Centella asiatica ameliorates scopolamine-induced cognitive impairment via acetylcholinesterase modulation and oxidative stress reduction

Abstract

Centella asiatica, a traditional medicinal herb, is well-known for its neuroprotective, antioxidant, and anti-inflammatory effects. This study investigated the impact of C. asiatica extract (CAE) on glutamate-induced neurotoxicity in HT22 cells and scopolamine-induced cognitive impairment in mice. CAE protected HT22 cells by attenuating oxidative stress and regulating apoptosis-related proteins, including Bcl-2 and Bax. Additionally, CAE was shown to upregulate the expression of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB), a critical transcription factor involved in neuronal differentiation. In scopolamine-induced mice, oral administration of CAE (30, 60, and 100 mg/kg) significantly enhanced behavioral performance in memory tests, with effects comparable to those of the positive control, donepezil. Furthermore, CAE elevated hippocampal acetylcholine levels, inhibited acetylcholinesterase activity, and enhanced antioxidant defenses. These findings demonstrate that CAE exerts neuroprotective and memory-enhancing effects via antioxidant, anti-apoptotic, and cholinergic modulation mechanisms, suggesting its potential as a functional food ingredient for cognitive maintenance.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-026-02132-w.

Introduction

Alzheimer’s disease (AD) is characterized by significant degeneration of basal forebrain cholinergic neurons, resulting in a substantial reduction in cortical innervation. This cholinergic deficit contributes to impaired synaptic transmission in both the cortex and hippocampus, regions essential for cognitive processing and memory consolidation (Savonenko et al., 2012). Inhibition of acetylcholinesterase (AChE) has become a key therapeutic approach for managing AD. By reducing AChE activity, synaptic levels of acetylcholine (ACh) are maintained, which enhances cholinergic neurotransmission and improves cognitive functions reliant on cholinergic signaling (Haider et al., 2016). Notably, AChE inhibitors decrease peri-synaptic ACh hydrolysis and sustain extrasynaptic ACh levels, thereby prolonging cholinergic tone and enabling extended activation of postsynaptic receptors (Aydin et al., 2016). Furthermore, cholinergic dysfunction, induced experimentally with muscarinic antagonists like scopolamine, not only causes acute memory deficits but also increases oxidative stress markers in the brain, including lipid peroxidation and reactive oxygen species (ROS) production (Bruel-Jungerman et al., 2011; Xu et al., 2016).

Concurrently, early-stage AD pathology is marked by a diminished activity of endogenous antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) (Arslan et al., 2020; Puertas et al., 2012). This reduction intensifies oxidative damage and heightens neuronal susceptibility. The ensuing imbalance between oxidants and antioxidants accelerates pathological processes, including protein misfolding, lipid peroxidation, and mitochondrial dysfunction, thereby contributing to the progression of neurodegenerative cascades (Butterfield, 2020; Cheignon et al., 2018). The cholinergic system plays a crucial role in the modulation of hippocampal neurogenesis, engaging neurogenic pathways that are mediated by brain-derived neurotrophic factor (BDNF) and the transcription factor cAMP response element-binding protein (CREB) (Bruel-Jungerman et al., 2011). Disruption of cholinergic signaling through the administration of scopolamine results in significant reductions in hippocampal BDNF expression and CREB phosphorylation, which are associated with deficits in memory function (Park et al., 2016). In clinical populations, reduced BDNF levels in the entorhinal cortex and hippocampus have been correlated with low Mini-Mental State Examination (MMSE) scores, thereby underscoring the relationship between neurotrophic support and cognitive decline in AD (Wu et al., 2016). Moreover, the elevated production of nitric oxide (NO) and other reactive nitrogen species in the AD brain can enhance AChE expression and activity, thereby exacerbating cholinergic dysfunction. This process may establish a feed-forward loop that amplifies synaptic deficits (Heneka et al., 2015; Xian et al., 2015).

Centella asiatica (L.) Urban is a perennial medicinal herb widely used in traditional medical systems across Asia. It has long been consumed as both a therapeutic agent and a dietary component, reflecting its broad ethnopharmacological relevance (Orhan, 2012). In recent years, extracts of C. asiatica have attracted increasing scientific interest due to their reported antioxidant, anti-inflammatory, and neuroprotective activities (Brinkhaus et al., 2000; Gray et al., 2015). Phytochemical studies indicate that the biological effects of C. asiatica are primarily associated with its triterpenoid-rich composition (Hashim et al., 2011; James and Dubery, 2009). In particular, pentacyclic triterpenes, including asiaticoside, madecassoside, asiatic acid, and madecassic acid, constitute the major bioactive constituents and have been implicated in diverse biological processes such as modulation of inflammation, promotion of tissue repair, and protection against oxidative stress (Sun et al., 2020). Consistent with this phytochemical profile, previous studies have demonstrated that C. asiatica extract (CAE) exhibits a broad range of biological activities. These include neuroprotective effects against oxidative stress induced by global cerebral ischemia/reperfusion injury, antioxidant activity against scopolamine-induced oxidative stress in the rat brain, mitigation of scopolamine-induced motor deficits, and antihyperglycemic effects in streptozotocin-induced diabetic rats. These findings substantiate its traditional medicinal use and highlight its anti-inflammatory properties (Hein et al., 2025; Sabaragamuwa et al., 2018; Wong et al., 2021). However, despite the documented antioxidant and neuroprotective effects of CAE, comprehensive investigations into its impact on hippocampal oxidative status and neurotrophic signaling in the context of scopolamine-induced cognitive impairment remain limited. Therefore, the present study aims to evaluate the neuroprotective potential of CAE against scopolamine-induced memory deficits and to elucidate its effects on biomarkers related to hippocampal oxidative stress and neurotrophic support, as reflected by hippocampal BDNF expression, in vivo.

