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Article

Neuroprotective Effect of the Combined Extract of Mentha piperita and Cornus officinalis Against Neuronal Cell Death and Scopolamine-Induced Memory Impairment

by
Kang-Il Oh
1,2,†,
Junhwan Jeong
1,2,†,
Hyesoo Jeong
3,
Yoonjoong Yong
3,
Subin Yeo
3,
Eunkuk Park
1,2,* and
Seon-Yong Jeong
1,2,*
1
Department of Medical Genetics, Ajou University School of Medicine, Suwon 16499, Republic of Korea
2
BK21 R&E Initiative for Advanced Precision Medicine, Department of Biomedical Sciences, Graduate School of Ajou University, Suwon 16499, Republic of Korea
3
Nine B Co., Ltd., Daejeon 34121, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Int. J. Mol. Sci. 2026, 27(5), 2508; https://doi.org/10.3390/ijms27052508
Submission received: 30 January 2026 / Revised: 6 March 2026 / Accepted: 6 March 2026 / Published: 9 March 2026
(This article belongs to the Special Issue Functions and Applications of Natural Products: 2nd Edition)
Browse Figures
Versions Notes

Abstract

Mild cognitive impairment (MCI) represents an intermediate stage between normal aging and Alzheimer’s disease. This study investigated the neuroprotective effects of a combined extract of Mentha piperita (MP) and Cornus officinalis (CO) (MC) using in vitro and in vivo models. In SK-N-SH cells, pretreatment with MC (50–150 μg/mL) significantly attenuated H2O2-induced cellular injury, as evidenced by a reduction in Annexin V-positive cells and an increase in brain-derived neurotrophic factor (BDNF) mRNA expression. Rosmarinic acid and loganin, the marker compounds of MP and CO, alone or combined at a 6:4 ratio, mitigated H2O2-induced decreases in cell viability and BDNF mRNA. In the in vivo study, male Sprague–Dawley rats were orally administered MC (50, 100, or 200 mg/kg/day) for 28 days, with phosphatidylserine (50 mg/kg/day) serving as a positive control. MC administration significantly improved cognitive performance in rats with scopolamine-induced memory impairment, as demonstrated by increased step-through latency in the passive avoidance test and reduced escape latency in the Morris water maze. Furthermore, in the probe trial, MC-treated rats spent significantly more time in the target quadrant, indicating enhanced spatial memory retention. Mechanistically, MC restored hippocampal acetylcholine levels and reversed the scopolamine-induced decrease in BDNF and its downstream signaling. Specifically, MC upregulated hippocampal BDNF expression and enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and cAMP response element-binding protein (CREB). In conclusion, these results demonstrate that the MC extract possesses potent neuroprotective and learning- and memory-enhancing effects, highlighting its potential as a therapeutic candidate for managing age-related cognitive decline and MCI.

Graphical Abstract

1. Introduction

Mild cognitive impairment (MCI) refers to more than normal cognitive decline with age, yet insufficient to warrant a diagnosis of dementia [1]. While cognitive decline is part of normal aging, patients with MCI exhibit pronounced deficits in processing speed, executive control, and memory [2]. Importantly, the annual conversion rates to dementia are approximately 10–15% in MCI versus 1–2% in cognitively normal older adults, positioning MCI as an intermediate stage and a target for interventions to delay dementia onset [3,4]. A comprehensive meta-analysis estimated an overall MCI prevalence of 15.56% (95% CI, 13.24–18.03%) among community-dwelling adults aged ≥50 years [5]. Given the lack of disease-modifying therapies for dementia, interventions that can stabilize or improve cognition at the MCI stage may substantially reduce future dementia incidence and associated healthcare costs. With an increase in population aging, MCI is emerging as a substantial global health and economic burden [6].
MCI involves converging pathological mechanisms—oxidative stress, excitotoxicity, and cholinergic dysregulation—that compromise neuronal integrity [7,8]. These factors reduce brain-derived neurotrophic factor (BDNF) expression and downstream signaling through extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and cAMP response element-binding protein (CREB), leading to synaptic dysfunction and neuronal loss [9,10]. Therefore, strategies that target these converging pathways may offer a rational approach to stabilize or improve cognition at the MCI stage [11]. Although several neuroprotective agents have been evaluated, their clinical benefits remain modest and typically target single pathological mechanisms. For instance, the free-radical scavenger edaravone attenuates oxidative injury but shows limited cognitive efficacy beyond niche indications [12], and the N-methyl-D-aspartic acid (NMDA) receptor antagonist memantine reduces excitotoxic stress yet demonstrates limited and inconsistent efficacy in altering cognitive trajectories in MCI [13,14]. These limitations highlight the need for multifaceted strategies that can address multiple converging pathways simultaneously. Accordingly, there is growing interest in herbal medicines and bioactive botanical combinations that can attenuate neuronal injury while preserving BDNF-related signaling [15].
Within this framework, Mentha piperita (MP) and Cornus officinalis (CO) offer complementary mechanisms relevant to oxidative stress, neurotrophic support, and cholinergic dysfunction. Both MP and CO, as well as the CO-derived iridoid loganin, have been reported to ameliorate scopolamine-induced cognitive deficits [16,17], supporting their relevance to cholinergic dysregulation implicated in MCI-related memory decline. MP has been reported to exert antioxidant and cytoprotective effects and to modulate acetylcholinesterase (AChE)- and inflammation-related pathways [18,19,20,21,22], whereas CO has been reported to support BDNF-related signaling and to modulate excitotoxicity, mitochondrial stress, and apoptosis in preclinical neurodegeneration models [23,24]. Nevertheless, the combined neuroprotective effects of MP and CO have not been systematically evaluated in MCI-relevant models. Because reduced BDNF signaling is closely linked to synaptic dysfunction and memory decline in MCI, we measured BDNF expression in both in vitro and in vivo experiments and assessed downstream ERK/AKT/CREB signaling in the in vivo model. Accordingly, we evaluated the neuroprotective effects of the combined MP and CO extract (MC) against reactive oxygen species (ROS)-induced cytotoxicity in SK-N-SH cells and scopolamine-induced cognitive deficits in rats. H2O2 exposure in SK-N-SH cells was used as an oxidative stress-driven neuronal injury model, whereas scopolamine administration in rats was used as an acute cholinergic dysfunction model to assess cognition-related outcomes and hippocampal molecular changes.

