Chromatography-guided purification and characterization of p-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, p-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 Mitf, a key transcriptional regulator of tyrosinase. These findings indicate that HP inhibits melanogenesis through transcriptional downregulation of Mitf, 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.