鮟鱇鱼皮硫酸皮肤素的提取、纯化、结构和活性研究
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摘要
本文旨在建立从鮟鱇鱼皮中提取、纯化硫酸皮肤素(DS)的方法,并对其结构和抗凝血活性进行研究。为鮟鱇鱼皮的利用与硫酸皮肤素的开发提供理论依据。
鮟鱇鱼皮硫酸皮肤素的提取采用酶解法,通过正交试验法和响应面分析法对提取参数进行优化,得到最佳提取工艺如下:鮟鱇鱼皮原料加入2.5%的复合酶(木瓜蛋白酶:胰酶=1:2,W/W),按液料比26:1(V/W)加入蒸馏水,76℃下提取19小时,3倍体积乙醇沉淀,冷冻干燥后得到粗品。
采用三氯乙酸(TCA)法、Sevag法和CPC法对提取所得DS进行纯化,结果显示Sevag法和CPC法纯化效果均较好,三氯乙酸法纯化效果较差。Sevag法操作简便,比CPC法更具优越性。经液相色谱法和醋酸纤维薄膜电泳法双重鉴定表明纯化后的DS为单一组分,纯度为99.15%。
通过化学法对鮟鱇鱼皮DS的基本组成进行分析,结果表明DS中氨基己糖含量为35.75±1.70%,己糖醛酸含量为29.02±2.90%,硫酸基含量为18.83±0.60%,硫酸基与羧酸基的比值为1.02±0.14:1。与来源于猪皮、鸡皮和牛蛙皮的DS相比,鮟鱇鱼皮DS中硫酸基含量最高且它在组成上有别于其它来源的DS。
根据高效凝胶渗透色谱(HPGPC)、气相色谱(GC)、毛细管液相色谱(CLC)、质谱(MS)、红外光谱(IR)的分析结果,结合其他理化性质,对鮟鱇鱼皮DS的化学结构进行了初步确认。鮟鱇鱼皮DS的分子量为1.59×10~9,其结构中存在木糖、半乳糖、葡萄糖醛酸、艾杜糖醛酸和N-乙酰氨基半乳糖五种单糖,摩尔比为0.07:0.04:0.07:1.12:1.19,其中木糖与半乳糖是存在于糖链与核心蛋白连接区的少量单糖。鮟鱇鱼皮DS可被硫酸软骨素ABC酶酶解,37℃反应4小时可完全酶解,其糖链是以艾杜糖醛酸-[1→3]-N-乙酰氨基半乳糖-4硫酸(△Di-4S(Ido))为主要二糖重复单位构成的,此外还含有少量的艾杜糖醛酸-[1→3]-N-乙酰氨基半乳糖-6硫酸(△Di-6S(Ido))和葡萄糖醛酸-[1→3]-N-乙酰氨基半乳糖(△Di-OS)二糖单位。△Di-OS、△Di-4S(Ido)与△Di-6S(Ido)三种不饱和二糖的摩尔比为0.01:1.71:0.01。鮟鱇鱼皮DS具有糖胺聚糖的特征红外吸收,其单糖环型为吡喃型。,
鮟鱇鱼皮DS的体外抗凝血活性实验以肝素为对照,研究了不同剂量DS对凝血时间(CT)和血浆凝血酶原时间(PT)的影响。结果表明,DS对凝血时间(CT)的影响较大,对血浆凝血酶原时间(PT)无显著性影响,说明其凝血机制主要是作用于内源性凝血途径。而肝素对CT和PT的影响均较显著,抗凝效果大于DS。在1-10 mg/mL的剂量范围内随着剂量的升高,DS的抗凝效果增强。DS的抗凝血效果虽然低于肝素,但由于其出血副作用较小,因此具有很大的应用潜力。
In this paper,the extraction,purification,structure and anticoagulant activity of dermatan sulfate(DS)from the skin of Lophius litulon was investigated.The study is to provide a theoretical basis for utilization of Lophius litulon skin and development of DS.
The method of enzymatic degradation had been used to extract the DS from Lophius litulon skin.The condition of extraction was optimized using orthogonal design and response surface techniques.The optimum conditions for DS extraction was a solvent-biomass ratio of 26 mE/g,at 76℃for 19h using 25mg enzyme(papain:pancreas=1:2,W/W)per gram of tissue.After centrifugation,3 volumes of ethanol were added to the supernatant,the precipitate was freeze-dried to afford crude DS.
