A Study On Matrix Metalloroteinase Inhibitors From Trifolium Pratense L And Their Antioxidant Activities

Matrix metalloproteinases(MMPs)are zinc-dependent endopeptidases,which are capable of collectively degrading all kinds of extracellular matrix proteins.It seems that the current literatures have provided adequate evidence that matrix metalloroteinase inhibitors have broad potentials in curing cancer,so research on screening inhibitors for matrix metalloproteinases has become a hotspot and we hope to develop some new anti-tumor agents from plant extracts of Chinese herbs.With the MMPs as the target,by using of silica gel column chromatography and preparative HPLC and the enzymologic techniques,the paper is aimed at tracing active ingredients which can inhibit MMP-16 from Trifolium pretense L and studying the antioxidant activity of extracts.The main contents include:1.Literature ReviewsIn this dissertation,we summarize the advance in domestic and international studies on Trifolium pratenseL,matrix metalloproteinase inhibitors and antioxidant activity.2.Screening of active ingredients from Chinese herbs for inhibiting the activity of MMP-16In an attempt to screen inhibitors to matrix metalloproteinases,a total of 7 extracts of natural plants,such as extracts of Trifolium pratense L,Ilexpubescens Hook.et Am.,Honeysuckle Flower et al were chosen.The results showed that the 48μg/mL 40%Trifolium pratense L extract can control 42%MMP-16,.It means that Trifolium pratense L has the inhibitory component to MMP-16.3.The inhibitory activity of the main isoflovones from Trifolium pratense L to MMP-16The main isoflovones of Trifolium pratense L include Biochanin A,Fomononetin, Genistein and Daidzein.The experimental result proved that prepared 73.47% Biochanin A has inhibitory activity on MMP-16 and is relatively strong,48μtg/mL of Biochanin A can inhibit 99%activity of MMP-16.The inhibition became weak with reduceing the amount of Biochanin A gradually.The IC₅₀s of 92.28%Biochanin A and synthetical Biochanin A for MMP-16 were 29.56μg/mL,117.74μg/mL,respectively, which means the inhibitory activity of Biochanin A on MMP-16 activity is relatively weak.The inhibitory activity of prepared Fomononetin and synthetical Fomononetin are unstabe.The inhibitory activity of Genistein is weak,the 20μg/mL Trifolium can control 3.52%MMP-16;Daidzein has nearly no inhibitory activity.So the main isoflovones of Trifolium pratense L are not the primary components with inhibitory activity to MMP-16.4.Screening and separation of inhibitors to MMP-16 from 40%Trifolium pratense LWe obtained 33 fractions from 40%Trifolium pratense L by silica gel column chromatography and found that fraction 13 has strong inhibitory activity on MMP-16 activity and is dose-dependent,for example,1μL can control 56%,2μL can control 80%.We obtained 5 components from fraction 13 by semi-preparative chromatography.Their chemical structures were identified by UV,ESI/MS.We confer that the solids 1,2,4,5 were Fomononetin,Texasin,Irilone,and biochanin A, respectively.Finally,we find that Irilone is a strong MMPI to MMP-16 for the first time,and IC₅₀is 25.17μmol/L.5.Study of antioxidant activity of Trifolium pratense LAfter researching the radical scavenging activities of 40%Trifolium pratense L extract,fraction13,prepared irisolone,respectively,we got the result that all the three products have antioxidant activities.The DC₅₀are 68.71μg/L,9.09μg/L,0.023μmol/L,respectively.The experiment result also shows that when the concentration of Vc is 0.023μmol/L,the radical scavenging activity is only 2.08%.Therefore,the result suggests that the radical scavenging activities of prepared irisolone is stronger than Vc,and it could be a good anti-oxidant.