Chemical Constituents And ɑ-glucosidase Inhibitory Activities Of Ribes Meyeri

In previous studies,it was found that the extract of Ribes meyeri L.had good hypoglycemic activity,but the active ingredients were not clear.In this thesis,the chemical constituents of the fruits,stems and leaves of Ribes meyeri were isolated and purified.The extraction and purification process of anthocyanins from the fruits of Ribes meyeri was optimized.Fourteen compounds were isolated and identified from the extracts of fruits,stems and leaves.These compounds were isolated from the plant for the first time.The inhibitory activity of monomeric compounds on α-glucosidase was studied by molecular docking,and the inhibitory activity of monomeric compounds on α-glucosidase was determined.The research content includes the following three aspects:1.Extraction,purification and identification of anthocyanins from Ribes meyeri fruit: The extraction process of anthocyanins from Ribes meyeri fruit was optimized by single factor test and orthogonal test.The optimized extraction conditions were as follows: ethanol concentration 40%,hydrochloric acid concentration 1%,extraction time 60 min,solid-liquid ratio 1:40(g/m L).Then,the adsorption and desorption characteristics of 26 macroporous resins on anthocyanins from Ribes meyeri fruit were studied.Among them,AB-8 macroporous resin showed the best adsorption and desorption ability.The adsorption process mechanism was analyzed by pseudo-first-order and pseudo-second-order models.The adsorption kinetics of AB-8 resin on anthocyanin from Ribes meyeri fruit was more in line with the pseudo-second-order model.Through the analysis of Langmuir and Freundlich isotherm models,the adsorption of anthocyanins by AB-8 resin on Ribes meyeri fruit conforms to the Langmuir isotherm adsorption model.The dynamic adsorption and desorption tests were carried out on AB-8 resin column.The results showed that the sample flow rate of 1 m L/min and the sample volume of 80 m L were the best parameters for adsorption.The elution flow rate of 1.5 m L/min and the desorption amount of 30 m L were the best parameters for desorption.The purification of anthocyanins was analyzed by liquid chromatography-mass spectrometry.The results showed that the impurity peak of anthocyanin was significantly reduced after purification with AB-8 macroporous resin.The anthocyanin fraction purified by AB-8 macroporous resin was separated by Toyopearl HW-40 C gel column and preparative liquid chromatography,and three compounds were obtained.Their structures were identified by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy as cyanidin,cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside.2.Isolation and identification of chemical constituents from stems and leaves of Ribes meyeri: The stems and leaves of Ribes meyeri were extracted with methanol water,and the extracts were extracted with petroleum ether,dichloromethane and ethyl acetate,respectively.The ethyl acetate and dichloromethane fractions of stems and leaves of Ribes meyeri were isolated and purified by silica gel column chromatography,MCI resin and gel column chromatography.The isolated compounds were identified by NMR and HR-LS/MS.Nine compounds were isolated and identified from the ethyl acetate fraction,which were epigallocatechin,caffeic acid,protocatechuic acid,gallic acid,catechin,kaempferol-3-O-rutinoside,sinapic acid,rutin and isoquercitrin.Two compounds were isolated and identified from the dichloromethane fraction,which were nobiletin and hydroxygenkwanin.3.The ɑ-glucosidase inhibitory activity of the chemical constituents of Ribes meyeri: There are two main methods for the determination of ɑ-glucosidase inhibitory activity.One is the traditional method,using p NPG as the substrate,α-glucosidase hydrolyzes it to generate p-nitrophenyl-α-Dglucopyranoside,and the enzyme activity is determined by its color reaction.However,the sample in this method has great interference with the test results and is easy to produce false positive results.In this thesis,a method for the determination of ɑ-glucosidase inhibitory activity with maltose as substrate was established.The traditional method was replaced by LC-MS method to determine the released glucose.This method has high specificity and more accurate results.In this thesis,two methods were used to determine the ɑ-glucosidase inhibitory activity of different extraction parts and monomer compounds of Ribes meyeri,and the ɑ-glucosidase inhibitory activity of 14 isolated compounds was verified by molecular docking.Among them,cyanidin,epigallocatechin and hydroxygenkwanin have the best inhibitory activity,which may be the hypoglycemic active ingredients in Ribes meyeri.