{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:38:18Z","timestamp":1785411498119,"version":"3.56.0"},"reference-count":40,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T00:00:00Z","timestamp":1783555200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NSRF via the Program Management Unit for Human Resources &amp; Institutional Development, Research and Innovation, Thailand","award":["B13F680065"],"award-info":[{"award-number":["B13F680065"]}]},{"name":"Program Management Unit for Human Resources &amp; Institutional Development, Research and Innovation, Thailand","award":["B38G670003"],"award-info":[{"award-number":["B38G670003"]}]},{"name":"Faculty of Pharmaceutical Sciences, Khon Kaen University","award":["-"],"award-info":[{"award-number":["-"]}]},{"award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"https:\/\/ror.org\/03cq4gr50","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Ubon Ratchathani University","award":["-"],"award-info":[{"award-number":["-"]}]},{"award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"https:\/\/ror.org\/045nemn19","id-type":"ROR","asserted-by":"publisher"}]},{"name":"BCG Economy and Sustainable Development Network through Center of Excellence Consortium under the Reinventing University System (Khon Kaen University),Visiting Professor Program 2025, Thailand","award":["-"],"award-info":[{"award-number":["-"]}]},{"award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"https:\/\/ror.org\/03cq4gr50","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background: Alzheimer\u2019s disease (AD) is a progressive neurodegenerative disorder characterized by oxidative stress, cholinergic dysfunction, and amyloid-\u03b2 (A\u03b2) 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-A\u03b2 aggregation assays, alongside neuroprotective evaluation in H2O2-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-A\u03b2 aggregation effects, with P. nigrum and O. tenuiflorum identified as the primary contributing components. Neuroprotective effects were confirmed in H2O2-induced SH-SY5Y cells, where TSB significantly improved cell viability across concentrations of 1\u2013100 \u00b5g\/mL. Acute oral toxicity assessment revealed an LD50 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 \u00b1 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.<\/jats:p>","DOI":"10.3390\/ph19071063","type":"journal-article","created":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T16:16:44Z","timestamp":1783613804000},"page":"1063","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Multi-Targeted Anti-Alzheimer\u2019s Effects of Tri-Sannibat-Phol: Biological Evaluation, Behavioral Validation, and LC-MS\/MS Phytochemical Profiling"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8358-0420","authenticated-orcid":false,"given":"Pitchayakarn","family":"Takomthong","sequence":"first","affiliation":[{"name":"Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pornthip","family":"Waiwut","sequence":"additional","affiliation":[{"name":"Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1116-4636","authenticated-orcid":false,"given":"Sumet","family":"Kongkiatpaiboon","sequence":"additional","affiliation":[{"name":"Drug Discovery and Development Center, Office of Advanced Science and Technology, Thammasat University (Rangsit Campus), Pathum Thani 12121, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-5759-6418","authenticated-orcid":false,"given":"Khemjira","family":"Phemphunananchai","sequence":"additional","affiliation":[{"name":"Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4065-6090","authenticated-orcid":false,"given":"Chantana","family":"Boonyarat","sequence":"additional","affiliation":[{"name":"Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand"},{"name":"Center for Research and Development of Herbal Health Products, Khon Kaen University, Khon Kaen 40002, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,9]]},"reference":[{"key":"ref_1","unstructured":"(2024). Alzheimer\u2019s Association Report 2024 Alzheimer\u2019s Disease Facts and Figures. Alzheimer\u2019s Dement. J. Alzheimer\u2019s Assoc., 20, 3708\u20133821."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"S177","DOI":"10.37765\/ajmc.2020.88482","article-title":"Economic Burden of Alzheimer Disease and Managed Care Considerations","volume":"26","author":"Wong","year":"2020","journal-title":"Am. J. Manag. Care"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"115522","DOI":"10.1016\/j.bcp.2023.115522","article-title":"Alzheimer\u2019s Disease: Insights and New Prospects in Disease Pathophysiology, Biomarkers and Disease-Modifying Drugs","volume":"211","author":"Monteiro","year":"2023","journal-title":"Biochem. Pharmacol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Breijyeh, Z., and Karaman, R. (2020). Comprehensive Review on Alzheimer\u2019s Disease: Causes and Treatment. Molecules, 25.","DOI":"10.3390\/molecules25245789"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Peng, Y., Jin, H., Xue, Y., Chen, Q., Yao, S., Du, M., and Liu, S. (2023). Current and Future Therapeutic Strategies for Alzheimer\u2019s Disease: An Overview of Drug Development Bottlenecks. Front. Aging Neurosci., 15.","DOI":"10.3389\/fnagi.2023.1206572"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"7299778","DOI":"10.1155\/2022\/7299778","article-title":"Potential Therapeutic Applications of Plant-Derived Alkaloids against Inflammatory and Neurodegenerative Diseases","volume":"2022","author":"Aryal","year":"2022","journal-title":"Evid.