Study On Extraction,isolation,and In Vitro Activity Of Isoflavones From Cicer Arietinum L. And Glycine Max L.merr.

Chickpea（Cicer arietinum L.）and black bean（Glycine max L.merr.）are leguminous seeds,which belong to annual herbs or perennial climbing herbs.They have been planted in China for more than 2000 years.Modern studies show that the main components of chickpeas and black beans are isoflavones,which have medicinal value,such as anti-inflammatory,antioxidant,and anti-tumor properties.At present,most studies on isoflavones from chickpeas and black beans focus on extraction,but there are few reports on isolation,purification,and activity of isoflavones.Therefore,this paper,focuses on the extraction,separation,purification and activity screening of chickpea and black bean isoflavones and lays a foundation for the development and utilization of these natural products.In this study,the isoflavones from chickpeas and black beans were extracted,isolated,and analyzed in vitro by using mathematical model,liquid chromatography mass spectrometry（LC-MS）,and chromatography based on an anti-inflammatory mechanism.Furthermore,a rapid screening method for isoflavones in leguminous chickpeas and black beans was established.Firstly,a response surface model combined with a genetic algorithm was used to optimize the extraction process of isoflavones from chickpeas.An LC/MS method was developed for identifying isoflavones.Ultrafiltration mass spectrometry（UF-LC/MS）was used to screen potential xanthine oxidase（XOD）inhibitors in chickpeas.Semi-preparative liquid chromatography combined with high-speed countercurrent chromatography（CCC）was used to isolate XOD inhibitors from chickpeas.The structure analysis and preliminary identification of the four isolated inhibitors were conducted by using mass spectrometry（MS）.Four XOD inhibitors were identified,namely,daidzin,daidzein calycosin,and biochanin A.The purities were 95.27%,90.08%,97.45%,and 97.24%,respectively.Secondly,a response surface model combined with particle swarm optimization was used to optimize the extraction process of isoflavones from black beans.An LC-MS method was developed to rapidly identify isoflavones in black beans.Five potential XOD inhibitors were screened by UF-LC-MS.Five active components from black beans were isolated and purified by semi-preparative liquid chromatography-gradient elution reflux chromatography.The purified fractions were daidzein（80.53%）,daidzein（96.17%）,genistin（94.13%）,daidzein（97.23%）,and genistein（98.44%）.Finally,enzyme kinetics was used to evaluate the inhibition of XOD.Seven active components were isolated from chickpeas and black beans,and the mechanism of action was determined by a Lineweaver-Burk（double reciprocal）plot.The results showed that chickpeas,black beans,and the 7 isolated compounds（daidzin,glycitein-7-O-glucoside,genistin,daidzein,genistein,calycosin,and biochanin A）had a good inhibitory effect on XOD,with IC₅₀ values of 23.11,25.17,35.08,56.22,30.76,68.79,95.37,72.14,and 67.48μg/m L,respectively.According to the IC₅₀ value,the inhibitory effect of XOD inhibitors on XOD was determined as follows:genistin>daidzin>glycitein-7-O-glucoside>biochanin A>daidzein>calycosin>genistein.Depending on the type of inhibition experiment,the compounds daidzein,genistin,daidzein,calycosin,and biochanin A all showed reversible inhibition effect on XOD.Daidzin,genistin,and calycosin were competitive inhibitors with inhibition constants（K_i）of 201.19,306.47,and 782.31μg/m L,respectively.Daidzein and calycosin showed noncompetitive inhibition with K_i of 373.69μg/m L and 466.09μg/m L,respectively.In conclusion,this paper takes chickpea and black bean as the research object,and combines mathematical model with extraction experiment.Ultrasonic assisted enzymatic hydrolysis,aqueous n-butanol extraction,and mathematical modelling were used to optimize the extraction process of isoflavones from chickpeas and black beans.The potential XOD inhibitors were screened by UF-LC-MS.HSCCC combined with semi-preparative HPLC was used to separate and purify the isoflavones.The inhibitory ability of isoflavones on XOD was evaluated by kinetic calculation of the enzymatic catalyzed reaction,which provided experimental data for the development of natural anti-inflammatory drugs for gout.