Study On The Anti-hepatic Fibrosis Effect Of Penthorum Chinense Pursh

Objective:To optimize the conditions for the extracting of Penthorum chinense P(PC);To develop a novel method with high performance liquid chromatographic for the determination of quercitrin in PC to validly control its quality;To explore the effect and mechanism of PC ethanol extracts on immunological hepatic fibrosis;To develop a simple HPLC method to determine the concentrations of quercitrin of the extract of PC,and evaluate the absolute bioavailability of quercitrin in rats.Methods:? Orthogonal design was utilized to optimize the preparing conditions including the amount of ethanol,extraction duration per time,extraction times and concentration of ethanol when quercitrin was selected as the evaluation index;a novel method with high performance liquid chromatographic was established for the determination of quercitrin in PC.? The rat model of immunological hepatic fibrosis was established by intraperitoneal injection of pig's serum repeatedly.We determined the content of N-terminal propeptide of type ? collagen(?PC),hyaluronic acid(HA),tissue inhibitor of metalloproteinase type(TIMP-1),tissue plasminagen activator(TPA)in serum by ELISA,determined the content of Hyp in liver tissue by HPLC.We observed Collagen deposition in liver tissue by masson staining,and measured the fibrosis area(S)by Motic Med 6.0 motic digital medical image analysis system.? The determination of the concentration of quercitrin in rat plasma was used high performance liquid chromatographic(HPLC)method with UV detection and it was validated to pharmacokinetic study after intravenous and oral administration of Penthorum chinense P.(i.g.1000 mg/kg,equally 23mg quercitrin and i.v.50mg/kg,equally 1.15mg quercitrin)to Sprague-Dawley rats.Blood samples were collected from the tail vein,nononcompartmental PK analysis of plasma qurcitrin concentration versus time data for each rat was carried out using DAS software package,and calculate the absolute bioavailability(F).Results:? The method was accurate,reliable and good reappearance for the determination of quercitrin in PC extracts.? The pre-column derivativation HPLC method is simple,accurate and sensitive to analyze Hyp content in liver tissue;Compared with the control group,the content of TIMP-1,HA,?PC and TPA in serum and Hyp in liver tissue evidently decreased of PC ethanol extracts group.The hepatic fibrosis area evidently decreased of PC ethanol extracts groups.? A simple and specific high performance liquid chromatographic(HPLC)method with UV detection was developed,validated it to determine the concentration of quercitrin in rat plasma and study its pharmacokinetics after intravenous and oral administration of Penthorum chinense P.(i.g.1000 mg/kg,equally 23mg quercitrin and i.v.50mg/kg,equally 1.15mg quercitrin)to Sprague-Dawley rats.This validated method was successfully applied to the determination of quercitrin in rat plasma for pharmacokinetic study.After single oral administration,the main pharmacokinetic parameters Tmax,Cmax,tl/2,z,MRTO-t,AUCO-t,AUC0-? were 1 h,331±57.3 ng/L,1.339±0.019 h,2.713±0.106h;1027.7±108.5 h-ng/L,1183.5±90.8 h ng/L,respectively.Our results showed a poor oral absorption with 2.5%bioavailability of quercitrin in rats.Conclusion:? The optimal extraction process is to extract two times using tenfold 80%ethanol and 0.5 h per time.? PC ethanol extracts have significant therapeutic effect on immunological hepatic fibrosis induced by pig's serum.It shows PC ethanol extracts have significant positive regulatory effect on matrix metalloproteinase(MMP),have passive regulatory effect on synthesis and deposition of extracellular matrix(ECM),and can promote cell regeneration after injury,can decrease the content of III PC and hepatic fibrosis area,so that it can slow down the process of liver fibrosis and recovery of liver function.? In the present study,a simple,sensitive and reliable RP-HPLC method to determine quercitrin in rat plasma was established.This developed method with efficient liquid liquid extraction as sample clean-up procedure,and was successfully applied for determination of quercitrin in rat plasma for pharmacokinetic study after intravenous andoral administration of PC extract.The results of pharmacokinetics obtained in the study demonstrate that oral bioavailability of quercitrin was poor,only get approximately 2.5%of dose.