鱼鳔类肝素提取工艺优化及抗凝血活性研究
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摘要
以鱼鳔为原料,采用酶法提取类肝素,研究其提取工艺参数和体外抗凝血活性。在单因素试验基础上,以加酶量、酶解时间和温度为影响因子,类肝素得率为响应值,采用响应曲面法优化鱼鳔类肝素(HSB)提取工艺,并通过测定活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和血浆凝血酶时间(TT)评价其抗凝血活性。结果显示,最优提取条件为2709碱性蛋白酶添加量5.4 mg/mL,酶解温度50℃,时间20h,在该条件下类肝素得率为(1.79±0.05)%,与预测值相差3.24%;同时,所提取的类肝素具有一定的抗凝血活性,并通过外源性凝血途径和共同凝血途径发挥作用。
A natural heparin-like compound was isolated from swim bladder and its anticoagulant activities were determined.The response surface analysis based on Box-Behnken design was adopted to examine effects of protease consumption,extracting time and temperature on heparin-like compound extraction.The anticoagulant activity of swim bladder heparin-like compound was evaluated by APTT,PT and TT method.Consequently,the optimum conditions were as follows.The concentration of protease from Bacillus licheniformis was 5.4 mg/mL,extracted at 50 ℃for 20 h.Under the control of these conditions, the predicted extraction rate by mathematical model was(1.79±0.05)%,and the standard error was3.24%.In addition,the heparin-like compound from swimming bladder(HSB)showed negligible in vitro anticoagulant activity,and played a role through the exogenous coagulation pathway and the common coagulation pathway.
A natural heparin-like compound was isolated from swim bladder and its anticoagulant activities were determined.The response surface analysis based on Box-Behnken design was adopted to examine effects of protease consumption,extracting time and temperature on heparin-like compound extraction.The anticoagulant activity of swim bladder heparin-like compound was evaluated by APTT,PT and TT method.Consequently,the optimum conditions were as follows.The concentration of protease from Bacillus licheniformis was 5.4 mg/mL,extracted at 50 ℃for 20 h.Under the control of these conditions, the predicted extraction rate by mathematical model was(1.79±0.05)%,and the standard error was3.24%.In addition,the heparin-like compound from swimming bladder(HSB)showed negligible in vitro anticoagulant activity,and played a role through the exogenous coagulation pathway and the common coagulation pathway.
引文
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[3]涂宗财,唐平平,郑婷婷,等.响应面优化鱼鳔胶原肽制备工艺及其抗氧化活性研究[J].食品与发酵工业,2017,43(5):160-166.
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[10]吴丽娟,赵峡,王伟.几种海洋类肝素多糖的制备及抗乙肝病毒活性初步研究[J].中国海洋药物,2016,35(6):31-37.
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[13]ALVES M G D C F,NOBRE L T D B,MOURA G E D D D,et al.Effects of heparinoids from algae on hemostasis and their action on the cycle cell[J].Biomedicine&Preventive Nutrition,2012,2(3):163-168.
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[15]孙诗清,孙永军,张儒.生物分离实验技术[M].北京:北京理工大学出版社,2017:13.
[16]张晓娟,熊双丽,周涛.肝素钠效价测定方法的筛选[J].食品工业科技,2014,35(12):66-69.
[17]BITTER T,MUIR H M,陈祥娥.一种改进的糖醛酸咔唑反应[J].食品与药品,2010,12(3):147-149.
[18]WAGNER W D.Amore sensitive assay discriminatinggalactosamine and glucosamine in mixture[J].Analytical Biochemistry,1979,94(2):394-396.
[19]DODGSON K S,PRICE R G.A note on the determination of the estersulphate content of sulphated polysaccharides[J].Biochemical Journal,1962,84(1):106.
[20]上官新晨,吴少福,蒋艳,等.不同浓度中性盐、醇溶液对籽粒苋蛋白质溶解度影响研究[J].江西农业大学学报,2000,22(5):76-78.
[21]ARUMUGAM M,SHANMUGAM A.Extraction of heparin and heparin-like substance from marine mesogastropod mollusc Turritella attenuata(Lamarck,1779)[J].Indian Journal of Experimental Biology,2004,42(5):529.
[22]BALAMURUGAN K,NAGAR A.Isolation and Identification of Heparin like Substances from the body Tissue of Conus musicus[J].International Journal of Pharmtech Research,2009,1(4):1 650-1 653.
[23]河北常山生化药业股份有限公司.肝素钠下脚料制备类肝素工艺:中国,201610598231.6[P].2016-11-23.
[24]杭州惠顺生物科技有限公司.一种从肝素副产物废蛋白中分离类肝素的方法:中国,201510394022.5[P].2015-11-11.
[25]OPOKU G,QIU Xiang-dong,DOCTOR V.Effect of oversulfation on the chemical and biological properties of kappa carrageenan[J].Carbohydrate Polymers,2006,65(2):134-138.
[26]文丹丹.岩藻糖化硫酸软骨素抗凝作用机制及构效关系研究[D].广州:中山大学,2012:23-26.
[27]SAYARI N,BALTI R,MANSOUR M B,et al.Anticoagulant properties andcytotoxic effect against HCT116human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster(Nephrops norvegicus)shell[J].Biomedicine&Pharmacotherapy,2016,80:322-330.
[28]ANDRADE G P,LIMA M A,DE SOUZA JUNIOR A A,et al.A heparin-like compound isolated from a marine crab rich in glucuronic acid 2-O-sulfate presents low anticoagulant activity[J].Carbohydrate Polymers,2013,94(1):647-654.
[29]GONZALES J N,KIM K,ZEMSKOVA M A,et al.Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner[J].Vascular Pharmacology,2014,62(2):63.