-Based Complement. Altern. Med."},{"key":"ref_7","first-page":"605","article-title":"Health Promotion in Thai Traditional Medicine Practice: Related Theories and Strategies","volume":"20","author":"Pimpa","year":"2022","journal-title":"J. Thai Tradit. Altern. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1016\/j.ctim.2015.10.003","article-title":"Traditional Chinese and Thai Medicine in a Comparative Perspective","volume":"23","author":"He","year":"2015","journal-title":"Complement. Ther. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5125","DOI":"10.3390\/molecules18055125","article-title":"Herb-Herb Combination for Therapeutic Enhancement and Advancement: Theory, Practice and Future Perspectives","volume":"18","author":"Che","year":"2013","journal-title":"Molecules"},{"key":"ref_10","first-page":"149","article-title":"The Promotion of Thai Traditional Medicine Policy in Government Hospitals: Myth or Reality","volume":"9","author":"Vadhnapijyakul","year":"2014","journal-title":"Isan J. Pharm. Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"798","DOI":"10.1016\/j.fct.2009.12.009","article-title":"Piperine, the Main Alkaloid of Thai Black Pepper, Protects against Neurodegeneration and Cognitive Impairment in Animal Model of Cognitive Deficit like Condition of Alzheimer\u2019s Disease","volume":"48","author":"Chonpathompikunlert","year":"2010","journal-title":"Food Chem. Toxicol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Abdul Manap, A.S., Wei Tan, A.C., Leong, W.H., Yin Chia, A.Y., Vijayabalan, S., Arya, A., Wong, E.H., Rizwan, F., Bindal, U., and Koshy, S. (2019). Synergistic Effects of Curcumin and Piperine as Potent Acetylcholine and Amyloidogenic Inhibitors With Significant Neuroprotective Activity in SH-SY5Y Cells via Computational Molecular Modeling and in Vitro Assay. Front. Aging Neurosci., 11.","DOI":"10.3389\/fnagi.2019.00206"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"6632","DOI":"10.1021\/jf501006y","article-title":"Assessment of Antioxidative, Chelating, and DNA-Protective Effects of Selected Essential Oil Components (Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus Officinalis Oil","volume":"62","author":"Horvathova","year":"2014","journal-title":"J. Agric. Food Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"154930","DOI":"10.1016\/j.phymed.2023.154930","article-title":"Eugenol Relieves the Pathological Manifestations of Alzheimer\u2019s Disease in 5 \u00d7 FAD Mice","volume":"118","author":"Jung","year":"2023","journal-title":"Phytomedicine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1225578","DOI":"10.1155\/2022\/1225578","article-title":"ROS: Basic Concepts, Sources, Cellular Signaling, and Its Implications in Aging Pathways","volume":"2022","author":"Dantas","year":"2022","journal-title":"Oxid. Med. Cell. Longev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4828","DOI":"10.3892\/mmr.2020.11548","article-title":"Monotropein Alleviates H2O2-Induced Inflammation, Oxidative Stress and Apoptosis via NF-\u03baB\/AP-1 Signaling","volume":"22","author":"Jiang","year":"2020","journal-title":"Mol. Med. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"899","DOI":"10.4162\/nrp.2023.17.5.899","article-title":"Neuroprotective Effects of Hesperetin on H2O2-Induced Damage in Neuroblastoma SH-SY5Y Cells","volume":"17","author":"Moon","year":"2023","journal-title":"Nutr. Res. Pract."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"14722","DOI":"10.1038\/s41598-023-41996-4","article-title":"A Quantitative Analysis of Spontaneous Alternation Behaviors on a Y-Maze Reveals Adverse Effects of Acute Social Isolation on Spatial Working Memory","volume":"13","author":"Kim","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1007\/s10339-011-0430-z","article-title":"The Novel Object Recognition Memory: Neurobiology, Test Procedure, and Its Modifications","volume":"13","author":"Antunes","year":"2012","journal-title":"Cogn. Process."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"848","DOI":"10.1038\/nprot.2006.116","article-title":"Morris Water Maze: Procedures for Assessing Spatial and Related Forms of Learning and Memory","volume":"1","author":"Vorhees","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1538\/expanim.21-0120","article-title":"Morris Water Maze: A Versatile and Pertinent Tool for Assessing Spatial Learning and Memory","volume":"71","author":"Othman","year":"2022","journal-title":"Exp. Anim."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"8022","DOI":"10.1039\/C4AY01088D","article-title":"Identification and Characterization of Stress Degradation Products of Piperine and Profiling of a Black Pepper (Piper nigrum L.) Extract Using LC\/Q-TOF-Dual ESI-MS","volume":"6","author":"Kotte","year":"2014","journal-title":"Anal. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"100995","DOI":"10.1016\/j.phyplu.2026.100995","article-title":"Synergistic Phytochemicals in Multi-Target Drug Discovery for Complex Diseases: A Narrative Review","volume":"6","author":"Ugwu","year":"2026","journal-title":"Phytomed. Plus"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"100021","DOI":"10.1016\/j.pscia.2023.100021","article-title":"Emerging Role of Antioxidants in Alzheimer\u2019s Disease: Insight into Physiological, Pathological Mechanisms and Management","volume":"2","author":"Kamaljeet","year":"2024","journal-title":"Pharm. Sci. Adv."