2. Results

2.1. Identification of MC Marker Compounds by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD)

HPLC-DAD analysis of the combined MP and CO extract (MC) confirmed the presence of both marker compounds. In the MC chromatogram (Figure 1A), rosmarinic acid and loganin eluted at 27.3 and 17.8 min, respectively, which matched the retention time and UV spectra of the reference standards (Figure 1B). In addition, a prominent peak at approximately 15–16 min was assigned as morroniside (from C. officinalis) based on comparison with an authentic reference standard (retention time and UV spectrum). The UV spectra of the rosmarinic acid and loganin peaks were identical to those of the corresponding standards. Together with single-extract analyses (Figure S1), these results established rosmarinic acid (MP) and loganin (CO) as quantitative markers for MC identity and batch-to-batch standardization.

2.2. MP and CO Combined Extract Mitigated H2O2-Induced Cytotoxicity and BDNF Downregulation in SK-N-SH Cells, with Superior Efficacy at 6:4 Ratio

We next evaluated the protective effects of MC using a H2O2-induced cytotoxicity model in SK-N-SH cells. Exposure to 0.3 mM H2O2 for 24 h markedly reduced cell viability and BDNF mRNA levels relative to those in the untreated control group, confirming the successful induction of oxidative cytotoxicity. Pretreatment with MC for 1 h followed by co-exposure to H2O2 for 24 h, at fixed MP:CO ratios (6:4, 5:5, 4:6) and concentrations (50, 100, 150 μg/mL) significantly increased the cell viability compared with the H2O2-treated (mock) group (Figure 2A). In contrast, the modulation of BDNF mRNA expression was ratio-dependent; the 6:4 MC combination significantly upregulated the levels of BDNF transcript at 50, 100, and 150 μg/mL, whereas no consistent increase was observed at other ratios (Figure 2B). At 150 μg/mL, the 6:4 MC formulation yielded higher cell viability than either MP or CO alone (Figure 2C) and produced greater BDNF mRNA induction than single-extract treatments (Figure 2D; see also Figure S2). In addition, MP and CO alone (50–150 μg/mL) did not show significant cytotoxicity in SK-N-SH cells (Figure S3), ensuring that the observed neuroprotective effects were not influenced by inherent extract toxicity. Collectively, these data indicated that the 6:4 combination of MP and CO most effectively mitigated the H2O2-induced cytotoxicity and preserved BDNF expression.

2.3. MP and CO Combined Extract at a 6:4 Ratio Mitigated H2O2-Induced Cell Death

To determine whether the optimized 6:4 MP:CO combination suppresses oxidative stress-induced cell death, we quantified apoptosis and necrosis by flow cytometry using Annexin V and propidium iodide (PI), respectively. In SK-N-SH cells, 24 h exposure to 0.3 mM H2O2 markedly increased the Annexin V- and PI-positive populations, confirming enhanced apoptosis and membrane-compromised necrosis. Treatment with the 6:4 MP:CO combination attenuated H2O2-induced apoptosis, significantly reducing both early (Annexin V-positive, PI-negative) and late (Annexin V-positive, PI-positive) apoptotic populations at all tested concentrations (50, 100, and 150 μg/mL) (Figure 3). The number of necrotic (Annexin V-negative/PI-positive) cells was modestly reduced. These findings suggest that the 6:4 formulation exerts neuroprotective effects under oxidative stress conditions, including a reduction in apoptotic cell death.

2.4. Rosmarinic Acid, Loganin and Their Combination Mitigated H2O2-Induced Cytotoxicity and BDNF mRNA Downregulation in SK-N-SH Cells

Next, to address whether the standard marker compounds—rosmarinic acid (RA, a marker for MP) and loganin (LO, a marker for CO)—are biologically active in oxidative stress–induced cellular model, we evaluated effects of marker compounds on H2O2-induced cytotoxicity and BDNF mRNA downregulation in SK-N-SH cells. As expected, exposure to H2O2 significantly reduced cell viability and BDNF mRNA expression in the mock group compared with the untreated control group (Figure 4A,B).
In the cell viability assay, RA significantly increased cell viability compared with the mock group at all concentrations, whereas LO significantly improved viability at 25, 50, and 100 µM (Figure 4A). Notably, the combined treatment of RA and LO at a 6:4 ratio (RL) significantly increased cell viability compared with the mock group in all concentrations (Figure 4A).
In addition, qRT-PCR analysis showed that RA significantly increased BDNF mRNA expression levels compared with the mock group at all concentrations, whereas LO significantly increased BDNF mRNA levels at 25, 50, and 100 µM (Figure 4B). The 6:4 RL combination significantly increased BDNF mRNA expression compared with the mock group at all concentrations (Figure 4B). Importantly, the 6:4 RL combination at 100 µM resulted in significantly greater improvements in both cell viability and BDNF mRNA expression than either RA or LO alone, suggesting enhanced effects of combined components (Figure 4A,B).

2.5. MC Ameliorated Scopolamine-Induced Memory Impairment Assessed Using Passive Avoidance and Morris Water Maze

To investigate whether the neuroprotective effects observed in vitro can be translated to cognitive benefits in vivo, we examined the effect of MC on scopolamine-induced memory impairment. Behavioral testing was performed in independent randomized cohorts with identical dosing schedules.
Seven-week-old male Sprague–Dawley rats received oral MC (50, 100, or 200 mg/kg/day), phosphatidylserine (PS, 50 mg/kg/day, positive control; PC in figures), or vehicle once daily for 28 days. General health was monitored daily and body weight was measured weekly (Figure S4). Rats underwent adaptation training in a passive avoidance apparatus on days 25 and 26. On day 27, scopolamine (1 mg/kg, i. p.) was administered, followed by a learning trial. A retention trial (passive avoidance test) was conducted on day 28. For the Morris water maze test, training trials were conducted on days 23–27, with scopolamine administered 30 min before each session. The probe trial was conducted on day 28.
In the passive avoidance test, MC-administered rats exhibited significantly increased step-through latency compared with the scopolamine-only negative control group (NC) (Figure 5A). In the Morris water maze, MC-administered rats showed significant improvements in escape latency beginning on training day 4 (Figure S5). On day 5, all MC treatment groups (50, 100, and 200 mg/kg/day) exhibited a significantly reduced escape latency compared with the NC group (Figure 5B,C). During the probe trial, MC-administered rats spent significantly more time in the target quadrant than the NC rats, indicating improved spatial memory retention (Figure 5D). Similarly, PS (PC) restored scopolamine-induced deficits in both behavioral paradigms. Following behavioral testing, hippocampal acetylcholine levels were measured. Scopolamine administration significantly reduced the acetylcholine levels compared with the untreated control group, whereas all MC treatment groups and the PS group showed significantly increased hippocampal acetylcholine levels relative to the NC group (Figure 5E).

