Screening Of Dipeptidyl Peptidase-Ⅳ Inhibition Components From Chrysanthemum Indicum L.based On Spectral Effect Relationship Analysis

Objective:To study the inhibition of dipeptidyl peptidase-IV（DPP-IV）activity of different plant parts and different polar extraction parts of Chrysanthemum indicum L.,and to study the differences of chemical components among these extracts screened the related chemicals.The compounds which may have inhibitory effect were predicted by general effect relationship analysis.The activity of these compounds was verified.Combined with blood component analysis to predict its oral absorption.Finally,compounds with inhibitory effect on DPP-IV activity were screened from Chrysanthemum indicum for further study.Methods:（1）In vitro DPP-IV study and UPLC-Q-TOF/MS analysis of extracts from various parts of Chrysanthemum indicum:（1）Extracts from root,stem,leaf and flower of Chrysanthemum indicumwere extracted by systematic solvent extraction method.（2）The inhibition of DPP-IV by different polar extracts of root,stem,leaf and flower of Chrysanthemum indicum was detected by an in vitro model of enzyme reaction with substrate（Glycine-proline-7-amino-4-methylcoumarin hydrobromate）,and the extracts were divided into high inhibition group and low inhibition group.（3）Mass Lynx 4.1 was used to collect the Mass spectral data of each extraction site by UPLC-Q-TOF/MS technique.（4）The mass spectrometry data were imported into the data processing software Progenesis QI for peak processing and peak alignment.（5）According to the grouping of activity inhibition rate,multivariate analysis of mass spectrometry data was performed using OPLS-DA in EZinfo 3.0 software to obtain variables importance projection（VIP）.（6）The ion fragments were analyzed in Progenesis QI software,which met the four conditions of P≤0.05,VIP>1,positive correlation with inhibition rate,and the maximum fold change between groups>5.（7）The potential active compound structure was predicted by comparing with the known chemical composition standard library of Chrysanthemum indicum.（2）Verification of potential inhibition of DPP-IV active components of Chrysanthemum indicum:Standard compounds were purchased according to the compound list speculated by spectral effect analysis,and the inhibitory effect of potential active ingredients on DPP-IV was detected in vitro inhibitory response model and Caco-2 cell model to verify the results of spectral effect analysis.（3）Isolation and purification of DPP-IV inhibitory components of Chrysanthemum indicum:（1）Gel column chromatography combined with C₁₈semipreparative liquid chromatography was used to separate and purify the compounds from the extracts with high activity.（2）In vitro inhibition model was used to detect the inhibitory effect of isolated monomer compounds on DPP-IV,and UPLC-Q-TOF-MS technology was used to obtain the molecular ion fragment information of these monomer compounds,And compared with the standard database to predict the structural formula.（4）Study on components absorbed into rat blood after oral administration of Chrysanthemum indicum:Male SD rats were used as animal models.Orbital blood was collected before drug administration as blank control.Total ethanol extract from four medicinal parts of Chrysanthemum indicum was given intragastric twice a day for 3 consecutive days.UPLC-Q-TOF-MS was used to analyze the differences between the comparative chemical components of blank serum and drug-containing serum of rats,and to screen the components,which into blood.By comparing with the database of known compounds of Chrysanthemum indicum,the structure of the components in blood of Chrysanthemum indicum was speculated.Results:（1）The methanol,ethyl acetate and dichloromethane fractions of Chrysanthemum indicum were the most active fractions.In spectrum-effect relationship between UPLC-Q-TOF/MS and inhibition of DPP-IV active compounds,a total of 38 compounds were identified,including 17 flavonoids,9 phenolic acids,7 terpenoids and 5 volatile oil compounds.（2）It was found that 16 compounds showed the inhibitory effect of DPP-IV activity on in vitro DPP-IV study.The inhibitory effects of 10compounds on DPP-IV activity were dose-dependent.The 10 compounds were ranked from low to high according to IC50,respectively.There were isoquercetin（52.21μM）,quercetin（101.22μM）,1,5-dicoffeylquinic acid（107.83μM）,3,5-dicoffeylquinic acid（113.89μM）,isorhamnetin（207.71μM）,1,3-dicoffeylquinic acid（216.83μM）,cryptochlorogenic acid（271.43μM）,neochlorogenic acid（430.11μM）,chlorogenic acid（515.95μM）,apigenin-7-O-β-D-glucoside（4856.19μM）.In the Caco-2 cell model,14 of the 16 compounds presented the dose-dependent inhibitory effects on DPP-IV activity.These 14 compounds was sequenced from low to high according to IC50 value as following:3,5-dicof-feanylquinic acid（24.11μM）,apigenin-7-O-β-d-glucoside（40.97μM）,1,5-dicoffeanylquinic acid（44.81μM）,apigenin（53.26μM）,1,3-dicoffeanylquinic acid（68.15μM）,luteolin（84.64μM）,neochlorogenic acid（88.68μM）,chlorogenic acid（90.79μM）,quercitrin（114.80μM）,cryptochlorogenic acid（147.21μM）,isoquercetin（181.99μM）,quercetin（252.90μM）,eupatilin（290.53μM）and isorhamnetin（2922.79μM）.（3）Fourteen monomers were purified by gel chromatography combined with C18 semi-preparative liquid chromatography.The purity of them reached85%,and the inhibitory rate of 11 monomers was greater than 10%at the concentration of 0.0167 mg/m L in DPP-IV inhibitory activity test.The structures of these 14 monomers were identified by UPLC-Q-TOF-MS analysis.The structures of these 14 monomers were showed a high degree of match compare with 1,5-dicafoylquinic acid,1,3-dicafoylquinic acid and3,5-dicafoylquinic acid.（4）A total of 37 compounds were identified from the blood.Base on the spectrum-effect relationship,15 active compounds can be inferred,including neochlorogenic acid,1,5-dicafeyl-quinic acid,1,3-dicafeylquinic acid,cryptochlorogenic acid,etc.Conclusions:The extracts of Chrysanthemum indicum presented the different inhibitory effects on DPP-IV.Among the 38 compounds inferred by spectrum-effect relationship analysis,14 compounds were verified to have DPP-IV inhibitory activity.The 14 monomers with strong inhibitory effect were prepared by gel chromatography column combined with C₁₈semi-preparation column.The structures of the 14 compounds were identified by UPLC-Q-TOF-MS technique,and the structures of the 14 compounds were showed a high degree of match compare to 1,3-dicafeyl-quinic acid,1,5-dicafeyl-quinic acid and 3,5-dicafeyl-quinic acid.The results of blood components of the root showed that the active compounds such as neochlorogenic acid,1,5-dicafeyl-quinic acid,1,3-dicafeylquinic acid and cryptochlorogenic acid were the blood components of the root.In this study,the compounds with inhibitory effect on DPP-IV were screened out from Chrysanthemum indicum,which could be used as lead compounds in DPP-IV inhibitor research.The results of this study provided a scientific basis for material basis research on the hypoglycemic effect of Chrysanthemum indicum.