Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by oxidative stress, cholinergic dysfunction, and amyloid-β (Aβ) 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-Aβ 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-Aβ 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 1-100 µg/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 ± 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.

Dry eye disease (DED) is a multifactorial ocular surface disorder characterized by inflammation, oxidative stress, tear film instability, and secretory dysfunction. This study investigated the protective effects of Lute-gen[®], a lutein and zeaxanthin-based formulation, in both in vitro and in vivo DED models. Human corneal epithelial (HCE-T) cells were stimulated with TNF-α, while dry eye was induced in female Sprague-Dawley rats using subcutaneous scopolamine (SCP) administration. In HCE-T cells, Lute-gen[®] showed no cytotoxicity, restored cell viability, reduced intracellular ROS, and was associated with increased expression of antioxidant-related markers (Nrf2, HO-1, SOD, CAT, and GPx), reduced expression of inflammatory mediators (TLR4/MyD88/NF-κB/NLRP3), and increased expression of AQP3 and AQP5. In SCP-induced rats, Lute-gen[®] significantly improved tear secretion and reduced corneal fluorescein staining. Histopathological analyses revealed restoration of conjunctival goblet cells, mucin staining, corneal epithelial integrity, acinar area and cell density, and lacrimal gland architecture, with reduced inflammatory infiltration. Immunofluorescence further demonstrated reduced TLR4 and MMP9 immunoreactivity and decreased CD68[+] inflammatory cell infiltration. Molecular analyses showed reduced expression of inflammatory cytokines and NF-κB/MAPK/MMP signaling-related inflammatory mediators, together with restoration of AQP1, AQP3, and AQP5 expression in corneal tissues. Collectively, these findings suggest that Lute-gen[®] treatment was associated with improvements in dry eye-related pathological changes, including restoration of antioxidant-related markers, attenuation of inflammatory responses, restoration of aquaporin expression, and preservation of ocular surface and lacrimal gland integrity. These preclinical findings support further mechanistic investigations and the future clinical evaluation of Lute-gen[®] as a potential nutritional intervention for dry eye disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder lacking effective disease-modifying therapies. A promising regenerative approach involves enhancing endogenous neurogenic capacity within the injured brain. Reactive astrocytes-stellate-like cells in the AD brain-may contribute to a pro-neurogenic environment through transcription factors (TFs) such as neurogenin 2 (NGN2) and SOX-11. This process is tightly regulated by epigenetic mechanisms, particularly SIRT-1, a neuroprotective histone deacetylase that modulates TF activity and neuronal fate. Vitamin A (VA), a key regulator of differentiation and epigenetic remodeling via its active metabolite retinoic acid, is stored in astrocytes and hepatic stellate cells (HSCs). We hypothesized that AD-related astrocyte activation depletes cerebral VA, mobilizes hepatic stores, contributes to liver fibrosis, and that VA supplementation may restore astrocytic function, activate endogenous TFs via SIRT-1, and drive cholinergic neuron regeneration. In a scopolamine (SCO)-induced AD rat model, VA biodistribution was traced using confocal microscopy. Brain and liver VA deficiency were confirmed via retinol-binding protein (RBP) and ALDH1A1 expressions. Rats received VA (1500, 3000, or 4500 IU/kg/day) or donepezil. Outcomes included neurogenesis (DCX), NGN2/SOX-11 expression, SIRT-1 activation, cholinergic regeneration, amyloid-β deposition, and serum tau. Liver fibrosis was assessed via TGF-β, hydroxyproline and histopathologically. AD induced systemic VA depletion and liver fibrosis. Medium-dose VA (VAMD) significantly enhanced neurogenesis, TF expression, SIRT-1 activation, cholinergic regeneration, and reversed liver fibrosis. VAMD demonstrated neuroregenerative and antifibrotic effects, indicating a possible therapeutic role in AD.

