FIGURE SUMMARY
Title

Identification of Novel Melanin Synthesis Inhibitors From Crataegus pycnoloba Using an in Vivo Zebrafish Phenotypic Assay.

Authors
Agalou, A., Thrapsianiotis, M., Angelis, A., Papakyriakou, A., Skaltsounis, A.L., Aligiannis, N., Beis, D.
Source
Full text @ Front Pharmacol

Screening for inhibitors of melanogenesis from Crataegus pycnoloba extracts. Crude extract (A) and fractions (C–J) were tested for anti-pigmentation ability by in vivo zebrafish assay. Synchronized 24 hpf embryos were treated with 0.04 mg/mL of the crude extract and 0.1 mg/mL (CPC-Fr6-8) or 0.01 mg/mL (CPC-Fr9-13) from the different fractions for 24 h. Images were obtained at 48 hpf using a stereomicroscope. Test compounds were dissolved in DMSO. (B) Control treated embryo. Scale bar: 100 μM.

Effects of isolated compounds from the dibenzofuran family on melanin synthesis in zebrafish. (A) Embryos were treated from 24 to 48 hpf with 0.01 mg/mL of the compounds and the effects on the pigmentation were assessed using a stereomicroscope. (B) Melanin content was quantified by a photometric method. PTU was used as positive control. Results shown are the mean of three independent experiments ± SEM. p < 0.001, versus DMSO control.

Dibenzofuran compounds inhibit melanogenesis in a dose dependent manner. Synchronized embryos were treated with test compounds at the indicated concentrations from 24 hpf for 24 h. The effects on the pigmentation of zebrafish were observed at 48 hpf under the stereomicroscope. Treatment with 0.002 mg/mL had no effect on pigmentation for any of the tested compounds (A,D,G,J). Mild effect on melanogenesis was observed at 0.005 mg/mL (B,E,K). At 0.01 mg/mL there was a strong inhibition of melanogenesis (C,F,L). Compound 3 had no effect at any of the tested concentrations (G–I). Scale bar: 100 μM.

Isolated compounds from the dibenzofuran family repress reversibly in vivo pigmentation in zebrafish. Embryos were pretreated at 24 hpf with 0.01 mg/mL test compounds for 24 h. At 48 hpf pigmentation levels were recorded (A–F) and the embryos were divided into two groups. One group was washed and bathed immediately in fresh medium, whereas the other group was incubated with dibenzofuran compounds for further 24 h. At 72 hpf, the effect on melanogenesis was assessed using a stereomicroscope for both the compound treatment stopped group (G–L) and the embryos that were continuously treated with the dibenzofuran compounds for 48 h (M–R). PTU (E,K,Q) and DMSO (F,L,R) were used as positive and negative controls, respectively. (S) A schematic representation for the schedule of pigmentation rescue study. Scale bar: 100 μM.

Treatment with the dibenzofuran derivatives does not affect the expression of sox10 in zebrafish embryos. Lateral views of transgenic embryos 72 hpf from line Tg(sox10:GFP) treated from 3 hpf with DMSO (A,B) or the compound 1 (C,D). Both treated and non-treated embryos show extensive GFP expression throughout forebrain (A), anterior midbrain (B), otic epithelium (C), and branchial arches (D) while in lateral views of the trunk there is increased GFP expression in oligodendrocytes (arrow) and Schwann cells (arrowhead).

Acknowledgments
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