Tyrosinase inhibitory activity of Leonurus sibiricus L. extract

Abstract

Background: Tyrosinase is a copper-containing enzyme that catalyzes key initial reactions of melanogenesis, including the hydroxylation of L-tyrosine and oxidation of L-DOPA. The discovery of natural tyrosinase inhibitors remains relevant to pharmaceutical, pharmacognostic, and cosmetic research. Species of the genus Leonurus contain diverse secondary metabolites, including phenolic compounds, phenylpropanoids, flavonoids, iridoids, alkaloids, and diterpenoids. Selected constituents from Leonurus species have previously demonstrated tyrosinase inhibitory properties. However, information on the tyrosinase inhibitory activity of chromatographically fractionated Leonurus sibiricus L. remains limited. Aim: To fractionate the n-butanol fraction of L. sibiricus by centrifugal partition chromatography (CPC), pool the obtained fractions according to their thin-layer chromatographic profiles, and evaluate the mushroom tyrosinase inhibitory activity of the pooled samples to identify the most active chromatographic region. Materials and Methods: The n-butanol fraction of L. sibiricus was separated by CPC, yielding 82 individual fractions. The fractions were analyzed by thin-layer chromatography (TLC) on silica gel using ethyl acetate–methanol (85:15, v/v) as the mobile phase. After development, the chromatograms were derivatized with NPG reagent and visualized under UV light. Consecutive fractions showing comparable TLC band patterns and fluorescence profiles were combined into 15 pooled samples. CPC fractions 59–64 were combined as Sample 11. Tyrosinase inhibitory activity was evaluated using a 96-well microplate spectrophotometric assay with mushroom tyrosinase and L-DOPA as the substrate. Samples were prepared in DMSO at 1 mg/mL. Each reaction contained 100 mM phosphate buffer (pH 6.8), 20 µL of sample solution, and 20 µL of mushroom tyrosinase (500 U/mL). After 10 min of pre-incubation, 80 µL of 8 mM L-DOPA was added, and the reaction was maintained at 25 °C for 20 min. Dopachrome formation was measured at 450 nm using a Promega GloMax Explorer microplate reader. Sample background absorbance was corrected using enzymeand substrate-free wells, and kojic acid at 1 mg/mL was used as a positive control. Results: The 15 pooled samples demonstrated different levels of tyrosinase inhibitory activity. Sample 11, corresponding to CPC fractions 59–64, showed the strongest inhibitory effect, with 35.89% inhibition. Samples 14, 5, and 3 showed 21.98%, 20.43%, and 16.96% inhibition, respectively, whereas the remaining pooled samples showed lower or negligible inhibition. Kojic acid showed 59.14% inhibition under the same assay conditions. The highest activity observed in Sample 11 suggests enrichment of one or more bioactive constituents within the chromatographic region corresponding to CPC fractions 59–64. Conclusion: The combination of CPC fractionation, TLC-guided pooling, and a mushroom tyrosinase–L-DOPA screening assay enabled localization of tyrosinase inhibitory activity within a defined fraction range of L. sibiricus. Sample 11, comprising CPC fractions 59–64, represents the priority material for subsequent LC-MS/MS characterization, bioactivity-guided subfractionation, isolation of active constituents, and concentration-dependent IC₅₀ evaluation.