Anisodus tanguticus (Maxim.) Pascher has been documented in Tibetan ethnomedicine. Traditional records indicate that A. tanguticus has traditionally been used to relieve pain, treat parasitic infections, and heal skin wounds caused by viral infections. A. tanguticus is rich in tropane alkaloids, including anisodamine, scopolamine, and atropine, which are pharmacologically active constituents with well-known parasympatholytic effects. These alkaloids have shown promise in controlling cancer cell proliferation and metastasis, with preclinical studies indicating antitumor activity against liver, breast, and colorectal cancers. The extraction of A. tanguticus traditionally involves methods such as juice pressing or boiling; however, modern techniques like ultrasonic, reflux, and supercritical fluid extraction have enhanced alkaloid yield and quality. While the pharmacological properties of A. tanguticus suggest that some constituents of A. tanguticus may have preclinical antitumor relevance, most studies remain preclinical, and clinical data is limited. This review highlights the need for further pharmacological, toxicological, and translational studies. Current evidence suggests that A. tanguticus and several of its constituents exhibit preclinical antitumor activity; however, their clinical relevance remains uncertain, and further pharmacological, toxicological, and clinical validation is required. This narrative review summarizes the traditional use, phytochemical composition, extraction methods, antitumor-related findings, and proposed mechanisms of A. tanguticus, while highlighting the need for further pharmacological, toxicological, and translational studies.

Drug-facilitated sexual assault is a major public health and forensic concern, frequently involving central nervous system depressants such as scopolamine. This tropane alkaloid is especially hazardous due to its strong amnestic, sedative, and hallucinogenic effects, combined with its lack of color, odor, and taste. Despite its toxicological importance, there are still few methods available for the rapid on-site detection of scopolamine. Therefore, portable, low-cost analytical tools capable of detecting scopolamine in a variety of matrices at the point-of-need are still lacking. Here, we developed an autonomous microfluidic paper-based analytical device for the rapid detection of scopolamine in pharmaceuticals, plant extracts, and beverages. The device is based on Scott's colorimetric reaction, in which Cobalt(II) thiocyanate forms a visible pink-to-blue complex with scopolamine under acidic conditions. The system consists of a cellulose substrate sandwiched between hydrophilic and hydrophobic pressure-sensitive adhesive layers and a poly(methyl methacrylate) cover, enabling capillary-driven fluid transport and direct visual readout. Calibration studies demonstrated a concentration-dependent color change, achieving limits of detection of 0.13 mg mL[-1] for pure scopolamine and 0.11 mg mL[-1] for commercial formulations such as Buscapina®. The device was tested in a wide range of alcoholic and non-alcoholic beverages and produced a clear response in extracts of Datura stramonium and Atropa belladonna. This microfluidic paper-based device provides a fast, reliable, and easy-to-use approach for the on-site detection of scopolamine and related compounds. Its low cost, portability, and visual readout make it particularly suitable for forensic screening and public health protection. The platform represents a significant advancement toward practical point-of-need detection of drug-facilitated assault agents.

Alzheimer's disease (AD) is associated with renin angiotensin system (RAS) overactivation that stimulates amyloid β-induced neurodegeneration. Activation of angiotensin II type 1 (AT1) receptor impairs the protective phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway. The combination between angiotensin converting enzyme 2 (ACE2)/ angiotensin 1-7 (Ang 1-7) /Mas receptor (MAS1) axis activation and PI3K/Akt stimulation counteracts AT1-induced apoptosis. Dapagliflozin (DAPA) alters the imbalance between the two RAS arms favoring the protective arm. Accordingly, the aim of the current study was to evaluate the possible neuroprotective effects of DAPA in guarding against scopolamine (SCO)-induced AD in rats via inhibiting RAS classical arm, and activating RAS protective arm, as well as PI3K/Akt pathway. DAPA administration suppressed ACE expression and reduced the levels of Ang II and AT1 in the hippocampi, while elevated the hippocampal levels of ACE2 and Ang 1-7 in addition to boosting MAS1 hippocampal expression. Of note, DAPA prominently augmented the level of PI3K. Also it enhanced the activity of protein kinase A. Furthermore, the hippocampal levels of caspase-3, cytochrome-c, interleukin-6 and nuclear factor kappa B were suppressed, while guanine nucleotide-binding protein alpha-3 level was elevated by DAPA administration. DAPA also attenuated the histopathological SCO-induced damage in rats and enhanced their recognition memory and learning in novel object recognition test and Y-maze spontaneous alternation test. Hence, the current results represent the first evidence for proving that DAPA-induced activation of the protective RAS arm suppressed the cognitive deficits in SCO-induced AD in rats.

