S. commixta bark was purchased from a local herbal market (Baekje Herb Market, Daejeon, Korea). The bark was powdered and extracted three times with 70% (v/v) ethanol under mild shaking (JSSI-200C; JSR, Daejeon, Korea) at 25 ± 1℃ for 8 h. To obtain SCBE, the extracts were passed through Whatman No. 2 filter paper (8 μm; GE Healthcare, Chicago, IL, USA), followed by vacuum concentration at 60℃ and subsequent lyophilization. The extraction yield was 23.4% (w/w). The dried extract was stored at 4 ± 1℃ until use.

AChE inhibitory activity was determined using a colorimetric assay based on Ellman et al. (1961). Whole brains from mice not subjected to behavioral testing were homogenized using a tissue homogenizer (Ultra-Turrax T25; IKA, Staufen, Germany) in cold 100 mM sodium phosphate buffer (pH 8.0) and centrifuged at 14,000 × g for 25 min at 4℃. The supernatants were used as the enzyme source. The assay was performed in the reaction mixture composed of 100 mM sodium phosphate buffer (pH 8.0, 120 μL), 0.71 mM acetylthiocholine iodide (10 μL), 0.5 mM 5,5’-dithiobis(2-nitrobenzoic acid) (10 μL), and either the sample extract or donepezil (30 μL). Following a 30 min pre-incubation at 25℃, 30 μL of the enzyme source was added to initiate the reaction, and the optical density was monitored at 412 nm with a microplate reader (SpectraMax M2; Molecular Devices, San Jose, CA, USA).

Male Institute of Cancer Research (ICR) mice were utilized for behavioral experiments following a 7-day acclimation period in a facility maintained at a constant temperature (23 ± 1℃) and 55 ± 5% relative humidity under a regulated 12-h light/dark cycle. Food and water were supplied ad libitum during the experimental period. Five-week-old mice were randomly assigned to six groups (n = 5 per group): normal control, scopolamine-treated, donepezil (5 mg/kg) + scopolamine, and SCBE (50, 100, or 500 mg/kg) + scopolamine. SCBE or donepezil was orally administered once daily for 2 weeks, and scopolamine (2 mg/kg, i.p.) dissolved in saline was injected 30 min before behavioral testing to induce acute memory impairment. All groups except the normal control received scopolamine. Behavioral assessments were performed within 30 min after injection, and mice were euthanized by decapitation. All experimental protocols involving animals received official approval from the Institutional Animal Care and Use Committee (IACUC) at Kyung Hee University (Accession No. KHUASP (SE)-17-126-1). Furthermore, all procedures involving animal management and testing were conducted following the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (Publication No. 80-23, revised 1996).

The Y-maze test was conducted to assess spatial working memory. The apparatus consisted of three black plastic arms (40 × 3 × 12 cm³). Mice were individually introduced into one arm of the Y-maze and permitted to explore the apparatus freely for 8 min. A spontaneous alternation was recorded when the animal made successive entries into all three distinct arms (for instance, ABC, CAB, or BCA). The spontaneous alternation rate was determined as the ratio of alternation events to the total arm entries minus two and expressed as a percentage. Scopolamine (2 mg/kg, i.p.), dissolved in 0.9% saline, was administered 30 min before testing to induce memory impairment and was injected once daily for 14 consecutive days. The normal control group was administered an equal volume of 0.9% saline.

ACh levels were measured in hippocampal homogenates using a commercial colorimetric assay kit (ab65345; Abcam, Cambridge, UK). The hippocampus from one hemisphere was homogenized on ice in assay buffer, and the optical density was measured at 570 nm using a microplate reader (SpectraMax M2; Molecular Devices, San Jose, CA, USA). ACh concentrations were quantified using standard curves and normalized to total protein (nmol/mg protein).

ACh levels were measured in the hippocampus, whereas AChE activity was assessed in the striatum, as these regions are known to reflect neurotransmitter levels and enzymatic activity of the cholinergic system, respectively.

