Anethole from Star Anise and Fennel Shows Promise Against Alzheimer-Like Memory Loss in Rats

Trans-anethole, the aromatic molecule that gives star anise, aniseed, and fennel their distinctive licorice-like scent, has emerged as a surprising candidate in the search for treatments that go beyond the symptomatic relief offered by current Alzheimer’s disease therapies. In a study published in Frontiers in Pharmacology, researchers from NGSM Institute of Pharmaceutical Sciences at Nitte (Deemed to be University) in Mangalore, India, working with a collaborator at Silicon Script Sciences in Nepal, report that this common food-grade compound significantly improved memory performance in a rat model of Alzheimer-like cognitive dysfunction while restoring the brain’s antioxidant defenses and cholinergic signaling. The findings, drawn from an unusually comprehensive integration of computational pharmacology, cell-based assays, and live animal experiments, position trans-anethole as a potential multi-target neuroprotective preclinical candidate worthy of further investigation.

The motivation behind the work reflects a persistent frustration in Alzheimer’s research. Current medications, including donepezil, rivastigmine, galantamine, and memantine, provide only symptomatic benefit and do nothing to halt the underlying neurodegenerative process. Because Alzheimer’s disease is multifactorial, characterized by amyloid-beta plaque accumulation, hyperphosphorylated tau tangles, oxidative stress, neuroinflammation, mitochondrial dysfunction, and progressive cholinergic neuron loss, many researchers argue that effective future treatments will need to engage multiple pathological pathways simultaneously rather than a single molecular target. Plants have long served as a source of such compounds, and trans-anethole, the most abundant and non-toxic isomer of anethole, possesses documented antioxidant, anti-inflammatory, antibacterial, antifungal, anticonvulsant, antinociceptive, and anticancer properties. Previous reports have suggested neuroprotective effects on cortical neuronal cells and anti-amnesic activity in behavioral tasks, hinting that the molecule might be beneficial in neurodegenerative disease.

To test this hypothesis rigorously, the team designed a three-tiered experimental strategy. The computational arm used network pharmacology to predict which human proteins and signaling pathways trans-anethole might engage in the context of Alzheimer’s disease. The in vitro arm evaluated cytotoxicity, antioxidant enzyme modulation, and cholinesterase inhibition in BV2 murine microglial cells, comparing trans-anethole against donepezil as a standard. The in vivo arm employed the intracerebroventricular streptozotocin model, in which a single unilateral injection of streptozotocin at 3 mg/kg into the lateral ventricle of adult Wistar rats induces a brain insulin-resistant state that progressively impairs cognition and memory, a condition that closely mirrors sporadic Alzheimer’s disease in humans. This model reproduces cholinergic dysfunction, tau hyperphosphorylation, insulin receptor impairment, mitochondrial dysfunction, oxidative stress, and disrupted energy metabolism, the hallmark features of the human condition, without relying on transgenic amyloid-overproducing animals.

The computational results were striking in their breadth. Trans-anethole satisfied Lipinski’s criteria with a molecular weight of 148.09 g/mol, a consensus logP of 2.79, one hydrogen bond acceptor, no hydrogen bond donors, and a very low topological polar surface area of 9.23 Ų, indicating excellent membrane permeability and likely blood-brain barrier penetration. Pharmacokinetic predictions showed high gastrointestinal absorption, no P-glycoprotein substrate liability, and a bioavailability score of 0.55, with no PAINS or Brenk structural alerts. When the researchers merged 237 predicted trans-anethole targets with 584 Alzheimer’s disease-associated targets drawn from OMIM, TTD, GeneCards, CTD, and DisGeNET, they identified 174 common targets. Protein-protein interaction analysis using STRING revealed a densely connected network of 171 nodes and 4,176 edges with an average node degree of 48.8 and a statistically significant enrichment p-value below 1.0 × 10⁻¹⁶. KEGG pathway enrichment pointed to apoptosis, AGE-RAGE signaling, TNF signaling, HIF-1 signaling, and PI3K-Akt signaling as the dominant pathways, all closely aligned with Alzheimer’s pathology. Gene Ontology analysis further highlighted oxidative stress response, hypoxia response, and regulation of apoptosis as the most enriched biological processes, with molecular functions concentrated in enzyme binding, kinase binding, and ubiquitin ligase binding, and cellular components enriched in membrane rafts, mitochondrial outer membrane, and secretory vesicles.