A muscarinic acetylcholine receptor (MAchR) mechanism at the hypoglossal motor nucleus (HMN) can suppress tongue motor activity, and anti MAchR agents are a component of pharmacotherapy for obstructive sleep apnea. However, the source and operation of cholinergic modulation of HMN activity are unknown in-vivo. Here we identify tongue motor responses to optical stimulation of cholinergic neurons at the HMN and intermediate reticular nucleus (IRt) in isoflurane-anesthetized transgenic mice (ChAT-ChR2(H134R)-EYFP) with and without pharmacological manipulation of the HMN. The IRt constitutes a major source of cholinergic input to the HMN and relay station for transmission of respiratory drive. In response to 2 sec pulsed (10ms at 10Hz, 0-20mW, n=15) or tonic ('square-wave', 0-20mW, n=9) optical stimulation of the HMN or IRt, there was a power-dependent increase in tongue motor responses with lesser responses from the IRt (all P<0.05). During tonic IRt stimulation we identified that MAchR antagonism at the HMN (scopolamine, 2mM, n=7) increased endogenous respiratory tongue motor activity at higher stimulation intensities (20mW, P=0.030) whereas addition of nicotinic (N) AchR antagonism (mecamylamine, 200µM) decreased respiratory tongue activity at lower stimulation intensities (5mW, P=0.012). These data identify opposing inhibitory and excitatory MAchR- and NAchR-dependent modulation of respiratory hypoglossal motor output. In contrast, MAchR antagonism alone or combined with NAchR antagonism at the HMN decreased evoked tonic motor activity (10 and 20mW, P<0.034), identifying reduction of a tonic excitatory effect. Together, these data identify cholinergic modulation of IRt-evoked HMN activity that shapes tonic and respiratory hypoglossal motor outputs in-vivo.

Alkaloid N-demethylation is a critical step in the production of many semi-synthetic alkaloid therapeutics and has traditionally relied on synthetic chemical routes. Microbial whole-cell bioproduction offers a promising alternate route for generating these valuable pharmaceuticals. This work demonstrated a greener N-demethylation platform applicable to morphinan and tropine alkaloid classes, employing an engineered yeast expressing a cytochrome P450 enzyme with N-demethylase activity, a cytochrome P450 oxidoreductase and purine permeases as transporters. This platform enabled the N-demethylation of the morphinan alkaloids, oripavine and thebaine with high activity, and the tropane alkaloids, atropine and scopolamine with moderate activity. The study systematically evaluated the contributions of substrate uptake and enzymatic conversion within the whole-cell system. Heterologous transporters enabled mass transfer across the cell membrane, with no significant difference detected for the four substrates, while in vitro enzyme catalysis differed between substrates and correlated with yield. Computational molecular docking, combined with deep learning-based protein structure prediction, found all four substrates had high affinity for the enzyme. This approach was also used to identify potential target substrates for future biotransformation. Process performance could be further improved with the use of larger scale, well controlled bioreactors.

