基于ABTS自由基的薄层色谱-生物自显影新方法的建立及其在中药抗氧化活性评价中的应用

目的：建立一种基于2,2-氨基-二（3-乙基-苯并噻唑啉-6-磺酸）二铵盐（ABTS）自由基清除能力活性筛选的薄层色谱-生物自显影新方法。方法：将含有14 mmol/L ABTS和4.9 mmol/L过硫酸钾混合水溶液10 ml,置于暗处12～16 h制备ABTS＋.,用75%乙醇将ABTS＋.溶液稀释至100 ml,作为显色剂。将点有样品（或展开）的薄层板喷显色剂,在可见光下检视或在734 nm下扫描。对显色剂、衍生化方法、薄层板、检测波长等参数进行优化使薄层板获得明显的蓝色背景色。结果：具有清除ABTS自由基活性的化合物在薄层板的蓝色背景下呈现白色斑点。采用建立的TLC-生物自显影法结合扫描仪测定6-羟基-2,5,7,8-四甲基苯并二氢吡喃-2-羧酸（Trolox）,叔丁基羟基茴香醚（BHA）,2,6-二叔丁基对甲酚（BHT）,特丁基对苯二酚（TBHQ）和没食子酸丙酯（PG）等抗氧化剂的ABTS自由基清除能力,结果抗氧化能力顺序为：PG〉BHA〉TBHQ〉麦角甾苷〉BHT〉Trolox〉野黄芩苷,与分光光度法测定结果一致;并采用TLC-生物自显影法对不同来源的中药肉苁蓉和灯盏花样品及其所含清除ABTS自由基活性成分进行了分析和评价,发现肉苁蓉和灯盏花中很多成分具有清除ABTS自由基作用,购自上海的肉苁蓉和购自云南文山的灯盏花的抗氧化活性强于其他来源样品。结论：该方法能够测定和评价单体化合物和复杂组分（如中药提取物）中化合物的ABTS自由基清除能力。

Establishment and Application of a New TLC-bioautography Technology in Antioxidant Activity Evaluation of Materia Medica

Objective： To develop a TLC-bioautographic method to detect compounds of scavenging ABTS radical capacity in plant extract.Methods： Mixture aqueous solution containing 14 mmol/L ABTS and 4.9 mmol/L Potassium persulfate,placed in the dark for 12-16 h to prepare ABTS＋·,then added 75% ethanol to dilute the ABTS＋· solution to 100 ml,as the ABTS reagent solution.After migration of the samples,the TLC plate was sprayed with ABTS reagent solution,detected in visible light or scanned under wavelength at 734 nm.In addition,the parameters including color developing agent,derivatization method,thin layer plate and wavelength were optimized to get a clear blue background colouration.Results： The compounds with scavenging ABTS radical activity were visualized as white spots on the TLC plates.The scavenging ABTS radical capacity of antioxidants detected by scanning the bands of Trolox,BHA,BHT,PG,TBHQ,acteoside and scutellarin on the plate were： PG＆gt;BHA＆gt;TBHQ＆gt;Acteoside＆gt;BHT＆gt;Trolox＆gt;Scutellarin,and the results were consistent with that of the spectrophotometry method.In addition,the new TLC-bioautographic method was used to analyze and evaluate antioxidant capacity of the active ingredients in Cistanches Herba and Erigerontis Herba.The antioxidant activity of Cistanches Herba bought from Shanghai and Erigerontis Herba bought from Yunnan is better than other.Conclusion： This method is able to detect and evaluate scavenging ABTS radical capacity in monomeric compound and materia medica.