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

Manju Rani, Dr. Umesh Kumar*

ABSTRACT

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.

Keywords: Epilepsy, Annona squamosa, Quercetin, ?-Caryophyllene, Anticonvulsant activity, Oxidative stress.