Study Of The Correlation Between Efficacy And Bioactive Components Extracted From Psoraleae Fructus And The Chemical's Safety

Aims:1.To determine the active ingredients in the ethenal extracts of Psoralea Fructus?PEs?on tyrosinase activity in vitro.2.Probe test in vitro and qRT-PCR was established to evaluate the inhibition and induction of psoralidin,isobavachalcone,neobavaisoflavone and daidzein in Psoralea Fructus on CYP2E1 and CYP3A4 enzymes.3.An ionic liquid-based ultrasonic-assisted method?ILUAE?coupled with Plackett–Burman?PB?design and Box–Behnken response surface design?BBD?for the extraction of psoralen,isopsoralen,neobavaisoflavone,bavachin,psoralidin,isobavachalcone,and bavachinin A from Psoralea Fructus was proposed.Methods:1.A high performance liquid chromatography?HPLC?method was established to obtain the specific chromatograms.Tyrosinase activity was assayed by the method of oxidation rate of levodopa in vitro.The spectrum-effect relationship and knock-out method were established to predict the active compounds,which were then identified with the high resolution mass spectra.2.The relevant metabolites were measured by HPLC,after probe substrates of 4-nitrophenol and testosterone as well as the drugs at different concentrations were added to the incubation systems,which were used to predict the inhibition on CYP2E1 and CYP3A4 enzymes.In addition,real-time RT-PCR was performed to determine the induction of psoralidin,neobavaisoflavone and daidzein on the mRNA expression of CYP3A4 in rat liver.3.Several ILs of different types and parameters,including the concentration of ILs,concentration of ethanol?EtOH?,solid–liquid ratio,particle size,ultrasonic time,centrifugal speed,and ultrasonic power,were optimized by the PB and BBD design.Results:1.Chromatographic peaks P2,P4,P9,P10,P11,P13,P21,P26,P28,and P30 were positively related to the activating effects on tyrosinase activity in PEs,whereas chromatographic peaks P1,P3,P6,P14,P16,P19,P22,and P29 were negatively related to the activating effects on tyrosinase in PEs.P2,P5,P13,P18,P20,P23 and P24 were daidzein,psoralen,corylin,psoralidin,isobavachalcone,and bavachinin A by HRMS identification,respectively.When the sample concentration was 0.5 g·mL^-1,equal to the amount of raw medicinal herbs,the target components were daidzein?P2?,psoralen?P5?,neobavaisoflavone?P13?,and psoralidin?P20?,which were consistent with the results of spectrum-effect relationships.2.The results suggested that psoralidin,isobavachalcone and neobavaisoflavone were Medium-intensity inhibitors of CYP2E1 with Ki values of 2.58,1.28 and 19.07?M,respectively,which could inhibit the increase of CYP2E1 and reduce diseases caused by lipid peroxidation.Isobavachalcone?Ki=37.52?M?showed a weak competitive inhibition on CYP3A4.Psoralidin and neobavaisoflavone showed obvious induction effects on CYP3A4 in the expression level of mRNA,which could accelerate the effects of drug metabolism and lead to the risk of inducing DDI and serious adverse reactions.3.The optimum extraction parameters adjusted by the integer of Psoralea Fructus were obtained as follows:the concentration of ILs was 0.6 M,concentration of EtOH was 75%,the solid–liquid ratio was 1:40,the particle size was 20meshes,the ultrasonic time was 20 min,the centrifugal speed was 3000 r/min,and the ultrasonic power was 300 W.Under this optimal condition,the total extraction yield of the seven compounds in Psoralea Fructus was 18.90 mg/g,and significantly greater than the conventional 75%EtOH solvent extraction.Conclusions:1.When the concentration of each sample was 0.5 g·mL^-1 equal to the amount of raw medicinal herbs,psoralen?P5?,corylin?P18?,psoralidin?P20?,isobavachalcone?P23?,and bavachinin A?P24?showed the inhibitory effects on tyrosinase activity,whereas daidzein?P2?and neobavaisoflavone?P13?showed the activation effects of tyrosinase activity.2.Psoralidin and neobavaisoflavone had the inhibition of CYP2E1 and induction of CYP3A4.In addition,isobavachalcone had the inhibition of CYP2E1 and CYP3A4.The effects of psoralidin,isobavachalcone,neobavaisoflavone and daidzein on the inhibition and induction of CYP2E1 and CYP3A4,which are the main constituents of Fructus Psoraleae,can reduce hepatotoxicity or induce DDI in clinic.3.Compared with traditional methods,the extraction yields of the present approach may increase by 28.40%.Thus,the ILUAE is a simple,rapid,and highly efficient extraction technique for the seven compounds—psoralen,isopsoralen,neobavaisoflavone,bavachin,psoralidin,isobavachalcone,and bavachinin A from Psoralea Fructus.