Male C57BL/6 mice (10 weeks, 25–27 g) were purchased from NARA biotech (Seoul, South Korea) and housed in a regulated environment (temperature, 21 ± 2 °C; 12-h light/dark cycle; light period starting at 8 AM) with free access to food and water. All experimental studies were submitted to and approved by Konkuk University’s Council Directive for the Care and Use of Laboratory Animals (KU11025). To test the effect of Rg3GE on cognitive function, the mice were randomly assigned to five treatment groups (
n = 10 per group, Fig.
1c): 1) non-induction + vehicle (deionized water), 2) vehicle + scopolamine (1 mg/kg/day, 100 μl/day), 3) Rg3GE (50 mg/kg/day, 150 μl/day) + scopolamine (1 mg/kg/day, 100 μl/day), 4) Rg3GE (100 mg/kg/day, 150 μl/day) + scopolamine (1 mg/kg/day, 100 μl/day), and 5) RGE (100 mg/kg/day, 150 μl/day) + scopolamine (1 mg/kg/day, 100 μl/day). Group 5 was the positive control. Rg3GE and RGE were suspended in deionized water and administered by oral gavage (p.o.) on days 1–8, prior to training for the Morris water maze test, and continued on days 9–14, during which the mice underwent the Morris water maze test. Scopolamine was dissolved in deionized water and administered by intraperitoneal (i.p.) injection on days 9–14. On days 9–14, Rg3GE and RGE were administered 60 min before the trial, and scopolamine was injected 30 min before the trial. Since scopolamine causes temporary cognitive deficit [
16,
17], animal models for amnesia using scopolamine have been developed by injecting scopolamine 30 to 60 min before measuring memory deficit. To investigate the anti-amnesic activity of drugs, the protocol which applies test drugs before the scopolamine injection is being used [
11,
18‐
26]. Mouse weights were measured during the treatments with Rg3GE, RGE, and scopolamine on days 1–14. On day 14, the mice were decapitated immediately following the Morris water maze test. The hippocampus was removed, dissected on ice, and frozen at −80 °C until analysis. Four or three mice hippocampi were randomly selected for acetylcholinesterase activity or western blot analysis, respectively. They were homogenized using Bullet Blender (Next Advance, Averill Park, NY, USA) in ice-cold Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL, USA) containing Phosphatase Inhibitor Cocktail (Sigma-Aldrich), 0.1 mM phenylmethanesulfonylfluoride, and 10 μg/ml leupetin. Lysates were centrifuged at 19,000 × g for 15 min, and the protein concentrations of the supernatants were determined using the Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). The experimental design is summarized in Fig.
1b.