Materials and methods

Sample preparation

CAE (Himalca®, Batch No. SCAL23560) was derived from C. asiatica leaves and supplied as a powdered extract by 3H LABS (Goyang, Gyeonggi-do, Korea). The raw plant material was cultivated under controlled agricultural conditions, harvested at maturity, and processed into a fine powder using standardized drying and milling procedures to ensure batch consistency. The extraction procedure was performed as previously described (Park et al., 2021a). Briefly, dried C. asiatica leaves were twice extracted with 50% (v/v) fermented ethanol at 80 °C for 8 h and 6 h, respectively. The combined extracts were filtered, concentrated under reduced pressure, and spray-dried using maltodextrin as a carrier to obtain the final CAE powder. The extraction yield was 29.03% (w/w) relative to the dried plant material. The resulting extract was stored at –20 °C until use. The final extract complied with the quality and safety standards established by the Korean Ministry of Food and Drug Safety (MFDS) for application in health functional foods.

HPLC analysis of asiaticoside in CAE

High-performance liquid chromatography (HPLC) was performed to characterize CAE and quantify asiaticoside as a marker compound, as previously described (Park et al., 2021b). Analysis was conducted using an Agilent 1260 Infinity system equipped with a diode-array detector (Agilent Technologies, Santa Clara, CA, USA). Separation was achieved on Cadenza C18 column (250 mm × 4.6 mm, 3 μm; IMTAKT, Portland, OR, USA) at a flow rate of 1.0 mL/min, with the column temperature maintained at 40 °C. The mobile phase consisted of distilled water (A) and acetonitrile (B), and a binary linear gradient was applied as follows: ratio of mobile phase A and B were changed after 0 min, 90:10 (v/v); 10 min, 80:20 (v/v); 40 min, 73:27 (v/v); 45 min, 80:20 (v/v); 51 min, 20:80 (v/v); and 55 min, 90:10 (v/v). The injection volume was 10 μL, and detection was performed at 206 nm. Quantification of asiaticoside was carried out using an external standard method with authentic asiaticoside (ChemFaces, Wuhan, Hubei, China). Identification was based on comparison of retention time and UV spectra with those of the reference standard. The asiaticoside content of CAE was determined to be 1.41% (w/w), and a representative HPLC chromatogram is shown in Supplementary Fig. 1.

Cell culture

The mouse hippocampal neuronal cell line HT-22 was cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; LM001-05; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂ and subcultured when they reached approximately 70% confluency.

Cell counting kit-8 (CCK-8) assay

CCK-8 assay was employed to assess the cell viability. HT22 cells were cultured in 96-well plates at a density of 3 × 10³ cells/well and incubated overnight. Subsequently, the cells were treated with varying concentrations of CAE. After 24 h, 6 mM glutamate (G5889; Sigma) was added to the wells. Following an additional 24 h incubation, CCK-8 solution (CK04-05, Dojindo Molecular Technologies, Rockville, MD, USA) was added to each well. The plates were then incubated for 2 h at 37 °C, and absorbance was measured at 450 nm using a SpectraMax i3 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The results are expressed relative to the untreated control group.

Measurement of reactive oxygen species (ROS)

Following CAE treatment, the cells were washed three times with phosphate-buffered saline and subsequently stained with the fluorescent probe H2DCFDA (C400; Invitrogen, Thermo Fisher Scientific, Inc, Waltham, MA, USA) to evaluate ROS levels. The staining was performed in the dark for 30 min at 37 °C. After incubation, the cells were examined and imaged using a fluorescence microscope (ECLIPSE Ti2; Nikon Corporation, Minato-ku, Tokyo) (Fig. 1).

Fig. 1.

