鲍鱼内脏多糖的结构和活性研究
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摘要
从鲍鱼内脏中提取活性多糖,为提高鲍鱼内脏的附加值,促进鲍鱼的综合利用和开发提供理论和技术支持。本论文采用水煮、醇沉的方法,得到鲍鱼内脏粗多糖CAVP, CAVP经冻融分级、酶-Sevag法联合脱蛋白、SepharoseCL-6B柱层析等方法进行纯化,得到水溶性多糖AVP,苯酚-硫酸法测总糖含量为91.24%。经SepharoseCL-6B柱层析和HPLC分析,AVP为均一性多糖,分子量约为3.5万。气相色谱法分析表明AVP主要由Man、Gal、Glc三种单糖组成,其摩尔比为2.7:0.7:6.5。
采用部分酸水解、高碘酸氧化、Smith降解、甲基化及IR、GC、GC-MS等方法对AVP的结构进行分析。AVP经部分酸水解反应,AVP的主链由Glc单糖组成,糖链末端或支链主要由Glc和Gal组成,支链或主链边缘主要是Man。A VP甲基化分析结果表明,AVP主链部分主要由(1→4)-Glc组成,分支点为Glc的6-O处,分支点残基为(1→4,6) -Glc;支链部分主要是(1→3,6)-Man;非还原末端残基是(1→)-Gal和(1→)-Glc。应用MTT法初步考察了AVP对小鼠体外淋巴细胞增殖的影响。结果表明,AVP在100-400μg/ml浓度下,均能促进淋巴细胞的增殖,并且随着浓度的增加,促进作用越明显;在100-200μg/ml时,AVP可以促进ConA诱导的T淋巴细胞的增殖,同时对LPS诱导的B淋巴细胞的增殖有明显影响。
We extracted active polysaccharide from Abalone viscera which can provide theoretical and technical support to increase the added value of abalone viscera, and to promote the utilization and development of abalone. The polysaccharide of CAVP was isolated from Abalone viscera by water.The water-extracted polysaccharide CAVP was fractionated by frozen-thawed, deproteinated with enzyme/Sevag method and purified by gel filtration chromatography on a column of Sepharose CL-6B. Then fraction were obtained and named as AVP. The content of polysaccharide was 91.24%. The molecular weight of it was 3.5×104 Da ,which measured by HPLC. GC analysis showed that AVP was composed of Man,Gal and Glc.The molar ratio was2.7:0.7:6.5.
The structure of AVP was elucidated by periodate oxidation, Smith degradation, methylation analysis and IR,GC,GC-MS. The partial acid hydrolysis of AVP showed that the main chain of AVP was composed of Glc .The end of sugar chain or branched-chain was composed of Gal and Glc. The methylation analysis show that AVP consisted of a backbone was composed of (1→4)-Glc The side chains was composed of (1→3,6)-linked–Man non-reducing terminal residues was composed of (1→)-Gal and (1→)-Glc.
The immunobiological activity of CAVP and AVP were detected by Lymphocyte proliferation assay with MTT. The bioassay showed that CAVP and AVP could induce Lymphocyte proliferation from 100μg/ml to 400μg/ml, and the higher the concentration, the effect is more obvious. In the dose of 100 and 200μg/ml, CAVP and AVP can promote the ConA-induced proliferation of T Lymphocytes, what is more, they did affect the LPS-induced proliferation of B lymphocytes.
采用部分酸水解、高碘酸氧化、Smith降解、甲基化及IR、GC、GC-MS等方法对AVP的结构进行分析。AVP经部分酸水解反应,AVP的主链由Glc单糖组成,糖链末端或支链主要由Glc和Gal组成,支链或主链边缘主要是Man。A VP甲基化分析结果表明,AVP主链部分主要由(1→4)-Glc组成,分支点为Glc的6-O处,分支点残基为(1→4,6) -Glc;支链部分主要是(1→3,6)-Man;非还原末端残基是(1→)-Gal和(1→)-Glc。应用MTT法初步考察了AVP对小鼠体外淋巴细胞增殖的影响。结果表明,AVP在100-400μg/ml浓度下,均能促进淋巴细胞的增殖,并且随着浓度的增加,促进作用越明显;在100-200μg/ml时,AVP可以促进ConA诱导的T淋巴细胞的增殖,同时对LPS诱导的B淋巴细胞的增殖有明显影响。
We extracted active polysaccharide from Abalone viscera which can provide theoretical and technical support to increase the added value of abalone viscera, and to promote the utilization and development of abalone. The polysaccharide of CAVP was isolated from Abalone viscera by water.The water-extracted polysaccharide CAVP was fractionated by frozen-thawed, deproteinated with enzyme/Sevag method and purified by gel filtration chromatography on a column of Sepharose CL-6B. Then fraction were obtained and named as AVP. The content of polysaccharide was 91.24%. The molecular weight of it was 3.5×104 Da ,which measured by HPLC. GC analysis showed that AVP was composed of Man,Gal and Glc.The molar ratio was2.7:0.7:6.5.
The structure of AVP was elucidated by periodate oxidation, Smith degradation, methylation analysis and IR,GC,GC-MS. The partial acid hydrolysis of AVP showed that the main chain of AVP was composed of Glc .The end of sugar chain or branched-chain was composed of Gal and Glc. The methylation analysis show that AVP consisted of a backbone was composed of (1→4)-Glc The side chains was composed of (1→3,6)-linked–Man non-reducing terminal residues was composed of (1→)-Gal and (1→)-Glc.
