Highly potent tyrosinase inhibitor,neorauflavane from Campylotropis hirtella and inhibitory mechanism with molecular docking

Tyrosinase inhibition may be a means to alleviate not only skin hyperpigmentation but also neurodegeneration associated with Parkinson’s disease. In the course of metabolite analysis from tyrosinase inhibitory methanol extract (80% inhibition at 20 μg/ml) of Campylotropis hirtella, we isolated fourteen phenolic compounds, among which neorauflavane 3 emerged as a lead structure for tyrosinase inhibition. Neorauflavane 3 inhibited tyrosinase monophenolase activity with an IC₅₀ of 30 nM. Thus this compound is 400-fold more active than kojic acid. It also inhibited diphenolase (IC₅₀ = 500 nM), significantly. Another potent inhibitor 1 (IC₅₀ = 2.9 μM) was found to be the most abundant metabolite in C. hirtella. In kinetic studies, compounds 3 showed competitive inhibitory behavior against both monophenolase and diphenolase. It manifested simple reversible slow-binding inhibition against monophenolase with the following kinetic parameters: K_i^app = 1.48 nM, k₃ = 0.0033 nM⁻¹ min⁻¹ and k₄ = 0.0049 min⁻¹. Neorauflavane 3 efficiently reduced melanin content in B16 melanoma cells with 12.95 μM of IC₅₀. To develop a pharmacophore model, we explored the binding mode of neuroflavane 3 in the active site of tyrosinase. Docking results show that resorcinol motif of B-ring and methoxy group in A-ring play crucial roles in the binding the enzyme.