2.6. MC Promoted Neuroprotection Through Activation of Hippocampal BDNF Signaling

To investigate whether MC modulates BDNF expression and its downstream signaling components, we performed Western blot analysis using the hippocampal tissue. Scopolamine administration significantly reduced BDNF protein levels compared with those in the control group. Treatment with PS or MC restored BDNF expression, with the effect being pronounced at higher MC doses (Figure 6A,B).
Further, we examined the expression of key components of the BDNF-related signaling cascade, including ERK, AKT, and CREB. Scopolamine administration markedly decreased the phosphorylation of ERK, AKT, and CREB compared with that in the untreated control group. Both PS and MC treatment significantly increased ERK, AKT, and CREB phosphorylation relative to the scopolamine-only NC, with MC demonstrating enhanced phosphorylation at higher doses (Figure 6A,C–E).

3. Discussion

This study investigated the neuroprotective and cognition-enhancing effects of the combined Mentha piperita (MP) and Cornus officinalis (CO) extract at a 6:4 ratio (MC). MC attenuated ROS-induced cytotoxicity and apoptosis while preserving BDNF mRNA expression in SK-N-SH cells. Moreover, MC ameliorated scopolamine-induced learning and memory impairments in rats, which was accompanied by restoration of hippocampal acetylcholine levels, increased BDNF expression, and enhanced ERK/AKT/CREB phosphorylation. This suggests that MC can effectively repair the damaged neurotrophic network in the early stages of mild cognitive impairment (MCI). Collectively, these results support MC as a multi-target botanical strategy acting across converging pathways relevant to cognitive decline.
Previous studies show that rosmarinic acid and loganin are strong antioxidants [25,26]. A combination of MP (6:4) effectively protected cells from oxidative damage, compared to a single compound, indicating broader protection. Indeed, MC consistently attenuated oxidative stress–induced injury and preserved BDNF expression in vitro, supporting a cytoprotective effect in ROS-driven conditions. Importantly, the behavioral improvements observed in the scopolamine model were accompanied by restoration of hippocampal acetylcholine levels and activation of BDNF-related signaling (BDNF and ERK/AKT/CREB), which provides a mechanistic basis linking cholinergic recovery and neurotrophic signaling to cognitive outcomes.
This study further investigated the contribution of marker compounds derived from extracts to clarify the effects of individual bioactive components. HPLC-DAD identification of rosmarinic acid (RA) in MP and loganin (LO) in CO as marker compounds supports extract identity and batch consistency. Previous studies have suggested the roles of phenolic antioxidants and iridoids in neuroprotection [27,28]. In this study, RA and LO, alone and in combination (RL; 6:4), mitigated H2O2-induced decreases in cell viability and BDNF mRNA expression in SK-N-SH cells, supporting the biological neuroprotective effects in oxidative stress–related cellular conditions. A mechanistic rationale is supported by prior evidence suggesting that the marker compounds may contribute to neuroprotection beyond serving as extract markers. Specifically, rosmarinic acid has been reported to suppress hippocampal inflammation and downregulate JNK signaling in an Alzheimer’s disease model with reduced tau phosphorylation and improved cognitive outcomes [29]. Similarly, loganin administration in 3xTg-AD mice (a triple-transgenic Alzheimer’s disease model expressing mutant APP, PS1, and Tau) enhanced cognitive performance and reduced Aβ deposition and tau phosphorylation, along with improvements in synaptic- and metabolism-related pathways and inflammatory responses [30]. In addition, a prominent peak at approximately 15–16 min in the MC chromatogram was assigned to morroniside, an iridoid glycoside derived from C. officinalis, by comparison with an authentic reference standard. Morroniside has shown neuroprotective effects in mouse models of Parkinson’s disease, by reducing oxidative stress and regulating ferroptosis-related pathways [31]. Therefore, multiple constituents, including rosmarinic acid, loganin, and potentially morroniside, may contribute to the overall neuroprotective effects of MC. Notably, the 6:4 RL combination showed significantly greater protective effects on cell viability and BDNF mRNA expression than either RA or LO alone. Consistently, the combined crude extracts of MP and CO at a 6:4 ratio more effectively attenuated H2O2-induced cytotoxicity, reduced apoptotic cell death, and mitigated H2O2-induced BDNF downregulation compared with the single MP or CO extracts, indicating the importance of ratio optimization for maximizing complementary benefits [32].
In contrast to prior studies that emphasized peppermint essential oils [20,21], this study highlights the ethanolic MP extract enriched in nonvolatile phenolics as a key contributor for preserving BDNF expression and behavioral benefits. This distinction is mechanistically relevant because ethanolic extraction yields a profile that extends beyond classical acetylcholinesterase modulation [33]. Regarding CO, previous studies have frequently attributed BDNF upregulation to isolated iridoids, such as loganin and morroniside [23,34,35,36]. In this study, crude ethanolic CO extract alone did not increase BDNF levels. These differences were probably due to variations in the extraction solvent, phytochemical composition, and dose. When interpreting these findings, differences in extraction and composition should be considered. Notably, the 6:4 mixture preserved BDNF transcript levels in vitro and increased hippocampal BDNF in vivo together with enhanced phosphorylation of the downstream effectors of the BDNF-related signaling cascade, including ERK, AKT, and CREB. Two nonexclusive mechanisms may contribute to the observed effects of the 6:4 mixture. First, MP-mediated cytoprotective and anti-apoptotic effects under oxidative stress conditions may establish a permissive redox environment that promotes BDNF signaling. Secondly, the combination may engage pathways that converge upon CREB activation to sustain BDNF expression.
Consistent with the behavioral improvements, hippocampal analyses indicated restoration of acetylcholine levels and activation of BDNF-related signaling. MC increased step-through latency in passive avoidance and reduced escape latency with greater time in the target quadrant in the Morris water maze, consistent with improved learning and memory processes that depend on hippocampal plasticity and BDNF-ERK-CREB signaling. Furthermore, MC restored hippocampal acetylcholine levels, supporting the partial recovery of cholinergic tone. By inhibiting AChE, the MC extract keeps acetylcholine levels high and may block secondary oxidative stress [37]. This dual effect explains why the MC extract significantly improved memory and learning in the scopolamine-treated animal model. Although this study does not focus on any single pathway, the alignment of anti-apoptotic protection under oxidative stress, BDNF pathway activation, and cholinergic restoration provides a coherent mechanistic framework for the observed cognitive benefits [38].
This study has several limitations that should be considered when interpreting the results. First, the sample size (n = 5) was determined to comply with the 3Rs’ (Replacement, Reduction, Refinement) ethical guidelines. While n = 5 is consistent with prior scopolamine-model studies [39], larger, appropriately powered studies will be required to confirm these findings. Notably, cognitive outcomes were evaluated using two complementary behavioral paradigms (passive avoidance and Morris water maze) performed in independent cohorts, providing convergent evidence for the observed effects. Second, MP-only and CO-only groups were not included in the in vivo study; therefore, additive or synergistic interactions cannot be directly assessed in this in vivo study. In addition, the present in vitro experiments were not specifically designed to evaluate interaction effects among the major constituents. Future studies that incorporate predefined mixture designs (e.g., binary mixtures centered on key constituents) in neuroprotection assays will enable a more direct evaluation of constituent-level pharmacodynamic interactions within the combined extract [40], which reported synergism between polyphenols and iridoids, consistent with our evaluation of rosmarinic acid (a polyphenol) and loganin (an iridoid). However, since the 6:4 ratio was selected through in vitro optimization, the in vivo study was designed to validate the final optimized candidate (MC) while minimizing unnecessary animal use. Third, brain analysis focused on the hippocampus as it is the critical region for spatial memory and the primary target of scopolamine-induced cholinergic impairment [41]. While we acknowledge that cognitive decline involves multiple regions, the hippocampal results provide a direct biological correlate to the improved behavioral outcomes. Nevertheless, this study has certain constraints. The scopolamine model may not fully reflect the long-term, complex pathology of chronic dementia [42,43]. Future studies will incorporate histopathological evaluations and multi-regional analyses, including the prefrontal cortex, to identify broader neuroprotective effects [44]. Finally, as we observed BDNF-related signaling changes, future research using loss-of-function approaches will be necessary to establish the causal mechanisms.
In summary, the optimized 6:4 MP-CO combination attenuated ROS-induced cytotoxicity and apoptosis while preserving BDNF mRNA expression in SK-N-SH cells, and ameliorated scopolamine-induced learning and memory impairments in rats, accompanied by increased hippocampal BDNF, ERK/AKT/CREB phosphorylation, and acetylcholine levels. Collectively, these findings indicate that MC exerts multifaceted neuroprotective and cognition-supporting effects by coupling antioxidant defenses with neurotrophic and cholinergic support. The optimized 6:4 combination of MP and CO shows great potential as a multi-target treatment. By reducing oxidative stress, improving cholinergic function, and improving BDNF levels at the same time, plant-based extract provides a promising and safe strategy for preventing and managing MCI and related memory disorders. These preclinical data justify further evaluation of MC as a candidate neuroprotective combination extract for enhancing cognitive functions during age-associated memory decline and in MCI. Given the traditional use and generally favorable safety profiles of MP and CO, and the multi-target actions demonstrated herein, MC warrants further preclinical studies to refine standardization, dosing, and safety. This can be followed by dose-optimization and pharmacokinetic studies, and ultimately, pilot clinical trials in at-risk elderly populations with early cognitive decline.

