响应面法优化北豆根粗多糖的除蛋白工艺及其神经保护活性研究
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摘要
为研究北豆根粗多糖的最佳除蛋白方法和工艺,探究北豆根粗多糖的神经保护活性。实验比较了酶法、Sevage法、酶-Sevage法、三氯乙酸(TCA)-正丁醇法、酶-TCA-正丁醇法除蛋白效果,筛选出了北豆根粗多糖的最佳除蛋白方法,并用响应面法对该方法进行优化。经响应面分析,以综合评分为指标,考查TCA与正丁醇体积比、北豆根粗多糖溶液与TCA-正丁醇溶液体积比和振摇时间对北豆根粗多糖TCA-正丁醇法除蛋白工艺的影响。用H_2O_2诱导PC12细胞损伤,探究了北豆根粗多糖的神经保护作用。结果表明,北豆根粗多糖的最佳除蛋白方法为TCA-正丁醇法。经响应面优化,确定最优除蛋白工艺为TCA:正丁醇=1∶10.4,北豆根粗多糖溶液:TCA-正丁醇溶液=1∶1.77,振摇时间为36.5 min。采用该工艺对北豆根多糖进行除蛋白,其蛋白清除率为73.1%,综合评分为92.1。神经保护研究结果表明,北豆根粗多糖对H_2O_2诱导的PC12细胞损伤具有明显的保护作用。本研究表明TCA-正丁醇法可以有效的除去北豆根粗多糖中的蛋白质且北豆根粗多糖具有神经保护活性。
To optimize the deproteinization process and to determine the neuroprotective activity of Rhizoma Menispermi crude polysaccharide,five methods were compared to screen out the best protein removing method for the Rhizoma Menispermi crude polysaccharide,including enzymatic method,Sevage method,enzyme-Sevage method,TCA-n-butanol method and enzyme-TCA-n-butanol method.The effects of the volume ratio of TCA to n-butanol 、the ratio of the polysaccharide solution to the TCA-n-butanol solution and the shake time on the deproteinization of Rhizome Menispermi crude polysaccharide in TCA-n-butanol method were investigated by single-factor experiment combined with central combination design method of Box-Behnken.PC12 cell injury was induced by H_2O_2 to study the neuroprotective activity of Rhizoma Menispermi crude polysaccharides.The results showed that the best method for deproteinization process of Rhizoma Menispermi crude polysaccharide was TCA-n-butanol method.The optimal conditions were as follows∶the ratio of TCA to n-butanol was 1∶10.4,the ratio of solution of Rhizoma Menispermi crude polysaccharide to TCA-n-butanol solution was 1∶1.77,and the shaking time was 36.5 min.Under this condition the remove rate of protein was 73.1 %,the comprehensive score was 92.1.Neuroprotective studies have showed that Rhizoma Menispermi crude polysaccharide has obvious protective effect on H_2O_2-induced PC12 cell injury.This study showed that the TCA-n-butanol method can effectively remove the protein in the Rhizoma Menispermi crude polysaccharides,and the Rhizoma Menispermi crude polysaccharides had neuroprotective activity.
To optimize the deproteinization process and to determine the neuroprotective activity of Rhizoma Menispermi crude polysaccharide,five methods were compared to screen out the best protein removing method for the Rhizoma Menispermi crude polysaccharide,including enzymatic method,Sevage method,enzyme-Sevage method,TCA-n-butanol method and enzyme-TCA-n-butanol method.The effects of the volume ratio of TCA to n-butanol 、the ratio of the polysaccharide solution to the TCA-n-butanol solution and the shake time on the deproteinization of Rhizome Menispermi crude polysaccharide in TCA-n-butanol method were investigated by single-factor experiment combined with central combination design method of Box-Behnken.PC12 cell injury was induced by H_2O_2 to study the neuroprotective activity of Rhizoma Menispermi crude polysaccharides.The results showed that the best method for deproteinization process of Rhizoma Menispermi crude polysaccharide was TCA-n-butanol method.The optimal conditions were as follows∶the ratio of TCA to n-butanol was 1∶10.4,the ratio of solution of Rhizoma Menispermi crude polysaccharide to TCA-n-butanol solution was 1∶1.77,and the shaking time was 36.5 min.Under this condition the remove rate of protein was 73.1 %,the comprehensive score was 92.1.Neuroprotective studies have showed that Rhizoma Menispermi crude polysaccharide has obvious protective effect on H_2O_2-induced PC12 cell injury.This study showed that the TCA-n-butanol method can effectively remove the protein in the Rhizoma Menispermi crude polysaccharides,and the Rhizoma Menispermi crude polysaccharides had neuroprotective activity.