TCA method,Sevag method and CPC method were used to purify the crude DS from Lophius litulon skin.Both Sevag and CPC method can effectively purify DS,while TCA method can not.Sevag method is easy to perform,so it is better than CPC method.The DS from Lophius litulon skin was verified to be mono-fraction by HPGPC and cellulose acetate electrophoretic method,its purity is 99.15%.
Chemical methods were used to anlysis the basic constituents of DS. It showed that the DS from Lophius litulon skin contains hexosamine (35.75±1.70%),uronic acid(29.02±2.90%)and SO_4~-(18.83±0.60%),and the rate of OSO_3~-:COO~- is 1.02±0.14:1.The SO_4~- content was most in the DS from Lophius litulon skin compared with the DS from pig skin, chicken skin and bloody noun skin.Chemical composition of the DS from Lophius litulon skin is different from others.
The structures of the DS from Lophius litulon skin were demermined by GPC,GC,CLC,MS and IR techniques.The molecular weight of the DS was 1.59×10~9,it is consisted of xylose,galactose,glucuronic acid, iduronic acid and N-acetyl-D-galactosamine,and their mole ratio is 0.07: 0.04:0.07:1.12:1.19.Xylose and galatose are the few monosaccharides between polysaccharide chains and core protein.The DS from Lophius litulon skin can be well digested by chondroitinase ABC at 37℃for 4 hours.Polysaccharide chains of the DS consists of a prevailing disaccharide unit IdUA-[1→3]-GalNAc-4S and some IdUA-[1→3]-GalNAc-6S and GlcUA-[1→3]-GalNAc,their mole ratio is 1.71:0.01:0.01.DS has a characteristic IR spectrum as glycosaminogly,its monosaccharide is pyranose.
The anticoagulant activity of the DS from Lophius litulon skin was invested in terms of effection of DS on coagulation time(CT)and prothrombin time(PT)with heparinum as control.The results showed that the DS has significant effect on CT and has no effect on PT which indicating that the DS mainly effect on intrinsic pathway.The anticoagulant activity of heparinum is better than the DS from Lophius litulon skin by possessing significant effect on both CT and PT.The anticoagulant activities of the DS were related to their differences in concentration.The anticoagulant activity of DS is worse than heparinum; however it has potential utilization because of its relatively low side effect of hemorrhage.
鮟鱇鱼皮硫酸皮肤素的提取采用酶解法,通过正交试验法和响应面分析法对提取参数进行优化,得到最佳提取工艺如下:鮟鱇鱼皮原料加入2.5%的复合酶(木瓜蛋白酶:胰酶=1:2,W/W),按液料比26:1(V/W)加入蒸馏水,76℃下提取19小时,3倍体积乙醇沉淀,冷冻干燥后得到粗品。
采用三氯乙酸(TCA)法、Sevag法和CPC法对提取所得DS进行纯化,结果显示Sevag法和CPC法纯化效果均较好,三氯乙酸法纯化效果较差。Sevag法操作简便,比CPC法更具优越性。经液相色谱法和醋酸纤维薄膜电泳法双重鉴定表明纯化后的DS为单一组分,纯度为99.15%。
通过化学法对鮟鱇鱼皮DS的基本组成进行分析,结果表明DS中氨基己糖含量为35.75±1.70%,己糖醛酸含量为29.02±2.90%,硫酸基含量为18.83±0.60%,硫酸基与羧酸基的比值为1.02±0.14:1。与来源于猪皮、鸡皮和牛蛙皮的DS相比,鮟鱇鱼皮DS中硫酸基含量最高且它在组成上有别于其它来源的DS。
根据高效凝胶渗透色谱(HPGPC)、气相色谱(GC)、毛细管液相色谱(CLC)、质谱(MS)、红外光谱(IR)的分析结果,结合其他理化性质,对鮟鱇鱼皮DS的化学结构进行了初步确认。鮟鱇鱼皮DS的分子量为1.59×10~9,其结构中存在木糖、半乳糖、葡萄糖醛酸、艾杜糖醛酸和N-乙酰氨基半乳糖五种单糖,摩尔比为0.07:0.04:0.07:1.12:1.19,其中木糖与半乳糖是存在于糖链与核心蛋白连接区的少量单糖。鮟鱇鱼皮DS可被硫酸软骨素ABC酶酶解,37℃反应4小时可完全酶解,其糖链是以艾杜糖醛酸-[1→3]-N-乙酰氨基半乳糖-4硫酸(△Di-4S(Ido))为主要二糖重复单位构成的,此外还含有少量的艾杜糖醛酸-[1→3]-N-乙酰氨基半乳糖-6硫酸(△Di-6S(Ido))和葡萄糖醛酸-[1→3]-N-乙酰氨基半乳糖(△Di-OS)二糖单位。△Di-OS、△Di-4S(Ido)与△Di-6S(Ido)三种不饱和二糖的摩尔比为0.01:1.71:0.01。鮟鱇鱼皮DS具有糖胺聚糖的特征红外吸收,其单糖环型为吡喃型。,