Molecular docking with AutoDock Vina across 34 target proteins identified AKT1, CDK5, and IDE as the top three binding partners, with binding energies of −6.5, −6.4, and −6.4 kcal/mol respectively. AKT1, the serine/threonine kinase central to the PI3K-Akt survival pathway, engaged trans-anethole through five π bond interactions with residues including TRP80, VAL270, LYS268, and LEU210, plus four van der Waals contacts. IDE, the insulin-degrading enzyme responsible for amyloid-beta clearance, formed one hydrogen bond with LEU204 and six π bond interactions. CDK5, the cyclin-dependent kinase implicated in tau hyperphosphorylation, engaged through eight π bond interactions. To verify that these static poses were physiologically meaningful, the team ran 200-nanosecond molecular dynamics simulations in GROMACS using the CHARMM36 force field. Backbone RMSD profiles for all three complexes stabilized within narrow ranges, radius of gyration values remained constant, and solvent-accessible surface area measurements confirmed that ligand binding did not induce destabilizing conformational expansion. Principal component analysis and free energy landscape mapping revealed deep, well-defined energy basins, indicating that the complexes occupied stable conformational states throughout the simulation. MMPBSA binding free energy calculations showed favorable van der Waals and gas-phase contributions, though the authors note these were partially counterbalanced by positive solvation energies, and they emphasize that the computational findings should be regarded as hypothesis-generating rather than definitive evidence of direct target engagement.

In the BV2 microglial cell experiments, trans-anethole demonstrated a favorable cytotoxicity profile with an IC25 of 168.9 nM after 24 hours, compared to 6.485 nM for donepezil. More importantly, trans-anethole outperformed donepezil in several antioxidant parameters. Superoxide dismutase activity reached 1.01 ± 0.09 U/mg protein at 10 µM, exceeding donepezil’s 0.83 ± 0.18 at the same concentration. Catalase activity was significantly higher than donepezil at most concentrations tested, peaking at 6,326.92 ± 7.77 U/mg protein at 10 nM. Reduced glutathione levels were elevated across multiple doses. However, the cholinesterase inhibition data told a different story. Donepezil, as expected, showed potent concentration-dependent acetylcholinesterase inhibition with maximal suppression at 1 µM, while trans-anethole exhibited only moderate AChE inhibition and weak, non-selective BChE inhibition at higher doses. The authors interpret this pharmacological divergence as establishing trans-anethole as a potential adjunctive antioxidant agent rather than a primary cholinergic modulator, with donepezil remaining the superior cholinergic restorer.

The in vivo behavioral results provided the most compelling evidence of neuroprotection. In the open field test, disease control rats showed markedly reduced center entries compared to vehicle controls, indicating increased anxiety-like behavior following streptozotocin administration. Trans-anethole at 100 mg/kg and 400 mg/kg, administered orally from day 8 to day 28, progressively increased exploratory activity from day 14 onwards, with the high-dose group reaching 1.40 ± 0.24 center entries by day 28 compared to 0.40 ± 0.24 in disease controls. In the Y-maze spontaneous alternation task, which assesses spatial working memory, disease controls showed persistent impairment with alternation rates dropping to 26.26% ± 2.37% on day 7 compared to 72.33% ± 8.43% in vehicle controls. The low-dose trans-anethole group showed progressive recovery, reaching 64.49% ± 8.96% by day 28, approaching vehicle control levels of 71.30% ± 5.28%. The novel object recognition test, which measures short-term recognition memory through a discrimination index, revealed that disease controls maintained negative discrimination indices throughout the experiment, while both trans-anethole dose groups transitioned to positive values by day 21 and day 28, with the low-dose group reaching 0.27 ± 0.18 and the high-dose group 0.28 ± 0.10 by day 28. The authors note that the open field findings were interpreted as attenuation of anxiety-like behavioral changes rather than direct evidence of cognitive improvement, and that locomotor activity was not separately quantified, so subtle activity-related effects cannot be completely excluded.

