牡荆总黄酮提取工艺优化及其对产气荚膜梭菌抑菌活性研究

Study on Optimization of Extraction Process and Antibacterial Activity to Clostridium Perfringens of Total Flavonoids from Vitex negundo L.var.cannabifolia(Sieb.et Zucc.)Hand.-Mazz.

【目的】探究牡荆总黄酮的提取工艺,并探讨牡荆提取物对产气荚膜梭菌的抑菌活性。【方法】以芦丁作为对照品,采用分光光度法对牡荆中的总黄酮含量进行测定;以总黄酮含量为考察指标,先通过单因素试验研究乙醇浓度、提取时间、提取温度、料液比4个单因素对牡荆总黄酮提取量的影响,确定单因素情况下最佳工艺的水平范围;在单因素试验基础上,通过设计L9(3^(4))正交试验,利用NaNO_(2)-AL(NO_(3))_(3)-NaOH显色法对牡荆总黄酮含量进行测定,绘制标准曲线,计算回归方程,通过精密度、稳定性、重复性、加样回收率进行方法学考察,验证标准曲线。通过将试验所得数据代入标准曲线,比较得出牡荆总黄酮的最佳制备工艺,并在此基础上通过微量二倍稀释法考察其提取物对产气荚膜梭菌的抗菌活性。【结果】牡荆总黄酮最佳提取工艺条件为:乙醇浓度60%、提取温度50℃、提取时间2.5 h、料液比1∶35。在此最优提取条件下,牡荆总黄酮含量最高(43.17 mg/g)。其因素影响顺序为料液比>提取时间>乙醇浓度>提取温度,料液比对牡荆总黄酮含量影响最大。最佳工艺下的牡荆提取物对产气荚膜梭菌的最小抑菌浓度(MIC)值为7.81μg/mL。【结论】在乙醇浓度60%、提取温度50℃、提取时间2.5 h、料液比1∶35的条件下提取的牡荆总黄酮含量最高,所制备提取物对产气荚膜梭菌具有较强的抑菌作用,为生产高质量的牡荆提取物提供了理论依据。

【Objective】This study was aimed to explore the extraction technology of total flavonoids from Vitex negundo L.var.cannabifolia(Sieb.et Zucc.)Hand.-Mazz.(V.negundo)and its antibacterial activity against Clostridium perfringens.【Method】Rutin was used as reference substance and the content of total flavonoids in V.negundo was determined by spectrophotometry.Taking the content of total flavonoids as the investigation index,the effects of ethanol concentration,extraction time,extraction temperature and solid-liquid ratio on the extraction amount of total flavonoids from V.negundo were studied through single factor test,and the level range of the best process under the condition of single factor was determined.On the basis of single factor test,through the design of L9(34)orthogonal test,the content of total flavonoids in V.negundo was determined by NaNO_(2)-AL(NO_(3))_(3)-NaOH color development method,the standard curve was drawn,the regression equation was calculated,and the standard curve was verified through the methodological investigation of precision,stability,repeatability and sample recovery.By substituting the data obtained by the experiment into the standard curve,the best preparation process of total flavonoids of V.negundo was obtained,and on this basis,the antibacterial activity of its extract against Clostridium perfringens was investigated by micro double dilution method.【Result】The optimum extraction conditions of total flavonoids from V.negundo were ethanol concentration of 60%,extraction temperature of 50℃,extraction time of 2.5 h and solid-liquid ratio of 1∶35.Under the optimum extraction conditions,the content of total flavonoids in V.negundo was the highest(43.17 mg/g).The order of influencing factors was solid-liquid ratio>extraction time>ethanol concentration>extraction temperature.The solid-liquid ratio had the greatest influence on the content of total flavonoids in V.negundo.The minimum inhibitory concentration(MIC)of V.negundo extract against Clostridium perfringens was 7.81μg/mL.【Conclusion】Under the conditions of ethanol concentration of 60%,extraction temperature of 50℃,extraction time of 2.5 h and solid-liquid ratio of 1∶35,the content of total flavonoids extracted from V.negundo was the highest.The prepared extract had strong antibacterial effect on Clostridium perfringens,which provided a theoretical basis for the production of high-quality V.negundo extract.