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.

Fig 1. The chemical structure of safflospermidine A and B [21].

Fig 2. Relative cell viability of B16F10 cells following treatment with various concentrations of (A) safflospermidines and (B) kojic acid for 24, 48, and 72 h, as determined by the MTT assay.

Data are presented as the mean ± SD from three independent experiments. Significant differences compared to the control group are shown as ** and *** for p < 0.01 and p < 0.001, respectively, (one-way ANOVA followed by Tukey’s post-hoc test for kojic acid-treated cells at all time points).

Fig 3. Effect of safflospermidines and kojic acid on B16F10 cells after 72 h treatment, showing the (A) intracellular and (B) extracellular melanin content.

Melanin content is expressed as a percentage of the control group (set at 100%). Data are presented as the mean ± SD from three independent experiments. Significant differences compared to the control group are shown as *, **, and *** for p < 0.05, p < 0.01, and p < 0.001, respectively, (one-way ANOVA followed by Tukey’s post-hoc test for intracellular melanin and Dunnett’s T3 test for extracellular melanin). The bottom panel shows the color intensity of (A) dissolved melanin after cell lysis and (B) melanin secreted into the CM.

Fig 4. Morphology of B16F10 cells after 72 h of exposure to safflospermidines at (A) 0 μg/mL (control), (B) 62.5 μg/mL, (C) 125 μg/mL, (D) 250 μg/mL, and (E) 500 μg/mL, and (F) 250 μg/mL of kojic acid; as observed at 200X magnification under light microscopy.

Each image is representative of three independent experiments. Scale bar = 50 μm.

Fig 5. Effect of safflospermidines and kojic acid treatment for 48 h on the cellular TYR activity of B16F10 cells.

Results are presented as the mean ± SD from three independent experiments. Significant differences from the control group are shown as * and *** for p < 0.05 and p < 0.001, respectively, (one-way ANOVA followed by Tukey’s post-hoc test).

Fig 6. Effects of safflospermidines and kojic acid treatment for 48 h on the relative transcript levels of genes involved in melanogenesis in B16F10 cells.

The mRNA expression level of TYR, TRP-1, and TRP-2, normalized to GAPDH, is shown relative to the untreated group. Results are presented as the mean ± SD of three independent experiments. Significant differences compared to the control group are shown as *, **, and *** for p < 0.05, p < 0.01, and p < 0.001, respectively, (one-way ANOVA followed by Tukey’s post-hoc test).

Fig 7. Effects of safflospermidines on zebrafish embryos: (A) Survival rate (%) and (B) relative melanin content (% of control) after exposure to safflospermidines at 3.91–62.50 µg/mL or 0.0015% (v/v) PTU. Significant differences from the control group are shown as * and *** for p < 0.05 and p < 0.001, respectively, (one-way ANOVA followed by Tukey’s post hoc test). (C) Dorsal view showing the morphology and melanin production after 48 h of treatment.