Simultaneous quantitative analysis of 12 methoxyflavones with melanogenesis inhibitory activity from the rhizomes of Kaempferia parviflora

A methanol extract from the rhizomes of Kaempferia parviflora Wall. ex Baker (Zingiberaceae) has shown inhibitory effects against melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC₅₀ = 9.6 μg/mL). Among 25 flavonoids and three acetophenones isolated previously (1–28), several constituents including 5-hydroxy-7,3′,4′-trimethoxyflavone (6, IC₅₀ = 8.8 μM), 5,7,3′,4′-tetramethoxyflavone (7, 8.6 μM), 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (12, 2.9 μM), and 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (13, 3.5 μM) showed inhibitory effects without notable cytotoxicity at the effective concentrations. Compounds 6, 7, 12, and 13 inhibited the expression of tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA, which could be the mechanism of their melanogenesis inhibitory activity. In addition, a quantitative analytical method for 12 methoxyflavones (1, 2, 4–11, 13, and 14) in the extract was developed using HPLC. The optimal condition for separation and detection of these constituents were achieved on an ODS column (3 μm particle size, 2.1 mm i.d. × 100 mm) with MeOH–0.1 % aqueous acetic acid solvent systems as the mobile phase, and the detection and quantitation limits of the method were estimated to be 0.08–0.66 ng and 0.22–2.00 ng, respectively. The relative standard deviation values of intra- and interday precision were lower than 0.95 and 1.08 %, respectively, overall mean recoveries of all flavonoids were 97.9–102.9 %, and the correlation coefficients of all the calibration curves showed good linearity within the test ranges. For validation of the protocol, extracts of three kinds of the plant’s rhizomes collected from different regions in Thailand (Leoi, Phetchabun, and Chiang Mai provinces) were evaluated. The results indicated that the assay was reproducible, precise, and could be readily utilized for the quality evaluation of the plant materials.