网格状与线状鼠尾藻多糖抗炎作用研究
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摘要
目的对鼠尾藻多糖进行分离纯化,并对其理化特性、微观结构和对炎症因子表达的影响进行研究。方法采用热水提取法及DEAE-Sepharose CL-6B和Sephacryl S-300HR柱层析分离纯化鼠尾藻多糖,利用高效空间排阻色谱法(HPSEC)、气相色谱法(GC)、红外光谱法(FTIR)和透射电子显微镜(TEM)研究理化特性及微观结构,实时荧光定量PCR法研究炎症相关因子的mRMA表达。结果得到两种鼠尾藻均一多糖STP-Ⅰ和STP-Ⅲ,相对分子质量分别是29和350kDa,糖含量均超过96%。透射电子显微镜观察到STP-Ⅰ是网格状结构,STP-Ⅲ是线状且螺旋缠绕结构。活性研究表明,STP-Ⅲ能更好地抑制脂多糖(LPS)诱导下小鼠巨噬细胞RAW264.7系炎症因子TNF-α、IL-6和COX-2的mRNA表达量,在150μg·mL~(-1)质量浓度下,STPⅢ对炎症因子TNF-α、IL-6和COX-2的mRNA表达抑制率均超过95%。结论鼠尾藻多糖具有抗炎症因子表达的作用,可以作为潜在的抗炎药物开发。线状缠绕的鼠尾藻多糖STP-Ⅲ较网格状的STP-Ⅰ能更好地抑制炎症相关因子的表达。
Objective To isolate polysaccharides fromSargassum thunbergii and study their physicochemical properties,microstructures and anti-inflammation effects.Methods Two polysaccharides(STP-I and STP-Ⅲ)were purified using water extraction method,DEAE-Sepharose CL-6 Band Sephacryl S-300 HR column chromatogram.The physicochemical properties and microstructures were studied using high-performance size-exclusion chromatography(HPSEC),gas chromatography(GC),Fourier transform infrared spectroscopy(FTIR)and transmission electron microscopy(TEM)analysis.Anti-inflammation activities of the polysaccharides were identified using real time quantitative PCR.Results STP-Ⅰ and STP-Ⅲ were all homogeneous,molecular weights(Mw)were 29 and 350 kDa,and the contents of total carbohydrates were more than 96%.TEM revealed further that STP-Ⅰ was mesh-shaped,and STP-Ⅲ was observed as linear microstructures with entangled chains.Due to the results of anti-inflammation activities of the polysaccharides,STP-Ⅲ showed the higher inhibitory effects of TNF-α,IL-6 and COX-2 mRNA expressions in LPS-stimulated RAW 264.7 mouse macrophage cells than others,and the inhibitory effects were more than 95%at 150μg·mL~(-1) concentrations.Conclusion The polysaccharides fromS.thunbergii have obvious anti-inflammation effects and may serve as potential anti-inflammatory agents,and linear STP-Ⅲ shows more noticeable anti-inflammation effects than the huge meshshaped STP-Ⅰ.
Objective To isolate polysaccharides fromSargassum thunbergii and study their physicochemical properties,microstructures and anti-inflammation effects.Methods Two polysaccharides(STP-I and STP-Ⅲ)were purified using water extraction method,DEAE-Sepharose CL-6 Band Sephacryl S-300 HR column chromatogram.The physicochemical properties and microstructures were studied using high-performance size-exclusion chromatography(HPSEC),gas chromatography(GC),Fourier transform infrared spectroscopy(FTIR)and transmission electron microscopy(TEM)analysis.Anti-inflammation activities of the polysaccharides were identified using real time quantitative PCR.Results STP-Ⅰ and STP-Ⅲ were all homogeneous,molecular weights(Mw)were 29 and 350 kDa,and the contents of total carbohydrates were more than 96%.TEM revealed further that STP-Ⅰ was mesh-shaped,and STP-Ⅲ was observed as linear microstructures with entangled chains.Due to the results of anti-inflammation activities of the polysaccharides,STP-Ⅲ showed the higher inhibitory effects of TNF-α,IL-6 and COX-2 mRNA expressions in LPS-stimulated RAW 264.7 mouse macrophage cells than others,and the inhibitory effects were more than 95%at 150μg·mL~(-1) concentrations.Conclusion The polysaccharides fromS.thunbergii have obvious anti-inflammation effects and may serve as potential anti-inflammatory agents,and linear STP-Ⅲ shows more noticeable anti-inflammation effects than the huge meshshaped STP-Ⅰ.