[2]PAL G K,NIDHEESH T,GOVINDARAJU K,et al.Enzymatic extraction andcharacterisation of a thermostable collagen from swim bladder of rohu(Labeo rohita)[J].Journal of the Science of Food&Agriculture,2016,97(5):1 451-1 458.
[3]涂宗财,唐平平,郑婷婷,等.响应面优化鱼鳔胶原肽制备工艺及其抗氧化活性研究[J].食品与发酵工业,2017,43(5):160-166.
[4]PAL G K,SURESH P V.Physico-chemical characteristics and fibril-forming capacity of carp swim bladder collagens and exploration of their potential bioactive peptides by in silico approaches[J].International Journal of Biological Macromolecules,2017,101(1):204-205.
[5]SUO Hua-yi,SONG Jia-le,ZHOU Ya-lin,et al.Induction of apoptosis in HCT-116colon cancer cells by polysaccharide of Larimichthys crocea swim bladder[J].Oncology Letters,2015,9(2):972-978.
[6]CHEN Lian-hong,SONG Jia-le,QIAN Yu,et al.Increased preventive effect on colon carcinogenesis by use of resistant starch(RS3)as the carrier for polysaccharide of larimichthys crocea swimming bladder[J].International Journal of Molecular Sciences,2014,15(1):17-29.
[7]JIANG Xian-hong,ZHAO Xin,LUO Hua-li,et al.Therapeutic effect of polysaccharide of large yellow croaker swim bladder on lupus nephritis of mice[J].Nutrients,2014,6(3):1 223-1 235.
[8]凌沛学,何兆雄,姬胜利.肝素[M].北京:中国轻工业出版社,2015.
[9]CHAVANTE S F,BRITO A S,LIMA M,et al.A heparin-like glycosaminoglycan from shrimp containing high levels of 3-O-sulfated D-glucosamine groups in an unusual trisaccharide sequence[J].Carbohydrate Research,2014,390(1):59.
[10]吴丽娟,赵峡,王伟.几种海洋类肝素多糖的制备及抗乙肝病毒活性初步研究[J].中国海洋药物,2016,35(6):31-37.
[11]DREYFUSS J L,REGATIERI C V,LIMA M A,et al.A heparin mimetic isolated from a marine shrimp suppresses neovascularization[J].Journal of Thrombosis&Haemostasis,2010,8(8):1 828-1 837.
[12]BRITO A S,ARIMATEIA D S,SOUZA L R,et al.Anti-inflammatory properties of a heparin-like glycosaminoglycan with reduced anti-coagulant activity isolated from a marine shrimp[J].Bioorganic&Medicinal Chemistry,2008,16(21):9 588-9 595.
[13]ALVES M G D C F,NOBRE L T D B,MOURA G E D D D,et al.Effects of heparinoids from algae on hemostasis and their action on the cycle cell[J].Biomedicine&Preventive Nutrition,2012,2(3):163-168.
[14]熊双丽,金征宇.硫酸化糖胺聚糖的简易分光光度测定法[J].光谱实验室,2005,22(6):1 294-1 297.
[15]孙诗清,孙永军,张儒.生物分离实验技术[M].北京:北京理工大学出版社,2017:13.
[16]张晓娟,熊双丽,周涛.肝素钠效价测定方法的筛选[J].食品工业科技,2014,35(12):66-69.
[17]BITTER T,MUIR H M,陈祥娥.一种改进的糖醛酸咔唑反应[J].食品与药品,2010,12(3):147-149.
[18]WAGNER W D.Amore sensitive assay discriminatinggalactosamine and glucosamine in mixture[J].Analytical Biochemistry,1979,94(2):394-396.
[19]DODGSON K S,PRICE R G.A note on the determination of the estersulphate content of sulphated polysaccharides[J].Biochemical Journal,1962,84(1):106.
[20]上官新晨,吴少福,蒋艳,等.不同浓度中性盐、醇溶液对籽粒苋蛋白质溶解度影响研究[J].江西农业大学学报,2000,22(5):76-78.
[21]ARUMUGAM M,SHANMUGAM A.Extraction of heparin and heparin-like substance from marine mesogastropod mollusc Turritella attenuata(Lamarck,1779)[J].Indian Journal of Experimental Biology,2004,42(5):529.
[22]BALAMURUGAN K,NAGAR A.Isolation and Identification of Heparin like Substances from the body Tissue of Conus musicus[J].International Journal of Pharmtech Research,2009,1(4):1 650-1 653.
[23]河北常山生化药业股份有限公司.肝素钠下脚料制备类肝素工艺:中国,201610598231.6[P].2016-11-23.
[24]杭州惠顺生物科技有限公司.一种从肝素副产物废蛋白中分离类肝素的方法:中国,201510394022.5[P].2015-11-11.
[25]OPOKU G,QIU Xiang-dong,DOCTOR V.Effect of oversulfation on the chemical and biological properties of kappa carrageenan[J].Carbohydrate Polymers,2006,65(2):134-138.
[26]文丹丹.岩藻糖化硫酸软骨素抗凝作用机制及构效关系研究[D].广州:中山大学,2012:23-26.
[27]SAYARI N,BALTI R,MANSOUR M B,et al.Anticoagulant properties andcytotoxic effect against HCT116human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster(Nephrops norvegicus)shell[J].Biomedicine&Pharmacotherapy,2016,80:322-330.
[28]ANDRADE G P,LIMA M A,DE SOUZA JUNIOR A A,et al.A heparin-like compound isolated from a marine crab rich in glucuronic acid 2-O-sulfate presents low anticoagulant activity[J].Carbohydrate Polymers,2013,94(1):647-654.
[29]GONZALES J N,KIM K,ZEMSKOVA M A,et al.Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner[J].Vascular Pharmacology,2014,62(2):63.