短梗五加多糖的提取分离与体外活性研究

Exaction, Separation and Activity of Polysaccharides from Acanthopanax sessiliflorus

目的:提取分离短梗五加多糖组分,并体外评价其生物活性。方法:水提醇沉法提取短梗五加粗多糖(ASP),离子交换色谱柱(DEAE-52)分离,苯酚-硫酸法、间羟基联苯法、考马斯亮蓝法测定其总糖、糖醛酸、蛋白质含量,高效液相色谱法(HPLC)分析单糖组成。噻唑蓝比色法(MTT)检测淋巴细胞的增殖,中性红比色法检测巨噬细胞的吞噬,硝酸还原酶法(Griess)和酶联免疫法(ELISA)检测一氧化氮(NO)释放和肿瘤坏死因子α(TNF-α)分泌,以评价其免疫调节作用;采用心肌细胞(H9c2)缺氧复氧(H/R)损伤模型,评估其对H9c2细胞H/R损伤的保护作用及机制。结果:ASP经分离得到中性多糖(ASP-Ⅰ,由甘露糖、葡萄糖、半乳糖、阿拉伯糖组成,摩尔比为1∶6.63∶2.72∶0.88)和酸性多糖(ASP-Ⅱ,由甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖组成,摩尔比为1∶2.28∶4.30∶1.18∶2.82∶2.81)。ASP-Ⅰ和ASP-Ⅱ的总糖、糖醛酸、蛋白质含量分数分别为87.61%,0,0.82%和72.33%,19.71%,0.36%。与空白对照组、刀豆蛋白(ConA)组、脂多糖(LPS)组相比,短梗五加多糖50μg/mL~200μg/mL各组可单独及协同ConA、LPS促进淋巴细胞的增殖。与空白对照组相比,短梗五加多糖50μg/mL~200μg/mL各组可促进淋巴细胞的增殖、增强巨噬细胞的吞噬,促进NO的释放和TNF-α的分泌。与模型对照组相比,短梗五加多糖50μg/mL~100μg/mL各组可抑制H9c2细胞H/R损伤模型中乳酸脱氢酶(LDH)、肌酸磷酸激酶(CK)活力、丙二醛(MDA)含量、半胱氨酸蛋白酶3(Caspase 3)蛋白表达的升高,促进超氧化物歧化酶(SOD)活力升高;上调微小RNA 451(micro RNA 451)表达,下调高迁移率族蛋白B1(HMGB1)表达。结论:短梗五加多糖具有免疫调节及保护H9c2细胞H/R损伤的作用,H9c2细胞H/R损伤与micro RNA 451/HMBG1表达有关。

Objective:To extract and separate the polysaccharides from Acanthopanax sessiliflorus(ASP)and analyze its activity in vitro.Method:Crude ASP was obtained by water extraction and alcohol precipitation,and different components were separated by ion exchange column chromatography(DEAE-52).Phenol-sulfuric acid method,m-hydroxybiphenyl method and Coomassie brilliant blue method were used to determine the content of total sugar,uronic acid and protein,respectively.The monosaccharide composition was analyzed by high performance liquid chromatography(HPLC).The lymphocyte proliferation,macrophage phagocytosis,nitric oxide(NO)release and tumor necrosis factor(TNF)-αsecretion were detected by MTT assay,neutral red assay,Griess and ELISA,respectively,which can characterize the immunomodulatory effect of ASP.The protective effect and mechanism of ASP against hypoxia/reoxygenation(H/R)injury in H9c2 cells were explored.Result:Neutral polysaccharide(ASP-I,composed of mannose,glucose,galactose and arabinose at a molar ratio of 1:6.63:2.72:0.88)and acidic polysaccharide(ASP-II,composed of mannose,rhamnose,galacturonic acid,glucose,galactose and arabinose at a molar ratio of 1:2.28:4.30:1.18:2.82:2.81)were fractionated from ASP.The content of total sugar,uronic acid and protein was 87.61%,0 and 0.82%in ASP-I and 72.33%,19.71%and 0.36%in ASP-II,respectively.Compared with blank control group,ConA group and LPS group,ASP(50μg/mL~200μg/mL)could promote the proliferation of lymphocytes alone or in conjunction with ConA and LPS.Compared with the blank control group,ASP(50μg/mL~200μg/mL)promoted the proliferation of lymphocytes,enhanced the phagocytosis of macrophages,and increased the release of NO and the secretion of TNF-α.Compared with the model control group,ASP(50μg/mL~100μg/mL)inhibited the increases in lactate dehydrogenase(LDH),creatine phosphokinase(CK),malondialdehyde(MDA)and cysteine protease 3(caspase-3)levels and promoted the increase in superoxide dismutase(SOD)level in the H/R injured H9c2 cells.Furthermore,ASP up-regulated the expression of microRNA-451 and down-regulated the expression of high mobility group protein B1(HMGB1).Conclusion:ASP has immunomodulatory and protective effects on H/R injury of H9c2 cells by regulating the expression of microRNA 451/HMBG1.