The immunobiological activity of CAVP and AVP were detected by Lymphocyte proliferation assay with MTT. The bioassay showed that CAVP and AVP could induce Lymphocyte proliferation from 100μg/ml to 400μg/ml, and the higher the concentration, the effect is more obvious. In the dose of 100 and 200μg/ml, CAVP and AVP can promote the ConA-induced proliferation of T Lymphocytes, what is more, they did affect the LPS-induced proliferation of B lymphocytes.
引文
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[3]Zhang W J. Biochemical Study Technology in Polysaccharide Compounds, Zhejiang University Press: Zhejiang. 1999, 193–197.
[4]HATANADA K,YOSHIDA T,MIYAHARA S. et al. Synthesis of new heparinoids with high anticoagulant activity[J]. Med Chem,1987,30(5):810-814
[5]刘芳,李卓能,庞雅琴.海带多糖对小鼠动脉血气影响及抗疲劳作用[J].中国公共卫生,2003,19(12):1462-1463.
[6]冯晓梅,韩玉谦,赵志强.牡蛎中糖蛋白成分的分离纯化及其性质研究[J].天然产物研究与开发,2008,20(4):709.
[7]黄建设,龙丽娟.海洋天然产物及其生理活性的研究进展[J].海洋通报,2001,20( 4) : 83 - 92.
[8]高续春.多糖提取纯化方法研究进展[J].榆林学院报,2009,19(4):65-67.
[9]盛家荣,曾令辉,翟春等.多糖的提取、分离及结构分析[J].广西师范学报,1999,16(4):49-54.
[10]杨悦,石森林,葛卫红多糖的化学研究概况.中国药师,2008,11(1):93-95.
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[12]秦俊哲,陈明,陈合,等.食药用真菌多糖的研究现状与展望[J].中国食用菌,2003,23(2):6-9.
[13]李峰,刘延吉,宗绪岩,等.草本刺嫩芽根多糖脱蛋白方法研究[J].安徽农业科学, 2006, 34(1): 9-10.
[14] Structural characterization of rhamnogalacturonan oligomers generated by controlled acid hydrolysis of sugar-beet pulp[J].Carbohydrate Research.1998,305:271-280.
[15] Linker A, Evans L R, Impallomeni G..The structure of a polysaccharide from infectious strains of Burkholderia cepacia. Carbohydrate Research 2001,335: 45–54.
[16] Takegawa K, Satoh K, Ramli N, et al. Production and characterization of extracellular uronic acid-containing glycoproteins from Fusarium oxysporum[J]. Journal of Fermentation and Bioengineering.1997,83(2): 197-200.
[17] Hanniffy O M, Shashkov A S, Moran A P, et al. Chemical structure of a polysaccharide from Campylobacter jejuni 176.83 (serotype O:41) containing only furanose sugars[J].Carbohydrate Research.1999, 319(1-4): 124-132.
[18] Li S P, Zhao K J, Ji Z N, et al. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide-inducedinjury. Life Sciences. 2003,73(19):2503–2513.
[19] Fu D, O’neill R A. Monosaccharide Composition Analysis of Oligosaccharides and Glycoproteins by High-Performance Liquid Chromatography[J]. Analytical Biochemistry.1995, 227: 377-384.
[20] Myung J Y, Jin K K, Seong-yeol P, Yunha K, Channy P, Eun SK. Potential anticancer properties of the water extract of Inontus obliquus by induction of apoptosis in melanoma B16-F10 cells. J Ethnopharmacology.2009, 12:221–228.
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[22]黄益丽,郑天凌.海洋生物活性多糖的研究现状与展望[J].海洋科学, 2004, 28(4): 58.
[23]苏文金,黄益丽,黄耀坚,等.海洋放线菌免疫活性多糖的筛选[J].海洋学报,2001(6): 114-119.
[24]WangHu,i Chiu LCM,OoiVEC, eta.l Seaweed polysaccharide with anticancer potential[J]. BotanicaMarina, 2008, 51(4): 313.
[25] Gerber P, Dutcher D, AdamsV, et al. Protective effect of seaweed extracts for chicken embryos infected with influenza B or mumps[J]·ProcSoc Exp Biol Med, 1958, 99:590~593.
[26]Hu J F, Geng M Y, Zhang J T, et al. An in vitro study of the brown algae to its antioxidant effect[J]. J Asian Nat ProdRes, 2001, 3 (4): 353-358.
[27]Kulicake W M. Correlation between immunological activity,molar mass,and molecular structure of different(1→3)-β-D-glucans[J].Carbohydr Res,1997,297:135.
[28]张坤,王令充,吴皓,等.活性海洋多糖的功能及结构研究概况中国海洋药物杂志2010年6月第29卷第3期
[29]赵峡,杨海,于广利海洋多糖保湿乳剂的制备研究[J].中国海洋大学学报第37卷第4期2007年7月
[30]王兵,许东晖,许实波,等.鲍鱼多糖对环磷酰胺的增效减毒作用[J ] .中药材,1999 ,22(4) :198 - 200.
[31]王兵,蒋建敏,许东晖,等.鲍鱼多糖对人鼻咽癌裸鼠抗癌作用的研究[J].中草药, 2000, 31(8): 597 - 599.
[32]陈倩超.鲍鱼多糖的提取及抗肿瘤实验.中国现代应用药学.第十五卷第1期.1998年2月
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