何首乌多糖的结构表征及其免疫调节活性研究
|
摘要
目的从何首乌Polygonum multiflorum中提取分离多糖,对其结构进行表征,并评价其免疫调节活性。方法采用水提、醇沉法从何首乌中提取粗多糖,经Q-Sepharose Fast Flow强阴离子交换色谱柱分离纯化得到何首乌多糖(PMT)。利用高效凝胶渗透色谱-十八角度激光光散射联用法(HPGPC-MALLS)测定PMT的绝对分子质量,采用PMP柱前衍生-高效液相色谱法(HPLC)测定单糖组成。采用二维核磁共振波谱(2D-NMR)对PMT的结构进行表征。采用四甲偶氮唑盐(MTT)法、中性红比色法和Griess法分别检测PMT对小鼠单核巨噬细胞RAW264.7的生长、吞噬活性和释放一氧化氮(NO)能力的影响。结果 PMT是一种α-1,4-葡聚糖,其绝对分子质量为3.96×105。PMT可显著促进RAW264.7细胞的增殖,促进细胞的吞噬能力,增加NO的释放量。结论何首乌来源的α-1,4-葡聚糖具有显著的免疫调节活性,具有药物开发潜力。
Objective To extract and separate a polysaccharide from Polygonum multiflorum, characterize its structural features and study its immunomodulatory activity. Methods The polysaccharide from P. multiflorum(PMT) was isolated and purified by water extraction and ethanol precipitation following Q-Sepharose Fast Flow ion exchange chromatography column. Molecular weight of PMT was determined by high performance gel permeation chromatography-multiple angle laser light scattering(HPGPC-MALLS),and monosaccharide composition was analyzed by HPLC with PMP(1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization,respectively. The structure of PMT was characterized by proton nuclear magnetic resonance spectrum(2 D-NMR). The immunomodulatory activities were tested by MTT, neutral red colorimetric assay and Griess method. Results PMT was a kind ofα-1,4-glucan, and its molecular mass was 3.96 × 105. PMT promoted the proliferation and phagocytosis of RAW 264.7 cells, and significantly induced the increase of NO production in a dose-dependent manner. Conclusion The polysaccharide from P.multiflorum is a linear α-1,4-glucan with potent immunomodulatory activity, which would be potentially developed as an effective drug.
Objective To extract and separate a polysaccharide from Polygonum multiflorum, characterize its structural features and study its immunomodulatory activity. Methods The polysaccharide from P. multiflorum(PMT) was isolated and purified by water extraction and ethanol precipitation following Q-Sepharose Fast Flow ion exchange chromatography column. Molecular weight of PMT was determined by high performance gel permeation chromatography-multiple angle laser light scattering(HPGPC-MALLS),and monosaccharide composition was analyzed by HPLC with PMP(1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization,respectively. The structure of PMT was characterized by proton nuclear magnetic resonance spectrum(2 D-NMR). The immunomodulatory activities were tested by MTT, neutral red colorimetric assay and Griess method. Results PMT was a kind ofα-1,4-glucan, and its molecular mass was 3.96 × 105. PMT promoted the proliferation and phagocytosis of RAW 264.7 cells, and significantly induced the increase of NO production in a dose-dependent manner. Conclusion The polysaccharide from P.multiflorum is a linear α-1,4-glucan with potent immunomodulatory activity, which would be potentially developed as an effective drug.
引文
[1]李时珍.本草纲目[M].北京:人民卫生出版社,1792.
[2]Yao S,Li Y,Kong L Y,et al.Preparative isolation and purification of chemical constituents from the root of Polygonum multiflorum by high-speed counter-current chromatography[J].J Chromatogr A,2006,1115(1/2):64-71.
[3]Lv L S,Gu X H,Tang J,et al.Antioxidant activity of stilbene glycoside from Polygonum multiflorum Thunb in vivo[J].Food Chem,2007,104(4):1678-1681.
