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.