Protective effects of CAE against glutamate-induced excitotoxicity in HT22 cells. Assessment of cell viability and morphology following CAE treatment. HT22 cells were exposed to 6 mM glutamate for 24 h in the presence or absence of CAE. A-B Cell viability was measured using the CCK-8 assay, and representative phase-contrast images revealed that glutamate induced significant cell death and morphological shrinkage, both of which were attenuated by CAE in a concentration-dependent manner. C-D Intracellular reactive oxygen species (ROS) generation was assessed by staining with 5 μM 2’,7’-dichlorofluorescein diacetate (DCFDA). Fluorescence intensity was measured using a microplate reader, and representative photographs are presented. E Representative Western blot images illustrating the expression of apoptosis-related proteins (Bcl-2 and Bax), neurotrophic signaling markers, BDNF, and p-CREB, in HT22 cells treated with glutamate (6 mM) in the presence or absence of CAE for 24 h. F Quantitative densitometric analysis of protein expression normalized to β-actin or total protein levels. Data are represented as mean ± SEM (n = 5). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. ##:P < 0.01 vs. the untreated control group; **:P < 0.01, and ***:P < 0.001 vs. the glutamate-treated group

Animal model for cognitive impairment

Wild-type (WT) male mice of the C57BL/6 J strain, aged 6 weeks and weighing 18-22 g, were procured from DooYeol Biotech (Seoul, Korea) and acclimated for one week in a temperature-controlled animal facility under a 12 h light/dark cycle. Group allocation was performed using randomization, and all behavioral and biochemical analyses were conducted under blinded conditions. The mice were randomly assigned to six groups (n = 8 per group) as follows: control (Con); scopolamine (1 mg/kg) with water (SCO); scopolamine (1 mg/kg) with CAE at 30 mg/kg (AL); scopolamine (1 mg/kg) with CAE at 60 mg/kg (AM); scopolamine (1 mg/kg) with CAE at 100 mg/kg (AH); and scopolamine (1 mg/kg) with donepezil (3 mg/kg) (DONE). CAE was administered orally once daily at the indicated doses for 21 consecutive days, whereas scopolamine (1 mg/kg) was administered intraperitoneally once daily for 14 consecutive days. The experimental schedule is illustrated in Fig. 2A. Only healthy animals that completed the full treatment protocol were included in the analyses. Exclusions occurred solely due to technical failure or sample loss during tissue processing, and no animals were excluded based on experimental outcomes. All procedures were conducted following the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Oriental Medicine (approval number #24–067).

Fig. 2.

Effect of CAE on spatial memory performance in scopolamine-induced mice using the Y-maze test. A Schedule of the overall in vivo experiment. B Spontaneous alternation behavior in the Y-maze was assessed as an additional measure of short-term spatial working memory. C Schematic representation of the Y-maze test design. During the training session, mice were placed in the maze with one arm closed and permitted to explore the two open arms for 8 min. Following a 1 h retention interval, all three arms were opened, and mice were allowed to explore for an additional 8 min. Preference for the novel arm was evaluated as an indicator of spatial recognition memory. D Quantitative assessments of locomotor activity and E percentage of distance in the novel arm were measured. Data are presented as mean ± SEM (n = 8). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. #:P < 0.05 and ###:P < 0.01 vs. the vehicle-treated control group; *:P < 0.05, **:P < 0.01, and ***:P < 0.001 vs. the scopolamine-treated group

Assessment of cognitive function:

Y-Maze test.

The Y-maze test was conducted following previously established protocols (Kraeuter et al., 2019; Pak et al., 2022b). The apparatus comprised three identical arms, each measuring 40 cm in length, 4 cm in width, and 15 cm in height, arranged at 120° angles relative to one another. Each mouse was placed at the end of one arm and permitted to explore the maze freely for 10 min. Spontaneous alternations were evaluated by recording sequential entries into all three arms. The percentage of alternation was calculated as follows: [spontaneous alternation/(total number of arm entries—2)] × 100.

Novel object recognition test (NOR test)

The novel object recognition test was performed according to established protocols (Leger et al., 2013; Lueptow, 2017; Pak et al., 2022a). The testing apparatus consisted of a square arena measuring 35 cm × 35 cm × 35 cm. During the habituation phase, mice were allowed to explore the empty arena for 10 min. In the familiarization phase, two identical objects were placed within the arena, and the mice were allowed to explore them for 10 min. During the test phase, one of the familiar objects was replaced with a novel object, and the mice were given 10 min to explore. The duration of exploration for each object was recorded, and a preference index was calculated.

Passive avoidance test (PAT)

The passive avoidance test was conducted using the Shuttle Box Avoidance Basic Test Package (Med Associates Inc., Fairfax, VT, USA), which comprises four reaction chambers. The apparatus consisted of a brightly illuminated compartment and a dark compartment, separated by an automated gate. The dark compartment featured a grid floor capable of delivering an electric shock. During the training session, each mouse was placed in the brightly lit compartment, and following a 30-s habituation period, the automated door opened to allow the mouse to enter the dark compartment. Upon the mouse’s entry into the dark compartment, the door closed, and a mild electric shock (0.2 mA, 2 s) was administered. The test session was performed the following day under identical conditions, but without shock delivery. Latency times, defined as the duration for the mouse to transition from the light compartment to the dark compartment, were recorded, with a maximum cutoff time of 3 min.

