Target guided isolation of potential tyrosinase inhibitors from Otholobium pubescens (Poir.) J.W. Grimes by ultrafiltration, high-speed countercurrent chromatography and preparative HPLC

Abstract Otholobium pubescens (Poir.) J.W. Grimes ( O. pubescens ) is a commercial and multiple therapeutic medicinal plant in South America, yet its phytochemicals are rarely studied. In the present study, a combination of affinity-based ultrafiltration, HSCCC, and preparative HPLC was applied for the efficient screening and isolation of ten tyrosinase inhibitors from the O. pubescens methanol extract and eight of them were successfully identified as daidzin, isoorientin, vitexin, isovitexin, isoorientin 3′-methyl ether, daidzein, genistein, and a novel compound 3-(5-hydroxybenzofuran-6-yl) propanoic acid by EI-MS and NMR; all of these compounds are first reported in O. pubescens and possessed tyrosinase inhibitory activities by in vitro tyrosinase inhibition assay. Among the inhibitors, vitexin, isovitexin, 3-(5-hydroxybenzofuran-6-yl) propanoic acid, and daidzein revealed higher tyrosinase inhibitory activities than the positive control β-arbutin, with IC 50 values of 0.35 mM, 1.73 mM, 1.33 mM, 1.57 mM, and 1.83 mM, respectively. Moreover, a long time tyrosinase inhibition duration, up to 150 min, was observed for compounds vitexin, daidzein, and particularly 3-(5-hydroxybenzofuran-6-yl) propanoic acid, which was assessed as a special mixed-type tyrosinase inhibitor because of its capability of inhibiting tyrosinase almost in an irreversible inhibition manner, through tyrosinase inhibition duration, inhibition kinetics, and dialysis studies. In addition, the major nonpolar compound from O. pubescens methanol extract was also purified by silica gel column chromatography and identified as bakuchiol. Results suggested that vitexin, isovitexin, daidzein, and particularly the novel compound 3-(5-hydroxybenzofuran-6-yl) propanoic acid are promising tyrosinase inhibitors with the potential for pharmaceutical, cosmetic, and food industrial applications.