AChE activity was determined in striatal homogenates using a colorimetric assay kit (ab138871; Abcam, Cambridge, UK), according to the manufacturer’s instructions. Briefly, the striatum was homogenized on ice in lysis buffer. Absorbance was measured at 410 nm using a microplate reader (SpectraMax M2; Molecular Devices, San Jose, CA, USA). Enzyme activity was calculated using standard curves, normalized to total protein, and expressed as mU/mg protein.

Hippocampal tissues were homogenized in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The homogenates were centrifuged at 12,000 × g for 15 min at 4℃ to remove insoluble debris, and the supernatants were collected for further analysis. Protein concentrations were quantified using a Bradford protein assay with a microplate reader (SpectraMax M2; Molecular Devices, San Jose, CA, USA). Equal amounts of protein (30 μg per sample) were resolved on 10–12% SDS-polyacrylamide gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Primary antibodies against BDNF (ab108319) and pro-BDNF (ab72440) were obtained from Abcam (Cambridge, UK), while antibodies against CREB (9198), phospho-CREB (p-CREB; 9197), and β-actin (3700) were obtained from Cell Signaling Technology (Danvers, MA, USA). After protein transfer, PVDF membranes were immersed in a blocking solution consisting of 5% skim milk prepared in TBS-T (Tris-buffered saline containing 0.05% Tween-20; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The membranes were subsequently incubated with primary antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After extensive washing with TBS-T (Sigma-Aldrich, St. Louis, MO, USA), the membranes were treated with HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse IgG; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Protein signals were detected using an enhanced chemiluminescence reagent (ECL; Thermo Fisher Scientific, Waltham, MA, USA) and captured with a chemiluminescence imaging system (Fusion Solo S; Vilber, Collégien, France). Densitometric analysis was performed using image analysis software, and band intensities were quantified using the predominant band corresponding to the expected molecular weight (~43 kDa for CREB and p-CREB; ~32 kDa for pro-BDNF; ~14 kDa for mature BDNF). Representative bands at the expected molecular weight were selected for analysis. All signals were normalized to β-actin.

Free-floating sections were incubated for 5 min in 1% cresyl violet acetate dissolved in tertiary distilled water, followed by sequential washing with 70–100% ethanol for 30 sec each and rinsing with tap water. The sections were then incubated for 3 min in xylene and mounted using DPX mounting medium. Microscopic images of the tissue sections were captured using a laser-scanning confocal microscope (K1-Fluo; Nanoscope Systems, Daejeon, Korea) in bright-field mode to obtain high-resolution images under conditions comparable to conventional light microscopy. Quantitative analysis was performed by measuring the optical density of cresyl violet staining in each region of interest [CA1, CA3, and dentate gyrus (DG)] using image analysis software. Regions of interest were defined based on anatomical landmarks, and staining intensity values were normalized to the normal group (set as 100%).

All experimental results were analyzed using one-way analysis of variance (ANOVA) with IBM SPSS Statistics (IBM Corp., Armonk, NY, USA). When significant differences were detected, Tukey’s multiple comparison test was applied for post hoc analysis. Groups sharing the same letter are not significantly different, whereas groups with different letters indicate significant differences at p < 0.05.

SCBE exhibited a concentration-dependent inhibitory effect on acetylcholinesterase (AChE) activity (Fig. 1). At 20, 100, 250, 500, 1000, and 2000 μg/mL, the inhibition rates were 8.12 ± 1.00%, 14.37 ± 2.50%, 28.50 ± 1.50%, 42.36 ± 1.83%, 73.20 ± 2.86%, and 73.55 ± 0.66%. Donepezil showed consistently potent inhibition across all tested concentrations (78.93 ± 0.73% to 87.40 ± 0.97%). A plateau in SCBE-mediated inhibition was observed at 1000–2000 μg/mL, although the maximal inhibition remained lower than that of donepezil (Fig. 1).

Fig. 1.  Inhibitory effects of SCBE on AChE activity in vitro.

SCBE and donepezil (20–2000 μg/mL). AChE inhibitory activity was measured using a colorimetric assay. Values are mean ± SD (n = 3). Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters (a-g) indicate significant differences among groups at p < 0.05, whereas groups sharing the same letter are not significantly different.