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"11854","DOI":"10.1021\/acs.jafc.4c01323","article-title":"Natural Antioxidants: An Effective Strategy for the Treatment of Alzheimer\u2019s Disease at the Early Stage","volume":"72","author":"Wang","year":"2024","journal-title":"J. Agric. Food Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"S210","DOI":"10.1080\/10408398.2019.1565489","article-title":"A Systematic Review on Black Pepper (Piper nigrum L.): From Folk Uses to Pharmacological Applications","volume":"59","author":"Takooree","year":"2019","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"s151","DOI":"10.4103\/pm.pm_539_20","article-title":"Antioxidant and Antiacetylcholinestrase Studies of In Vitro Regenerated and Transformed Hairy Roots of Ocimum sanctum (L.)","volume":"17","author":"Toppo","year":"2021","journal-title":"Pharmacogn. Mag."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e50","DOI":"10.1192\/j.eurpsy.2025.2446","article-title":"The Effect of Scopolamine on Memory and Attention: A Systematic Review and Meta-Analysis","volume":"68","author":"Miravalles","year":"2025","journal-title":"Eur. Psychiatry"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.ejpb.2016.03.029","article-title":"In Vitro Blood\u2013Brain Barrier Permeability Predictions for GABAA Receptor Modulating Piperine Analogs","volume":"103","author":"Eigenmann","year":"2016","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Falsafi, S.K., Deli, A., H\u00f6ger, H., Pollak, A., and Lubec, G. (2012). Scopolamine Administration Modulates Muscarinic, Nicotinic and NMDA Receptor Systems. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0032082"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1393","DOI":"10.1002\/hipo.22615","article-title":"Detecting and Discriminating Novel Objects: The Impact of Perirhinal Cortex Disconnection on Hippocampal Activity Patterns","volume":"26","author":"Kinnavane","year":"2016","journal-title":"Hippocampus"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.neurobiolaging.2023.06.003","article-title":"Perirhinal Cortex Is Associated with Fine-Grained Discrimination of Conceptually Confusable Objects in Alzheimer\u2019s Disease","volume":"130","author":"Frick","year":"2023","journal-title":"Neurobiol. Aging"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Mak, E., Kantarci, K., and Arvanitakis, Z. (2025). Sex Differences in in Vivo Biomarkers of Neurodegenerative Dementia. Front. Dement., 4.","DOI":"10.3389\/frdem.2025.1706177"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.2174\/1567205015666180613111504","article-title":"Sex and Gender Differences in the Brain Cholinergic System and in the Response to Therapy of Alzheimer Disease with Cholinesterase Inhibitors","volume":"15","author":"Giacobini","year":"2018","journal-title":"Curr. Alzheimer Res."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Luca, S.V., Gawe\u0142-B\u0119ben, K., Strz\u0119pek-Gom\u00f3\u0142ka, M., Czech, K., Trifan, A., Zengin, G., Korona-Glowniak, I., Minceva, M., Gertsch, J., and Skalicka-Wo\u017aniak, K. (2021). Insights into the Phytochemical and Multifunctional Biological Profile of Spices from the Genus Piper. Antioxidants, 10.","DOI":"10.3390\/antiox10101642"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Takomthong, P., Waiwut, P., Yenjai, C., Wangboonskul, J., Plekratoke, K., Arsito, P.N., Ballatore, C., and Boonyarat, C. (2025). Kaempferia Parviflora Extract and Its Methoxyflavones as Potential Anti-Alzheimer Assessing in Vitro, Integrated Computational Approach, and in Vivo Impact on Behaviour in Scopolamine-Induced Amnesic Mice. PLoS ONE, 20.","DOI":"10.1371\/journal.pone.0316888"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/0006-2952(61)90145-9","article-title":"A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity","volume":"7","author":"Ellman","year":"1961","journal-title":"Biochem. Pharmacol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1021\/acschemneuro.5b00214","article-title":"Curcumin Attenuates Amyloid-\u03b2 Aggregate Toxicity and Modulates Amyloid-\u03b2 Aggregation Pathway","volume":"7","author":"Thapa","year":"2016","journal-title":"ACS Chem. Neurosci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1016\/j.foodchem.2011.04.089","article-title":"Curcumin Protects Mouse Neuroblastoma Neuro-2A Cells against Hydrogen-Peroxide-Induced Oxidative Stress","volume":"129","author":"Zhao","year":"2011","journal-title":"Food Chem."},{"key":"ref_40","unstructured":"(2001). OECD Guideline for Testing of Chemicals, OECD. Test No. 420: Acute Oral Toxicity\u2014Fixed Dose Procedure."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/7\/1063\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T04:11:14Z","timestamp":1784175074000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/7\/1063"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,9]]},"references-count":40,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ph19071063"],"URL":"https:\/\/doi.org\/10.3390\/ph19071063","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,9]]}}}