Chromatography-guided purification and characterization of <i>p-</i> hydroxyphenethyl anisate as a potent anti-melanogenesis component from the concentrated Chinese medicine, Qiang Huo

Melanogenesis is tightly regulated by tyrosinase, and its inhibition represents a key strategy in developing skin-whitening agents. In this study, <i>p</i>-hydroxyphenethyl anisate (HP) was identified as a potent melanogenesis inhibitor through a zebrafish-based screening without affecting embryo viability at concentrations up to 10 μM. The biological activity of HP was further validated in isobutyl-1-methylxanthine (IBMX)-stimulated B16-F1 murine melanoma cells. HP treatment significantly suppressed IBMX-induced melanin synthesis without reducing cell viability. Mechanistically, HP markedly inhibited tyrosinase activity and reduced tyrosinase protein levels. Furthermore, HP suppressed tyrosinase mRNA expression, as well as the expression of <i>Mitf</i>, a key transcriptional regulator of tyrosinase. These findings indicate that HP inhibits melanogenesis through transcriptional downregulation of <i>Mitf</i>, hence its downstream tyrosinase, leading to reduced protein expression and enzymatic activity. Seeking the source of HP, we applied the purification process guided by HPLC peak-tracing and successfully isolated HP from a concentrated Chinese medicine Qiang Huo. Starting from herbal powder, a methanol extract was fractionated by LH-20 column chromatography, and the target compound was purified using preparative C18 HPLC. Collectively, our study identifies HP as a promising natural compound with potent and nontoxic anti-melanogenic activity, supporting its potential application in cosmetic or therapeutic skin depigmentation.