Scopolamine administration significantly reduced spontaneous alternation in the Y-maze test (42.20 ± 1.31%) compared with the normal control group (69.10 ± 1.64%). The donepezil-treated group showed significantly increased alternation behavior (65.09 ± 3.52%) relative to the scopolamine group. SCBE treatment significantly attenuated scopolamine-induced deficits in a dose-dependent manner, with spontaneous alternation values of 59.75 ± 5.86%, 63.61 ± 4.66%, and 68.96 ± 1.51% at 50, 100, and 500 mg/kg, respectively. The 500 mg/kg SCBE group showed no significant difference compared with the normal control group (Fig. 2).

Fig. 2.  Effect of SCBE on spontaneous alternation behavior in the Y-maze test in scopolamine-treated mice.

Groups: saline (Normal); scopolamine (2 mg/kg, i.p.; Control); donepezil (5 mg/kg, p.o.) + scopolamine; and SCBE (50, 100, or 500 mg/kg, p.o.) + scopolamine. Values are mean ± SD (n = 3). Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters (a-c) indicate significant differences among groups at p < 0.05, whereas groups sharing the same letter are not significantly different.

Scopolamine administration significantly decreased hippocampal acetylcholine (ACh) levels (0.82 ± 0.05 nmol/mg protein) compared with the normal control group (1.33 ± 0.09 nmol/mg protein). Donepezil treatment increased ACh levels to 1.32 ± 0.03 nmol/mg protein. In the SCBE-treated groups, hippocampal ACh levels were 0.96 ± 0.06, 1.30 ± 0.04, and 1.31 ± 0.05 nmol/mg protein at doses of 50, 100, and 500 mg/kg, respectively. The 100 and 500 mg/kg SCBE groups showed no significant differences compared with the normal or donepezil-treated groups (Fig. 3A).

Fig. 3.  Effect of SCBE on cholinergic markers in scopolamine-treated mice.

Groups: saline (Normal); scopolamine (2 mg/kg, i.p.; Control); donepezil (5 mg/kg, p.o.) + scopolamine; and SCBE (50, 100, or 500 mg/kg, p.o.) + scopolamine. (A) Hippocampal ACh levels and (B) brain AChE activity. ACh levels are expressed as nmol/mg protein and AChE activity as mU/mg protein. Values are mean ± SD (n = 5). Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters (a-c) indicate significant differences among groups at p < 0.05, whereas groups sharing the same letter are not significantly different.

Conversely, scopolamine treatment significantly increased brain acetylcholinesterase (AChE) activity (35.69 ± 2.59 mU/mg protein) compared with the normal control group (21.14 ± 2.31 mU/mg protein). Donepezil treatment decreased AChE activity to 23.31 ± 2.68 mU/mg protein. SCBE administration reduced AChE activity in a dose-dependent manner to 31.95 ± 2.14, 24.01 ± 1.55, and 23.64 ± 2.61 mU/mg protein at 50, 100, and 500 mg/kg, respectively. The 100 and 500 mg/kg SCBE groups showed significantly lower AChE activity compared with the scopolamine-treated group (Fig. 3B).

Scopolamine significantly decreased hippocampal BDNF protein expression to 53.78 ± 5.85% of the normal control level. SCBE increased BDNF expression in a dose-dependent manner to 64.67 ± 2.61%, 86.01 ± 6.01%, and 90.53 ± 5.63% at 50, 100, and 500 mg/kg, respectively. Pro-BDNF expression was also reduced by scopolamine (46.83 ± 4.37%). SCBE administration increased pro-BDNF levels to 46.95 ± 1.55%, 76.01 ± 2.63%, and 76.45 ± 2.45% at 50, 100, and 500 mg/kg, respectively. Pro-BDNF levels in the donepezil group were higher than those in the SCBE-treated groups, although all SCBE groups showed significant increases compared with the scopolamine-treated group (Fig. 4).

Fig. 4.  Effect of SCBE on hippocampal BDNF expression in scopolamine-treated mice.

