Takomthong_2026_Pharmaceuticals.(Basel)_19_

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by oxidative stress, cholinergic dysfunction, and amyloid-beta (Abeta) aggregation. Tri-Sannibat-Phol (TSB), a classical Thai polyherbal formulation comprising Piper retrofractum fruit, Ocimum tenuiflorum root, and Piper nigrum root, has been traditionally used for its medicinal properties, yet its anti-AD potential has never been scientifically evaluated. Methods: The therapeutic potential of TSB was investigated through in vitro bioassays including antioxidant, acetylcholinesterase (AChE) inhibitory, and anti-Abeta aggregation assays, alongside neuroprotective evaluation in H(2)O(2)-induced SH-SY5Y neuroblastoma cells. Acute oral toxicity was assessed in male ICR mice in accordance with OECD Guideline 420. Cognitive-enhancing effects were evaluated using the modified Y-maze, Novel Object Recognition, and Morris Water Maze tests in a scopolamine-induced amnesic mouse model. LC-MS/MS analysis was performed for phytochemical characterization and chemical standardization of the formulation. Results: TSB demonstrated significant antioxidant activity, AChE inhibitory activity, and anti-Abeta aggregation effects, with P. nigrum and O. tenuiflorum identified as the primary contributing components. Neuroprotective effects were confirmed in H(2)O(2)-induced SH-SY5Y cells, where TSB significantly improved cell viability across concentrations of 1-100 microg/mL. Acute oral toxicity assessment revealed an LD(50) exceeding 2000 mg/kg, indicating a favorable safety profile. In vivo behavioral studies demonstrated that TSB at medium-to-high doses significantly reversed scopolamine-induced cognitive deficits across all three behavioral tests. LC-MS/MS analysis identified thirteen piperidine alkaloids, with piperine as the dominant constituent at 17.61 +/- 0.80% w/w, proposed as the primary bioactive driver and chemical marker for future quality standardization. Conclusions: These findings suggest that TSB exerts multi-targeted anti-AD effects through complementary mechanisms, supporting its potential as a traditional medicine-based therapeutic candidate for further preclinical and clinical investigation.