Depression is a global mental health crisis, and the rapid-acting antidepressant scopolamine (Scop) is limited by systemic administration drawbacks. This study developed a scopolamine-loaded bilayer dissolving microneedle (Scop-DMN) system for transdermal delivery and evaluated its antidepressant efficacy in a chronic restraint stress (CRS)-induced mouse model. Bilayer DMNs were fabricated via sequential centrifugal molding, with a polyvinyl alcohol (PVA) backing layer and a dextran‑sodium hyaluronate drug-loaded tip matrix. Physicochemical properties, biocompatibility, and in vivo therapeutic effects were systematically characterized. Scop-DMNs showed uniform conical morphology, high drug loading homogeneity, and complete dissolution within 2 min in porcine skin. In C57BL/6 J mice, Scop-DMNs significantly ameliorated CRS-induced depressive-like behaviors in open field, sucrose splash tests, and tail suspension tests 24 h post-administration, outperforming conventional intraperitoneal scopolamine. Mechanistically, Scop-DMNs robustly activated the brain-derived neurotrophic factor / mammalian target of rapamycin (BDNF/mTOR) pathway and upregulated synaptic proteins (postsynaptic density protein 95 (PSD95), Synapsin I (SynI)) in the medial prefrontal cortex (mPFC), reversing stress-induced neurotrophic and synaptic deficits. This Scop-DMN system provides a safe, minimally invasive, and effective transdermal platform for scopolamine delivery, offering a promising clinical strategy for rapid management of acute depressive episodes.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder and the major cause of dementia globally. One of the major barriers to curing AD is the lack of experimental models that fully recapitulate the complexity of human AD pathology. Evidence supports the notion that neuroinflammation is a key pathological contributing factor of AD. Therefore, various external stimuli are used to recapitulate the AD-like pathology, i.e., LPS, scopolamine, and aluminum chloride. Although PHA is reported for its pro-inflammatory activity, using PHA to recapitulate the AD-like pathology is relatively new. The current study was designed to evaluate the PHA potential to recapitulate the AD-like pathology in primary neuronal culture as well as in vivo AD modeling. Since LPS is widely used, in the current study, it was used to compare it with the experimental findings using PHA. Confirmation of AD-like pathology was carried out through gene and protein expression in the in vitro study, while in vivo results were verified through behavioral and histopathological assays. It was observed that PHA mimics the AD-like pathology at the cellular level by aggregating, shrinking, and disrupting synaptic plasticity of primary neuronal cells. An elevated level of BACE1 and amyloid-β and hyperphosphorylated tau protein expression, along with reduced levels of the ADAM10 gene, evidently proves that PHA induces the AD-like pathology. Disrupted cognitive and memory patterns confirm the initiation of AD pathogenesis in the in vivo study, which is further confirmed by observing the amyloid plaques stained by Congo red (CR) dye. Based on these findings, it is concluded that PHA is a potent inducer to recapitulate neuroinflammatory-mediated AD-like pathology both in vitro and in vivo.

This study investigated the 3,3'-Diindolylmethane (DIM) memory-enhancing and neuroprotective effects in Wistar rats against scopolamine (Scop)-induced neurotoxicity. Rats were divided into six groups: healthy control, Scop (1 mg/kg), DIM (25, 50, and 100 mg/kg), and donepezil (5 mg/kg). All groups except the control received Scop for 14 days, with DIM or DPZ administered concurrently. NORT and Y-maze tests were employed to assess cognition and memory. Activity of acetylcholinesterase (AChE), acetylcholine (ACh) levels, oxidative stress parameters (MDA, ROS, SOD, CAT, NRF2, HO-1), inflammatory mediators (TNF-α, NF-κB, IL-6, IL-10), and apoptotic factors (cytochrome c, and caspases) were measured in brain samples, and H&E staining was performed. DIM significantly improved cognition and memory as demonstrated by enhanced spontaneous alternations (%) and recognition index from behavioural analysis. DIM reduces AChE activity and increases ACh levels, indicating that improvement in cholinergic function is the reason for improved cognition and memory. Additionally, DIM treatment enhanced the antioxidant markers like CAT, SOD, NRF2, and HO-1, while reducing the total ROS and MDA levels in the brain, indicating its antioxidant potential. Similarly, DIM attenuated neuroinflammatory mediators IL-6, TNF-α, NF-κB, and elevated IL-10, an anti-inflammatory cytokine, suggesting DIM acts as an anti-inflammatory agent. Moreover, DIM reduced the neuronal apoptosis markers, cytochrome C, caspase 9 and 3, and improved the hippocampal CA1 neuronal architecture, indicating that it preserved the neuronal architecture. Correlation analysis between memory and other biochemical parameters provides a stronger negative correlation with AchE and neuroinflammation and a positive correlation with NRF2, indicating that DIM enhanced memory and neuroprotection involved multiple pathways. In conclusion, DIM enhances cognition and memory by improving cholinergic dysfunction and exhibits neuroprotective effects by reducing oxidative stress, inflammation, and apoptosis against scopolamine-induced neurotoxicity.