引文
[1]Dung N T,Bajpai V K,Yoon J I,et al.Anti-inflammatory effects of essential oil isolated from the buds of Cleistocalyx operculatus(Roxb.)Merr and Perry[J].Food Chem Toxicol,2009,47:449-453.
[2]Kern T S.Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy[J].Exp Diabetes Res,2007,1(1):1-14.
[3]Schroder K,Sweet M J,Hume D A.Signal integration between IFNc and TLR signaling pathways in macrophages[J].Immunobiology,2006,211(68):511-524.
[4]Kaz+owska K,Hsu T,Hou C C,et al.Anti-inflammatory properties of phenolic compounds and crude extract from Porphyradentata[J].J Ethnopharmacol,2010,128:123-130.
[5]Mueller M,Hobiger S,Jungbauer A.Anti-inflammatory activity of extracts from fruits,herbs and spices[J].Food Chem,2010,122:987-996.
[6]汤志宏,刘振羽,王军,等.鼠尾藻微卫星标记开发及青岛群体遗传多样性分析[J].中国海洋大学学报,2015,45(1):35-40.
[7]张而贤,俞丽君.鼠尾藻多糖清除氧自由基作用的研究:IIUvc鼠尾藻对多糖抗氧化作用的影响[J].中国海洋药物,1997,16(3):1-4.
[8]Zhuang C,Itoh H,Mizuno T,et al.Antitumor active fucoidan from the brown seaweed,umitoranoo(Sargassum thunbergii)[J].Biosci Biotech Bioch,1995,59:563-567.
[9]黄智璇,梅雪婷,许东晖,等.螺旋藻多糖和鼠尾藻多糖对四氧嘧啶致糖尿病大鼠血管的保护作用[J].中国中药杂志,2005,30(3):211-215.
[10]魏玉西,李敬,赵爱云,等.鼠尾藻多糖的制备及其抗凝血活性的初步研究[J].中国海洋药物,2006,25(2):41-44.
[11]Wang K,Li W,Rui X,et al.Chemical modification,characterization and bioactivity of a released exopolysaccharide(r-EPS1)from Lactobacillus plantarum 70810[J].Glycoconjugate J,2015,32(7):17-27.
[12]Luo D H,Fang B S.Structural identification of ginseng polysaccharides and testing of their antioxidant activities[J].Carbohydr Polym,2008,72(3):376-381.
[13]Dubois M,Gillis K A,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances[J].Anal Chem,1956,28(3):350-356.
[14]Bradford M M.Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Chem,1976,72(1/2):248-254.
[15]Bitter T,Muir H M.A modified uronic acid carbazole reaction[J].Anal Biochem,1962,4:330-334.
[16]Wang Z J,Luo D H.Antioxidant activities of different fractions of polysaccharide purified from Gynostemma pentaphyllum Makino[J].Carbohydr Polym,2007,68(1):54-58.
[17]Wang Z J,Zeng Y W,Luo D H.Structure elucidation of a non-branched and entangled heteropolysaccharide from Tremella sanguinea Peng and its antioxidant activity[J].Carbohydr Polym,2016,152:33-40.
[18]周容,曹森,罗巅辉.7种不同产地石耳鉴定及其粗多糖抗氧化和抗炎作用研究[J].中国药学杂志,2018,53(19):38-43.
