SYNTHESIS OF ΑLPHA-PINENE HYDROPEROXIDES AND TOXICITY ASSESSMENT VIA ZEBRAFISH EMBRYO

Abstract

Alpha-pinene is a naturally occurring monoterpene present in the essential oils of various plants. The oxidation of α-pinene with singlet oxygen [1O2(1Δg)] yields hydroperoxide and hydroxide derivatives that possess potential biological activities. Considering the known anxiolytic properties of α-pinene, these oxidized derivatives may exhibit comparable or enhanced effects, thereby justifying further investigation, prompting the present study. The photo-oxidation reaction was carried out by combining 1 cm³ (0.858 g) of α-pinene with 0.001 g of methylene blue as the photosensitizer in 50 mL of chloroform. The mixture was exposed to continuous oxygen flow and irradiated for 6 hours using a 200 W tungsten halogen lamp as the light source. Reaction progress and purification were monitored via thin-layer and column chromatography, with structural characterization by NMR and FT-IR spectroscopy. Toxicity was evaluated by exposing Zebrafish (Danio rerio) embryos to α-pinene and its hydroperoxide at varying concentrations and observing developmental endpoints over a 96-hour period. α-pinene hydroperoxide formation was verified by the 1H NMR peaks at 5.52 (-OOH), 4.49, 4.25, 1.27, 0.64 ppm, and FT-IR absorptions at 3340.2 (-OH stretching) and 844.09 cm⁻ ¹ (O-O). α-pinene and its hydroperoxide exhibited dose-dependent toxicity, with 100% mortality at ≥300 mg/L, while lower concentrations (37.50 and 18.75 mg/L) showed reduced toxicity. The LC₅₀ value of α-pinene hydroperoxide was 69 mg/L. Exposure to α-pinene hydroperoxide caused significant developmental malformations, including pericardial and yolk sac edema. Additionally, spinal curvature was frequently observed during the early stages of embryonic development. However, this malformation showed signs of gradual correction over time and was notably resolved by 96 hours post-exposure (hpe), suggesting a potential for developmental recovery under sublethal exposure conditions. However, further studies are required to confirm these observations. α-Pinene completely inhibited hatching (0%) at concentrations of 75-600 mg/L, whereas α pinene hydroperoxide caused complete inhibition at 150-600 mg/L and improved hatching rates (40-80%) at lower concentrations (18.75-75 mg/L). In conclusion, spectral data confirms the hydroperoxide formation, and the Zebrafish Embryo Toxicity (ZET) assay highlights the dose-dependent toxicity of α-pinene hydroperoxide in zebrafish embryos.