Toxicity of Spherulites Paenoy Superior Retinol and SymRenew™ HPR to zebrafish embryos. (A) The irritation experiment was carried out by exposing 24 hpf zebrafish embryos to 50 μg/mL Spherulites Paenoy Superior Retinol and 5 μg/mL SymRenew™ HPR for 15 min and self-rotation frequencies were recorded, compared with blank control (B). The acute toxicity test was performed by exposing 6 ~ 8 hpf zebrafish embryos to Spherulites Paenoy Superior Retinol and SymRenew™ HPR gradations respectively till 48 hpf. The blank control showed no dead or malformed embryos, the positive control DCA caused 30% mortality and 100% malformation to embryos. (C) Representative photos of embryos at the end of the experiment. (D, E) The mortality and teratogenicity responses and LC50 and TC50 values calculated by regression analysis. Compared with blank control, “**” denotes p < 0.01, “***” denotes p < 0.001.

The skin care efficacies of Spherulites Paenoy Superior Retinol were investigated using zebrafish embryos. Spherulites Paenoy Superior Retinol of 5 μg/mL was used in all seven experiments. (A, B) The anti-ROS experiment exposed 48 hpf embryos to Spherulites Paenoy Superior Retinol solution for 24 h, H2DCFDA stained with fluorescence signal intensity measured. The positive control 0.1 mg/mL glutathione reduced 13% (p = 0.0011) and Spherulites Paenoy Superior Retinol reduced 7.4% (p = 0.028) ROS compared with the blank control. (C, D) The anti-inflammation experiment treated 72 hpf zebrafish embryos with Spherulites Paenoy Superior Retinol solution in the presence of 10 μM CuSO4 (inflammation inducer) for 40 min, Sudan Black B stained, with neutrophils (stained brown dots) in the lateral midline region (red dash line circled) counted. The positive control 10 μM indomethacin reduced 30% (p < 0.001) and Spherulites Paenoy Superior Retinol reduced 20% (p < 0.001) neutrophil aggregation compared with the model control. (E, F) The blood circulation experiment exposed 72 hpf zebrafish embryos to Spherulites Paenoy Superior Retinol solution for 2 h, with dorsal aorta blood circulation speed measured. The positive control 50 μg/mL caffeine increased 28% (p < 0.001) and Spherulites Paenoy Superior Retinol increased 14% (p = 0.0063) blood circulation speed compared with the blank control. (G, H) The whitening experiment exposed 6 ~ 8 hpf zebrafish embryos to Spherulites Paenoy Superior Retinol solution till 48 hpf with embryo transmittance signal measured to correlate melanin level. The 30 μg/mL PTU inhibited embryos melanin development completely (100% whitening efficacy, p < 0.001), positive control 2.5 mg/mL kojic acid reduced 45% (p < 0.001) and Spherulites Paenoy Superior Retinol reduced 10% (p < 0.001) melanin development. (I, J) The wound-healing experiment exposed tail fin amputated zebrafish embryos to Spherulites Paenoy Superior Retinolsolution for 48 h, with tail fin length measured. The positive control 0.1% Rehmannia glutinousa Libosch extract enhanced 31% (p < 0.001) and Spherulites Paenoy Superior Retinol enhanced 9.0% (p = 0.030) tail fin regeneration. (K, L) The skin barrier protection experiment exposed 72 hpf zebrafish embryos to Spherulites Paenoy Superior Retinol solution for 4 h with the 1.1 mg/mL lactic acid and 0.02 mg/mL crystal violet were added to the solution for 20 min, using fluorescence area in the embryo tail region as the measurement. The positive control 150 μg/mL ectoin reduced 33% (p < 0.001) and Spherulites Paenoy Superior Retinol reduced 72% (p < 0.001) crystal violet penetrating through the skin barrier. (M, N) The gene regulation experiment exposed 4 dpf zebrafish embryos to Spherulites Paenoy Superior Retinol solution for 24 h, and the relative gene expression level by qRT-PCR was analyzed using β-Actin as the house-keeping gene. Type I collagen genes (col1a1a, col1a1b and col1a2) and elastin gene (elna) expression analysis showed that both positive control acetyl hexapeptide-8 (0.8 mg/mL) and Spherulites Paenoy Superior Retinol significantly (p < 0.05) increased the expression of these genes, and 5α-reductase genes (czsrd5a1, zsrd5a2a and zsrd5a2b) expression analysis revealed that positive control dutasteride (1 μM) and Spherulites Paenoy Superior Retinol significantly (p < 0.05) reduced the expression of these genes. Data was presented as mean ± SEM. “###” denotes compared with blank control, p < 0.001. Compared with blank control or model wherever model included, “*” p < 0.05, “**” p < 0.01, “***” p < 0.001.