Hematoxylin and eosin (H&E) staining and histological analysis

Hematoxylin and eosin (H&E) staining was performed to assess neuronal pathology in the hippocampus. Mouse brains were extracted and post-fixed in formalin, followed by paraffin embedding. Coronal brain Sects. (10 μm thick) corresponding to the hippocampal region (anteroposterior coordinate: -1.5 mm) were prepared using a rotary microtome. Sections were stained with H&E solution for 5 min, rinsed in distilled water, mounted with mounting medium, and imaged using a light microscope (Olympus Corporation, Tokyo, Japan). Quantitative histological analysis was conducted using a two-dimensional image-based morphometric approach. The U-shaped region of the hippocampal CA3 subfield was defined as a fixed region of interest (ROI) based on anatomical landmarks. For each animal, multiple non-overlapping sections were analyzed under identical imaging conditions. Dead cells were visually identified and counted according to predefined morphological criteria, including hypereosinophilic (dark pink) cytoplasm and loss of hematoxylin-positive (blue-purple) nuclear staining.

Estimation of acetylcholine levels and acetylcholinesterase activity

The hippocampus was dissected from each brain, weighed, homogenized in lysis buffer, and centrifuged at 12,000 × g for 20 min at 4 °C. The supernatants were collected, and protein concentrations were determined using the BCA assay. Equal amounts of protein were used for subsequent analyses. Acetylcholine (ACh) levels and acetylcholinesterase (AChE) activity were measured using the Amplex™ Acetylcholine/Acetylcholinesterase Assay Kit (A12217; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Fluorescence was measured using a SpectraMax i3 microplate reader with excitation at 530–560 nm and emission detection at approximately 590 nm. ACh concentrations were calculated from a standard curve and expressed as μM/mg protein. AChE activity was calculated relative to the activity of 0.2 U/mL AChE provided in the kit and expressed as fold change.

Evaluation of antioxidant effects and oxidative stress

To evaluate the antioxidant effects and oxidative stress response of CAE, the following assay kits were employed: Superoxide Dismutase (SOD) Assay Kit (706,002; Cayman Chemical, Ann Arbor, MI, USA), Glutathione (GSH) Assay Kit (703,002; Cayman Chemical, Ann Arbor, MI, USA), and Catalase Assay Kit (BO-CAT-400; BIOMAX, Seoul, Korea). The activities of SOD, GSH, and catalase in the hippocampus of the brain were measured from tissue lysates prepared according to the manufacturers’ protocols. The obtained data were then analyzed using the respective standard curves for each assay and subsequently quantified.

Western blot analysis

Cellular and isolated mouse hippocampal tissues were lysed on ice for 30 min using a lysis buffer composed of TBS (pH 7.4) supplemented with 2% Triton X-100, 0.1% SDS, 2 mM EDTA, 2 mM Na₃VO₄, along with 1% protease and phosphatase inhibitors. The lysates were centrifuged at 13,000 rpm for 15 min. Protein concentrations in the supernatants were determined using Pierce™ BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein were resolved by 10–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking in 5% BSA, membranes were incubated overnight at 4 °C with primary antibodies, including anti-β-actin (#sc47778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Bcl-2(#3498, Cell Signaling Technology, Danvers, MA, USA), anti-Bax (#14,796, Cell SignalingTechnology, Danvers, MA, USA), BDNF (#47,808, Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated-CREB (#9198, Cell Signaling Technology, Danvers, MA, USA), and anti-CREB (#9197, Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with appropriate anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature. Protein expression was detected using an enhanced chemiluminescence detection system (LAS 500; GE Healthcare Bio-Sciences AB, 751 25, Uppsala, Sweden) and quantified using Image J software (v1.52a).

Immunohistochemistry

Slides were rehydrated sequentially in xylene, followed by 100%, 90%, and 70% ethanol at room temperature. Antigen retrieval was performed by incubating the slides in 10 mM sodium citrate buffer using a microwave for 15 min. Endogenous peroxidase activity was then inhibited by treating the slides with 3% hydrogen peroxide at RT. After washing, the sections were blocked for 1 h with blocking solution and subsequently incubated overnight at 4 °C with BDNF antibody (#47,808, Cell Signaling Technology, Danvers, MA, USA). After washing, the slides were incubated with a peroxidase-conjugated secondary antibody and visualized using the Dako REAL EnVision detection system (K5007, Dako, Agilent Technologies, Santa Clara, CA, USA). The slides were then dehydrated through graded ethanol solutions (70%, 90%, and 100%) and cleared twice in xylene for 10 min each. Finally, the slides were mounted with mounting medium and imaged at 20 × magnification using a microscope (Olympus Corporation, Tokyo, Japan). BDNF-positive cells within the hippocampal region were quantified using ImageJ software (v1.52a).