鮟鱇鱼皮DS的体外抗凝血活性实验以肝素为对照,研究了不同剂量DS对凝血时间(CT)和血浆凝血酶原时间(PT)的影响。结果表明,DS对凝血时间(CT)的影响较大,对血浆凝血酶原时间(PT)无显著性影响,说明其凝血机制主要是作用于内源性凝血途径。而肝素对CT和PT的影响均较显著,抗凝效果大于DS。在1-10 mg/mL的剂量范围内随着剂量的升高,DS的抗凝效果增强。DS的抗凝血效果虽然低于肝素,但由于其出血副作用较小,因此具有很大的应用潜力。
In this paper,the extraction,purification,structure and anticoagulant activity of dermatan sulfate(DS)from the skin of Lophius litulon was investigated.The study is to provide a theoretical basis for utilization of Lophius litulon skin and development of DS.
The method of enzymatic degradation had been used to extract the DS from Lophius litulon skin.The condition of extraction was optimized using orthogonal design and response surface techniques.The optimum conditions for DS extraction was a solvent-biomass ratio of 26 mE/g,at 76℃for 19h using 25mg enzyme(papain:pancreas=1:2,W/W)per gram of tissue.After centrifugation,3 volumes of ethanol were added to the supernatant,the precipitate was freeze-dried to afford crude DS.
TCA method,Sevag method and CPC method were used to purify the crude DS from Lophius litulon skin.Both Sevag and CPC method can effectively purify DS,while TCA method can not.Sevag method is easy to perform,so it is better than CPC method.The DS from Lophius litulon skin was verified to be mono-fraction by HPGPC and cellulose acetate electrophoretic method,its purity is 99.15%.
Chemical methods were used to anlysis the basic constituents of DS. It showed that the DS from Lophius litulon skin contains hexosamine (35.75±1.70%),uronic acid(29.02±2.90%)and SO_4~-(18.83±0.60%),and the rate of OSO_3~-:COO~- is 1.02±0.14:1.The SO_4~- content was most in the DS from Lophius litulon skin compared with the DS from pig skin, chicken skin and bloody noun skin.Chemical composition of the DS from Lophius litulon skin is different from others.
The structures of the DS from Lophius litulon skin were demermined by GPC,GC,CLC,MS and IR techniques.The molecular weight of the DS was 1.59×10~9,it is consisted of xylose,galactose,glucuronic acid, iduronic acid and N-acetyl-D-galactosamine,and their mole ratio is 0.07: 0.04:0.07:1.12:1.19.Xylose and galatose are the few monosaccharides between polysaccharide chains and core protein.The DS from Lophius litulon skin can be well digested by chondroitinase ABC at 37℃for 4 hours.Polysaccharide chains of the DS consists of a prevailing disaccharide unit IdUA-[1→3]-GalNAc-4S and some IdUA-[1→3]-GalNAc-6S and GlcUA-[1→3]-GalNAc,their mole ratio is 1.71:0.01:0.01.DS has a characteristic IR spectrum as glycosaminogly,its monosaccharide is pyranose.
The anticoagulant activity of the DS from Lophius litulon skin was invested in terms of effection of DS on coagulation time(CT)and prothrombin time(PT)with heparinum as control.The results showed that the DS has significant effect on CT and has no effect on PT which indicating that the DS mainly effect on intrinsic pathway.The anticoagulant activity of heparinum is better than the DS from Lophius litulon skin by possessing significant effect on both CT and PT.The anticoagulant activities of the DS were related to their differences in concentration.The anticoagulant activity of DS is worse than heparinum; however it has potential utilization because of its relatively low side effect of hemorrhage.
引文
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