[19]曹森,周容,聂开颖,等.一个鼠尾藻异多糖的结构特征及其抗氧化和抗结肠癌细胞增殖作用[J].天然产物研究与开发,2017,29(10):1635-1641.
[20]Chen L,Liu J,Zhang Y,et al.Structural,thermal,and anti-inflammatory properties of a novel pectic polysaccharide from alfalfa(Medicago sativa L.)stem[J].J Agr Food Chem,2015,63(11):3219-3228.
[2]Kern T S.Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy[J].Exp Diabetes Res,2007,1(1):1-14.
[3]Schroder K,Sweet M J,Hume D A.Signal integration between IFNc and TLR signaling pathways in macrophages[J].Immunobiology,2006,211(68):511-524.
[4]Kaz+owska K,Hsu T,Hou C C,et al.Anti-inflammatory properties of phenolic compounds and crude extract from Porphyradentata[J].J Ethnopharmacol,2010,128:123-130.
[5]Mueller M,Hobiger S,Jungbauer A.Anti-inflammatory activity of extracts from fruits,herbs and spices[J].Food Chem,2010,122:987-996.
[6]汤志宏,刘振羽,王军,等.鼠尾藻微卫星标记开发及青岛群体遗传多样性分析[J].中国海洋大学学报,2015,45(1):35-40.
[7]张而贤,俞丽君.鼠尾藻多糖清除氧自由基作用的研究:IIUvc鼠尾藻对多糖抗氧化作用的影响[J].中国海洋药物,1997,16(3):1-4.
[8]Zhuang C,Itoh H,Mizuno T,et al.Antitumor active fucoidan from the brown seaweed,umitoranoo(Sargassum thunbergii)[J].Biosci Biotech Bioch,1995,59:563-567.
[9]黄智璇,梅雪婷,许东晖,等.螺旋藻多糖和鼠尾藻多糖对四氧嘧啶致糖尿病大鼠血管的保护作用[J].中国中药杂志,2005,30(3):211-215.
[10]魏玉西,李敬,赵爱云,等.鼠尾藻多糖的制备及其抗凝血活性的初步研究[J].中国海洋药物,2006,25(2):41-44.
[11]Wang K,Li W,Rui X,et al.Chemical modification,characterization and bioactivity of a released exopolysaccharide(r-EPS1)from Lactobacillus plantarum 70810[J].Glycoconjugate J,2015,32(7):17-27.
[12]Luo D H,Fang B S.Structural identification of ginseng polysaccharides and testing of their antioxidant activities[J].Carbohydr Polym,2008,72(3):376-381.
[13]Dubois M,Gillis K A,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances[J].Anal Chem,1956,28(3):350-356.
[14]Bradford M M.Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Chem,1976,72(1/2):248-254.
[15]Bitter T,Muir H M.A modified uronic acid carbazole reaction[J].Anal Biochem,1962,4:330-334.
[16]Wang Z J,Luo D H.Antioxidant activities of different fractions of polysaccharide purified from Gynostemma pentaphyllum Makino[J].Carbohydr Polym,2007,68(1):54-58.
[17]Wang Z J,Zeng Y W,Luo D H.Structure elucidation of a non-branched and entangled heteropolysaccharide from Tremella sanguinea Peng and its antioxidant activity[J].Carbohydr Polym,2016,152:33-40.
[18]周容,曹森,罗巅辉.7种不同产地石耳鉴定及其粗多糖抗氧化和抗炎作用研究[J].中国药学杂志,2018,53(19):38-43.
[19]曹森,周容,聂开颖,等.一个鼠尾藻异多糖的结构特征及其抗氧化和抗结肠癌细胞增殖作用[J].天然产物研究与开发,2017,29(10):1635-1641.
[20]Chen L,Liu J,Zhang Y,et al.Structural,thermal,and anti-inflammatory properties of a novel pectic polysaccharide from alfalfa(Medicago sativa L.)stem[J].J Agr Food Chem,2015,63(11):3219-3228.