The in cellular and in vivo melanogenesis inhibitory activity of safflospermidines from Helianthus annuus L. bee pollen in B16F10 murine melanoma cells and zebrafish embryos.

Abstract

Melanin, synthesized by tyrosinase (TYR), is a natural pigment essential for skin protection and pigmentation. However, excessive melanin production can cause dermatological disorders. Safflospermidines, comprised of safflospermidine A and B isomers isolated from sunflower (Helianthus annuus L.) bee pollen, were shown to exhibit a strong in vitro TYR inhibitory activity against mushroom TYR. However, their anti-melanogenesis activity in cellular and in vivo models remains unexplored. This study firstly evaluated the effects of these safflospermidines on melanogenesis in α-melanocyte stimulating hormone-stimulated B16F10 cells, using kojic acid as a positive control. Cytotoxicity was evaluated using the MTT assay, while TYR activity and melanin content were measured to assess melanogenesis inhibition. The expression of key melanogenesis-related genes was analyzed through quantitative real-time reverse transcription (RT-q)PCR to elucidate the molecular mechanisms involved. Secondly, the melanogenic activity and potential toxicity of the compounds were confirmed in zebrafish embryos, with phenylthiourea (PTU) as a reference. The results revealed that a mixture of these two safflospermidines exhibited no cytotoxicity across the treated concentration range (0-500 µg/mL). At 62.5 µg/mL, safflospermidines significantly reduced the intracellular melanin content by 21.78 ± 4.01% and the TYR activity by 25.71 ± 3.08% in B16F10 cells through downregulation of TYR, TYR-related protein 1 (TRP-1), and TRP-2 gene expression. Additionally, the safflospermidines induced a noticeable reduction in dendritic cell structures, which likely contributed to the marked decrease in extracellular melanin levels. Kojic acid (250 μg/mL) significantly reduced the melanin content and TYR activity by suppressing all three melanogenesis-related genes. In zebrafish embryos, safflospermidines showed no toxicity or morphological abnormalities within a concentration range of 0-62.5 µg/mL, and at a concentration of 15.63 µg/mL the melanin production in zebrafish embryos was significantly reduced by 28.43 ± 9.17%. In comparison, 0.0015% (v/v) PTU decreased melanin production by 53.20 ± 3.75%. These findings suggest that safflospermidines are safe, effective melanin inhibitors with potential applications in pharmaceuticals and cosmetics for managing hyperpigmentation.