Combined Ethanol Extract of Piper guineense Leaf and Zingiber officinale Rhizome Attenuates Aluminium Chloride Induced Dementia in Wistar Rats by Inhibiting NADPH Oxidase Driven Oxidative Stress and Neuroinflammation

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder with limited treatment options. Neuroinflammation and oxidative stress, driven by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, play central roles in AD pathogenesis. This study evaluated the safety profile and neuroprotective efficacy of a combined ethanol extract of Piper guineense leaf and Zingiber officinale rhizome in an aluminium chloride (AlCl3)-induced dementia model in Wistar rats. Methods: Acute toxicity was assessed using a modified Lorke's method. Thirty male Wistar rats were divided into six groups (n=5): normal control, AlCl₃ control (120 mg/kg), extract control (800 mg/kg), low-dose extract (400 mg/kg + AlCl₃), high-dose extract (800 mg/kg + AlCl3), and donepezil (5 mg/kg + AlCl3). The extract doses (400 and 800 mg/kg) were selected based on neuroprotective doses reported in previous studies on the individual plants, and the LD₅₀ was determined to confirm safety. After 21 days, brain homogenates were analysed for NADPH oxidase, nitric oxide metabolites (NOx), nitrate, malondialdehyde (MDA), antioxidant enzymes (SOD, CAT, GPx), and cytokines (TNF-α, IL-6, IL-1β). Results: The LD₅₀ was 7745.97 mg/kg, indicating a wide safety margin. AlCl3 administration significantly upregulated NADPH oxidase (73.66±1.17 vs. 47.96±0.99 pg/mg in control), elevated total NOx, depleted nitrate levels, reduced SOD (from 26.84±0.90 to 12.58±0.26 u/g), CAT (from 11.67±0.29 to 5.71±0.12 u/g), and GPx (from 38.96±0.97 to 20.17±0.75 u/g), and increased MDA three-fold (0.60±0.03 vs. 0.20±0.01 mmol/g). Pro-inflammatory cytokines were markedly elevated. High-dose extract (800 mg/kg) significantly inhibited NADPH oxidase (50.00±0.26 pg/mg), restored nitrate levels (3.48±0.14 µmol/g), normalized antioxidant enzymes (SOD: 24.48±0.40 u/g; CAT: 10.24±0.24 u/g; GPx: 34.31±0.66 u/g), reduced MDA (0.28±0.01 mmol/g), and suppressed cytokine levels, with efficacy comparable to donepezil. Conclusion: The combined extract demonstrates a favourable safety profile and exerts neuroprotective effects by inhibiting NADPH oxidase, restoring redox homeostasis, and suppressing neuroinflammation, supporting its potential as a multi-target therapeutic agent for neurodegenerative conditions.