建立基于96孔板检测的FRAP法和DPPH法及其在橘核抗氧化活性研究中的应用

Establishment of Improved FRAP and DPPH Methods based on 96-well Plate Detection and Its Application in Evaluating the Antioxidant Capacity ofCitrus Seeds

目的建立基于96孔板检测的样品消耗小、实验结果准确稳定的改良微型铁离子还原/抗氧化力测定法(ferric reduction ability plasma assay,FRAP)和1,1-二苯基-2-三硝基苯肼自由基清除能力测定法(2,2diphenyl-1-picrylhydrazyl assay,DPPH),并将之应用于橘核抗氧化活性评价。方法首先,分别考察不同反应温度、反应体系以及反应时间对于FRAP法和DPPH法检测抗坏血酸(Aa)抗氧化能力的影响,选择Aa的浓度和抗氧化能力之间线性关系最佳的反应条件作为最适反应条件并建立这两种方法的微量模型。其次,通过精密度、回收率以及标准品对照实验验证微量模型的稳定性及准确性。最后,将微型FRAP,DPPH法应用于橘核提取物(Cse)及其主要活性成分柠檬苦素(Lim)、诺米林(Nom)、橙皮苷(Hes)的抗氧化活性评价。结果微型FRAP法将150μl TPTZ工作液与5μl待测抗氧化剂溶液作为反应体系且选择在30℃温育反应,反应时间为5 min。DPPH法将160μl DPPH溶液与40μl待测抗氧化剂溶液作为反应体系且选择于25℃保温显色,显色时间为30 min。用这2种改良微型法获得的Aa,Cse和Hes的AEAC值分别为1.00,187.42,19.31g·L-1,EC50值分别为0.06,24.83,4.62 g·L-1。结论基于96板检测的微型FRAP,DPPH法样品消耗量小,实验结果稳定可靠且筛选效率高。Aa的抗氧化能力>Hes>Cse,而Lim,Nom不具抗氧化能力,故该实验认为橘核中抗氧化作用的主要物质基础为Hes。

Objective To establish improved FRAP）and DPPH methods based on 96 -well plate detection with less sample con- sumption but more accurate and stable experimental results. Then we apply them to evaluate the antioxidant capacity of citrus seeds. Methods The effects of different reaction temperature,reaction system ,as well as reaction time were investigated in order to detect the accuracy,linearity and stability of miniature FRAP and DPPH methods,then these two improved methods were ap- plied to evaluate the antioxidant capacity of citrus seed extracts and its main active ingredients such as [imonin, nomilin and hes- peridin. Results 150 pJ TPTZ working fluid and 5 μl ascorbic acid solution were added into 96 - well plate and incubated at 30 ℃for 5 rain. 160μl DPPH solution and 40 bd aseorbic acid solution were added into 96 - well plate and incubated at 25 ℃for 30 rain. The AEAC values of aseorbie acid,citrus seed extracts and hesperidin were 1.00,187.42,19.31 g·L-1 ,and the EC50 values were 0. 06 ,24. 83 ,4. 62 g ~ L- ~ respectively. Conclusion The improved FRAP and DPPH methods have the advantages of less sample consumption and more stable, reliable efficiency. From the AEAC values and the EC50 values, we concluded that the antioxidant capacity of ascorbic acid 〉 hesperidin 〉 citrus seed extracts. Limonin and nomilin do not show any antioxidant capacity in this experiment,so the material basis of antioxidant capacity is hesperidin.