[4]罗瑞芝,贾伟,赵利斌,等.何首乌研究进展[J].中草药,2005,36(7):1097-1100.
[5]Lin L F,Ni B,Lin H M,et al.Traditional usages,botany,phytochemistry,pharmacology and toxicology of Polygonum multiflorum Thunb:A review[J].JEthnopharmacol,2015,159(15):158-183.
[6]Zong A Z,Cao H Z,Wang F S,et al.Anticancer polysaccharides from natural resources:A review of recent research[J].Carbohydr Polym,2012,90(4):1395-1410.
[7]Bounda G A,Feng Y U.Review of clinical studies of Polygonum multiflorum Thunb and its isolated bioactive compounds[J].Pharmacogn Res,2015,7(3):225-236.
[8]Um M Y,Choi W H,Aan J Y,et al.Protective effect of Polygonum multiflorum Thunb on amyloidβ-peptide25-35 induced cognitive deficits in mice[J].JEthnopharmacol,2006,104(1/2):144-148.
[9]Franz G.Polysaccharides in pharmacology,current application and future concepts[J].Plant Med,1989,55(6):493-497.
[10]宋士军,李芳,岳华.何首乌的抗衰老作用研究[J].河北医科大学学报,2003,24(2):90-91.
[11]Lv L S,Cheng Y H,Zheng T S,et al.Purification,antioxidant activity and antiglycation of polysaccharides from Polygonum multiflorum Thunb[J].Carbohydr Polym,2014,99(2):765-773.
[12]Chen Q,Zhang S Z,Ying H Z,et al.Chemical characterization and immunostimulatory effects of a polysaccharide from Polygoni Multiflori Radix Praeparata in cyclophosphamide-induced anemic mice[J].Carbohydr Polym,2012,88(4):1476-1482.
[13]Bitter T,Muir H M.A modified uronic acid carbazole reaction[J].Anal Biochem,1962,4(4):330-334.
[14]Zhang X,Zhao X L,Lang Y Z,et al.Low anticoagulant heparin oligosaccharides as inhibitors of BACE-1,the Alzheimer’sβ-secretase[J].Carbohydr Polym,2016,151(18):51-59.
[15]Shan X D,Liu X,Hao J J,et al.In vitro and in vivo hypoglycemic effects of brown algal fucoidans[J].Int JBiol Macromol,2016,82(11):249-255.
[16]Yang Y,Zhao X L,Li J,et al.Aβ-glucan from Durvilaea Antarctica has immunomodulatory effects on RAW 264.7macrophages via toll-like receptor 4[J].Carbohydr Polym,2017,191(1):255-265.
[17]Jordan T,Schmidt S,Liebert T,et al.Molten imidazole-a starch solvent[J].Green Chem,2014,16(4):1967-1973.
[18]Shi X D,Li O Y,Yin J Y,et al.Structure identification ofα-glucans from Dictyophora echinovolvata by methylation and 1D/2D NMR spectroscopy[J].Food Chem,2018,271(15):338-344.
[19]Beutler B.Innate immunity:An overview[J].Mol Immunol,2004,40(12):845-859.
[20]Sun H X,Zhang J,Chen F Y,et al.Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms[J].Carbohydr Polym,2015,121(5):388-402.
[21]Fang W S,Bi D C,Zheng R J,et al.Identification and activation of TLR4-mediated signalling pathways by alginate-derived guluronate oligosaccharide in RAW 264.7macrophages[J].Sci Rep,2017,7(1):1-13.
[22]Li M,Yan Y Y,Yu Q T,et al.Comparison of immunomodulatory effects of fresh garlic and black garlic polysaccharides on RAW 264.7 macrophages[J].JFood Sci,2017,82(3):765-771.
[2]Yao S,Li Y,Kong L Y,et al.Preparative isolation and purification of chemical constituents from the root of Polygonum multiflorum by high-speed counter-current chromatography[J].J Chromatogr A,2006,1115(1/2):64-71.
