Evaluation Of The Aprotinin Potency And The Antioxidant And Inhibition Of Alpha-glucosidase Activities Of Isoflavones By Capillary Electrophoresis

Capillary electrophoresis has been widely used in the measurement of enzyme potency,in the evaluating and screening of antioxidant substances and enzyme inhibitors because of its low reagent and sample consumption and high separation efficiency.At the same time,the capillary can be used as a reaction container.In this study,capillary electrophoresis-ultraviolet detection methods for the measurement of aprotinin potency and for evaluation of antioxidant and inhibitory ?-glucosidase activity of isoflavones were developed.The established methods were simple and fast,and the main research works are as follows:(1)A new method for the measurement of aprotinin potency by capillary electrophoresis ultraviolet detection was established for the first time.The on-line mixing of substrate,trypsin and aprotinin using at-inlet technology was realized by the established method.Enzymatic reaction,separation and detection of substrate and product can be performed simultaneously online.The aprotinin potency can be measured within 4 min.The response surface methodology was used to optimize the incubation conditions of trypsin and substrate,and the optimized conditions were obtained under 17.39 mM phosphate buffer at pH 7.6 and 1.40 min of incubation time.The repeatability of proposed method was evaluated in three different systems of capillary zone electrophoresis:(i)only substrate;(ii)trypsin and substrate;(iii)aprotinin,trypsin and substrate,and the RSDs of migration times and peak areas of substrate were less than 2.7% and 3.1%,respectively.The RSDs of migration times and peak areas of product were less than 2.1% and 3.0%,respectively.A formula was also developed to calculate the aprotinin potency in this method.In a word,the established capillary zone electrophoresis-ultraviolet method was convenient,fast and environmentally friendly for the measurement of aprotinin potency.(2)A new capillary electrophoresis method for the rapid separation and determination of six isoflavones was established with ?-cyclodextrin as the modifier for the first time,and its antioxidant activity was also evaluated combined with 1,1-diphenyl-2-picrylhydrazyl.The response surface design was used to optimize the effects of the concentration and the pH of borax,the concentration of ?-cyclodextrin and the content of methanol on the separation and determination of the six isoflavones,with the resolution and total migration time as the response indicators.The optimum conditions were obtained under 25 mM borax,5 mM ?-cyclodextrin and 20 % methanol at pH 8.9,and the total migration time was within 8 min.Finally,the established method was verified and applied to the determination of isoflavones in Astragalus extracts,Astragalus essence oral liquid and Astragalus injection,and it was also applied to the evaluation of antioxidant activity of the Astragalus injection and the Astragalus extracts.(3)The electrophoretically mediated microanalysis filling technique was used to evaluate the inhibitory activity of nine isoflavones on ?-glucosidase,and the molecular docking was also used to predict and explain the interaction mode between the nine isoflavones and ?-glucosidase.The relationship between structure of the isoflavones and inhibitory activity of ?-glucosidase was preliminarily explored.In this study,the single rotation method was used to optimize the separation conditions of the substrate p-nitrophenol-?-D-glucoside and the product p-nitrophenol.The optimum conditions were obtained under 20 mM borax at pH 8.5 and the applied voltage at 20 kV.The enzymatic reaction conditions including sequence of injection,concentration of incubation buffer,injection length of the incubation buffer and incubation time were also optimized.Finally,the inhibitory activities of the nine isoflavones were evaluated by the optimized method,and the peak height changes of the nine isoflavones as inhibitors in the enzymatic reaction were studied,which provided the possibility for the development of high-throughput capillary electrophoresis method to evaluate the activity of inhibitors.