PFE was collected from Miryang, from August to September of 2008. Specimens were freeze-dried (SFDSM06, Samwon Co., Pusan, Korea) and extracted three times with 20 volumes of 100% methanol at room temperature for 24 h. The extract was then obtained using a rotary evaporator and the yield was 23.43%. RA, one of the major constituents of PFE [13], was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The methanol extracts from PFE and RA were dissolved in phosphate-buffered saline and dimethylsulfoxide (DMSO), respectively.

Aβ_25-35 and malondialdehyde (MDA) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). DMSO and NaCl were purchased from Bio Basics Inc. (Ontario, Canada). Thiobarbituric acid (TBA) was obtained from Lancaster Synthesis (Ward Hill, MA, USA). Phosphoric acid and butanol were acquired from Samchun Pure Chemical Company (Gyeonggi, Pyeongtaek, Korea).

Male 5-week-old ICR mice (Orient Inc., Seongnam, Korea) weighing 25-27 g were housed in plastic cages with free access to food and water and maintained in a controlled environment (20 ± 2℃, 50 ± 10% relative humidity, 12-h light/dark cycle). Mice were divided into four groups comprising eight individuals in each of four cages. The groups were defined as follows: normal group, which received 0.9% NaCl injection plus oral administration of water; control group, which received Aβ_25-35 injection plus oral administration of water; PFE group, which received Aβ_25-35 injection plus oral administration of PFE (50 mg/kg/day); and RA group, which received Aβ_25-35 injection plus oral administration of RA (0.25 mg/kg/day) for 14 days using a sonde. There were no significant differences in initial body weight among groups. Animal experiments were conducted according to the Guidelines for Care and Use of Laboratory Animals published by the Pusan National University Institutional Animal Care and Use Committee (IACUC, approval number PNU-2009-0042).

Aβ_25-35 was aggregated according to the procedure outlined by Maurice et al. [14]. In brief, the peptide was dissolved and diluted in 0.9% NaCl to achieve a concentration of 5 nmol. Aβ_25-35 was incubated at 37℃ for 3 days before injection to induce aggregation. Aggregated Aβ_25-35 was injected into mice according to the procedure established by Laursen and Belknap [15]. Mice were lightly anesthetized with CO₂, after which the solution was injected 0.8 mm posterior to the bregma and 1.5 mm lateral to the sagittal suture. All injections were made using a 10 µl Hamilton microsyringe fitted with a 26 gauge needle that was inserted 2.2 mm beneath the surface of the brain. Animals were injected with 5 µl of 0.9% NaCl or 5 nmol Aβ_25-35 aggregate in each cerebral lateral ventricle at a rate of 1 µl/min and the needle was left in the injection site for 1 min. The behavioral experimental schedule of mice injected with Aβ_25-35 is shown in Fig. 1.

The T-maze test was conducted according to the procedure established by Montgomery [16]. The maze apparatus was T-shaped, and the walls were made of black boards (length of start and goal stems = 50 cm, width = 13 cm, height = 20 cm) that were glued to a square black board bottom. The maze consisted of a start box, left arm, and right arm with a door to separate the two sides. The mice were placed at the start box, and the number of touches and exploration times of the right arm of the T-maze were recorded during a 10-min period (training session). The mice were then placed back into the same apparatus 24 h after the training session. Animals were allowed to explore the right and left sides of the maze freely for 10 min, and the number of touches and exploration times were recorded (test session). Space perceptive ability (percent) was calculated as the ratio of the number of left or right maze entries to the number of total maze entries multiplied by 100.

The object recognition test was performed in a square black open-field apparatus (40 × 30 × 20 cm) [17]. Two identical objects (plastic bottles) were placed at fixed distances within the square field. The mice were then placed at the center of the square field, and the number of touches of each object was recorded during a 10 min period (training session). Next, the mice were placed back into the same field 24 h after the training session, but one of the objects used during the training session was replaced with a new object (another plastic bottle). The mice were allowed to explore freely for 10 min, and the number of touches was recorded (test session). Object cognitive ability (percent), a ratio of the amount of time spent exploring any one of the two original objects (training session) or the novel object (test session) over the total time spent exploring both objects was used to measure cognitive function.

The Morris water maze test was conducted according to the procedure established by Morris, with slight modifications [18]. The apparatus used in the Morris water maze test consisted of a dark plastic circular pool, 80 cm in diameter, surrounded by a 40-cm-high wall randomly divided into quadrants. White poster color was added to the pool water to make it opaque, and the water temperature was maintained at 22 ± 1℃. A platform 8 cm in diameter was placed 1 cm below the water surface in the middle of one quadrant. The position of the platform was unchanged during the training session. Four posters on the walls of the apparatus provided visual cues for navigation. Three training trials per day were conducted for 3 days. During the training trials, mice were placed at random in the water facing the pool wall and allowed to swim for a maximum of 60 s. The latency time required to find the platform was recorded. Mice that found the platform were allowed to rest there for 15 s. If they failed to find the platform within 60 s, they were allowed to stay on the platform for 15 s to help them remember. A probe trial of the Morris water maze test was performed 1 day after the completion of training. In the primary test, the experiment was performed as before. In the secondary test, the trial was performed without the platform. The mice were placed in the pool and swam for 60 s while looking for the platform, and the latency time that the mice spent in the position previously occupied by the platform was recorded. In the tertiary test, the water was transparent and the number of trials that it took the mouse to reach the platform, which was visible 1 cm above the surface of the water, was counted. Occupancy of the target quadrant was calculated as the percentage of time spent in the target quadrant during a 60 s trial.