Groups: saline (Normal); scopolamine (2 mg/kg, i.p.; Control); donepezil (5 mg/kg, p.o.) + scopolamine; and SCBE (50, 100, or 500 mg/kg, p.o.) + scopolamine. (A) Representative Western blots of pro-BDNF (～32 kDa), BDNF (～14 kDa), and β-actin. (B) Quantification normalized to β-actin. Values are mean ± SD (n = 3). Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters (a-e) indicate significant differences among groups at p < 0.05, whereas groups sharing the same letter are not significantly different.

CREB phosphorylation, expressed as the p-CREB/CREB ratio, was significantly reduced by scopolamine (59.08 ± 4.41% of the normal control). SCBE treatment increased this ratio in a dose-dependent manner to 63.40 ± 1.76%, 72.77 ± 6.64%, and 79.87 ± 4.65% at 50, 100, and 500 mg/kg, respectively. Although the p-CREB/CREB ratio in the SCBE groups remained lower than that in the donepezil group (97.29 ± 6.47%), all SCBE-treated groups showed significantly higher values compared with the scopolamine-treated group (Fig. 5).

Fig. 5.  Effect of SCBE on hippocampal CREB phosphorylation in scopolamine-treated mice.

Groups: saline (Normal); scopolamine (2 mg/kg, i.p.; Control); donepezil (5 mg/kg, p.o.) + scopolamine; and SCBE (50, 100, or 500 mg/kg, p.o.) + scopolamine. (A) Representative Western blots of p-CREB (～43 kDa), CREB (～43 kDa), and β-actin. (B) p-CREB/CREB ratio. Values are mean ± SD (n = 3). Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters (a-f) indicate significant differences among groups at p < 0.05, whereas groups sharing the same letter are not significantly different.

Cresyl violet staining was performed to evaluate neuronal morphology and staining intensity in the CA1, CA3, and DG regions of the hippocampus. Compared with the normal group, the scopolamine-treated group showed reduced staining intensity and partial disruption of neuronal layer organization, without clear evidence of overt neuronal loss or degeneration under the present experimental conditions (Fig. 6).

Fig. 6.  Effects of SCBE on hippocampal neuronal morphology and staining intensity in scopolamine-treated mice.

Groups: saline (Normal); scopolamine (2 mg/kg, i.p.; Control); donepezil (5 mg/kg, p.o.) + scopolamine; and SCBE (50, 100, or 500 mg/kg, p.o.) + scopolamine. (A) Representative cresyl violet-stained coronal hippocampal sections (-1.70 to -2.30 mm from bregma) showing CA1, CA3, and DG. Scale bar = 100 μm. (B-D) Staining intensity in CA1 (B), CA3 (C), and DG (D), expressed as % of the normal group (mean ± SD, n = 3). Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters (a-c) indicate significant differences among groups at p < 0.05, whereas groups sharing the same letter are not significantly different.

Quantitative analysis showed that scopolamine significantly decreased staining intensity to 87.26 ± 3.73% in CA1, 81.60 ± 8.75% in CA3, and 87.31 ± 0.81% in DG, relative to the normal group (set at 100%). SCBE treatment modulated these histological alterations in a region-dependent manner. In CA1, staining intensity remained comparable to that of the normal group (SCBE50: 99.63 ± 0.71%; SCBE100: 101.23 ± 3.25%; SCBE500: 104.22 ± 5.82%) (Fig. 6A).

In CA3, SCBE partially restored staining intensity (SCBE50: 90.24 ± 4.11%; SCBE100: 91.84 ± 6.09%; SCBE500: 105.74 ± 5.38%), although the lower doses remained below or near the normal level (Fig. 6B).

In DG, SCBE increased staining intensity above the scopolamine-treated control level (SCBE50: 110.30 ± 4.30%; SCBE100: 112.86 ± 6.09%; SCBE500: 115.32 ± 3.72%), showing a pattern similar to that of the donepezil-treated group (116.12 ± 4.19%) (Fig. 6C).

At the highest dose (500 mg/kg), SCBE exhibited staining patterns and neuronal layer organization comparable to those of the donepezil-treated group across all hippocampal regions.