基于Box-Behnken结合总评“归一值”优化贝加尔唐松草黄酮成分的提取工艺及其抗氧化和抑菌活性研究

Optimize the Extraction Process of Flavonoids From Thalictrum baicalense Turcz.ex Ledeb.Based on Box-Behnken Design Combined with Overall Desirability and Investigate Its Antioxidant and Antibacterial Activities

为优化贝加尔唐松草黄酮成分的提取工艺,考察总黄酮的抗氧化和抑菌活性,采用热回流提取技术,在单因素实验基础上,以贝加尔唐松草中总黄酮得率、蒙花苷得率及其二者的总评“归一值(OD)”为综合评价指标进行Box-Behnken响应面试验,优化贝加尔唐松草黄酮成分的最佳提取工艺;以总黄酮对1,1-二苯基-2-三硝基苯肼(DPPH)自由基、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS^(+)·)和羟自由基(·OH)的清除率为指标,评价其体外抗氧化活性,并用抑菌圈法评价其对6种供试菌的抑菌活性。贝加尔唐松草黄酮成分的提取工艺参数为:乙醇浓度58%,料液比1:28(g:m L),提取时间34 min,提取温度70℃,在此条件下,蒙花苷得率为5.23±0.04 mg·g^(-1),总黄酮得率为40.78±0.12 mg·g^(-1);贝加尔唐松草总黄酮清除DPPH、ABTS^(+)·和·OH 3种自由基的半抑制浓度(IC_(50))值分别为0.341 mg·mL^(-1)、0.115 mg·mL^(-1)和0.481 mg·mL^(-1),对6种供试菌的最低抑菌浓度(MIC)在12.5~50.0μg·mL^(-1);在实验浓度范围类,总黄酮对3种自由基的清除率和抑菌活性与其浓度均呈一定的关系。在Box-Behnken响应面优化的参数下,贝加尔唐松草总黄酮和蒙花苷的提取工艺稳定、提取得率高。并且发现总黄酮表现出了良好的清除自由基能力和抑菌活性,具有良好的应用潜力。本研究为贝加尔唐松草活性成分的深入研究和资源的综合利用奠定了良好的基础。

To optimize the extraction process of flavonoids from Thalictrum baicalense Turcz.ex Ledeb.and investigate the antioxidant and antibacterial activities of total flavonoids.On the basis of single factor experiment,a Box-Behnken design experiment was carried out by using the extraction yield of total flavonoids,the extraction yield of Linarin and their overall desirability (OD) values as comprehensive evaluation indicators to optimize the extraction process of flavonoids from T baicalense.The antioxidant activities were evaluated by the scavenging rate of total flavonoids to three free radicals including 1,1-diphenyl-2-picrylhydrazyl (DPPH),2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS^(+)·) and·OH.The antimicrobial effect of flavonoids to six test strains was evaluated by K-B paper AGAR diffusion method.The optimum extraction parameters were as follows:ethanol concentration was 59%,ratio of sample to solvent was 1:30 (g:mL),extraction time was 70 min and extraction temperature was 70℃.Under the conditions,the optimum extraction yield of total flavonoids as high as 40.78±0.12 mg·g^(-1) and 5.23±0.04 mg·g^(-1) of Linarin was extracted from T.Baicalense.Compared to the positive drugs VC and BHT,the total flavonoids of T.Baicalense showed stronger antioxidant activities in DPPH,ABTS^(+)·and·OH assay.The IC_(50) values of it for 3 free radicals were 0.341 mg·mL^(-1),0.115mg·mL^(-1) and 0.481mg·mL^(-1),respectively.And the flavonoids had excellent antimicrobial activity in vitro.The minimum inhibitory concentration (MIC) of the flavonoids ranged from 12.5-50μg·mL^(-1).In the experimental concentration range,the scavenging rate of total flavonoids on the three free radicals and its antibacterial activity showed a certain dose effect relationship with the sample concentration.Under the optimized parameters of the Box-Behnken experiment,the extraction process was stable and the extraction yield of total flavonoids and Linarin from T.Baicalense was high.And it is found that the extracted flavonoids showed good antioxidant and antibacterial activity.This research has laid a good foundation for the in-depth study of the active ingredients and provide a new direction for the comprehensive development of the T.Baicalense resources.