MDA levels were measured by the method described by Ohkawa et al. [19]. After completion of the behavioral observations, mice were anesthetized with CO₂ and their brain, liver, and kidneys were removed immediately and placed on ice. The dissected tissue was then homogenized with saline solution and mixed with 1% phosphoric acid and 0.67% TBA solution. Following boiling for 45 min, the solution mixture was cooled in an ice bath, after which 2 ml of butanol was added and the samples were centrifuged at 3,000 rpm for 10 min. The absorbance values of the supernatant were measured at 535 and 520 nm and the level of lipid peroxidation was calculated using a MDA standard curve.

The nitric oxide (NO) concentration in tissues was determined as described by Schmidt et al. [20]. Briefly, 150 µl of supernatant from the lipid peroxidation procedure was mixed with 130 µl of distilled water, after which 20 µl of the dilution was added to the same amount of phosphoric acid and 0.1% n-(1-naphthyl) ethylenediamine dihydrochloride. The absorbance value of the mixture was measured at 540 nm and the NO yield was calculated using a sodium nitrite (NaNO₂) standard curve.

Results are expressed as the mean ± SD. Statistical significance was determined by one way ANOVA followed by Duncan's post hoc tests. Significance was set at P < 0.05.

The effects of PFE and RA on short-term memory were investigated using a T-maze test and measured by the number of entries into the different arms (Fig. 2). The results revealed that mice in the normal group showed a significant preference for the new arm, whereas those in the Aβ_25-35-treated control group did not show differences in the number of arm entries between old and new arms. However, mice in the PFE and RA groups displayed a significantly higher number of entries in the new arm. These results suggested that PFE and RA improve spatial learning memory against Aβ_25-35.

The effects of PFE and RA on Aβ_25-35-induced memory impairment were investigated using a novel object recognition test (Fig. 3). The same two objects were explored during the training session. After 24 h, one of the familiar objects was replaced with a novel object. The control group showed no significant difference between recognition of the familiar and novel objects. However, groups administered PFE and RA touched the novel object more times than the familiar object, and mice treated with PFE (50 mg/kg/day) had the highest novel object cognition ability. According to our results, mice orally administered PFE and RA showed higher recognition ability toward novel objects in the Aβ_25-35-induced cognitive impairment model.

To investigate whether PFE and RA improved the memory deficit induced by Aβ_25-35, the Morris water maze test was conducted. As shown in Fig. 4, all groups showed decreased time to reach the platform during the training session. After 3 days of training, the PFE and RA groups showed significantly decreased time to reach the platform relative to that of the Aβ_25-35-injected control group. On the final day, the hidden platform was removed and the time spent in the target quadrant was measured. The Aβ_25-35-injected control group showed low occupancy of the target quadrant compared with the normal group. However, the PFE and RA groups increased their occupancy of the target quadrant (46.1% and 51.7%, respectively), as shown in Fig. 5. Conversely, in the visible platform test, no significant differences in latency to reach the platform were observed among groups, indicating that visual and exercise ability did not affect the cognitive and memory function in the Aβ_25-35-induced AD model (Fig. 6).

Table 1 shows the inhibitory effects of PFE and RA against lipid peroxidation induced by Aβ_25-35. Our results indicated that injection of Aβ_25-35 significantly increased MDA levels in the brain, liver, and kidney. However, the PFE and RA administration groups showed reduced MDA levels in the kidney relative to the Aβ_25-35-induced control group. Furthermore, the liver MDA levels of the PFE group were noticeably lower than those of the control group. In particular, oral administration of PFE and RA markedly attenuated MDA levels in the brain by 21.07% and 51.04%, respectively, when compared with the Aβ_25-35-injected control group. These results indicate that PFE and RA inhibited MDA generation in the brain, liver, and kidney.

The NO scavenging effect of PFE and RA in the brain, liver, and kidney is shown in Table 2. When compared with the normal group, there was a significant increase in NO levels in the brains of Aβ_25-35-injected mice. However, PFE and RA administration resulted in decreased NO levels. Specifically, PFE at a dose of 50 mg/kg/day significantly decreased NO levels in the brain relative to the control group. Moreover, the NO levels in the livers decreased significantly in response to treatment with PFE and RA when compared with the Aβ_25-35-induced control group. These findings suggested that PFE and RA treatment attenuated the NO production induced by Aβ_25-35 in these tissues.