Statistical analysis

In these studies, results are presented as the mean ± SEM and were analyzed using GraphPad Prism 5. Experiments were conducted in at least triplicate. For experiments involving more than two groups, statistical significance was evaluated using one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparisons test, in which each treatment group was compared exclusively with the corresponding control group. For experiments involving only two groups, comparisons were performed using Student's t-test. A p-value less than 0.05 was considered statistically significant.

Results and discussion

Centella asiatica protects against glutamate-induced cytotoxicity and oxidative stress in HT22 cells

HT22 cells were treated with varying concentrations of C. asiatica extract (CAE) for 24 h, and cell viability was evaluated via the CCK-8 assay. CAE exhibited no cytotoxicity at concentrations between 15 and 100 μg/mL (Fig. 1A). Glutamate, the principal excitatory neurotransmitter in the central nervous system, is critical for synaptic plasticity, learning, and memory processes (Magdaleno Roman and Chapa Gonzalez, 2024). However, excessive glutamate accumulation elevates intracellular Ca²⁺ levels and reactive oxygen species (ROS) production, leading to oxidative stress and neuronal cell death (Li et al., 2024; Vongthip et al., 2024). To assess the neuroprotective potential of CAE, HT-22 cells were pretreated with CAE for 24 h prior to exposure to 6 mM glutamate for an additional 24 h. Glutamate treatment reduced cell viability to 54.7 ± 1.1% compared to the untreated control. Conversely, CAE treatment significantly restored cell viability in a dose-dependent manner: 65.8 ± 1.7% (15 μg/mL), 81.0 ± 1.7% (30 μg/mL), 88.0 ± 2.3% (60 μg/mL), and 91.8 ± 2.7% (100 μg/mL), compared to 54.9 ± 1.0% in the vehicle-treated group (Fig. 1B). Consistent with the CCK-8 assay, qualitative microscopic observation indicated improved overall cell appearance and viability in CAE-treated HT-22 cells compared with glutamate-treated cells. Given that glutamate-induced excitotoxicity is associated with oxidative stress, intracellular ROS levels were quantified using the DCFDA assay. Glutamate exposure significantly increased fluorescence intensity compared to controls, whereas CAE treatment markedly attenuated ROS production (Fig. 1C). Fluorescence microscopy further confirmed reduced ROS accumulation in CAE-treated cells (Fig. 1D). Collectively, these findings suggest that CAE mitigates glutamate-induced neuronal damage by reducing oxidative stress and promoting neuronal cell survival.

Centella asiatica modulates apoptotic and neurotrophic signaling pathways in HT22 Cells

To elucidate the neuroprotective mechanism of CAE, we investigated its effects on apoptotic and neurotrophic signaling pathways in HT22 hippocampal neuronal cells exposed to glutamate. The apoptosis-related proteins B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) are pivotal regulators of mitochondrial-dependent apoptosis, with the Bax/Bcl-2 ratio serving as a key indicator of neuronal vulnerability to cell death via modulation of cytochrome c release and caspase activation (Chen et al., 2020). As illustrated in Fig. 1E-F, glutamate exposure significantly increased Bax expression while decreasing Bcl-2 expression, resulting in a markedly elevated Bax/Bcl-2 ratio compared to glutamate-treated controls. Treatment with CAE effectively restored the Bax/Bcl-2 balance by downregulating Bax and upregulating Bcl-2 expression, indicating a pronounced anti-apoptotic effect.

Concurrently, we examined the impact of CAE on neurotrophic signaling molecules, specifically BDNF and CREB. Treatment with CAE resulted in a significant increase in BDNF expression relative to glutamate-treated controls. Additionally, CAE enhanced the phosphorylation of CREB without affecting the total levels of CREB protein. These findings suggest that CAE selectively activates CREB signaling pathways, thereby promoting the upregulation of BDNF expression, which is implicated in neuronal survival, synaptic plasticity, and cognitive function (Gupta et al., 2025). Collectively, these findings suggest that CAE exerts a dual protective effect by inhibiting apoptotic signaling through modulation of the Bax/Bcl-2 axis and concurrently enhancing neurotrophic pathways via CREB-mediated induction of BDNF. This integrated regulatory mechanism implies that CAE not only prevents neuronal loss but also promotes neuronal survival and functional plasticity.

