| Objective:To isolate and identify the active constituents and evaluate their tyrosinase inhibition activities from the methylene chloride fraction of Forsythia suspensa. Method:The active constituents were found through fraction collecting and tyrosinase inhibitory activity by bioassay-linked HPLC method, and they were isolated by silica gel column chromatography, Sephadex LH-20chromatography and semi-preparative HPLC method. These constituents’chemical structures were identified with UV, MS,1H-NMR and13C-NMR spectra data. The tyrosinase inhibition activities of the isolated compounds were evaluated by mushroom tyrosinase inhibitory test, MTT colorimetric assay and B16melanoma cells tyrosinase inhibitory test. The contents of the active constituents were analyzed by HPLC. Result:6constituents were isolated and identified as Betulinic acid, ursolic acid, phillygenin, phillyrin, Forsythoside A, suspensaside A. The6constituents showed tyrosinase inhibitory activities. Suspensaside A expressed the strongest mushroom tyrosinase inhibition than the known tyrosinase inhibitor, Arbutin (IC5099.05μmol/L) with IC50value of41.17μmol/L. IC50values of Betulinic acid was100.30μmol/L, similar with Arbutin. Ursolic acid and phillygenin showed strong B16cell inhibitory activities.Their IC50value were0.75μmol/L,44.64μmol/L, whereas for Arbutin it was35.53μmol/L. Betulinic acid, ursolic acid, Forsythoside A and suspensaside A displayed strong tyrosinase inhibition of B16melanoma cells, with IC50values of6.18μmol/L,0.05μmol/L,0.55μmol/L and5.77μmol/L, respectively. Ursolic acid was stronger than that of Arbutin (0.07μmol/L). The contents of Forsythoside A, suspensaside A, phillyrin, phillygenin, Betulinic acid, ursolic acid were1.57%,0.29%,0.19%,0.06%,0.30%,1.21%, respectively. Conclusion:Bioassay-linked HPLC method was provided for rapid determination the active constituents from Forsythia suspensa. Forsythia suspensa showed good application prospect in tyrosinase inhibition. |
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