4. Materials and Methods

4.1. Extraction and Sample Preparation

M. piperita (MP) and C. officinalis (CO) powders, along with their 6:4 (w/w) combination (MC), were obtained from Nine B Co., Ltd. (Daejeon, Korea). MP and CO were extracted using 30% and 20% ethanol, respectively; these ethanol concentrations were selected based on process optimization, balancing extraction yield with stable spray-dried powder formation. The resulting extracts underwent a sequential process of filtration, vacuum evaporation, and spray-drying with dextrin. For ratio screening, MP and CO were first tested in vitro at three nearby w/w ratios (6:4, 5:5, and 4:6) centered on an equal proportion (5:5). Based on this screening, the formulation used for the in vivo experiments was prepared by mixing the spray-dried MP and CO powders at a 6:4 (w/w) weight ratio.

4.2. Chemical Analysis by HPLC

Chemical analysis of the extract was performed using an Agilent 1260 HPLC system (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was carried out on a Hypersil GOLD™ C18 column (4.6 × 250 mm, 5 μm; Thermo Scientific, Waltham, MA, USA). The mobile phase consisted of 0.1% trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B), delivered at a flow rate of 0.6 mL/min. The gradient elution program was as follows: 5% B at 0 min, linearly increased to 40% B over 30 min, returned to 5% B at 33 min, and maintained at 5% B until 40 min. The injection volume was 10 μL, and the column temperature was maintained at 30 °C. Detection was performed at 240 nm using a UV–diode array detector equipped with the Agilent 1260 system (Agilent Technologies, Palo Alto, CA). Rosmarinic acid and loganin—standard compounds for MP and CO, respectively—were identified by comparing their characteristic retention times and UV absorbance profiles with those of authentic reference standards (Figure 1 and Figure S1). HPLC-grade acetonitrile was purchased from Burdick & Jackson (Muskegon, MI, USA), and trifluoroacetic acid was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rosmarinic acid (≥98% purity) and loganin (95–99% purity), used as reference standards and as test compounds for cellular assays, were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), respectively.

