Red adzuki beans (500 g) harvested in Tokachi, Hokkaido, Japan, were immersed in distilled deionized (dd) water (2 L) at room temperature overnight, and then the mixture was filtrated through cotton. After discarding the water, a 40% ethanol solution (4 L) was added to the red beans and the mixture allowed to stand at room temperature for 36 hours. After filtration of the ethanol extract with cotton, it was concentrated in vacuo at 45℃ to yield the concentrate (22 g). The obtained ethanol adzuki bean extract was stored at −20℃ for further use.

The Aβ solution was prepared according to the method in a previous report [17]. Briefly, synthetic Aβ_1-42 (PEPTIDE INSTITUTE, Osaka, Japan) was dissolved in 0.1% ammonia solution at a final concentration of 250 µM and sonicated in ice-cold water for a total of 5 min (1 min × 5 times) to avoid pre-aggregation. For preparation of the Aβ solution, aliquots of Aβ were diluted to 25 µM in 50 mM phosphate buffer (pH 7.5) and 100 mM NaCl.

The thioflavin T (ThT) fluorescence assay was performed as previously described [18]. Aβ solution (8 µL) was mixed with the adzuki extract and the mixture then added to 1.6 mL of ThT solution containing 5 µM ThT and 50 mM NaOH- glycine-buffer (pH 8.5). The final concentration of the adzuki extract in the mixture ranged from 0 to 1 mg/mL. The samples were incubated at 37℃ and the fibrillogenesis rate was monitored by using ThT fluorescence assays. The samples ThT fluorescence levels were evaluated by using an FP-6200 spectrofluorometer (JASCO, Tokyo, Japan). The respective excitation and emission wavelengths were 446 nm and 490 nm.

Analysis of transmission electron microscopy (TEM) results was performed as previously described [18]. The Aβ solution was incubated with or without Adzuki at 37℃ for 24 h. The final concentration of the adzuki extract was ranged from 0 to 1 mg/mL. Aliquots (10 µL) from the samples were placed on 200 mesh copper grids (Nisshin EM., Tokyo, Japan) for 5 min, after which, excess fluid was removed, and the grids stained with 10 µL of TI blue (Nisshin EM) for 5 min. TI blue is an alternative to uranyl acetate for TEM staining. Again, the excess fluid was removed and the grids dried. The prepared samples were examined with a JEM-1400 microscope (JEOL, Tokyo, Japan) and an accelerating voltage of 80 kV.

A UAS/GAL4 system was utilized for inducing transgene expression. The elav-GAL4 driver (Bloomington Drosophila stock center, IN, USA) was used to express transgenes in the brain starting early during development. The UAS transgene was human Aβ₄₂ (a generous gift from Dr. Koichi Iijima, National Center for Geriatrics and Gerontology, Japan). Flies were kept at 25℃. Adzuki extract dissolved in ddH2O was added to the standard cornmeal-molasses medium (1 mg/mL) and the mixture incubated at 60℃ for 15 min. Crosses were performed with flies either on standard cornmeal-molasses medium (control) or on medium supplemented with adzuki extract.

Aβ₄₂ levels were determined using Wako High Sensitive Human β Amyloid (1–42) ELISA kits (Wako, Osaka, Japan) according to the manufacturer's instructions. In brief, 20 fly heads were homogenized in 50 µL of ice-cold 1× RIPA buffer. The homogenates were diluted 10-fold with 450 µL of sample diluent and then cleared by centrifugation at 15,000 r/min for 5 min at 4℃. The supernatant (200 µL) of each sample was added to the ELISA plate.

Fly climbing assays were performed as previously described [19]. Approximately 15 flies were placed in an empty 2.5 cm by 9.5 cm (diameter by height) plastic vial, gently tapped to the vial bottom, and videotaped for 18 s. The vial was separated into three areas; “top”, “middle”, and “bottom” by clear and readable lines with each are including a distance of 3 cm. The percentages of flies that climbed to the top, middle, and bottom of the test vial were calculated. There was a minimum of 6 separate climbing trials per condition.

Standard single-cycle olfactory conditioning was performed as previously described [20]. Briefly, approximately 100 flies were trained by exposing them to two odors (3-octanol or 4-methylcyclohexanol), one of which was paired with a series of aversive electrical shocks (65 V, 1.5 s pulses every 5 s) for 1 min. For testing, flies were placed at a choice point located between the two odors for 1.5 min. Learning and memory retention were quantified as a performance index calculated by subtracting the percentage of flies that chose the shock-paired odor from the percentage that chose the unpaired odor. A performance index (PI) was calculated so that a 50:50 distribution (no memory) yielded a PI of 0 while a 0:100 distribution away from the shock-paired odor yielded a PI of 100. Individual performance indices were the average of two experiments in which the shock-paired and unpaired odor presentations were alternated.

