(10 kD)组成;两者均由鼠李糖(Rha)、葡萄糖(Glu)、半乳糖(Gal)、阿拉伯糖(Ara)和半乳糖醛酸(GalA)5种单糖组成,APS-Ⅰ中Rha,Gal A,Glu,Gal,Ara的比例为0.1∶0.39∶17.2∶13.4∶1;在APS-Ⅱ中为0.14∶0.14∶24.04∶9.6∶1;红外光谱显示APS-Ⅰ以β型糖苷键为主;APS-Ⅱ同时包含α型糖苷键和β型糖苷键;甲基化分析显示两者单糖残基的主要连接方式不同,分别为:→4)-D-Glu-(1→,→6)-D-Gal-(1→,→4)-L-Ara-(1→和→4)-D-Glu-(1→,→3,5)-D-Glu-(1→,→3,4)-D-Gal-(1→。MTT实验表明APS、APS-Ⅰ和APS-Ⅱ在0-100μg/ml质量浓度范围内无细胞毒性。与空白对照相比,1μg/ml LPS可显著上调巨噬细胞株RAW264.7中NO、TNF-α和IL-10的表达(P<0.05)。应用APS、APS-Ⅰ和APS-Ⅱ干预,可抑制促炎因子NO和TNF-α表达上调,促进抑炎因子IL-10表达上调(P<0.05),体外抗炎活性顺序为APS-Ⅰ>APS>APS-Ⅱ。结论APS-Ⅰ的体外抗炎活性优于APS-Ⅱ,可作为潜在的抗炎药物进行开发。Objective To extract and purify two polysaccharides from the Astragalus root,and investigate their structural characteri-stics and anti-inflammatory activity.Methods APS was extracted by water extraction and alcohol precipitation.APS-Ⅰand APS-Ⅱwere obtained by membrane separation.The relative molecular weight was determined by high performance gel permeation chromatography(HPGPC).Monosaccharide composition was determined by high performance liquid chromatography(HPLC-UV).The structures of APS-Ⅰand APS-Ⅱwere characterized by infrared analysis and methylation analysis.RAW264.7 macrophage cell line was cultured in medium with different concentrations of APS,APS-Ⅰand APS-Ⅱ,and then the appropriate concentration was screened by MTT assay.The RAW264.7 cell inflammation model was constructed by treatment with 1μg/ml lipopolysaccharide(LPS),and APS,APS-Ⅰand APS-Ⅱwere used to treat macrophage cell line RAW264.7 activated by LPS.The expression levels of inflammatory cytokines NO,interleukin-10(IL-10)and tumor necrosis factor(TNF-α)in cell culture supernatants were measured by enzyme linked immunosorbent assay.Results APS was composed of APS-Ⅰ(>2000 kD)and APS-Ⅱ(10 kD).Both of APS-Ⅰand APS-Ⅱwere composed of rhamnose,glucose,galactose,arabinose and galacturonic acid.The molar ratio of rhamnose,glucose,galactose,arabinose and galacturonic acid was 0.1∶0.39∶17.2∶13.4∶1 for APS-Ⅰand 0.14∶0.14∶24.04∶9.6∶1 for APS-Ⅱ.The results of infrared spectrum analysis showed that APS-Ⅰcontained mainlyβ-glycosidic bonds,and that APS-Ⅱcontainedβ-glycosidic bonds andα-glucosidic bonds.Methylation analysis showed that APS-Ⅰand APS-Ⅱhad different glycosidic linkages.The glycosidic linkages of APS-Ⅰand APS-Ⅱwere→4)-D-Glu-(1→,→6)-D-Gal-(1→,→4)-L-Ara-(1→and→4)-D-Glu-(1→,→3,5)-D-Glu-(1→,→3,4)-D-Gal-(1→,respectively.Astragalus polysaccharides had no cytotoxicity in the range of 0-100μg/ml.Compared with blank control,1μg/ml lipopolysaccharide significantly increased

关 键 词：黄芪多糖 分离纯化 结构表征 APS-Ⅰ APS-Ⅱ 抗炎活性 

分 类 号：R282.5[医药卫生—中药学]