4.3. Cell Culture and Viability Assay

SK-N-SH human neuroblastoma cells (KCLB No. 30011) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea) and cultured in Dulbecco’s Modified Eagle Medium High Glucose (DMEM; Welgene, Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Cell line identity was verified by the supplier (KCLB) before distribution. Cells were used at passages 5–10. In addition, mycoplasma contamination was tested prior to experiments using the BioMycoX® Mycoplasma PCR Detection Kit (CellSafe, Yongin, Republic of Korea), and cells were confirmed to be mycoplasma-negative. For the experiments, cells were seeded in 96-well plates at a density of 1 × 104 cells per well. The experimental groups were as follows: (1) untreated control, (2) H2O2-treated mock (0.3 mM), and (3) pretreatment groups. The cells were pretreated with extracts (MC, MP, or CO) or purified marker compounds (rosmarinic acid, loganin, or their 6:4 combination) at indicated concentrations for 1 h, followed by exposure to 0.3 mM H2O2 (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The H2O2 concentration (0.3 mM) was selected based on prior studies [45,46], using an established oxidative injury model [47,48], and was further optimized in the present experimental conditions to approximate the IC50 (Supplementary Figure S6). The concentration range of extracts was selected based on previous studies demonstrating antioxidant activity of Mentha piperita extracts at 8.33–166.7 μg/mL [49], and was consistently applied across all extract formulations to enable direct comparison. Specific concentrations and ratios for each experiment are detailed in the respective Results sections (Section 2.2, Section 2.3 and Section 2.4). Cell viability was assessed using the EZ-Cytox Enhanced Cell Viability Assay Kit (DOGEN, Seoul, Republic of Korea) according to the manufacturer’s instructions. Briefly, 10 μL of the WST reagent was added to each well and incubated for 1 h at 37 °C. Following this, the absorbance was measured at 450 nm using a microplate reader (BERTHOLD Technologies GmbH, Bad Wildbad, Germany).

4.4. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

SK-N-SH cells were homogenized in 1 mL QIAzol lysis reagent (QIAGEN, Hilden, Germany), and total RNA was isolated in accordance with the manufacturer’s instructions. cDNA was synthesized from the extracted RNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Hanover, NH, USA). qRT-PCR was performed using the SYBR Green I qPCR kit (Takara, Kusatsu, Japan) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the following cycling conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primers used in this study were as follows: forward 5′-TGA GGA CCA GAA AGT TCG GC-3′ and reverse 5′-GAG GCT CCA AAG GCA CTT GA-3′ for human BDNF, forward 5′-GAG TCA ACG GAT TTG GTC GT-3′ and reverse 5′-GAC AAG CTT CCC GTT CTC AG-3′ for human GAPDH. The expression level of BDNF mRNA was normalized to that of human GAPDH, and the results were calculated using the 2−ΔΔCt method, with fold changes expressed relative to the “Mock” condition.

4.5. Annexin V/PI-Based Apoptotic Cell Death Assay

To assess the effect of MC on H2O2-induced apoptosis in SK-N-SH cells, flow cytometry was performed using the FACSCalibur system (BD Biosciences, Franklin Lakes, NJ). Cells were pretreated with MC at concentrations of 50, 100, or 150 μg/mL for 1 h, followed by exposure to 0.3 mM H2O2 for 24 h. After treatment, the cells were detached using trypsin-EDTA (0.05%), collected via centrifugation, and washed twice with phosphate-buffered saline (PBS). The cell pellets were resuspended in 300 μL of PBS, and 5 μL each of FITC Annexin V and PI buffer (BD Biosciences) were added. After incubation for 20 min in the dark at room temperature, 1 × 104 cells were analyzed by flow cytometry. Data acquisition and analysis were performed using the FACS Diva software (BD Biosciences, Franklin Lakes, NJ; Version 8.0.2).

4.6. Scopolamine-Induced Memory Impairment Model in Rats

Seven-week-old male Sprague–Dawley rats were obtained from ORIENT BIO Inc. (Seongnam-si, Republic of Korea) and acclimated for seven days before experimentation. Animals were housed under controlled conditions at 22 ± 2 °C with a 12 h light/dark cycle and 40–60% relative humidity, with standard laboratory chow and water provided ad libitum. The rats were randomly assigned to six groups (n = 5 per group): (1) normal control (vehicle only; water for injection, JW Pharmaceutical, Gwacheon, Republic of Korea), (2) scopolamine-treated NC (1 mg/kg), (3) scopolamine + positive control phosphatidylserine 50 mg/kg/day, (4) scopolamine + MC 50 mg/kg/day, (5) scopolamine + MC 100 mg/kg/day, and (6) scopolamine + MC 200 mg/kg/day. MC and phosphatidylserine were administered by oral gavage once daily for 28 consecutive days. The 28-day dosing period was chosen to provide sufficient exposure prior to behavioral testing and to maintain dosing throughout the behavioral assessment phase. Rats in the normal and scopolamine-treated negative control groups received an equivalent volume (3 mL) of water for injection by oral gavage. Scopolamine (1 mg/kg, Sigma-Aldrich) was administered intraperitoneally 30 min before behavioral testing according to the experimental schedule [42]. At the end of the experiments, rats were euthanized under isoflurane anesthesia by exsanguination via the abdominal aorta. The brain was rapidly removed and the hippocampus was dissected and collected for biochemical analyses; hippocampal sampling was performed without restricting to a specific cerebral hemisphere. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech Co., Ltd. (Cheongju, Republic of Korea; Approval No. 190511, 17 September 2019) and conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

4.7. Behavioral Studies

The passive avoidance test and Morris water maze test were performed independently using separate cohorts of animals to avoid potential confounding effects of sequential behavioral testing.

