苦参抗水产病原哈氏弧菌活性物质的提取、鉴定及作用机制  

Extraction,Identification and Mechanism of Action of Antibacterial Substances of Chinese Herb Medicine Sophora flavescens Against Aquatic Pathogen Vibrio harveyi

作　　者：郭斯崖 郑慧芳 张淼 蔡静 王鑫彤 郭雷 GUO Siya;ZHENG Huifang;ZHANG Miao;CAI Jing;WANG Xintong;GUO Lei(Jiangsu Key Laboratory of Marine Bioresources and Environment,Co-Innovation Center of Jiangsu Marine Bio-industry Technology,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Key Laboratory of Marine Biotechnology,School of Ocean Food and Biological Engineering,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening,School of Pharmacy,Jiangsu Ocean University,Lianyungang 222005,China)

机构地区：[1]江苏海洋大学江苏省海洋生物产业技术协同创新中心,江苏省海洋生物资源与环境重点实验室,江苏连云港222005 [2]江苏海洋大学海洋食品与生物工程学院,江苏省海洋生物技术重点实验室,江苏连云港222005 [3]江苏海洋大学药学院,江苏省海洋药物活性分子筛选重点实验室,江苏连云港222005

出　　处：《水产学杂志》2023年第4期29-37,共9页Chinese Journal of Fisheries

基　　金：江苏省高校自然科学基金项目(19KJB350001);江苏省高校优势学科建设工程资助项目(PAPD).

摘　　要：利用单因素实验、Box-Behnken试验设计结合响应面分析法,从提取溶剂(蒸馏水、50%甲醇、甲醇、50%乙醇、乙醇)、乙醇浓度(60%~100%)、提取温度(60℃、70℃、80℃、90℃、100℃)、提取时间(60 min、90 min、120 min、150 min、180 min)和液料比等对抑菌活性的影响几方面优化苦参抗水产病原哈氏弧菌(Vibrio harveyi)活性物质(ASSF)的提取工艺;采用高效液相色谱和标准品比对技术,鉴定ASSF的抗菌活性化合物;从对哈氏弧菌的生长、细胞膜的完整性和通透性、细胞壁的通透性等方面探讨ASSF的作用机理。结果表明:ASSF的最佳提取条件是:无水乙醇为溶剂,液料比30∶1 mL/g,提取温度79℃,提取146 min。在此条件下,提取物抑菌圈的实际值为(17.64±0.22)mm,与预测值无显著性差异。固态ASSF的得率为(21.17±0.91)%。鉴定出苦参酮是ASSF中的抗哈氏弧菌活性化合物。ASSF和苦参酮的最小抑菌浓度分别为0.125 mg/mL和0.0625 mg/mL。ASSF对于哈维氏弧菌的抑制作用主要是通过破坏细胞膜的完整性和通透性、细胞壁的通透性抑制细胞的增殖。本研究结果可为进一步开发苦参酮在水产病害防治中的应用提供依据。The aim of this study was to optimize the extraction technology of anti-Vibrio harveyi active substances of Chinese herb medicine Sophora flavescens(ASSF),identify its antibacterial active ingredients and investigate its mechanism of action.The extraction process of ASSF was optimized from extraction solvent(distilled water,50%methanol,methanol,50%ethanol,ethanol),ethanol concentration(60%~100%),extraction temperature(60℃,70℃,80℃,90℃,100℃),extraction time(60 min,90 min,120 min,150 min,180 min)and the liquid-to-material ratio and other influences on the antibacterial activity by single factor experiment,Box-Behnken design combined with response surface methodology.The antibacterial active compounds of ASSF were identified by using high performance liquid chromatography and standard comparison technology,and the mechanism of ASSF was investigated in terms of the growth of V.harveyi,the integrity and permeability of the cell membrane,and the permeability of the cell wall.The best extraction of ASSF was observed under conditions of absolute ethanol as the solvent,extraction temperature of 79℃,extraction time of 146 min,and the liquid-to-material ratio of 30∶1 mL/g.Under these conditions,the average value of the inhibition zone of the extracts was found to be(17.64±0.22)mm,without significantly different from the predicted value,with solid ASSF yield of(21.17±0.91)%.Kurarinone was identified as the main antibacterial active compound against V.harveyi of ASSF which had the minimal inhibitory concentration(MIC)values of 0.125 mg/mL and kurarinone of 0.0625 mg/mL against V.harveyi.The inhibitory effect of ASSF on V.harveyi was primarily involved in destruction of the integrity and permeability of the cell membrane,and the permeability of the cell wall,thereby inhibiting cell proliferation.In conclusion,kurarinone has significant inhibitory activity on the aquatic pathogenic V.harveyi.The finding provides a basis for further development of kurarinone in the prevention and treatment of aquatic animal diseas

关 键 词：苦参 哈氏弧菌 抗菌活性 提取工艺 苦参酮 

分 类 号：S943[农业科学—水产养殖]