雪灵芝多糖的分离纯化及免疫活性评价
|
摘要
为分离纯化雪灵芝(Arenaria kansuensis)多糖,并对纯化组分进行分子量测定、单糖组分分析及免疫活性评价。实验采用水提醇沉法提取雪灵芝粗多糖(Arenaria kansuensis crude polysaccharide, AKCP);以DEAE-52纤维素柱对AKCP进行分离纯化,获得5个雪灵芝多糖组分AKP-1～AKP-5,进一步采用葡聚糖凝胶G-75柱对AKP-2进行分离纯化获得AKP-2a多糖组分。苯酚-硫酸法测定AKCP、AKP-2及AKP-2a的总糖含量分别为52%、70%和79%;凝胶渗透色谱-十八角度激光光散射(GPC-MALS)法检测AKP-2a的重均分子量Mw为2.07×10~5Da、数均分子量Mn为9.838×10~4Da;HPLC法检测AKP-2a是由半乳糖醛酸、甘露糖、核糖、鼠李糖、葡萄糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖、岩藻糖10种单糖组成,其摩尔比为1∶0.25∶0.01∶0.20∶0.11∶0.25∶0.61∶0.07∶0.21∶0.12;以MTT法检测体外培养小鼠脾淋巴细胞增殖,AKCP、AKP-2及AKP-2a各浓度组SI水平,均明显高于对照组(P<0.05)。经NO释放实验及IFN-γELISA检测,AKP-2a各浓度组小鼠腹腔巨噬细胞培养上清中二者的水平,较对照组呈浓度依赖性增高(P<0.01)。综上结果,本研究通过分离纯化,获得了总糖含量较高的雪灵芝多糖AKP-2a组分,初步确定其分子量范围及单糖组成,并证实其具有激活淋巴细胞增殖、促进巨噬细胞功能的生物活性。
In order to isolate and purify Arenaria kansuensis polysaccharide from natural Arenaria kansuensis,and study its molecular weight,monosaccharide component and immune activity,Arenaria kansuensis crude polysaccharide(AKCP) was extracted by water extraction and alcohol precipitation,and separated and purified into AKP-1-AKP-5 by DEAE cellulose 52 column.AKP-2 was further purified using Sephadex G-75 column and AKP-2 a polysaccharide component was obtained.The total sugar content of AKCP,AKP-2 and AKP-2 a was measured as 52%,70% and 79% respectively by phenol-sulfuric acid method.The GPC-MALS was applied to measure the molecular weight of AKP-2 a.Results showed that the weight-average molecular weight(Mw) of AKP-2 a was 2.07× 10~(5 )Da,and the number-average molecular weight(Mn) was 9.838× 10~4 Da.The 10 main monosaccharides in AKP-2 a detected by HPLC are galacturonic acid,mannose,ribose,rhamnose,glucuronic acid,glucose,galactose,xylose,arabinose and fucose,the molar ratio of which was 1∶0.25∶0.01∶0.20∶0.11∶0.25∶0.61∶0.07∶0.21∶0.12 respectively.Compared with control group,the AKCP,AKP-2 and AKP-2 a treatment groups with high,medium and low concentrations had a promoting effect on the proliferation of mice spleen lymphocytes by MTT in vitro(P<0.05).By NO release experiment and ELISA detection,AKP-2 a showed a concentration dependent activation effect on mice peritoneal macrophages in vitro(P<0.01). In conclusion,this study obtained AKP-2 a polysaccharide through isolation and purification of Arenaria kansuensis,which has a high total sugar content.The multiple monosaccharides and the molecular weight of AKP-2 a were identified,and the preliminary results showed that AKP-2 a had the biological activities on lymphocyte proliferation andmacrophage function.
In order to isolate and purify Arenaria kansuensis polysaccharide from natural Arenaria kansuensis,and study its molecular weight,monosaccharide component and immune activity,Arenaria kansuensis crude polysaccharide(AKCP) was extracted by water extraction and alcohol precipitation,and separated and purified into AKP-1-AKP-5 by DEAE cellulose 52 column.AKP-2 was further purified using Sephadex G-75 column and AKP-2 a polysaccharide component was obtained.The total sugar content of AKCP,AKP-2 and AKP-2 a was measured as 52%,70% and 79% respectively by phenol-sulfuric acid method.The GPC-MALS was applied to measure the molecular weight of AKP-2 a.Results showed that the weight-average molecular weight(Mw) of AKP-2 a was 2.07× 10~(5 )Da,and the number-average molecular weight(Mn) was 9.838× 10~4 Da.The 10 main monosaccharides in AKP-2 a detected by HPLC are galacturonic acid,mannose,ribose,rhamnose,glucuronic acid,glucose,galactose,xylose,arabinose and fucose,the molar ratio of which was 1∶0.25∶0.01∶0.20∶0.11∶0.25∶0.61∶0.07∶0.21∶0.12 respectively.Compared with control group,the AKCP,AKP-2 and AKP-2 a treatment groups with high,medium and low concentrations had a promoting effect on the proliferation of mice spleen lymphocytes by MTT in vitro(P<0.05).By NO release experiment and ELISA detection,AKP-2 a showed a concentration dependent activation effect on mice peritoneal macrophages in vitro(P<0.01). In conclusion,this study obtained AKP-2 a polysaccharide through isolation and purification of Arenaria kansuensis,which has a high total sugar content.The multiple monosaccharides and the molecular weight of AKP-2 a were identified,and the preliminary results showed that AKP-2 a had the biological activities on lymphocyte proliferation andmacrophage function.
