Studies On The Chemical Components Of Ilex Latifolia Thunb. And Its Interaction With Proteins

Ilex latifolia Thunb.(Aquifoliaceae),one of the species in Ilex genus,is an evergreen plant which is one of the maintream products of kudingcha.The separation,purification and structural identification of the chemical constituents of Ilex latifolia Thunb.have been reported,and the activity studies focused on crude extract or total saponins,but very few studies on the activity of monomeric compounds.In particular,the study of the binding mechanism of monomer compounds and proteins in the Ilex latifolia Thunb.has not been reported.Therefore,to obtain the monomeric compound,this paper systematically studied the chemical constituents of Ilex latifolia Thunb..The mechanism of the interactions of Latifolosides G,Latifolosides C and Kudinsoide G with BSA respectively had been investigated by spectroscopic methods.Used the spectroscopic methods to investigate the mechanism of the interactions of quercetin,rutin and kaempferol with xanthine oxidase respectively,which could provide the reference basis for the research of a new drug for treating gout.In this paper,we had systematically investigated the chemical constituents from the n-butanol extraction of 70% ethanol extract of Ilex latifolia Thunb.,20 compounds had been isolated,they were identified on the basis of spectral analysis and the physico-chemical characters.The structures of these compounds were elucidated as Latifolosides A,Latifolosides B,Latifolosides C,Latifolosides D,Latifolosides E,Latifolosides F,Latifolosides G,Latifolosides H,Latifolosides I,Kudinsoide A,Kudinsoide D,Kudinsoide F,Kudinsoide G,Ilekudinsoide E,IlexosideXV,Ursolic Acid and Oleanic acid.Ilexoside XV was isolated from this plant for the first time.The content of Latifoloside C was the highest,reaching 2.38%.In this paper,the interaction mechanism of Kudinoside G,Latifoloside C and Latifoloside G and BSA was studied by fluorescence spectroscopy and circular dichroism.They can cause BSA endogenous fluorescence to quench.At the temperature of 298 K,the fluorescence quenching degree of Kudinoside G,Latifoloside C and Latifoloside G to BSA was 22%,19%,22.7%.At the temperature of 310 K,the quenching degree was 17.7%,24.1%,27.1%.The quenching type was static quenching,and the 3 saponins formed complexes with BSA,and had one binding site.When the temperature is 298 K,the quenching constant Ka(L mol-1)of the 3 saponins and BSA were: 265.05,73.01,586.21.When the temperature is 310 K,the quenching constant Ka(L mol-1)of the 3 saponins and BSA were: 179.21,2000.32,1951.64.The thermodynamic equation shows that the interaction between Kudinoside G and BSA is hydrogen bond and van der Waals force.Latifoloside C,Latifoloside G and BSA are combined with hydrophobic forces.The results of circular dichroism show that,after binding to BSA,the degree of extension of BSA protein peptide chain was decreased.The hydrophobic group is structurally compact with ?-helix content increased.The results showed that the oleanolic-type Latifoloside C was more likely to bind to BSA than the ussulane-type Kudinoside G,and the reason may be related to the C-19 hydroxyl activity and the surrounding steric hindrance in its parent nucleus.In this paper,the interaction mechanism of quercetin,rutin,kaempferol and xanthine oxidase was studied by fluorescence spectroscopy and ultraviolet spectroscopy.All three compounds can make XO endogenous fluorescence obviously quenched.The quenching form is combined quenching,but mainly in static.Since there are two binding sites,a stable complex is formed.At the temperature of 288 K,respectively,The binding constant Ka(107L mol-1)of the three compounds to XO are,16.39,8.27,9.76.At the temperature of 310 K,respectively,the binding constant Ka(107L mol-1)of the three compounds to XO are 14.78,7.44,8.733.Thermodynamic studies have shown that the action of the three compounds with XO is entropy-driven exothermic.By analyzing the structure and binding ability of compounds,the results show that,quercetin with more active hydroxyl groups has stronger ability to bind than kaempferol,the protein conformation changed significantly.Because of the glycosyl substitution,rutin was not easy to penetrate into the XO protein structure.So rutin and XO had a lower degree of binding.The results of UV spectra show that quercetin and kaempferol can cause the XO secondary structure to change.Quercetin formed a more stable complex with XO.XO active center was not easy to expose.Molecular simulation docking preliminary study results can further confirm the spectral results.According to the results,the bioavailability of saponins is low,and the flavonoids are the active constituents that inhibit XO.