SDS-聚丙烯酰胺凝胶电泳法同时测定不同产地大蒜药材中的蒜酶与凝集素
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摘要
目的:建立SDS-聚丙烯酰胺凝胶电泳同时测定不同产地大蒜药材中蒜酶与凝集素相对含量和相对分子质量的方法。方法:通过考察溶媒方法、样品前处理方法、供试品溶液浓度等因素,确定SDS-聚丙烯酰胺凝胶电泳法测定蒜酶与凝集素的最佳条件:以超纯水为溶媒,静置10 min为前处理方法,供试品溶液质量浓度为28 mg·m L-1;分别配制5%浓缩胶、12%分离胶,凝胶厚度为1 mm,浓缩胶电压为80 V,分离胶电压为100 V,进行不连续体系垂直板电泳,借助Gel-pro Analyzer软件分析凝胶电泳图谱。以标准蛋白为marker,建立标准曲线,将蒜酶和凝集素的比移值代入标准曲线方程,计算蒜酶和凝集素的相对分子质量。结果:标准曲线方程为lgM=-1.369 1X+2.184 7,r=0.966 6。大蒜药材中蒜酶相对分子质量为52.1~54.0 k Da,凝集素相对分子质量为11.0~11.8 k Da。不同产地大蒜药材中蒜酶和凝集素总相对含量为18.1%~77.2%,其中蒜酶的相对含量为5.60%~27.1%,凝集素的相对含量为12.5%~68.2%。新疆巴里坤市大蒜样品中蒜酶的相对含量最高为27.1%,新疆阿克苏拜城县大蒜样品中凝集素的相对含量最高为68.2%。不同产地大蒜药材中蒜酶、凝集素的相对含量存在差异,凝集素相对含量普遍高于蒜酶相对含量。结论:与其他测定蛋白质含量的方法相比,本文建立的方法操作简便,样品用量少,能够准确测定大蒜药材中蒜酶及凝集素的相对分子质量与相对含量。不同产地大蒜中蒜酶与凝集素相对总含量均达50%以上,证明蒜酶和凝集素是大蒜中的主要蛋白质。大蒜中的蒜酶与蒜氨酸相对含量的相关性并不显著。
Objective:To establish a method that simultaneously determine relative content and molecular mass of alliinase and lectin in garlic from the different origins by SDS-polyacrylamide gel electrophoresis. Methods:The optimal conditions of the method were confirmed by investigating the factors of solvent methods,sample pretreatment method and the test solution concentrations. The relative content and the molecular mass of alliinase and lectin in garlic were measured by SDS-polyacrylamide gel electrophoresis. The optimal conditions were as follows:ultra-pure water as solvent,stationary for 10 minutes as pretreatment method,the sample solution concentration was 28 mg·m L-1. And 12% separation gel,5% concentrated gel were separately prepared. The gel thickness was 1 mm. The concentrated gel voltage was 80 V,and the concentrated gel voltage was 100 V. Then the discontinuous system and vertical plate electrophoresis were applied. The map of SDS-polyacrylamide gel electrophoresis was analyzed by gel-pro analyzer software. The standard curve was established with standard protein as a marker. The Rf values of alliinase and lectin were substituted for the standard curve equation to calculate their molecular mass. Results:The standard curve equation was lgM=-1.369 1 X+2.184 7,r=0.966 6. In the garlic herbs,the relative molecular mass of alliinase was 52.1-54.0 KDa,lectin was 11.0-11.8 KDa. The total relative content of alliinase and lectin was 18.1%-77.2% in garlic from different origins. The relative content of alliinase was 5.60%-27.1% and lectin was 12.5%-68.2%. The garlic sample from Xinjiang Balikun had the highest relative content of alliinase that was 27.1%. The garlic sample from Xinjiang Akesu Baicheng County had the highest relative content of lectin that was 68.2%. The contents of alliinase and lectin in garlic were different among different origins. The relative content of lectin was generally higher than alliinase. Conclusions:Compared with other reported methods of determining the protein content,this method has the advantages of convenient operation,small amount of required sample and can accurately measure the relative molecular mass and the relative content of alliinase and lectin in the garlic. The total relative contents of alliinase and lectin were more than 50% from difference origins of garlic,which proved that alliianse and lectin were the main proteins in garlic. The preliminary results indicate that there is no significant correlation between the amount of alliinase and alliin.