[3]Lv L S,Gu X H,Tang J,et al.Antioxidant activity of stilbene glycoside from Polygonum multiflorum Thunb in vivo[J].Food Chem,2007,104(4):1678-1681.
[4]罗瑞芝,贾伟,赵利斌,等.何首乌研究进展[J].中草药,2005,36(7):1097-1100.
[5]Lin L F,Ni B,Lin H M,et al.Traditional usages,botany,phytochemistry,pharmacology and toxicology of Polygonum multiflorum Thunb:A review[J].JEthnopharmacol,2015,159(15):158-183.
[6]Zong A Z,Cao H Z,Wang F S,et al.Anticancer polysaccharides from natural resources:A review of recent research[J].Carbohydr Polym,2012,90(4):1395-1410.
[7]Bounda G A,Feng Y U.Review of clinical studies of Polygonum multiflorum Thunb and its isolated bioactive compounds[J].Pharmacogn Res,2015,7(3):225-236.
[8]Um M Y,Choi W H,Aan J Y,et al.Protective effect of Polygonum multiflorum Thunb on amyloidβ-peptide25-35 induced cognitive deficits in mice[J].JEthnopharmacol,2006,104(1/2):144-148.
[9]Franz G.Polysaccharides in pharmacology,current application and future concepts[J].Plant Med,1989,55(6):493-497.
[10]宋士军,李芳,岳华.何首乌的抗衰老作用研究[J].河北医科大学学报,2003,24(2):90-91.
[11]Lv L S,Cheng Y H,Zheng T S,et al.Purification,antioxidant activity and antiglycation of polysaccharides from Polygonum multiflorum Thunb[J].Carbohydr Polym,2014,99(2):765-773.
[12]Chen Q,Zhang S Z,Ying H Z,et al.Chemical characterization and immunostimulatory effects of a polysaccharide from Polygoni Multiflori Radix Praeparata in cyclophosphamide-induced anemic mice[J].Carbohydr Polym,2012,88(4):1476-1482.
[13]Bitter T,Muir H M.A modified uronic acid carbazole reaction[J].Anal Biochem,1962,4(4):330-334.
[14]Zhang X,Zhao X L,Lang Y Z,et al.Low anticoagulant heparin oligosaccharides as inhibitors of BACE-1,the Alzheimer’sβ-secretase[J].Carbohydr Polym,2016,151(18):51-59.
[15]Shan X D,Liu X,Hao J J,et al.In vitro and in vivo hypoglycemic effects of brown algal fucoidans[J].Int JBiol Macromol,2016,82(11):249-255.
[16]Yang Y,Zhao X L,Li J,et al.Aβ-glucan from Durvilaea Antarctica has immunomodulatory effects on RAW 264.7macrophages via toll-like receptor 4[J].Carbohydr Polym,2017,191(1):255-265.
[17]Jordan T,Schmidt S,Liebert T,et al.Molten imidazole-a starch solvent[J].Green Chem,2014,16(4):1967-1973.
[18]Shi X D,Li O Y,Yin J Y,et al.Structure identification ofα-glucans from Dictyophora echinovolvata by methylation and 1D/2D NMR spectroscopy[J].Food Chem,2018,271(15):338-344.
[19]Beutler B.Innate immunity:An overview[J].Mol Immunol,2004,40(12):845-859.
[20]Sun H X,Zhang J,Chen F Y,et al.Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms[J].Carbohydr Polym,2015,121(5):388-402.
[21]Fang W S,Bi D C,Zheng R J,et al.Identification and activation of TLR4-mediated signalling pathways by alginate-derived guluronate oligosaccharide in RAW 264.7macrophages[J].Sci Rep,2017,7(1):1-13.
[22]Li M,Yan Y Y,Yu Q T,et al.Comparison of immunomodulatory effects of fresh garlic and black garlic polysaccharides on RAW 264.7 macrophages[J].JFood Sci,2017,82(3):765-771.