Centella asiatica enhances memory function without altering locomotor activity in the Y-maze test

To examine the effects of CAE on scopolamine-induced cognitive impairment, a series of Y-maze behavioral tests was performed. The experimental design of the animal study is illustrated in Fig. 2A. Administration of CAE at all tested doses (AL, AM, AH) significantly improved spontaneous alternation behavior (Fig. 2B), indicating an enhancement of working memory capacity. The Y-maze spontaneous alternation task was initially utilized to assess short-term spatial memory. As shown in Fig. 2C-D, mice treated with scopolamine demonstrated a significant decrease in distance traveled in the newly opened arm compared to the control (CON) group, indicating marked deficits in spatial memory formation. In contrast, oral administration of CAE at low (AL), medium (AM), and high (AH) doses significantly increased exploration distance in the novel arm relative to the SCO group, restoring performance to levels comparable to those observed in the donepezil (DONE)-treated positive control group. Notably, no significant differences were observed in the total distance traveled across all experimental groups (Fig. 2E), suggesting that the effects were not due to alterations in locomotor activity. Collectively, these findings demonstrate that CAE effectively ameliorates scopolamine-induced impairments in both spatial and working memory without affecting overall locomotor activity, thereby supporting its potential as a cognitive-enhancing agent.

Centella asiatica ameliorates scopolamine-induced recognition memory and long-term memory deficits.

To evaluate the effect of CAE on recognition memory, the novel object recognition (NOR) test was employed. During the training phase, all experimental groups exhibited comparable exploration times for identical objects, indicating no baseline preference (Fig. 3A-B). In the test phase, mice treated with scopolamine exhibited impaired recognition memory, as reflected by a significantly reduced discrimination index and decreased exploration time for the novel object compared to the CON group. In contrast, oral administration of CAE at all tested doses (AL, AM, AH) significantly increased exploration of the novel object and yielded higher discrimination indices relative to the SCO group (Fig. 3C). The memory-enhancing effects of CAE were comparable to those observed in the DONE-treated positive group. Importantly, no significant differences were found in the total exploration time across groups, suggesting that the observed improvements were attributable to enhanced recognition memory rather than alterations in general exploratory activity.

Fig. 3.

Effect of CAE on recognition memory and long-term memory in scopolamine-induced cognitive impairment using the Novel Object Recognition Test (NORT). A Schematic representation of the NORT procedure. The experiment was conducted over three consecutive days. On day 1 (habituation), mice were allowed to freely explore an empty open-field arena (35 × 35 × 35 cm³) for 10 min. On day 2 (training), two identical objects (familiar) were placed 15 cm apart within the arena, and mice were permitted to explore for 10 min. On day 3 (testing), one familiar object was replaced with a novel object, and mice were allowed to explore both objects for 10 min. The duration of exploration directed toward the familiar and novel objects was recorded using Smart v2.5 (Panlab S.L.U., Barcelona, Spain). Quantitative assessments were conducted during the familiarization (B), and the discrimination index (C) was calculated. D Schematic representation of PAT design. The PAT was conducted using a two-compartment shuttle box (MED-PC, Med Associates Inc., St. Albans, England) to evaluate long-term memory retention. During the training session, each mouse was placed in the illuminated compartment, and upon entering the dark compartment, it received a mild foot shock. After 24 h, a retention trial was performed in which the mice were again placed in the illuminated compartment, and E the latency to enter the dark compartment was recorded as an indicator of memory retention. Data are presented as mean ± SEM (n = 8). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. #:P < 0.05 and ###:P < 0.01 vs. the vehicle-treated control group; *:P < 0.05, **:P < 0.01, and ***:P < 0.001 vs. the scopolamine-treated group

To further assess the effects of CAE on aversive learning and long-term memory, a passive avoidance test was conducted. On the training day, all experimental groups displayed comparable latency times when attempting to enter the dark compartment, suggesting no baseline differences in exploratory behavior (Fig. 3D). During the test session, the CON group exhibited the longest latency to enter the dark compartment (60.1 ± 13 s), reflecting intact memory retention. In contrast, scopolamine-treated mice exhibited a pronounced reduction in latency time, indicative of impaired long-term memory (Fig. 3E). Notably, oral administration of CAE significantly increased latency time in a dose-dependent manner (AL: 25.2 ± 7 s; AM: 35.5 ± 14.5 s; AH: 36.8 ± 11.9 s). The latency observed in the AM and AH groups was comparable to that of the DONE-treated positive group (42.6 ± 15.8 s). Collectively, these results demonstrate that CAE effectively ameliorates scopolamine-induced deficits in aversive long-term memory.

Scopolamine-induced amnesia is extensively employed as a pharmacological model for AD, characterized by impairments in cholinergic neurotransmission, oxidative stress, and neuronal apoptosis (Klinkenberg and Blokland, 2010). Given that scopolamine impairs various forms of memory, administration of CAE significantly improved short-term spatial memory, as assessed by the Y-maze test, recognition memory through the NORT test, and long-term aversive memory evaluated via the PAT test. These findings suggest that CAE exerts a wide-ranging positive effect on cognitive functions.