引文
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2 Zhang Y,Peng YB,Chen XZ,et al.Bisbenzylisoquinoline alkaloids from the rhizome of Menispermum Dauricum[J].Mod Chin Med(中国现代中药),2016,18:951-955.
3 Li SS,Song X,Chai X,et al.Chemical constituent from the Rhizome of Menispermum Dauricum DC.[J].Nat Prod Res Dev(天然产物研究与开发),2013,25(1):60-63.
4 Gu FF,Li J,Yang CD,et al.Fractionation,structural characterization,and immunomodulatory activity of polysaccharide isolated from Ganoderma lucidum[J].Chin Tradit Herbal Drugs(中草药),2018,49:2359-2364.
5 Sun YL,Lvu SW,Wu JL,et al.Optimization of polysaccharide extraction from Bai-hu decoction and its hypoglycemic activity[J].Centr South Pharm(中南药学),2018,16:495-499.
6 Pan XP,Xing ZL,Jia WG,et al.Identification and preparation of a glycol-protein fraction AN from tea extracts and its in vivo and in vitro anti-hyperglycosemia activity[J].Chin J Chin Mater Med(中国中药杂志),2018,43:736-742.
7 Cheng XX,Zhang XH,Yang HW,et al.Isolation,purification and anti-oxidant activity of polysaccharides from Acanthopanax trifoliatus[J].Chin Tradit Herb Drugs(中草药),2017,48:4219-4223.
8 Yu LL,Huang SY.Study on antioxidation of polysaccharides from Lingyun,Guangxi wild Dendrobium officinale[J].Lishizhen Med Mater Med Res(时珍国医国药),2014,25:2842-2845.
9 Wan Q,Xiao W,Wang ZZ,et al.Study on the process of removing protein from Ligustrum lucidum polysaccharide[J].Chin Tradit Herb Drugs(中草药),2010,41:407-410.
10 Fang SP,Wang WX,Luo XL.Study on the method of removing protein from Chuanxiong polysaccharide[J].Lishizhen Med Mater Med Res(时珍国医国药),2009,20:2176-2177.
11 Hu HG,Zhao QL,Pang ZC,et al.Investigation of deproteinzation and decoloration technology for polysaccharide from mango pomace[J].Sci Tech Food Ind(食品工业科技),2018,39:183-188.
12 Liu Q,Song S,Guo J,et al.Protective effects of polysaccharide from seed watermelon on the oxidative damage of PC12 cells induced by H2O2[J].Nat Prod Res Dev(天然产物研究与开发),2015,27:338-343.
13 Wang FX,Liu Q,Wang W,et al.A polysaccharide isolated from Cynomorium songaricum Rupr.protects PC12 cells against H 2 O 2-induced injury[J].Int J Biol Macromol,2016,87:222-228.
14 Yuan F,Zhang JP,Ma L.Protective effects of polysaccharide from Vitis Vinifera L Aβ25-35 induced injury in PC12 cells[J].Sci & Tec Rev(科技导报),2012,30(4):47-50.
15 Gu CY,Chen QL,Li HT.Protective effects and mechanism of polysaccharide from platycodon grandiflorum on damage of PC12 cells induced by H2O2[J].J Nanjing Univ Tradit Chin Med(南京中医药大学学报),2017,33:268-272.
16 Shi DH,Liu WW,Liu YJ,et al.The protective effects of polysaccharides from Enteromorp haprolifera on the PC12 cells injury induced by H2O2 [J].Chin J Biochem Pharm(中国生化药物杂志),2012,33(1):40-42.