Anti-melanogenic effects of Medicago sativa seed extracts on melanocytes

Objective: Alfalfa (Medicago sativa) is a prominent herbal treatment among Asian countries and its antioxidant and anti-inflammatory effects have already been generally elucidated. Excessive melanin synthesis is one of the major troubles in the cosmetics industry, thus such research has been extensively described. Here, we investigated the anti-melanogenic effects and molecular mechanisms of two types of alfalfa extracts: alfalfa ethanol precipitate (AEP) and alfalfa ethanol supernatant (AES). Methods: The chemical composition of AEP and AES was analysed using HPAEC-PAD and LC–MS/MS. B16F10 cells and MNT-1 cells were used to demonstrate the inhibitory effect of two alfalfa seed extracts on melanin synthesis. The gene expression and protein levels of tyrosinase, tyrosinase-related protein 1 (TRP1), DCT and microphthalmia-associated factor (MITF) were confirmed using semi-quantitative RT-PCR, western blot and immunocytochemistry. Furthermore, the underlying mechanisms of these factors were elucidated in B16F10. The inhibitory effect on melanogenesis was validated using 3D artificial skin (MelanoDerm). Results: Both AEP and AES reduced melanin content in B16F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH) and subsequently decreased mRNA and protein levels of the melanogenesis-related targets, tyrosinase, TRP1 and MITF, as shown by semi-quantitative RT-PCR and immunocytochemistry. In addition, AEP and AES reduced protein levels of the MITF upstream regulators such as extracellular signal-mitogen-activated protein kinases (ERK), cAMP response element-binding protein (CREB) and β-catenin. Similar inhibition of melanin production and decreased expression of tyrosinase protein and MITF mRNA and protein were also confirmed in MNT-1 human melanoma cells. Using artificial human skin tissue (MelanoDerm), a significant reduction in melanin content was observed. Conclusion: Alfalfa seed extracts exert an inhibitory effect on the melanin production process by modulating the activity of ERK, CREB and β-catenin, thereby suppressing MITF and reducing the levels of tyrosinase, TRP1 and DCT. Collectively, these findings suggest that alfalfa extracts may be a promising avenue for further research and development in the fields of cosmetics and pharmaceuticals.