Construction Of Complex System Of Trilobatin With Steviol Glycosides And Its Effect On Tyrosinase Activity

Trilobatin(TLB),which is mainly derived from Lithocarpus litseifolius(Hance)Chun,is a dihydrochalcone with C₆-C₃-C₆ skeleton.It has various pharmacological activities including hypoglycemic,anti-inflammatory,neuroprotective and anti-HIV-1,but its poor water solubility limits its application.The traditional methods for the solubilization of flavonoids were complicated and carriers have potential toxicity.Steviol glycosides(STE)is a kind of sweetener derived from stevia leaves.STE is an amphiphilic molecular,which could self-assemble into micelles and then enhance the solubility and stability of some insoluble compounds.In this paper,STE-TLB micelle(STE-TLB MC)and STE-TLB solid dispersion(STE-TLB SD)were prepared with STE as solubilizer respectively.The quality evaluation of STE-TLB complex systems and their effects on antioxidant and tyrosinase activity were investigated.The mechanism of tyrosinase(TYR)and TLB were also investigated.The main conclusions are as follows:1.Construction and characterization of STE-TLB MC and STE-TLB SD.The preparation technology of STE-TLB complex systems were optimized.With350 mg of STE as solubilizer,90 mg of TLB could be dissolved in 5 m L water under magnetic stirring at 70?for 30 min and STE-TLB MC solution was transparent.STE-TLB SD was prepared with 250 mg of STE and 90 mg of TLB,and STE-TLB SD was dissolved in 5 m L water at room temperature.In addition,Rebaudioside A(RA)in STE would self-aggregate in water,which weakened the solubilization ability of STE in STE-TLB complex systems.The average particle size of STE-TLB MC was 262.8±3.4 nm by using the laser particle size and potential measuring instrument.The particle size distribution of STE-TLB MC was not only about 5 nm small particle size,but also 100 nm-1000 nm large particle size.The formation of STE-TLB SD was determined by Fourier transform infrared spectroscopy,DSC thermal analysis,X-ray diffraction analysis and scanning electron microscopy.The results showed that the TLB was an amorphous state in the solid dispersion and there was no chemical interaction between STE and TLB.2.Quality evaluation of STE-TLB MC and STE-TLB SD.The p H stability experiments illustrated that TLB was unstable under alkaline condition.The retention rates of pure TLB,STE-TLB MC and STE-TLB SD at p H9.0 for 24 h were 62.81±0.81%,68.52±0.40%and 62.92±0.27%,respectively.MC and SD could enhance the p H stability of TLB within 24 h,but the retention rate at 24h of TLB solubilized by STE based SD was not significantly different from that of pure TLB at p H 9.0.It might be that TLB in STE-TLB SD was directly dissolved in water in an amorphous state and the micellar encapsulation of TLB by STE micelle could protect TLB well.The degradation kinetics of pure TLB and the TLB in the two complex systems were well fitted with the first-order kinetics model.The digestion stability,release rate and release kinetics of pure TLB and TLB solubilized by STE based MC and based SD were investigated by in vitro digestion and release.The results showed that STE-TLB MC and STE-TLB SD exhibited good stability in digestive fluid,and the release rate of TLB was significantly improved by STE solubilization.The cumulative release rates of pure TLB,STE-TLB SD and STE-TLB MC in artificial gastric fluid(SGF)were 27.99%,55.81%and 40.98%,respectively,and in artificial intestinal fluid(SIF)were 2.10%,19.24%and 8.58%,respectively.The in vitro release of pure TLB,STE-TLB MC and STE-TLB SD were fitted by the equation,and the results showed that the release of TLB in the three systems were fitted the Retger-Peppas equation best.3.The interaction between TLB and TYR.The effects of TLB on tyrosinase activity and their binding mechanism were investigated by Ultraviolet(UV)and fluorescence spectra.TLB as a substrate has been verified by TYR substrate assay and high-performance liquid chromatography(HPLC).The results showed that TLB inhibited both monophenolase and diphenolase activity of tyrosinase.IC50 of diphenolase value was 100?mol/L.TLB combined with tyrosinase via hydrogen bonds and van der Waals forces,which quenched the intrinsic fluorescence of TLB and reduced its hydrophobicity.The results of UV and HPLC suggested that TLB not only inhibited the catalytic oxidation of L-dopa by TYR,but also was a substrate of TYR.Molecular docking showed that TLB enter the hydrophobic active center of TYR and competed with L-dopa for the active site,so that inhibited the activity of TYR.Interestingly,these findings indicated that TLB was both a tyrosinase inhibitor and substrate.4.Investigating the performance of TLB before and after solubilization.DPPH and ABTS scavenging experiments were used to evaluate the antioxidant activity of STE-TLB complex systems.The results showed that STE also had antioxidant activity.The antioxidant activity of the complex systems was stronger than that of pure TLB because of the increased interaction between TLB solubilized by STE and free radicals.TYR inhibition assay and fluorescence spectrum assay showed that the interaction between TLB and TYR was weakened after STE solubilization,and the interaction between TLB and TYR was the weakest in STE-TLB MC,which might be because the micellar encapsulation was not favorable for TLB binding to TYR.