4.7.1. Passive Avoidance Test

The passive avoidance test was performed using an apparatus consisting of illuminated (white) and dark compartments separated by a guillotine door, with overall dimensions of 490 × 250 × 300 mm (Daejong Inc., Busan, Republic of Korea), as described previously [50] with minor modifications (Figure 7). On day 25 after MC administration, rats were placed in the white compartment with the LED lights off and allowed to acclimate for 60 s. The LED was then turned on and the guillotine door opened simultaneously, allowing the rats to explore the dark compartment for 180 s. This acclimation procedure was repeated on day 26 until the latency to enter the dark compartment stabilized at approximately 30 s. On day 27, the acquisition trial was conducted 1 h after MC administration and 30 min after the intraperitoneal injection of scopolamine (1 mg/kg). Rats were placed in the white compartment with the LED turned off; after 10 s, the LED was turned on and the guillotine door was opened. Upon entry into the dark compartment (all four paws inside), the door was closed and a foot shock (0.5 mA, 3 s) was delivered through the floor grid. The retention trial was conducted 24 h later (day 28), 1 h after MC administration. Rats were placed in the white compartment with the LED turned off; after 10 s, the LED was turned on and the door was opened. The latency to enter the dark compartment (step-through latency) was recorded. The test was terminated when the rat did not enter the dark compartment within 300 s.

4.7.2. Morris Water Maze

The Morris water maze test was conducted as previously described [51] with slight modifications (Figure 8). The apparatus consisted of a circular pool (diameter, 150 cm; depth, 30 cm) divided into four equal quadrants. A white plastic escape platform was placed 1 cm below the water surface in the southwest quadrant. Visual-spatial cues in the form of geometric shapes were affixed to the inner wall of the maze to enable spatial orientation. The pool was filled with opaque water containing black paint (Kidsmomart, Uijeongbu, Republic of Korea) to conceal the platform. After 23 days of MC treatment, the Morris water maze test was performed for five consecutive days (days 23–27). On each test day, MC was administered by oral gavage and the rats were returned to their home cages for 30 min. Scopolamine (1 mg/kg, Sigma-Aldrich) was then administered intraperitoneally, and the rats were placed in their home cages for another 30 min before testing. Each rat underwent four training trials per day initiated from different starting positions (Table 1), with the rat facing the wall at the start of each trial. The rats were allowed up to 60 s to locate the hidden platform, and if a rat failed to find the platform within 60 s, it was gently guided or placed onto the platform. Once on the platform, the rats were allowed to remain there for 10 s before being removed. The inter-trial interval was 15–30 s. The escape latency (time to reach the platform) was recorded for each trial using the SMART video tracking system (Smart 3.0; Panlab Harvard Apparatus, Barcelona, Spain), and the mean escape latency for the four trials was calculated for each day. The platform was removed on day 6 (probe trial). Rats received MC and scopolamine on training days and were then placed in the pool opposite to the target quadrant. The time spent in the target quadrant (southwest) during a 60-s probe trial was recorded.

4.8. Acetylcholine Analysis

A portion of the hippocampal tissue was weighed and transferred to a microcentrifuge tube, and 10 volumes (w/v) of phosphate-buffered saline (PBS) were added. The tissue was homogenized using a disposable pestle, followed by complete lysis using a sonicator. The homogenate was centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatant was collected into a new tube. The hippocampus was dissected, and acetylcholine levels were analyzed with the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (Molecular Probes, Eugene, OR). The fluorescence was recorded at 540 nm (excitation) and 595 nm (emission) using a multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany).

4.9. Western Blotting

For Western blot analysis, a portion of the hippocampal tissue was homogenized in RIPA buffer (BIOSESANG, Seongnam, Republic of Korea) using a disposable pestle. The buffer contained phenylmethylsulfonyl fluoride (Sigma-Aldrich) and a phosphatase inhibitor cocktail (Sigma-Aldrich). After homogenization, lysates were processed to obtain total protein extracts. Protein samples were boiled for 10 min at 100 °C in 5X SDS loading buffer (BIOSESANG), separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto 0.45-µm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% bovine serum albumin (Bovogen, Melbourne, Australia) for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against BDNF (ab108319; Abcam, Cambridge, UK), total (t)-AKT (#9272; Cell Signaling Technology, Danvers, MA, USA), phosphorylated (p)-AKT (#9275; Cell Signaling Technology), t-ERK (#9102; Cell Signaling Technology), p-ERK (#9101; Cell Signaling Technology), t-CREB (#9197; Cell Signaling Technology), p-CREB (#9198; Cell Signaling Technology), and β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA). Thereafter, the membranes were incubated with either HRP-conjugated goat anti-mouse IgG (A90-116 P; Bethyl Laboratories, Montgomery, TX, USA) or HRP-conjugated goat anti-rabbit IgG (A120-101 P; Bethyl Laboratories) secondary antibodies at room temperature for 1 h. Immunoreactive bands were detected using the West-Q Pico Dura ECL Solution (GenDEPOT, Katy, TX, USA).

4.10. Statistical Analysis

Statistical analysis was performed using the GraphPad Prism 9.0.0 software (GraphPad Software, San Diego, CA, USA), and data were presented as the mean ± standard error of the mean or mean ± standard deviation, as indicated in each figure legend. Data distribution was assessed for normality. Statistically significant differences between two groups were assessed using Student’s t-test. A p-value < 0.05 was considered statistically significant. All in vitro experiments (cell viability, qRT-PCR, flow cytometry, and marker compound studies) were independently repeated at least three times to ensure reproducibility. For in vivo behavioral studies, each experimental group consisted of n = 5 rats.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052508/s1.

Author Contributions

K.-I.O. and J.J.: writing—original draft, investigation, data curation; H.J.: Visualization, software, investigation; Y.Y. and S.Y.: investigation, data curation, validation, methodology; E.P. and S.-Y.J.: writing—review and editing, supervision, project administration, funding acquisition, conceptualization. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Korea Health Technology R&D Project through the Korea Health Industry Development Institution (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea, grant numbers RS-2022-KH130303, RS-2021-KH113820, and RS-2025-25303051.

