A Study On The Quality Control Methods Of Ilex Pubescens Hook.Et Arn. And Pharmacokinetics Study Of Its Main Active Components

ObjectiveTo establish a quality control method for qualitative and quantitative analysis of the main active compound in Ilex pubescens Hook.et Arn.(MDQ).Evaluated the mechanism of drug intestinal absorption and transshipment using sigle pass intestinal perfusion model.Systematically and comprehensively analyzed the characteristics of drug absorption,distribution and metabolism of the main active compounds in MDQ in order to provid a scientific basis for the further research and utilization of MDQ.Methods1.Quality control methodAn ultraviolet spectrophotometric method was applied to determinate the contents of total phenolic and saponins in MDQ.Simultaneously determinate the 6 triterpenoid saponins(ilexgenin A,C1;ilexsaponin A1,C2;ilexsaponin B1,C3;ilexsaponin B2,C4;Ilexsaponin B3,DC1;ilexoside O,DC2)in MDQ using a quantitative analysis of multi-components with single marker(QAMS)method by HPLC and UPLC.The chromatographic fingerprint evaluation of 16 batches MDQ was achieved by high performance liquid chromatography-photo-diode array(HPLC-PDA)method.2.Determination of apparent oil/water partition coefficient and rat intestinal absorption in situ of Ilex pubescens Hook.et Arn.To determine the apparent oil/water partition coefficient of the 6 saponins in MDQ and to verify their absorption property by in situ perfused rat intestinal model.Monomer and extract were oral administrated respectively to evaluate the absorption characteristic and this research also pay attention to the effect of P-glycoprotein(P-gp)inhibitors,pinocytosis inhibitor and absorption accelerant on the intestinal absorption.3.Pharmacokinetics and distribution study of MDQIn this study,a novel Liquid chromatography-mass spectrometry(LC-MS)method with high sensitivity and selectivity was developed and validated for the simultaneous quantification of 6 saponins in rat plasma and tissues.Results1.Quality control methodThe content of total saponins and phenolic were 4.75%to 10.99%and 0.36%to 0.51%respectively.The relative correction factor(RCF)was revealed to be excellent system sutability under series of instruments,columns and concentration of the samples.The results calculated by QAMS were similar to that by external standard method.Linear regression method(LRG method)optimized from the traditional one(the average method,AVG method)showed better precision.Differentiation of each target compounds was found between batches,but little difference of the total contents of 6 saponins,about 10?15 mg/g.The phenolic in MDQ has better chromatographic retention ability than saponins under a RP-HPLC elution program.2.Determination of apparent oil/water partition coefficient and rat intestinal absorption in situ of Ilex pubescens Hook.et Arn.The Kow of 6 ingredients in MDQ was between-1 to 2,which mean good lipophilicity and permeability.C1,C2,C3 were verified to be excellent absorption in intestine of rat(Peff?1.20×10-3cm·min-1)with no significant difference between all intestine segments(P>0.05).However,C4,DC1 and DC2 were found to be nonabsorbent when given in monomer.On the contrary,C4,DC1 and DC2 unexpectedly showed appropriate intestinal permeability when administrated in extract form.The absorption rate and permeability of the tested saponins showed concentration-dependent trend in jejunum,ileum and duodenum at dosage of the extract less than the middle concentration level and consistent at high concentration level with no significant absorption priority in tested intestinal segments.The absorption capacity of C2,C3 and DC1 was raised when given verapamil and the EDTA also contributed to the improvement of drug absorption.Inhibition function was observed when given amantadine couple with the extract of MDQ.3.Pharmacokinetics(PK)and distribution study of MDQThe PK study indicated that the PK characteristics of 6 saponins in MDQ have been proposed to be similar to each other with a rapid absorption and elimination.The Tmax were between 0.45 to 2.77 h and the t1/2 were between 1.16 to 4.59 h.It was found that all ingredients main distributed in small intestine and thoracic aorta,where the amount maintained a relative higher level at 0.25 h and 8 h,and eliminated at 12 h.ConclusionIn this study,two kinds of applicable quantitative method were developed to evaluate the main active compounds of MDQ systematically.A sigle pass intestinal perfusion model was applied to evaluate the absorption property of 6 saponins in small intestine by a precise HPLC method.The newly developed LC-MS method for quantitative assessment was effective and comprehensive.The PK and tissue distribution study of 6 saponins will be helpful to better understand the pharmacological and clinical effects of MDQ.