Unveiling the antioxidant and anti-epileptic potential of Castanea sativa against pentylenetetrazole in mice: a comparative LC-MS analysis of two ethanol-based fractions

Abstract

Epilepsy is a chronic neurological disorder in which oxidative stress and neuroinflammation play key pathogenic roles. There is growing interest in natural antioxidants with neuroprotective properties as complementary therapies. Castanea sativa (C. sativa) fruits, rich in phenolic acids and flavonoids, may offer such potential. The current study aimed to assess the effect of Castanea sativa fractions on pentylenetetrazol (PTZ)-induced epilepsy in mice via modulation of brain Gamma-Aminobutyric acid (GABA), Excitatory amino acid transporter 2 (EAAT2), Tumor Necrosis Factor-alpha(TNF-α), and Malondialdehyde(MDA). Fruits were sequentially extracted using 70% and 100% ethanol to yield two fractions, which were phytochemically profiled via liquid chromatography-mass spectrometry (LC-MS). In vitro antioxidant capacity was assessed using DPPH, ABTS, and FRAP assays. In vivo efficacy was evaluated using a PTZ-induced seizure mouse model, with animals receiving vehicle, diazepam (1 mg kg⁻¹), or C. sativa extracts (400 mg kg⁻¹) orally, 30 minutes before PTZ injection (60 mg kg⁻¹). LC-MS profiling revealed over 70 metabolites, primarily phenolic acids (ellagic and gallic derivatives), flavonoid glycosides (quercetin and kaempferol), and several novel lignans and iridoid glycosides. The 100% ethanol fraction exhibited greater chemical diversity and peak intensities, alongside markedly stronger antioxidant activity, as reflected by lower IC₅₀ values (193.65 µg mL⁻¹ for DPPH and 128.87 µg mL⁻¹ for ABTS) and nearly fivefold higher FRAP capacity (26.59 µg TE mg⁻¹ fraction) compared to the 70% ethanol fraction. In vivo, the 100% extract delayed seizure onset 2.8-fold and reduced seizure duration and mortality, showing efficacy close to diazepam. Additionally, both extracts enhanced brain GABA, EAAT2, and reduced Glutathione(GSH) levels, while decreasing MDA, TNF-α, and Nuclear Factor kappa-B(NF-κβ) compared to the PTZ group. These findings support the antioxidant and neuroprotective potential of C. sativa fruit extracts, especially the 100% ethanol fraction, highlighting their promise as functional food ingredients or nutraceutical adjuncts in epilepsy management.