ISOLATION, CHARACTERIZATION, AND EVALUATION OF ANTICONVULSANT POTENTIAL OF QUERCETIN AND Β-CARYOPHYLLENE FROM HYDROETHANOLIC EXTRACT OF ANNONA SQUAMOSA LINN. LEAVES

Epilepsy is a prevalent neurological disorder affecting around 50 million people globally, with nearly 30–40% of patients exhibiting resistance to existing antiepileptic drugs (AEDs) and suffering from significant side effects. The present study was undertaken to scientifically validate the traditional anticonvulsant use of Annona squamosa Linn. leaves by evaluating the phytochemical profile, safety, and therapeutic potential of its hydroethanolic extract and major isolated bioactive compounds, quercetin and β-caryophyllene. Fresh mature leaves (2000 g) collected from Green Earth Nursery, Gajraula, Uttar Pradesh, India, in July 2024, were shade-dried, powdered, and subjected to Soxhlet extraction with 70% hydroethanol, yielding 15% crude extract. Preliminary phytochemical screening confirmed the presence of flavonoids, terpenoids, alkaloids, phenols, tannins, saponins, glycosides, carbohydrates, and proteins. Quercetin and β-caryophyllene were successfully isolated through successive liquid-liquid partitioning and silica gel column chromatography, achieving purity greater than 95%. The isolated compounds were characterized using HPLC, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Quantitative analysis revealed substantial enrichment of total flavonoid content (950 ± 12.5 mg QE/g) and total terpenoid content (910 ± 10.2 mg CE/g) in the purified fractions. In vitro DPPH radical scavenging assay demonstrated potent antioxidant activity of quercetin with an IC₅₀ value of 18.2 µg/mL. Acute (OECD 423) and sub-acute (OECD 407) toxicity studies in Swiss albino mice confirmed an excellent safety profile, with no mortality or severe adverse effects observed up to 2000 mg/kg. In vivo anticonvulsant activity was assessed in maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizure models. Both compounds exhibited significant dose-dependent protection. Notably, their combination (25 mg/kg each) produced synergistic effects, achieving 85% seizure protection, markedly reduced seizure duration and severity, and efficacy comparable to standard drugs (phenytoin and diazepam). Biochemical investigations revealed that the combination treatment effectively attenuated oxidative stress (reduced MDA levels) and restored antioxidant defense (SOD, catalase, and GSH), while normalizing GABA and glutamate neurotransmitter balance in brain tissue. These findings provide strong scientific evidence supporting the ethnomedicinal use of Annona squamosa leaves and highlight quercetin and β-caryophyllene as promising, safe, multi-targeted natural candidates for the development of novel anticonvulsant therapeutics.