Biochemical analysis of brain homogenates on day 29 confirmed the antioxidant mechanism. Disease control animals showed significantly depleted superoxide dismutase at 19.12 ± 5.33 U/mg protein, reduced glutathione at 10.89 ± 2.02 µmol/mg protein, and catalase at 24.14 ± 4.81 U/mg protein, all dramatically lower than vehicle controls at 98.74 ± 9.19, 82.69 ± 11.01, and 99.58 ± 10.35 respectively. Malondialdehyde, the lipid peroxidation marker, was elevated to 63.48 ± 28.26 nmol/mg protein in disease controls compared to 3.97 ± 4.67 in vehicle controls. Trans-anethole treatment at both doses significantly restored SOD to 54.18 ± 7.96 and 55.18 ± 8.24 U/mg protein, GSH to 15.21 ± 4.31 and 19.95 ± 3.35 µmol/mg protein, and catalase to 37.62 ± 5.27 and 54.68 ± 7.45 U/mg protein, while reducing MDA to 30.44 ± 13.35 and 35.75 ± 22.34 nmol/mg protein. Cholinergic dysfunction was similarly attenuated: disease control AChE activity of 13.77 ± 3.42 µmol/min/mg tissue was reduced to 3.86 ± 1.12 and 3.45 ± 1.17 with low and high dose trans-anethole, while BChE activity dropped from 11.12 ± 2.47 to 3.35 ± 0.30 and 2.66 ± 0.51 respectively. Histopathological examination of the hippocampus revealed qualitative preservation of neuronal morphology, with increased neuronal density in the dentate gyrus and CA3 regions and an absence of necrotic neurons in both trans-anethole dose groups, contrasting sharply with the disease control slides showing decreased cellularity, thinning of the granule cell layer, and pyknotic nuclei in CA1 pyramidal neurons.

The authors are careful to acknowledge the limitations of their study. The predicted molecular targets AKT1, CDK5, and IDE were not experimentally validated at the protein or gene level using Western blot, RT-PCR, or immunohistochemistry. Amyloid-beta, amyloid precursor protein, total tau, and phosphorylated tau were not measured. Only the BV2 microglial model was used in vitro, which is not a neuronal cell line, so the findings should not be interpreted as direct evidence of neuronal protection. The histopathological assessment was qualitative rather than quantitative, and no blinded histological scoring was performed. Critically, no pharmacokinetic or brain exposure data were collected, leaving uncertain whether the administered oral doses produce sufficient concentrations of trans-anethole or its metabolites within the brain to engage the computationally predicted targets. The intracerebroventricular streptozotocin model, while reproducing several features of sporadic Alzheimer’s disease, does not capture the full pathological complexity of the human condition. Nevertheless, the convergence of computational target prediction, cellular antioxidant activity, behavioral improvement across multiple memory domains, biochemical restoration of redox balance and cholinergic function, and qualitative preservation of hippocampal architecture collectively supports the potential neuroprotective properties of trans-anethole and provides a foundation for further preclinical investigation of its efficacy, molecular mechanism, pharmacokinetic profile, and long-term safety in cognitive impairment.

Subject of Research: Neuropharmacology

Article Title: Trans-anethole attenuates streptozotocin-induced cognitive dysfunction by modulating oxidative stress and cholinergic activity

Article References: Mole, S. S., Dwivedi, P. S. R., Lobo, W. L., Shastry, C. S., & Khanal, P. (2026). Trans-anethole attenuates streptozotocin-induced cognitive dysfunction by modulating oxidative stress and cholinergic activity. Frontiers in Pharmacology, 17, Article 1954658. https://doi.org/10.3389/fphar.2026.1954658

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1954658

Keywords: Alzheimer's disease, trans-anethole, oxidative stress, cholinergic activity, streptozotocin, neuroprotection, Frontiers in Pharmacology, cognitive dysfunction, network pharmacology, molecular docking, attenuates, streptozotocin-induced