IntroductionThe human skin, hair, and eyes derive their colours from a pigment called melanin. Melanin is synthesised within specialised cells known as melanocytes, specifically inside intracellular organelles called melanosomes, in a granular form. The melanosomes are subsequently transported and deposited in neighbouring keratinocytes1. Melanogenesis, the process of producing melanin, is complex, controlled by a network of critical signalling cascades and transcription factors2. The various factors involved in melanogenesis can be divided into three groups, namely, the structural proteins of melanosomes, enzymes required for melanin synthesis, and proteins required for melanosome transport and distribution3. Melanocyte dysfunction can disrupt the functions of many pigmentation-related factors that cause hypopigmentation disorder, affecting the colour of the skin, hair, and eyes, as in vitiligo and canities4,5.Attempts have been made to search for natural products that enhance melanogenic activity in order to overcome hypopigmentation disorders. Plant flavonoids, a group with diverse chemical structures and bioactivities, are of particular interest in this regard. Flavonoids have been classified into many subclasses, including flavonols, flavones, flavanones, isoflavones, chalcones, and anthocyanins, based on the core flavonoid structure. In the literature, flavonoids with melanogenesis-stimulating activity, such as naringenin among the flavanones6; quercetin, kaempferol, and rhamnetin among the flavonols7,8,9; luteolin and chrysin among the flavones7; and genistein, pratensein, and duartin among the isoflavones7,10, have been reported. Among these, naringenin, pratensein, and duartin have been reported to increase melanin production and tyrosinase (TYR) activity by inhibiting the function of two-pore channel 2 (TPC2) in human MNT-1 cells10. TPC2 is an ion-transport protein that functions as a counteractive controller of pigmentation by heightening the potential and acidity of the melanosomal membrane11,12,13.In addition to the above-mentioned flavonoid diversity, another group of flavones uniquely contains methoxylated substituents; they are methoxylated flavones or O-methylated flavones. They have been found in the rhizome extracts of Kaempferia parviflora14. Our research group at Chulalongkorn University has isolated 13 O-methylated flavones and completely elucidated their structures based on nuclear magnetic resonance (NMR) spectroscopy analysis15. We had also previously reported that the extracts of K. parviflora and Dalbergia parviflora are the only 2 among 40 Thai medicinal plant extracts that exert the strongest effect on stimulating melanin production in B16F10 mouse melanoma cells10. As little information is available on the effect of O-methylated flavones on melanogenesis in human melanocytes, we tested the melanogenesis-enhancing activity of 13 poly-O-methylated compounds isolated from the rhizome of K. parviflora in human melanoma cells MNT-1. The most potent one was then selected for further in-depth studies to understand the molecular mechanisms involved in melanin synthesis. The findings of this study may contribute to the development of therapeutic agents for hypopigmentation disorders, such as vitiligo, and the formulation of cosmetic products aimed at enhancing skin pigmentation or preventing premature hair greying.ResultsPMF from K. parviflora stimulated high melanin production in B16F10 and MNT-1 melanoma cellsThirteen O-methylated flavonoids (KP-1–KP-13) were isolated from the rhizome of K. parviflora, as described previously15. The isolates showed diversity in their structures based on the number and position of the -OCH3 groups attached to the flavonoid core structure. As shown in Fig. 1a, the -OCH3 group could be found in the mono- (KP-2, KP-13), di- (KP-1, KP-7, KP-10), tri- (KP-3, KP-5, KP-6, KP-11, KP-12), tetra- (KP-4, KP-8), and even penta-substituted forms (KP-9) of the flavonoids. Among them, three poly-O-methylated flavones, namely KP-9 (PMF), KP-7 (DMF), and KP-6 (TMF), were isolated as the major constituents, with high melanogenesis-stimulating activity in both B16F10 mouse melanoma (Fig. 1b) and MNT-1 human melanoma cells (Fig. 1c). PMF was found to be as potent as forskolin, the positive control, followed by DMF and TMF, in both cell types, whereas the other -OCH3 flavonoids appeared to show lower stimulatory activity. The presence of the two -OCH3 substituents at positions 5 and 7 of the A-ring (e.g., DMF) was found to be the most important factor for enhancing melanogenic activity. The other -OCH3 substitutions at position 4′ of the B-ring and position 3 of the C-ring seemed relatively less important; however, they did contribute to the enhancement of melanogenic activity, to a certain extent, as seen for PMF. To validate the relationship between the structures and activities shown above, we attempted docking of KP-5 to KP-12 into TPC2 using the HADDOCK 2.4 platform. However, despite extensive preprocessing, including ligand energy minimisation, manual PDB editing, and standardisation of residue numbering and atom names, all docking attempts failed at the topology generation step. These issues may have been related to limitations in HADDOCK’s compatibility with the chemical complexity of our ligand series and the absence of a high-resolution TPC2–ligand complex structure. Thus, we were unable to generate interpretable docking models. Therefore, PMF was subsequently used to investigate its mechanism of action in MNT-1 human melanoma cells.Fig. 1Structure and melanogenic activity of various O-methylated flavonoids isolated from Kaempferia parviflora. (a) Distribution of -OCH2 groups in the flavonoids, ranging from mono- to penta-substituted forms. (b, c) Melanogenic activity of the isolated O-methylated flavonoids in B16F10 and MNT-1 cells. Forskolin was used as a positive control. Data represent means ± SEM from three independent experiments. *p