| Aikeqing(AKQ)is a novel formulated Chinese medicine for adjuvant HIV/AIDS therapy.It is developed by the Institute of Tropical Medicine,Guangzhou University of Chinese Medicine,based on the symptoms and characteristics of HIV/AIDS patients.AKQ is composed of ten medicinal herbs:Radix Aconiti Lateralis Preparata,Herba Epimedii,Rhizoma Zingiberis,Radix Glycyrrhizae,Radix Ginseng,Radix Salviae Miltiorrhizae,Rhizoma Polygoni Cuspidati,Poria,Cortex Phellodendri,and Radix Scutellariae.Clinical studies showed efficacy of AKQ in the treatment of HIV patients in China,by enhancing and stabilized the immunologic function,improving the symptoms and signs,and improving the survival quality.However,the bioactive constituents and pharmacological mechanism of AKQ are still unclear.Characterization the bioactive constituents of AKQ is very necessary,which would be helpful to demonstrate mechanisms related to ameliorating incomplete immune reconstitution and increasing the antiviral effect of the chemical drugs.Objective:To investigate the chemical composition and potential bioactive compounds in vivo of AKQ,basing on an ultra-high performance liquid chromatography equipped with electrosprayionization quadrupole time-of-flight mass spectrometry(UPLC-ESI-QTOF-MS)method.To study the inhibition of AKQ and its active components on cytochrome P450 for evaluating the interaction of AKQ with HAART.To investigate the effect of AKQ on the pharmacokinetics of LPV and RTV in rats.Methods:1.A UPLC-ESI-QTOF-MS method was established to clarify the chemical composition of AKQ.Compouds were identified with reference standards,or tentatively characterized by matching their mass spectra recorded with the published references.2.A UPLC-ESI-QTOF-MS method was applied to analysize compounds in rat plasma,bile,urine and fece after oral administration.The absorbed chemical components and metabolites were identified by comparing with the data of standards and AKQ.3.The in vitro inhibition experiments were performed by incubation of the AKQ and probes in human liver microsomes.The metabolites of the 8 probes were observed by LC-MS/MS to obtain inhibitory rate(%)of and calculate the IC50.The level of protein expression of CYP3A4 and CYP2D6 in rat liver was detected by Western blot after oral administration.4.Molecular docking program SYBYL-X 2.1.1 and Discovery Studio 2016 were used for predicting the inhibitors of CYP3A4 and CYP2D6 in AKQ.The inhibition of candidate compouds on CYP3A4 and CYP2D6 were investigated in human liver microsomes.5.A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of lopinavir(LPV)and ritonavir(RTV)in rat plasma.A one-step plasma protein precipitation method with acetonitrile was used for sample preparation.Separation was achieved using an Agilent ZORAX Eclipse Plus C18(3.5 pm,50*4.6 mm).The mobile phase consisted of 35%A(0.1%formic acid)-65%B(0.1%formic acid-acetonitrile).LPV and RTV were quantified on a triple-quadrupole mass spectrometer under multiple reactions monitoring(MRM).The assay was applied to investigate the effect of AKQ on the pharmacokinetics of LPV and RTV in rats.Result:1.A total of 123 compounds were identified or tentatively characterized,including 33 alkaloids,57 flavonoids,12 triterpene saponins,5 anthraquinones and 3 phenolic acids.Originalities of all the compounds from the individual herbs of AKQ were assigned.2.The UPLC-ESI-QTOF-MS method was performed to identify the absorbed constituents and their metabolites in rat biosamples(i.e.,plasma,bile,urine,and feces).After oral administration of AKQ,52 parent compounds and 8 metabolites in rat plasma were detected and identified.36 parent compounds and 8 metabolites were detected in rat bile.45 parent compounds in rat urine and 53 parent compounds in rat feces were also identified.3.At a concentration range from 0 to 0.5 mg/mL,the extracts of AKQ could inhibited CYP2C8,CYP 2E1,CYP 2C19,CYP 1A2,CYP 2B6,CYP 2C9,CYP 2D6 and CYP3A4 with analogous effectiveness.Especially,the inhibitory effects on CYP2D6 were more obvious with the IC50 values of 0.0077 mg/mL.The results indicated that AKQ could inhibit the protein expression of in rat liver.4.Inhibition of 18 compounds on CYP3A4 and CYP2D6 were conducted in human liver microsomes after molecular docking.Glycyrrhizic acid,Salvianolic acid A and Baohuoside I showed moderate inhibition on CYP3A4 with the IC50 values of 3.36,6.05,9.47 ?M,respectively.2"-O-rhamnosyl icariside I can inhibit CYP 3A4 weakly with the IC50 values of 3.50 ?M.Salvianolic acid A showed moderate inhibition on CYP2D6 with the IC50 values of 8.49 ?M.Berberine showed weak inhibition on CYP2D6 with the IC50 values of 13.45 ?M.5.A rapid and efficient HPLC-MS/MS method were developed to determine LPV and RTV in rat plasma,and applied to investigate the effect of AKQ on the pharmacokinetics of LPV and RTV in rats.LPV and RTV had good linearity within the detection range(30-10000 ng/mL and 3-1000 ng/mL).The LLOQ for LPV and RTV were 30 ng/mL and 3 ng/mL,respectively.The inter-and intra-day precision(RSD%)for the detection of LPV and RTV were less than 15%.The accuracy(Bias%)values of LPV and RTV were in the ranges of 95.3%?107%and 96.7%?104%,respectively.There was no significant matrix effect observed.LPV and RTV were found to be stable.There were no statistically significant difference in the noncompartmental model parameters of AUC(0-t),AUC(0-?),MRT(0-t),t1/2z,Tmax,Vz/F,CLz/F and Cmax for LPV between experimental group(AKQ+kaletra treatment group)and control group(kaletra treatment group),whereas the MRT(0-?)showed statistically significant difference.There were no statistically significant difference in the noncompartmental model parameters for RTV between experimental group and control group in rat.Conclusion:In this paper,we analyzed and identified the chemical constituents of AKQ systematically by UPLC-ESI-Q-TOF-MS method and applied the established method to screen and identificate metabolites of AKQ in vivo,which provides a basis for improving the quality control level of AKQ.In vitro and in vivo pharmacological experiments suggestted that AKQ can inhibit C YP3A4 and CYP2D6.Coadministered with HARRT drugs could affect the metabolic characteristics of the drug by inhibiting the activity of CYP3A4 and CYP2D6.The components of AKQ such as Glycyrrhizic acid,Salvianolic acid A and Baohuoside,2"-O-rhamnosyl icariside I,Salvianolic acid A and berberine contributed to the inhibition of CYP3A4 and CYP2D6 of AKQ.We investigated the effects of AKQ on pharmacokinetics of lopinavir/ritonavir in rat.There was no significant effect on the pharmacokinetics parameters of lopinavir/ritonavir when Coadministered with AKQ. |
PDF Full Text Request