Khongkarat Phanthiwa, Kitipaspallop Wannakarn, Puthong Songchan, Pimtong Wittaya, Phuwapraisirisan Preecha, Chanchao Chanpen
Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand.
PLoS One. 2025 Jun 24;20(6):e0325264. doi: 10.1371/journal.pone.0325264. eCollection 2025.
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.
黑色素由酪氨酸酶（TYR）合成，是一种对皮肤保护和色素沉着至关重要的天然色素。然而，黑色素产生过多会导致皮肤病。从向日葵（Helianthus annuus L.）蜂花粉中分离出的由萨夫洛 spermidine A和B异构体组成的萨夫洛 spermidines，对蘑菇TYR表现出很强的体外TYR抑制活性。然而，它们在细胞和体内模型中的抗黑色素生成活性仍未得到探索。本研究首先以曲酸为阳性对照，评估了这些萨夫洛 spermidines对α-黑素细胞刺激激素刺激的B16F10细胞中黑色素生成的影响。使用MTT法评估细胞毒性，同时测量TYR活性和黑色素含量以评估黑色素生成抑制。通过定量实时逆转录（RT-q）PCR分析关键黑色素生成相关基因的表达，以阐明其中涉及的分子机制。其次，以苯硫脲（PTU）为参考，在斑马鱼胚胎中证实了这些化合物的黑色素生成活性和潜在毒性。结果显示，在处理浓度范围（0-500 µg/mL）内，这两种萨夫洛 spermidines的混合物未表现出细胞毒性。在62.5 µg/mL时，萨夫洛 spermidines通过下调TYR、TYR相关蛋白1（TRP-1）和TRP-2基因表达，显著降低了B16F10细胞内的黑色素含量21.78±4.01%，并降低了TYR活性25.71±3.08%。此外，萨夫洛 spermidines使树突状细胞结构明显减少，这可能导致细胞外黑色素水平显著降低。曲酸（250 μg/mL）通过抑制所有三个黑色素生成相关基因，显著降低了黑色素含量和TYR活性。在斑马鱼胚胎中，萨夫洛 spermidines在0-62.5 µg/mL的浓度范围内未显示出毒性或形态异常，在15.63 µg/mL的浓度下，斑马鱼胚胎中的黑色素生成显著降低了28.43±9.17%。相比之下，0.0015%（v/v）的PTU使黑色素生成降低了53.20±3.75%。这些发现表明，萨夫洛 spermidines是安全、有效的黑色素抑制剂，在治疗色素沉着过度的药物和化妆品中具有潜在应用价值。