引文
1 Luo DS.Chinese Herbal Medicine(中华藏本草)[M].Beijing:China National Press,1997:6.
2 Northwest Institute of Plateau Biology,Chinese Academy of Sciences.Tibetan Medicine(藏药志)[M].Xining:Qinghai People’s Publishing House,1991:449.
3 Liu ZG,Kang HL,Cui Y,et al.Research and investigation in germplasm resources of Kansu sandwort herbs in Qinghai province[J].Lishizhen Med Mate Med Res(时珍国医国药),2016,27:2996-2999.
4 Gao XX,Wang G,Zhang H,et al.Activation effect of crude polysaccharide from Arenaria kansuensis on proliferation and function of murine immunocyte in vitro[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:1188-1192.
5 Gao XX,Wang G,Yang XR,et al.Effects of crude polysaccharide from Arenaria kansuensison immune functions of immunosuppression mice induced by cyclophosphamide[J].Immunol J(免疫学杂志),2017,33:191-196.
6 Yang XR,Wang G,Ji LB,et al.Effects of Arenaria kansuensis aqueous extract on immune function of H22 tumor-bearing mice[J].Cancer Res Prev Treat(肿瘤防治研究),2015,42:662-665.
7 Wang G,Wang HY,Zhang H,et al.Effects of Arenaria kansuensisaqueous extract on lymphocytes proliferation and Th1 cytokine production[J].Chin J Immun(中国免疫学杂志),2013,29:905-909.
8 Wang G,Zhang H,Yang XR,et al.Effect of Arenaria kansuensis aqueous extract on proliferation and cell cycle of human gastric cancer cell line MGC-803[J].Cancer Res Prev Treat(肿瘤防治研究),2013,40:821-825.
9 Wang L,Wang C,Gao X,et al.Purification,characterization and anti-aging capacity of mycelia zinc polysaccharide by Lentinusedodes,SD-08[J].Bmc Complem Altern M,2015,15:111.
10 Li Q,Zhao T,Bai S Q,et al.Water-soluble polysaccharides from leaves of Abelmoschusesculentus:purification,characterization,and antioxidant activity[J].Chem Nat Compounds,2017,53:412-416.
11 Liu L,Shang Q,Liu F,et al.Polysaccharides from the leaves of Lonicera japonica[J].Chem Nat Compounds,2016,52(2):1-2.
12 Peng GH,Zhu Y,Xu QX.Infrared spectrum and gas chromatography analysis of the polysaccharides separation from Arenaria kansuensis maxim[J].China Pharmacist(中国药师),2009,12:1557-1560.
13 Killick J,Morisse G,Sieger D,et al.Complement as a regulator of adaptive immunity[J].Semin Immunopathol,2018,40(1):37-48.
14 Wang Y,Qi Q,Li A,et al.Immuno-enhancement effect of Yifei Tongluo Gramules on cyclophosphamide-induced immunosuppression in Balb/c mice[J].J Ethnopharmacol,2016,194:72-82.
2 Northwest Institute of Plateau Biology,Chinese Academy of Sciences.Tibetan Medicine(藏药志)[M].Xining:Qinghai People’s Publishing House,1991:449.
3 Liu ZG,Kang HL,Cui Y,et al.Research and investigation in germplasm resources of Kansu sandwort herbs in Qinghai province[J].Lishizhen Med Mate Med Res(时珍国医国药),2016,27:2996-2999.
4 Gao XX,Wang G,Zhang H,et al.Activation effect of crude polysaccharide from Arenaria kansuensis on proliferation and function of murine immunocyte in vitro[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:1188-1192.
5 Gao XX,Wang G,Yang XR,et al.Effects of crude polysaccharide from Arenaria kansuensison immune functions of immunosuppression mice induced by cyclophosphamide[J].Immunol J(免疫学杂志),2017,33:191-196.
6 Yang XR,Wang G,Ji LB,et al.Effects of Arenaria kansuensis aqueous extract on immune function of H22 tumor-bearing mice[J].Cancer Res Prev Treat(肿瘤防治研究),2015,42:662-665.
7 Wang G,Wang HY,Zhang H,et al.Effects of Arenaria kansuensisaqueous extract on lymphocytes proliferation and Th1 cytokine production[J].Chin J Immun(中国免疫学杂志),2013,29:905-909.
8 Wang G,Zhang H,Yang XR,et al.Effect of Arenaria kansuensis aqueous extract on proliferation and cell cycle of human gastric cancer cell line MGC-803[J].Cancer Res Prev Treat(肿瘤防治研究),2013,40:821-825.
9 Wang L,Wang C,Gao X,et al.Purification,characterization and anti-aging capacity of mycelia zinc polysaccharide by Lentinusedodes,SD-08[J].Bmc Complem Altern M,2015,15:111.
10 Li Q,Zhao T,Bai S Q,et al.Water-soluble polysaccharides from leaves of Abelmoschusesculentus:purification,characterization,and antioxidant activity[J].Chem Nat Compounds,2017,53:412-416.
11 Liu L,Shang Q,Liu F,et al.Polysaccharides from the leaves of Lonicera japonica[J].Chem Nat Compounds,2016,52(2):1-2.
12 Peng GH,Zhu Y,Xu QX.Infrared spectrum and gas chromatography analysis of the polysaccharides separation from Arenaria kansuensis maxim[J].China Pharmacist(中国药师),2009,12:1557-1560.
13 Killick J,Morisse G,Sieger D,et al.Complement as a regulator of adaptive immunity[J].Semin Immunopathol,2018,40(1):37-48.
14 Wang Y,Qi Q,Li A,et al.Immuno-enhancement effect of Yifei Tongluo Gramules on cyclophosphamide-induced immunosuppression in Balb/c mice[J].J Ethnopharmacol,2016,194:72-82.