New Constituent from Podocarpus macrophyllus var. macrophyllus Shows Anti-tyrosinase Effect and Regulates Tyrosinase-Related Proteins and mRNA in Human Epidermal Melanocytes

A new biflavonoid, 2,3-dihydro-4′,4′′′-di-O-methylamentoflavone (5), and five known compounds, (−)-catechin (1), quercetin (2), 2,3-dihydrosciadopitysin (3), sciadopitysin (4), and isoginkgetin (6), were isolated from Podocarpus macrophyllus var. macrophyllus (Podocarpaceae). These compounds were evaluated their ability to inhibit cellular tyrosinase activity and for their melanin inhibitory activity in human epidermal melanocytes (HEMn). In the melanin synthesis assay, 2,3-dihydro-4′,4′′′-di-O-methylamentoflavone (5) showed a potent anti-tyrosinase effect with IC50=0.098 mM in HEMn. It also significantly decreased both protein and mRNA levels of the tyrosinase-related protein-2 (TRP-2) by Western blot and quantitative real-time PCR (qRT-PCR) analysis. These findings suggest that the new compound, 2,3-dihydro-4′,4′′′-di-O-methylamentoflavone (5), is the most active component of P. macrophyllus var. macrophyllus in inhibiting pigmentation and that this inhibition is exerted through inhibition of transcription of the genes encoding TRP2.