*2.3. Spontaneous Alteration Test*

A spontaneous alternation test was conducted in accordance with our previous report [7]. The Y-maze is a three-arm maze with equal angles between all arms (25 cm long × 5 cm wide × 20 cm high). The maze walls were constructed from dark black, polyvinyl plastic. Each mouse was initially placed in one arm, and the sequence and number of arm entries were counted for 8 minutes. The alternation score (%) for each mouse was defined as the ratio of the actual number of alternations to the possible number (defined

as the total number of arm entries minus two) multiplied by 100 as follows: % Alternation = ((Number of alternations) / (Total arm entries − 2)) × 100.

To evaluate the effects of the samples on scopolamine-induced memory impairment, 6-week-old Crl:CD1 male mice were orally administered distilled water with or without peptides to be tested 1 h before evaluation. Forty minutes after the oral administration, memory impairment was induced by the intraperitoneal administration of 0.85 mg/kg scopolamine dissolved in saline. One hour after the oral administration, mice were subjected to the Y-maze test. In some experiments, mice were administered 0.85 mg/kg scopolamine and 0.05 mg/kg SCH-23390 (dopamine D1 antagonist) intraperitoneally at 40 min after the oral administration of 0 or 1 mg/kg WY peptide and subjected to the Y-maze test at 1 h after the oral administration. There were 10 mice per group.

In other experiments, C57BL/6J mice were orally administered a daily dose of 0 or 1 mg/kg WY peptide for 14 days. One hour after the last administration, mice were subjected to a Y-maze trial.