Objective:To establish a method that simultaneously determine relative content and molecular mass of alliinase and lectin in garlic from the different origins by SDS-polyacrylamide gel electrophoresis. Methods:The optimal conditions of the method were confirmed by investigating the factors of solvent methods,sample pretreatment method and the test solution concentrations. The relative content and the molecular mass of alliinase and lectin in garlic were measured by SDS-polyacrylamide gel electrophoresis. The optimal conditions were as follows:ultra-pure water as solvent,stationary for 10 minutes as pretreatment method,the sample solution concentration was 28 mg·m L-1. And 12% separation gel,5% concentrated gel were separately prepared. The gel thickness was 1 mm. The concentrated gel voltage was 80 V,and the concentrated gel voltage was 100 V. Then the discontinuous system and vertical plate electrophoresis were applied. The map of SDS-polyacrylamide gel electrophoresis was analyzed by gel-pro analyzer software. The standard curve was established with standard protein as a marker. The Rf values of alliinase and lectin were substituted for the standard curve equation to calculate their molecular mass. Results:The standard curve equation was lgM=-1.369 1 X+2.184 7,r=0.966 6. In the garlic herbs,the relative molecular mass of alliinase was 52.1-54.0 KDa,lectin was 11.0-11.8 KDa. The total relative content of alliinase and lectin was 18.1%-77.2% in garlic from different origins. The relative content of alliinase was 5.60%-27.1% and lectin was 12.5%-68.2%. The garlic sample from Xinjiang Balikun had the highest relative content of alliinase that was 27.1%. The garlic sample from Xinjiang Akesu Baicheng County had the highest relative content of lectin that was 68.2%. The contents of alliinase and lectin in garlic were different among different origins. The relative content of lectin was generally higher than alliinase. Conclusions:Compared with other reported methods of determining the protein content,this method has the advantages of convenient operation,small amount of required sample and can accurately measure the relative molecular mass and the relative content of alliinase and lectin in the garlic. The total relative contents of alliinase and lectin were more than 50% from difference origins of garlic,which proved that alliianse and lectin were the main proteins in garlic. The preliminary results indicate that there is no significant correlation between the amount of alliinase and alliin.
引文
[1]马丽娜,李峰杰,陈坚,等.大蒜主要活性成分及药理作用研究进展[J].中国药学通报,2014,33(6):760MA LN,LI FJ,CHEN J,et al.Progress of garlic main active ingredients and pharmacological research[J].Chin Pharm Bull,2014,33(6):760
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[10]陈德西,何忠全,向运佳,等.大蒜叶凝集素基因ASAL的克隆与表达载体的构建[J].西南农业学报,2016,29(10):2445CHEN DX,HE ZQ,XIANG YJ,et al.Cloning of ASAL gene and construction of its plant expression vector[J].Southwest China JAgric Sci,2016,29(10):2445
[11]刘琴,喻艳华,陈伟达,等.菜豆凝集素抑制剂的设计与凝血活性[J].高等学校化学学报,2017,38(7):1185LIU Q,YU YH,CHEN WD,et al.Design of common bean lectin in inhibitor and tts hemagglutination activity[J].Chem J Chin Univ,2017,38(7):1185
[12]SANTOSH KU,RAHUL S,SINGH R,et al.Purification and characterization of a lectin with high hemagglutination property isolated from Allium altaicum[J].Protein J,2011,30:374
[13]冀爱青,韩红艳,杨红雁,等.核桃凝集素的提取及其性质[J].食品科学,2014,35(20):23JI AQ,HAN HY,YANG HY,et al.Extraction and characterization of walnut(Juglans regia)kernel lectin[J].Food Sci,2014,35(20):23
[14]SANTOSH KU,SINGH PK.receptors of garlic(Allium sativum)Lectins and their role in insecticidal action[J].Protein J,2012,31:439
[15]BOSE S,LAHA B,BANERJEE S.Quantification of allicin by high performance liquid chromatography-ultraviolet analysis with effect of post-ultrasonic sound and microwave radiation on fresh garlic cloves[J].Phcog Mag,2014,10(38):288