Effects of Centella asiatica on hippocampal acetylcholine levels and acetylcholinesterase activity

A reduction in hippocampal ACh levels, accompanied by elevated AChE activity, reflects disruption of the central cholinergic system and is closely associated with cognitive impairment (Bartus et al., 1982; Rogers and Kesner, 2004). To elucidate the mechanism underlying the effects of CAE on memory function, we investigated hippocampal ACh levels and AChE activity. In the scopolamine-treated group, AChE activity was significantly increased compared with the CON group, accompanied by a marked reduction in hippocampal ACh levels, indicative of impaired cholinergic neurotransmission (Fig. 4). Oral administration of CAE significantly mitigated the scopolamine-induced increase in AChE activity in a dose-dependent manner, with the most pronounced inhibitory effect observed in the AH group (Fig. 4A). Correspondingly, hippocampal ACh levels were significantly restored in CAE-treated mice, particularly within the AH group, which demonstrated values comparable to those of the DONE-treated positive group (Fig. 4B).

Fig. 4.

Effect of CAE on acetylcholine (ACh) levels and acetylcholinesterase (AChE) activity in the hippocampus of scopolamine-induced mice. To evaluate the effects of CAE on cholinergic neurotransmission, hippocampal tissues were collected, homogenized in RIPA buffer, and analyzed using the Amplex Red ACh/AChE Assay Kit (Cat. No. A12217, Thermo Fisher Scientific). Equal protein quantities were incubated for 90 min in the dark with a reactive mixture comprising Amplex Red reagent, horseradish peroxidase (HRP), choline oxidase, and acetylcholinesterase. Fluorescence intensity was subsequently measured using a Spectra Max i3 (Molecular Devices, USA). Measurement included levels of A AChE activity and B ACh concentration within the hippocampus. Data are presented as mean ± SEM (n = 5). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. ##:P < 0.01 and ###:P < 0.01 vs. the vehicle-treated control group; **:P < 0.01 and ***:P < 0.001 vs. the scopolamine-treated group

Thus, the cognitive improvements observed in the present study are likely mediated by the restoration of cholinergic functions, as evidenced by reduced hippocampal AChE activity and increased ACh levels. These findings are consistent with previous studies demonstrating that triterpenoid compounds derived from C. asiatica, particularly asiaticoside, exert inhibitory effects on AChE and enhance cholinergic signaling in experimental models of AD (Liang et al., 2025; Veerendra Kumar and Gupta, 2003). Beyond its role in cholinergic modulation, asiaticoside has gained recognition as a pleiotropic neuroprotective agent targeting multiple pathological processes relevant to AD. Experimental evidence indicates that asiaticoside attenuates amyloid-β (Aβ)-induced neurotoxicity and improves cognitive performance while concomitantly suppressing neuroinflammatory signaling through inhibition of the TLR4/MyD88/TRAF6/NF-κB pathway and downregulation of pro-inflammatory cytokine expression (Song et al., 2018). In addition, asiaticoside enhances neurotrophic signaling by promoting the phosphorylation of CREB and increasing BDNF levels. It has been reported to elevate neurogenesis-associated markers, including doublecortin (DCX), particularly under conditions of pathological stress (Wang et al., 2020; Zhou et al., 2025). Collectively, these findings suggest that the cognitive benefits induced by CAE may arise from the integrated actions of asiatic acid on cholinergic, anti-inflammatory, neurotrophic, and neurogenic pathways. Nevertheless, given that CAE is a multi-component extract, it is likely that synergistic interactions among several constituents contribute to the overall neuroprotective effect.

Centella asiatica protects against scopolamine-induced neurodegeneration by preserving hippocampal neurons and enhancing antioxidant defense

We next investigated whether CAE protects hippocampal neurons from scopolamine-induced neurodegeneration. Histological analysis of hippocampal sections revealed that scopolamine administration induced significant neuronal degeneration, characterized by shrunken and pyknotic neurons, especially within the CA3 region. Conversely, treatment with CAE significantly attenuated neuronal death and preserved the cytoarchitecture of the hippocampus in a dose-dependent manner (Fig. 5A). These findings suggest that CAE exerts neuroprotective effects against scopolamine-induced hippocampal neurodegeneration.

Fig. 5.

CAE mitigates scopolamine-induced hippocampal neurodegeneration and restores antioxidant defenses in mice. A Representative histological images of hippocampal sections stained with H&E in the CA3 region are shown (n = 3). Administration of scopolamine induced marked neuronal degeneration, characterized by shrunken and pyknotic neurons (indicated by arrows), whereas CAE treatment attenuated neuronal cell death and preserved hippocampal cytoarchitecture in a dose-dependent manner. Scale bar = 100 μm. B Quantitative analysis of oxidative stress markers in hippocampal tissues. Data are presented as mean ± SEM (n = 5). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. #:P < 0.05 and ###:P < 0.01 vs. the vehicle-treated control group; *:P < 0.05 and **:P < 0.01 vs. the scopolamine-treated group

