*2.2. Animals*

The experimental protocol and process were admitted by the Animal Care and Use Committee (Permission number: 2019-05-003) of ChemOn Inc. (YoungIn, Korea). Eightweek-old male C57BL/6N mice were offered (Orient Bio, Seongnam, Korea) and housed under regulated conditions (temperature: 23 ± 3 ◦C, 12 h light/dark cycle, 55 ± 15% humidity, lighting intensity 150–300 Lux). They freely utilized food and drinking water and had an acclimation period for a week before the experiment started.

### *2.3. Design of Experiment*

The experimental mice were randomized into 5 groups after 1-week adaptation (*n* = 8 per group): Normal group, SCO-treated group, SCO with Brainon (30 or 100 mg/kg/day). Ginkgo Biloba Extract (GBE, 50 mg/kg/day), which was found to have an improving effect on memory deficit [13,14], was used as a positive control agent. 0.5% CMC was used to dissolve Brainon and GBE and administered (10 mL/kg/day) for 28 days via gastric gavage. The passive avoidance test was performed for 3 days (days 15–17), whereas the MWM task was performed for 7 days (days 22–28). Memory deficiency was caused by SCO injection (1 mg/kg, i.p.) within 30 min following the oral administration of Brainon and GBE extract. Behavior test was conducted for 30 min after the SCO injection.

### *2.4. Step-Through Passive Avoidance Performance*

The device (Twin County Med Associates, Hudson, NY, USA) for the passive avoidance test consisted of two identical compartments, separated into light and dark areas with an automated door and electric floor. Experiments were conducted at the same time every day for 3 days. The mice were placed in the dark compartments for 120 s before being moved back to the lit compartments. If the mice moved to a dark area, they were immediately transferred back to the lit compartment (day 15). During the acquisition trial (day 16), mice could move freely without lighting for 60 s for familiarization. After adaptation training, they were permitted to enter the two compartments and move freely for 120 s and, when moved to the dark area, a 0.20 mA scrambled shock was treated. Animals that did not move were removed from the tests. On the retention trial (day 17), the mice were located in the light section and the automated board was opened, and the transfer time to the dark section was analyzed.

### *2.5. MWM Trial*

To confirm the long-term spatial memory ability of the mice, an MWM test was conducted from days 22 to 28. The MWM tasks consisted of a training session for 2 days (days 22 and 23), a behavioral session for the following 5 days (days 24–27), and a probe trial session for 1 day (day 28). Mice were allowed 60 s to find the hidden platform located at one of the water pool's release points. If the mice could not locate the platform, they were manually located on the platform for 30 s. All mice were tested in two trials per day and the platform location in the water pool was randomly altered between trials. Learning and memory capabilities were confirmed by measuring the escape latency. The platform was eliminated from the pool and probe trials were performed to confirm the spatial memory ability by checking the platform crossing number of the mice for 60 s at day 28. Figure 1 is a schematic of the experimental design.

**Figure 1.** Experiment process of scopolamine-treated memory deficiency in mice.

### *2.6. AChE Activity and Contents of ACh*

The hippocampi were homogenized with cold phosphate-buffered saline and centrifuged (1500× *g*) for 15 min at 4 ◦C. Centrifuged supernatant was gathered to confirm AChE activity and ACh levels utilizing commercial kits (Abcam, Cambridge, UK), in accordance with the manufacturer's manuals.

### *2.7. Preparation of Total RNA and Quantitative RT-PCR*

Mice hippocampi were pulverized, and the total RNA was isolated utilizing a RNeasy Mini kit following the manufacturer's manuals (Qiagen GmbH, Hilden, Germany). cDNA synthesis was carried out utilizing a CellScriptTM All-in-One cDNA Master Mix (Cell-Safe, Yongin, Korea) at 25 ◦C (5 min) and 42 ◦C (15 min). qPCR was conducted using a QuantStudio 3 Real-time PCR device (Thermo Fisher Scientific Inc., Waltham, MA, USA). The synthesized primers for PCR are shown in Table 1. Conditions of cycling were 95 ◦C (10 min), followed by 50 cycles of 95 ◦C (15 s) and 72 ◦C (60 s). The target gene levels were

determined by the comparative Ct method utilizing LightCycler 96 Software 1.1 (Roche Diagnostics, Basel, Switzerland).


**Table 1.** Real-time PCR primer sequences.

### *2.8. Extraction of Protein and Western Blot Analysis*

The hippocampi were homogenized with RIPA reagen<sup>t</sup> (DYNE Bio, Seongnam, Korea) and the homogenates were centrifuged (10,000× *g*) for 15 min at 4 ◦C. Supernatant of hippocampal tissue was harvested, and protein concentrations were quantified utilizing a commercially available BCA protein assay kit (Thermo, Waltham, MA, USA). 30 μg of total protein was electrophoresed on SDS-PAGE and moved onto membranes (Millipore Corp., Bedford, MA, USA). For blocking, the membranes were kept for 1 h at 23 ◦C then incubated with the first antibodies for SOD-1, SOD-2, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax, Bcl-2, cleaved PARP, cleaved caspase-9, phosphor-CREB, CREB (Cell Signaling Technology, Inc., Danvers, MA, USA), and BDNF (Abcam, Cambridge, UK) at a 1:1000 dilution at 23 ◦C for 1 h. The incubated membranes were washed and further reacted with second antibodies (1:1000; GenDEPOT, Barker, TX, USA) at 23 ◦C for 1 h. The membranes were processed for detection using ECL solution (Atto, Tokyo, Japan) and the band intensity was measured utilizing Image-Pro Plus (Media Cybernetics, Inc., Rockville, MD, USA).

### *2.9. Statistical Analysis*

Statistical analysis between groups was conducted using Student's *t*-test and one-way analysis of variance followed by multiple comparisons Dunnett's post-hoc test utilizing Origin 7.0 (OriginLab, Northhampton, MA, USA). Results are expressed as the means ± standard error of the mean. Significant differences between groups were considered statistically significant at *p* < 0.05 and *p* < 0.01.