[15]GUO T,SU D,HUANG Y,et al.Ultrasound-assisted aqueous twophase system for extraction and enrichment of Zanthoxylum armatum Lignans[J].Molecules,2015,20:15273
[17]王旭,陈坚,李新霞.新疆4种地产大蒜的高效薄层氨基酸分析[J].新疆中医药,2010,28(2):51WANG X,CHEN J,LI XX.Analysis of 4 kinds of amino acids by HPTLC of Xinjiang estate garlic[J].Xinjiang J Tradit Chin Med Pharm,2010,28(2):51
[18]曹红,袁耀佐,陈坚.大蒜蒜酶/蒜氨酸催化动力学及临床应用研究[J].时珍国医国药,2009,20(7):1659CAO H,YUAN YZ,CHNE J.Enzymatic properties and kinetic charactristics of homogeneous alliinase from garlic[J].Lishizhen Med Mater Med Res,2009,20(7):16592017 7 28
[2]BORLINGHAUS J,ALBRECHT F,GRUHLKE M CH,et al.Allicin:chemistry and biological properties[J].Molecules,2014,19,12591
[3]李新霞,赵东升,关明,等.有关2010年版中国药典大蒜质量标准的几点建议[J].药物分析杂志,2012,32(4):682LI XX,ZHAO DS,GUAN M,et al.Suggestions,about the specification of garlic in Chinese Pharmacopeia(2010)[J].Chin JPharm Anal,2012,32(4):682
[4]魏宁漪,吴建敏,张启明.关于大蒜和大蒜素质量标准的商榷[J].药物分析杂志,2011,31(1):80WEI NY,WU JM,ZAHNG QM.Discussion on drug specification of garlic and alltride[J].Chin J Pharm Anal,2011,31(1):180
[5]赵东升,李新霞,张海波,等.以大蒜辣素为指标研究大蒜质量标准[J].药物分析杂志,2013,33(9):1587ZHAO DS,LI XX,ZHANG HB,et al.Study on quality specification of garlic with allicin as index components[J].Chin J Pharm Anal,2013,33(9):1587
[6]APPLE E,EBERHARD AV,RABINKOV A,et al.Therapy of murine pulmonary aspergillosis with antibody-alliinase conjugates and alliin[J].Antimicrob Agents CH,2010,54(2):898
[7]APPLE E,RABINKOV A,NEEMAN M,et al.Conjugates of daidzein-alliinase as a targeted pro-drug enzyme system against ovarian carcinoma[J].J Drug Target,2011,19(5):326
[8]SHIMON LINDA JW,RABINKOV A,SHIN I,et al.Two structures of alliinase from Alliium sativum L.:apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the PLPcofactor[J].J Mol Biol,2007,336:615
[9]TCHEMYCHEV B,RABINKOV A,MIRELMAN D,et al.Natural antibodies to dietary proteins:the existence of natural antibodies to alliinase(Alliin lyase)and mannose-specific lectin from garlic(Allium sativum)in human serum[J].Immunol Lett,1995(47):53
[10]陈德西,何忠全,向运佳,等.大蒜叶凝集素基因ASAL的克隆与表达载体的构建[J].西南农业学报,2016,29(10):2445CHEN DX,HE ZQ,XIANG YJ,et al.Cloning of ASAL gene and construction of its plant expression vector[J].Southwest China JAgric Sci,2016,29(10):2445
[11]刘琴,喻艳华,陈伟达,等.菜豆凝集素抑制剂的设计与凝血活性[J].高等学校化学学报,2017,38(7):1185LIU Q,YU YH,CHEN WD,et al.Design of common bean lectin in inhibitor and tts hemagglutination activity[J].Chem J Chin Univ,2017,38(7):1185
[12]SANTOSH KU,RAHUL S,SINGH R,et al.Purification and characterization of a lectin with high hemagglutination property isolated from Allium altaicum[J].Protein J,2011,30:374
[13]冀爱青,韩红艳,杨红雁,等.核桃凝集素的提取及其性质[J].食品科学,2014,35(20):23JI AQ,HAN HY,YANG HY,et al.Extraction and characterization of walnut(Juglans regia)kernel lectin[J].Food Sci,2014,35(20):23
[14]SANTOSH KU,SINGH PK.receptors of garlic(Allium sativum)Lectins and their role in insecticidal action[J].Protein J,2012,31:439
[15]BOSE S,LAHA B,BANERJEE S.Quantification of allicin by high performance liquid chromatography-ultraviolet analysis with effect of post-ultrasonic sound and microwave radiation on fresh garlic cloves[J].Phcog Mag,2014,10(38):288
[15]GUO T,SU D,HUANG Y,et al.Ultrasound-assisted aqueous twophase system for extraction and enrichment of Zanthoxylum armatum Lignans[J].Molecules,2015,20:15273
[17]王旭,陈坚,李新霞.新疆4种地产大蒜的高效薄层氨基酸分析[J].新疆中医药,2010,28(2):51WANG X,CHEN J,LI XX.Analysis of 4 kinds of amino acids by HPTLC of Xinjiang estate garlic[J].Xinjiang J Tradit Chin Med Pharm,2010,28(2):51
[18]曹红,袁耀佐,陈坚.大蒜蒜酶/蒜氨酸催化动力学及临床应用研究[J].时珍国医国药,2009,20(7):1659CAO H,YUAN YZ,CHNE J.Enzymatic properties and kinetic charactristics of homogeneous alliinase from garlic[J].Lishizhen Med Mater Med Res,2009,20(7):16592017 7 28