Institutional Review Board Statement

Institutional Animal Care and Use Committee is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC; Approval No. 190511, Date of Approval: 17 September 2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in this article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare that they have no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
BDNFBrain-derived neurotrophic factor
COCornus officinalis
CREBcAMP response element-binding protein
ERKExtracellular signal-regulated kinase
MCMP and CO
MCIMild cognitive impairment
MPMentha piperita
NCNegative control
PBSPhosphate-buffered saline
PIPropidium iodide
ROSReactive oxygen species

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Ijms 27 02508 g001
Figure 1. High-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis of the combined Mentha piperita and Cornus officinalis extract (MC) and standards. (A) HPLC chromatogram of MC. (B) HPLC chromatogram of rosmarinic acid and loganin standard mixture.
Figure 1. High-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis of the combined Mentha piperita and Cornus officinalis extract (MC) and standards. (A) HPLC chromatogram of MC. (B) HPLC chromatogram of rosmarinic acid and loganin standard mixture.
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Figure 2. Effects of the combined MC extract on cell viability and BDNF mRNA expression in H2O2-exposed SK-N-SH cells. Cells were pretreated with MC at fixed ratios (6:4, 5:5, and 4:6) and concentrations (50, 100, and 150 μg/mL) for 1 h and then exposed to 0.3 mM H2O2 for additional 24 h. (A) Cell viability was assessed using a commercial assay kit. (B) BDNF mRNA expression was quantified by qRT-PCR. MC (6:4, 150 μg/mL) was compared with single extracts for (C) viability and (D) BDNF mRNA expression. Data are presented as mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Mock). Control: untreated cells. Mock: 0.3 mM H2O2 only.
Figure 2. Effects of the combined MC extract on cell viability and BDNF mRNA expression in H2O2-exposed SK-N-SH cells. Cells were pretreated with MC at fixed ratios (6:4, 5:5, and 4:6) and concentrations (50, 100, and 150 μg/mL) for 1 h and then exposed to 0.3 mM H2O2 for additional 24 h. (A) Cell viability was assessed using a commercial assay kit. (B) BDNF mRNA expression was quantified by qRT-PCR. MC (6:4, 150 μg/mL) was compared with single extracts for (C) viability and (D) BDNF mRNA expression. Data are presented as mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Mock). Control: untreated cells. Mock: 0.3 mM H2O2 only.
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Figure 3. Flow cytometric analysis of apoptosis and necrosis in SK-N-SH cells under H2O2-induced oxidative stress with the optimized 6:4 MP:CO mixture. SK-N-SH cells were exposed to H2O2 (24 h, 0.3 mM) and stained with Annexin V and propidium iodide (PI) to quantify apoptotic and necrotic populations. Representative dot plots showing Annexin V-positive and PI-positive cells in the control, mock, and 6:4 MP:CO treatment groups (top). Quantification of Annexin V-positive cells demonstrating a concentration-dependent reduction at 100 and 150 μg/mL with 6:4 MP:CO (bottom). Data are presented as mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Mock). Control: untreated cells. Mock: 0.3 mM H2O2 only.
Figure 3. Flow cytometric analysis of apoptosis and necrosis in SK-N-SH cells under H2O2-induced oxidative stress with the optimized 6:4 MP:CO mixture. SK-N-SH cells were exposed to H2O2 (24 h, 0.3 mM) and stained with Annexin V and propidium iodide (PI) to quantify apoptotic and necrotic populations. Representative dot plots showing Annexin V-positive and PI-positive cells in the control, mock, and 6:4 MP:CO treatment groups (top). Quantification of Annexin V-positive cells demonstrating a concentration-dependent reduction at 100 and 150 μg/mL with 6:4 MP:CO (bottom). Data are presented as mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Mock). Control: untreated cells. Mock: 0.3 mM H2O2 only.
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Figure 4. Effects of rosmarinic acid (RA), loganin (LO), and their combination (RL) on cell viability and BDNF mRNA expression in H2O2-exposed SK-N-SH cells. Cells were pretreated with RA, LO, or RL (6:4) at the indicated concentrations (10, 25, 50, and 100 µM) for 1 h and then exposed to 0.3 mM H2O2 for an additional 24 h. (A) Cell viability was assessed using a commercial assay kit. (B) BDNF mRNA expression was quantified by qRT-PCR. Data are presented as mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Mock). Control: untreated cells. Mock: 0.3 mM H2O2 only.
Figure 4. Effects of rosmarinic acid (RA), loganin (LO), and their combination (RL) on cell viability and BDNF mRNA expression in H2O2-exposed SK-N-SH cells. Cells were pretreated with RA, LO, or RL (6:4) at the indicated concentrations (10, 25, 50, and 100 µM) for 1 h and then exposed to 0.3 mM H2O2 for an additional 24 h. (A) Cell viability was assessed using a commercial assay kit. (B) BDNF mRNA expression was quantified by qRT-PCR. Data are presented as mean ± standard error of the mean. One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. Mock). Control: untreated cells. Mock: 0.3 mM H2O2 only.
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Figure 5. Effects of MC on scopolamine-induced cognitive impairment in rats. Rats received oral MC (50, 100, or 200 mg/kg/day) or phosphatidylserine (50 mg/kg/day, positive control) once daily for 28 days. Memory impairment was assessed using the passive avoidance test and Morris water maze. (A) Step-through latency was measured in the passive avoidance test during retention trial (day 28). (B) Escape latency was recorded daily during Morris water maze training (days 1–5) with scopolamine given 30 min before each session. (C) Escape latency on training day 5 was measured to assess acquisition performance by the treatment group. (D) During the probe trial (day 28) with the platform removed, time spent in the target quadrant was measured to evaluate spatial memory retention. (E) Hippocampal acetylcholine levels were quantified using the acetylcholinesterase assay kit. Data are presented as mean ± standard error of the mean (n = 5 per group). One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC). NC: negative control. PC: positive control (phosphatidylserine). MC: combination of Mentha piperita and Cornus officinalis (6:4).
Figure 5. Effects of MC on scopolamine-induced cognitive impairment in rats. Rats received oral MC (50, 100, or 200 mg/kg/day) or phosphatidylserine (50 mg/kg/day, positive control) once daily for 28 days. Memory impairment was assessed using the passive avoidance test and Morris water maze. (A) Step-through latency was measured in the passive avoidance test during retention trial (day 28). (B) Escape latency was recorded daily during Morris water maze training (days 1–5) with scopolamine given 30 min before each session. (C) Escape latency on training day 5 was measured to assess acquisition performance by the treatment group. (D) During the probe trial (day 28) with the platform removed, time spent in the target quadrant was measured to evaluate spatial memory retention. (E) Hippocampal acetylcholine levels were quantified using the acetylcholinesterase assay kit. Data are presented as mean ± standard error of the mean (n = 5 per group). One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC). NC: negative control. PC: positive control (phosphatidylserine). MC: combination of Mentha piperita and Cornus officinalis (6:4).
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Figure 6. MC activates the BDNF signaling pathway in the hippocampus of scopolamine-treated rats. Rats received oral MC (50, 100, or 200 mg/kg/day) or PS (50 mg/kg/day) for 28 days. (A) Representative Western blots of BDNF, phosphorylated and total ERK, AKT, and CREB; β-actin served as the loading control. (B–E) Quantification of relative band intensities using the ImageJ software (Version 1.53g). Data are presented as the mean ± standard error (n = 5 per group). One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC). NC: negative control. PC: positive control (phosphatidylserine). MC: combination of Mentha piperita and Cornus officinalis (6:4). BDNF: brain-derived neurotrophic factor. ERK: extracellular signal-regulated kinase. AKT: protein kinase B. CREB: cAMP response element-binding protein.
Figure 6. MC activates the BDNF signaling pathway in the hippocampus of scopolamine-treated rats. Rats received oral MC (50, 100, or 200 mg/kg/day) or PS (50 mg/kg/day) for 28 days. (A) Representative Western blots of BDNF, phosphorylated and total ERK, AKT, and CREB; β-actin served as the loading control. (B–E) Quantification of relative band intensities using the ImageJ software (Version 1.53g). Data are presented as the mean ± standard error (n = 5 per group). One-way analysis of variance was performed, followed by Tukey’s honest significant difference post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC). NC: negative control. PC: positive control (phosphatidylserine). MC: combination of Mentha piperita and Cornus officinalis (6:4). BDNF: brain-derived neurotrophic factor. ERK: extracellular signal-regulated kinase. AKT: protein kinase B. CREB: cAMP response element-binding protein.
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Figure 7. Experimental timeline for the passive avoidance test. Rats received MC (50, 100, or 200 mg/kg/day) or phosphatidylserine (PS, 50 mg/kg/day) by oral gavage for 28 days. Adaptation trials were conducted on days 25–26. On day 27, scopolamine (1 mg/kg; intraperitoneally, i.p.) was administered 30 min before the learning trial. The retention trial was performed on day 28. MC: combination of Mentha piperita and Cornus officinalis (6:4).
Figure 7. Experimental timeline for the passive avoidance test. Rats received MC (50, 100, or 200 mg/kg/day) or phosphatidylserine (PS, 50 mg/kg/day) by oral gavage for 28 days. Adaptation trials were conducted on days 25–26. On day 27, scopolamine (1 mg/kg; intraperitoneally, i.p.) was administered 30 min before the learning trial. The retention trial was performed on day 28. MC: combination of Mentha piperita and Cornus officinalis (6:4).
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Figure 8. Experimental timeline for the Morris water maze test. Rats received MC (50, 100, or 200 mg/kg/day) or phosphatidylserine (PS, 50 mg/kg/day) by oral gavage for 28 days. Training trials were conducted on days 23–27, with scopolamine (1 mg/kg; intraperitoneally, i.p.) administered 30 min before each daily session. The probe trial was performed on day 28. MC: combination of Mentha piperita and Cornus officinalis (6:4).
Figure 8. Experimental timeline for the Morris water maze test. Rats received MC (50, 100, or 200 mg/kg/day) or phosphatidylserine (PS, 50 mg/kg/day) by oral gavage for 28 days. Training trials were conducted on days 23–27, with scopolamine (1 mg/kg; intraperitoneally, i.p.) administered 30 min before each daily session. The probe trial was performed on day 28. MC: combination of Mentha piperita and Cornus officinalis (6:4).
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Table 1. Starting positions of rats for Morris water maze training trials.
Table 1. Starting positions of rats for Morris water maze training trials.
DayTrial 1Trial 2Trial 3Trial 4
1SWNWSE
2NWSSEW
3SENWWS
4WSESNW
5SNWWSE
S: south. W: west. NW: northwest. SE: southeast. At the beginning of each trial, rats were placed in the pool facing the tank wall at the designated starting position.
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MDPI and ACS Style

