红芪多糖“脱蛋白-大孔吸附树脂”的纯化富集工艺考察  

Study on purification and enrichment technology of hedysarum polysaccharide"deproteinization-macroporous adsorption resin"

作　　者：赵沙沙 何海 张小荣 郭玫[1,2,3,4] 崔治家 邵晶[1,2,3,4] Sha-Sha ZHAO;Hai HE;Xiao-Rong ZHANG;Mei GUO;Zhi-Jia CUI;Jing SHAO(Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Northwest Collaborative Innovation Center for Traditional Chinese Medicine,Lanzhou 730000,China;Gansu Province Key Laboratory of Pharmacology and Toxicology of Traditional Chinese Medicine,Lanzhou 730000,China;Research Center of Traditional Chinese Medicine Pharmaceutical Technology and Engineering of Gansu Province,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000 [2]西北中藏药协同创新中心,兰州730000 [3]甘肃省中药药理与毒理学重点实验室,兰州730000 [4]甘肃省中药制药工艺工程研究中心,兰州730000

出　　处：《药物流行病学杂志》2023年第6期679-688,共10页Chinese Journal of Pharmacoepidemiology

基　　金：甘肃省科技计划自然科学基金项目(17JR5RA163、21JR11RA136);兰州市城关区科技计划项目(2020-2-2-2);甘肃省教育厅双一流重大科研项目(GSSYLXM-05);甘肃省中药制药工艺工程研究中心开放课题(ZYGY202004);中医药公共卫生服务补助专项子课题(2305191901)。

摘　　要：目的研究红芪多糖(HPS)标准提取物,筛选优化其“脱蛋白-大孔吸附树脂纯化富集”的工艺方法与工艺参数。方法以蛋白脱除率和多糖损失率为指标,比较4种不同方法的脱蛋白效果,并对筛选出的方法进行工艺参数优化;以吸附量和吸附率为指标,考察4种型号大孔吸附树脂(AB-8、D-101、NKA-9、X-5)对HPS的纯化富集效果,并进行相关工艺参数优化;采用苯酚硫酸法和考马斯亮兰法分别测定多糖和蛋白质含量;采用紫外分光光度(UV)和红外光谱(IR)法比较纯化前后多糖的基本特征。结∶果筛选出的脱蛋白方法为Sevag法,优选的树脂为AB-8大孔吸附树脂。通过工艺参数优选,确定的纯化工艺为:10 mg·mL^(-1)粗多糖水溶液,以1/2倍体积氯仿^(-1),∶v/v)混合试剂进行脱蛋白操作2次;除去残留有机溶剂后,以质量浓度为1.5 mg·mL^(-1)的水溶液,上预处理好的AB-8大孔吸附树脂柱[树脂量(g)与上样体10]∶,以2.0 mL·min^(-1)流速循环上样2次,动态吸附后再静态吸附6~8 h,后以30%乙醇1.0 mL·min^(-1)进行洗脱,至洗脱液无明显碱性酒石酸铜反应,收集洗脱液浓缩至与原药1冲洗沉淀,即得HPS。结论验证试验表明,经本工艺纯化富集后,所得HPS样品纯度提高了1.27倍;UV在260,280 nm处未见蛋白质吸收峰;IR表现出更突出的多糖结构特征吸收峰,说明纯化过程有效且未对HPS主要结构产生影响。优选出的HPS脱蛋白和大孔树脂纯化工艺稳定可行,重复性较好,使多糖得到较好的纯化富集,为HPS标准提取物的形成以及其能成为制剂科学化原料药提供了研究基础。Objective To study the standard extract of hedysarum polysaccharide(HPS),and to screen and optimize its"deproteinization-macroporous adsorption resin purification and enrichment"process method and process parameters.Methods Taking the protein removal rate and polysaccharide loss rate as indexes,the deproteinization effects of four different methods were compared,and the process parameters of the selected methods were optimized.Taking adsorption capacity and adsorption rate as indicators,the purification and enrichment effects of four types of macroporous adsorption resins(AB-8,D-101,NKA-9,X-5)on HPS were investigated,and the related process parameters were optimized.Phenol sulfuric acid method and Coomassie brilliant blue method were used to determine the content of polysaccharide and protein.The basic characteristics of polysaccharides before and after purification were compared by UV and IR methods.Results Sevag method was selected for deproteinization,and AB-8 macroporous adsorption resin was the preferred resin.Through the optimization of process parameters,the purification process was determined as follows:10 mg·mL^(-1) crude polysaccharide aqueous solution,deproteinization twice with 1/2 volume of chloroform-n-butanol(4∶1,v/v)mixed reagent;after the residual organic solvent was removed,the pretreated AB-8 macroporous adsorption resin column was loaded with 1.5 mg·mL^(-1) aqueous solution[the ratio of resin amount(g)to loading volume(v)was 1∶10],and the sample was loaded twice at a flow rate of 2.0 mL·min^(-1).After dynamic adsorption,static adsorption was carried out for 6-8 h,and then elution was carried out with 1.0 mL·min^(-1) of 30%ethanol until the eluate had no obvious basic copper tartrate reaction,the eluate was collected and concentrated to 1∶1 with the original drug,anhydrous ethanol was added to 80%alcohol content,the precipitate was precipitated for 10 h,then filtrated it and the precipitate was rinsed with anhydrous ethanol and acetone and the HPS was extracted.Conclusion The verifica

关 键 词：红芪多糖 脱蛋白 大孔吸附树脂 纯化工艺 

分 类 号：R283[医药卫生—中药学]