Black soybean extract protects against TMT-induced cognitive defects in mice

Abstract

To find a neuroactive compound with a potent inhibitory effect on acetylcholinesterase (AChE) and in vivo anti-amnesic activity from natural resources, we evaluated anthocyanins and nonanthocyanins from black soybean extract. Nonanthocyanins from black soybean extract were the most potent and dose-dependent AChE inhibitors. Intracellular reactive oxygen species accumulation resulting from H₂O₂ treatment was significantly decreased compared with cells treated with H₂O₂ only. Nonanthocyanins were also neuroprotective against H₂O₂ treated neurotoxicity by 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. Finally, nonanthocyanins from black soybean in the preadministration group attenuated trimethyltin (TMT)-induced memory injury in both in vivo tests. AChE, prepared from mice brain tissues, was inhibited by nonanthocyanins from black soybean in a dose-dependent manner. Malondialdehyde generation in the brain homogenates of mice treated with nonanthocyanins from black soybean was decreased. We concluded that nonanthocyanins from black soybean had an efficacious in vitro AChE inhibitory activity, and protected against H₂O₂-induced neurotoxicity. In addition, our findings suggest that nonanthocyanins from black soybean may improve the TMT-induced learning and memory deficit because of AChE inhibition of mice brain tissue. Consequently, these results demonstrate that the nonanthocyanins from black soybean could possess a wide range of beneficial activities for neurodegenerative disorders.

Trial registration: ClinicalTrials.gov NCT01651741.

Figures

FIG. 1.

Inhibitory effect of phenolics from black soybean against acetylcholinesterase (AChE). The AChE inhibition was expressed as a percentage of enzyme activity inhibited compared with the control value. Values represent the mean±SD (n=3). abcdefDifferent superscripts indicate significant difference among groups at P<.05.

FIG. 2.

Effect of nonanthocyanins from black soybean on reactive oxygen species production determined in the presence and absence of H2O2 in PC12 cell. Each value represents the mean±SD of triplicates. abcdefDifferent superscripts indicate significant difference among groups at P<.05.

FIG. 3.

Cell protective effect of nonanthocyanins from black soybean on H2O2-induced neurotoxicity in PC12 cell. (A) PC12 cells were pretreated for 48 h with various concentrations of nonanthocyanins. After 48 h, the cells were treated with 200 μM H2O2 for 3 h by MTT reduction assay. (B) Inhibition of lactate dehydrogenase release of the nonanthocyanins on H2O2-induced membrane injury in PC12 cell. Each value represents the mean±SD (n=3). abcdefDifferent superscripts indicate significant difference among groups at P<.05.

FIG. 4.

Effect of nonanthocyanins from black soybean on spontaneous alternation behavior in trimethyltin (TMT)-induced amnesic mice. The Y-maze test was performed 2 days after the TMT injection (2.5 mg/kg of body weight). Sample groups (N5, N10, and N20) were injected with TMT followed by feeding with the nonanthocyanins (5, 10, and 20 mg/kg of body weight, respectively). The spontaneous alternation behavior (A) and number of arm entries (B) were measured over 8 min. Results shown are mean±SD (n=8). abValues with the same letter are not significantly different (P<.05).

FIG. 5.

Effect of nonanthocyanins from black soybean on TMT-induced memory impairment mice. Passive avoidance test was performed to check the step-through latency for 300 s. Each behavioral test was conducted 3 days after TMT injection (2.5 mg/kg of body weight). Sample group (N5, N10, and N20) were injected with TMT followed by feeding with nonanthocyanin fraction from black soybean extract (5, 10, and 20 mg/kg of body weight, respectively). The results shown are mean±SD (n=8). abcdDifferent superscripts indicate significant difference among groups at P<.05.

FIG. 6.

Effect of nonanthocyanins from black soybean on AChE activity from TMT-induced mice brain homogenate. Sample group (N5, N10, and N20) were injected with TMT followed by feeding with nonanthocyanin fraction from black soybean (5, 10, and 20 mg/kg of body weight, respectively). The results shown are mean±SD (n=8). abcDifferent superscripts indicate significant difference among groups at P<.05.

FIG. 7.

Effect of nonanthocyanins from black soybean on lipid peroxidation in TMT-induced mice brain homogenate. Sample group (N5, N10, and N20) were injected with TMT followed by feeding with nonanthocyanin fraction from black soybean (5, 10, and 20 mg/kg of body weight). The results shown are mean±SD (n=8). abcDifferent superscripts indicate significant differences among groups at P<.05.

FIG. 8.

High-performance liquid chromatography chromatograms of standards (A) and the nonanthocyanins (B) at 280 nm. (A) Retention time 7.205 min, protocatechuic acid; 9.610 min, cathechin; 12.078 min, syringic acid. Color images available online at www.liebertpub.com/jmf