Lifespan assays were performed as described by Schriner et al. [21]. Approximately 10 flies were placed in a food vial (with or without adzuki extract) and fresh food was given every other day. Any flies that died due to food or that flew away during the study were censored from the longevity assay. Flies were checked every day during the light portion of the light:dark cycle and the total number of live flies was recorded in each vial each day. For each diet, at least 200 flies were assayed. Kaplan-Meier plots were derived, and the median survival time for each group was determined. Survival curves were compared by using a log-rank test.

Student's t-test was applied to determine the statistical significance of differences in the ThT assay and ELISA data. Multiple group comparisons were performed by performing one-way analysis of variance (ANOVA) followed by a Tukey-Kramer test to detect intergroup differences in the climbing and longevity assay data. All statistical analyses were performed using GraphPad Prism 5.0 Software (GraphPad Software, San Diego, CA, USA). Data are expressed as Means±standard error of the mean, with at least three repeats in each experimental group. Test results were considered significant at P < 0.05.

First, we conducted ThT assays to evaluate the effect of the adzuki bean extract on Aβ₄₂ aggregation. Setting the fluorescence of samples containing only Aβ₄₂ at 100%, the relative fluorescence of samples containing Aβ₄₂ and 1 mg/mL or 0.1 mg/mL of adzuki extract were both approximately 20% while the relative fluorescence of samples containing Aβ₄₂ and 0.01 mg/mL of adzuki extract was 75% (Fig. 1A). Next, the effect of adzuki extract on Aβ₄₂ aggregate formation was evaluated by TEM. While the fibrils formed by Aβ₄₂ alone were abundant, large, and broadly ribbon-like, the fibrils formed by Aβ₄₂ in the presence of adzuki extract (0.01–1 mg/mL) were less abundant and of a smaller diameter and length (Fig. 1B). These results indicate that the adzuki extract suppresses Aβ₄₂ aggregate formation in a concentration-dependent manner.

Aβ₄₂-overexpressing flies were used to evaluate in vivo the effect of the adzuki extract on the symptoms of Alzheimer's disease. As a first step, the Aβ₄₂ level in fly brain was determined by using ELISA. The Aβ₄₂ level was significantly lower in the group fed the regular medium containing adzuki extract than in the group fed only the regular medium (Fig. 2).

Next, to evaluate the effect of the adzuki extract on motor function in Aβ₄₂-overexpressing flies, we used climbing assays. In the fly group fed the regular medium, approximately 10% of 15-day-old flies, 40% of 25-day-old flies, and 70% of 30-day-old flies showed decreases in motor function (Fig. 3). However, in the group fed the regular medium containing adzuki extract, only 30% of flies showed a decrease in motor function at 30 and 35 days of age. These results indicated that the intake of adzuki extract delayed the decrease in motor function in Aβ₄₂ flies.

To evaluate the effect of the adzuki extract on cognitive function of flies, we used single-cycle olfactory conditioning assays. We observed a significant decrease in memory function and learning ability in Aβ₄₂-overexpressing flies older than 25 days fed the regular medium compared to control flies (carrying the Aβ₄₂ transgene but not expressing it) fed the regular medium; however, that decrease was suppressed in Aβ₄₂-overexpressing flies fed the regular medium containing adzuki extract (Fig. 4).

Lastly, to evaluate the effect of the adzuki extract on the lifespan of Aβ₄₂-overexpressing flies, we used survival assays. The survival rates in the two diet groups were calculated by applying the Kaplan-Meier method, and the difference was analyzed by using the log-rank test. The average life expectancy was calculated by summing the life expectancy (in days) of all individuals in a group and dividing it by the total number of individuals in that group. In the group of Aβ₄₂-overexpressing flies fed the regular medium, a rapid decrease in survival rate was observed between 30 and 40 days of age. The survival rates of that group and the group fed the regular medium containing adzuki extract were not significantly different until day 30 of the survival assays, but from day 30 to day 50 day, the decrease in survival was more gradual in the Aβ₄₂-overexpressing flies fed the regular medium containing adzuki extract than in those fed the regular medium only (Fig. 5). The average life expectancy was 32.8 days in the Aβ₄₂-overexpressing flies fed the regular medium and 45.4 days in the Aβ₄₂-overexpressing flies fed the regular medium containing adzuki extract. Thus, a greater than 10-day life expectancy increase was obtained by including adzuki extract in the diet.