Isolation and Identification of Condensed Tannins from Ziziphus jujuba Leaves and Their Inhibition Mechanism for Melanogenesis

Objective: To explore the structural characteristics of condensed tannins from Ziziphus jujuba leaves and their effect on tyrosinase and the inhibition mechanism for melanogenesis. Methods: The condensed tannins from Z. jujuba leaves were isolated and purified on the basis of Sephadex LH-20 gel column chromatography, and their structural features were identified by thiolysis coupled with high-performance liquid chromatography-electrospray ionization mass spectrometry (Thiolysis-HPLC-ESI-MS). Besides, the inhibitory mechanism of Z. jujuba leaves condensed tannins on tyrosinase was also systematically investigated by enzyme kinetics, UV-vis absorption and fluorescence spectroscopy. Finally, CCK-8 assay, L-DOPA oxidation assay, NaOH cleavage assay and real-time fluorescence quantitative PCR were employed to probe into the effects of Z. jujuba leaves condensed tannins on the proliferation, tyrosinase activity, melanogenesis and the level of melanogenesis-related gene expression of mouse melanoma cells (B16F10). Results: The condensed tannins from Z. jujuba leaves were mainly composed of (epi)catechin and (epi)gallocatechin. Z. jujuba leaves condensed tannins were found to strongly inhibit on both monophenolase and diphenolase of tyrosinase, and the inhibition against diphenolase was proved to be reversible and mixed-type. It was also observed that the condensed tannins from Z. jujuba leaves could effectively inhibit the cell viability, tyrosinase activity, melanin content, as well as the mRNA expression of TYR, TRP-1 and MITF genes in B16F10 cells. Conclusion: Based on the comprehensive analysis of the biological effects of condensed tannins from Z. jujuba leaves at enzyme level and cell level, we could clarify the molecular inhibition mechanism of Z. jujuba leaves condensed tannins on melanogenesis in B16F10 cells. This study would provide a theoretical basis for the later development of Z. jujuba leaves condensed tannins as new whitening cosmetic additives and preservatives for fruits and vegetables.