A Safe Natural Alternative to Phenylthiourea: Ethyl Acetate Extract of Alchemilla vulgaris for Zebrafish Embryo Depigmentation

Background: Zebrafish (Danio rerio) embryos are transparent in early stages of embryonic development; however, pigment formation at later stages hinders internal organ visualization during imaging. Chemicals such as 1-phenyl-2-thiourea (PTU) and kojic acid, used to block pigmentation, pose significant toxicity risks to human health. Therefore, effective and risk-free depigmentation agents are needed. This study investigates the efficacy of Alchemilla vulgaris (Lady’s mantle) as a safe, natural alternative for zebrafish embryo depigmentation. Methods: A. vulgaris was extracted using four solvents of varying polarities and evaluated for depigmentation efficacy and toxicity. Gas chromatography–mass spectrometry (GC–MS) was used to identify major constituents of the extract. Results: Ethyl acetate extract was more effective at removing pigments than all other extracts, and exhibited the lowest toxicity compared to PTU and kojic acid. Ethyl acetate extract of A. vulgaris remained effective even when administered 48 h post fertilization (post-pigmentation), making it suitable for long-term experiments requiring optical clarity. GC-MS revealed that this extract was rich in linoleic acid, various fatty acid esters, and phenolics, which likely contributed to its depigmentation activity. Conclusions: Based on these findings, we propose that ethyl acetate extract of A. vulgaris is a safer, natural alternative to PTU and kojic acid for depigmenting zebrafish embryos, particularly in long-term imaging experiments. The extract exhibits high efficacy at low concentrations, accompanied by a favorable toxicity profile, demonstrating potential as a depigmentation agent during early zebrafish development. However, further studies are needed to elucidate its mechanism of action.