Oxidative stress is widely recognized as a significant risk factor in the development of cognitive disorders and is associated with the etiology of AD (Cheignon et al., 2018; Tonnies and Trushina, 2017), implying that antioxidants may be useful in the prevention of AD (Wojtunik-Kulesza et al., 2016). To evaluate whether the neuroprotective effects of CAE are associated with its antioxidant properties, various oxidative stress markers were measured in the hippocampal tissues. Administration of scopolamine resulted in a significant reduction in superoxide dismutase (SOD) activity and glutathione (GSH) levels, accompanied by a decrease in catalase activity, indicating enhanced oxidative stress. In contrast, treatment with CAE markedly reversed these alterations, with higher doses of CAE restoring antioxidant enzyme activity and elevating GSH levels, as well as recovering catalase activity (Fig. 5B). These results indicate that the cognitive benefits of CAE are closely associated with its capacity to enhance endogenous antioxidant systems and mitigate oxidative stress, thereby preserving neuronal integrity. These results are consistent with the growing body of evidence suggesting that C. asiatica and its bioactive compounds activate antioxidant pathways and improve mitochondrial function, thus alleviating ROS-mediated neuronal injury (Gray et al., 2018, 2016). Taken together, these findings suggest that CAE enhances endogenous antioxidant defense mechanisms and mitigates oxidative stress associated with scopolamine-induced neurotoxicity.

Centella asiatica modulates apoptotic and neurotrophic pathways in the hippocampus

Apoptotic imbalance represents a critical mechanism underlying scopolamine-induced neurotoxicity. Reduced expression of Bcl-2 and increased levels of Bax are associated with hippocampal neuronal loss and compromised synaptic integrity (Ola et al., 2011). We next investigated whether CAE exerts neuroprotective effects by modulating apoptosis- and neurotrophin-related signaling pathways within the hippocampus. Western blot analysis demonstrated a significant downregulation of the anti-apoptotic protein Bcl-2 in the SCO group relative to the CON group. Treatment with CAE significantly restored Bcl-2 expression in a dose-dependent manner, with the most pronounced increase observed in the high-dose CAE group (Fig. 6A). In contrast, Bax protein levels exhibited an upward trend in the SCO group, whereas CAE treatment attenuated this elevation, although the differences did not achieve statistical significance (Fig. 6A). Furthermore, the expression of BDNF was significantly reduced in the SCO group compared to the CON group (Fig. 6B). Administration of CAE markedly increased BDNF protein levels, as confirmed by immunohistochemical staining of the hippocampal CA3 region, which revealed a higher number of BDNF-positive neurons in the CAE-treated groups (Fig. 6C). Notably, CAE restored BDNF and CREB signaling in the hippocampus, which are essential regulators of synaptic plasticity and memory consolidation. Dysfunction of BDNF and CREB signaling has been implicated in AD and other neurodegenerative conditions (Bekinschtein et al., 2014; Lu et al., 2013). Thus, the prevention of scopolamine-induced downregulation of these pathways by CAE suggests that its cognitive benefits extend beyond neuroprotection to the active enhancement of neuroplasticity.

Fig. 6.

CAE modulates apoptotic and neurotrophic signaling pathways in the hippocampus of scopolamine-induced mice. A Representative Western blot images and quantitative analysis of Bcl-2 and Bax protein expression in hippocampal tissues. scopolamine administration significantly decreased the expression of the anti-apoptotic protein Bcl-2, whereas CAE treatment restored Bcl-2 levels in a dose-dependent manner. Bax protein levels were elevated in the SCO group but were attenuated following CAE treatment, although the changes were not statistically significant. B Representative Western blot images and quantitative analysis of BDNF protein expression. Scopolamine markedly reduced BDNF levels, while CAE treatment significantly restored BDNF protein expression in a dose-dependent manne. C Representative immunohistochemical staining of BDNF in the hippocampal CA3 region. Scale bars = 50 μm. Data are presented as mean ± SEM (n = 5). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. ###:P < 0.01 vs. the vehicle-treated control group; *:P < 0.05, **:P < 0.01, and ***:P < 0.001 vs. the scopolamine-treated group

Taken together, the present study demonstrates that CAE ameliorates cognitive impairments through a synergistic mechanism that involves (1) the restoration of cholinergic function, (2) the enhancement of intrinsic antioxidant defenses, (3) the inhibition of apoptotic processes, and (4) the activation of neurotrophic signaling pathways. This multifaceted mode of action is particularly valuable for complex disorders such as AD, which is characterized by the convergence of multiple pathological mechanisms. However, further studies are necessary to isolate and characterize the bioactive constituents responsible for these effects and to clarify the molecular crosstalk among cholinergic, antioxidant, and neurotrophic pathways. These directions will be essential for establishing CAE as a viable therapeutic candidate for cognitive impairment and neurodegenerative disorders.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

3H LABS Corp, ERT2411220, Kyungho Kim

Declarations

Conflict of interest

The authors declare no competing interests. The sponsor’s involvement was strictly limited to extract preparation and analytical characterization and did not influence the study design, data analysis, interpretation, or manuscript preparation.