Oh, K.-I.; Jeong, J.; Jeong, H.; Yong, Y.; Yeo, S.; Park, E.; Jeong, S.-Y. Neuroprotective Effect of the Combined Extract of Mentha piperita and Cornus officinalis Against Neuronal Cell Death and Scopolamine-Induced Memory Impairment. Int. J. Mol. Sci. 2026, 27, 2508. https://doi.org/10.3390/ijms27052508

AMA Style

Oh K-I, Jeong J, Jeong H, Yong Y, Yeo S, Park E, Jeong S-Y. Neuroprotective Effect of the Combined Extract of Mentha piperita and Cornus officinalis Against Neuronal Cell Death and Scopolamine-Induced Memory Impairment. International Journal of Molecular Sciences. 2026; 27(5):2508. https://doi.org/10.3390/ijms27052508

Chicago/Turabian Style

Oh, Kang-Il, Junhwan Jeong, Hyesoo Jeong, Yoonjoong Yong, Subin Yeo, Eunkuk Park, and Seon-Yong Jeong. 2026. "Neuroprotective Effect of the Combined Extract of Mentha piperita and Cornus officinalis Against Neuronal Cell Death and Scopolamine-Induced Memory Impairment" International Journal of Molecular Sciences 27, no. 5: 2508. https://doi.org/10.3390/ijms27052508

APA Style

Oh, K.-I., Jeong, J., Jeong, H., Yong, Y., Yeo, S., Park, E., & Jeong, S.-Y. (2026). Neuroprotective Effect of the Combined Extract of Mentha piperita and Cornus officinalis Against Neuronal Cell Death and Scopolamine-Induced Memory Impairment. International Journal of Molecular Sciences, 27(5), 2508. https://doi.org/10.3390/ijms27052508

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