An investigation into the ameliorating effect of black soybean extract on learning and memory impairment with assessment of neuroprotective effects

BACKGROUND The physiological effects of the non-anthocyanin fraction (NAF) in a black soybean seed coat extract on Aβ-induced oxidative stress were investigated to confirm neuroprotection. In addition, we examined the preventive effect of NAF on cognitive defects induced by the intracerebroventricular (ICV) injection of Aβ. METHODS Levels of cellular oxidative stress were measured using 2',7'-dichlorofluorescein diacetate (DCF-DA). Neuronal cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. To investigate in vivo anti-amnesic effects of NAF by using Y-maze and passive avoidance tests, the learning and memory impairment in mice was induced by Aβ. After in vivo assays, acetylcholinesterase (AChE) activity and level of malondialdehyde (MDA) in the mouse brain were determined to confirm the cognitive effect. Individual phenolics of NAF were qualitatively analyzed by using an ultra-performance liquid chromatography (UPLC) Accurate-Mass Quadrupole Time of-Flight (Q-TOF) UPLC/MS. RESULTS A NAF showed cell protective effects against oxidative stress-induced cytotoxicity. Intracellular ROS accumulated through Aβ1-40 treatment was significantly reduced in comparison to cells only treated with Aβ1-40. In MTT and LDH assay, the NAF also presented neuroprotective effects on Aβ1-40-treated cytotoxicity. Finally, the administration of this NAF in mice significantly reversed the Aβ1-40-induced cognitive defects in in vivo behavioral tests. After behavioral tests, the mice brains were collected in order to examine lipid peroxidation and AChE activity. AChE, preparation was inhibited by NAF in a dose-dependent manner. MDA generation in the brain homogenate of mice treated with the NAF was decreased. Q-TOF UPLC/MS analyses revealed three major phenolics from the non-anthocyanin fraction; epicatechin, procyanidin B1, and procyanidin B2. CONCLUSIONS The results suggest that the NAF in black soybean seed coat extracts may improve the cytotoxicity of Aβ in PC12 cells, possibly by reducing oxidative stress, and also have an anti-amnesic effect on the in vivo learning and memory deficits caused by Aβ. Q-TOF UPLC/MS analyses showed three major phenolics; (-)-epicatechin, procyanidin B1, and procyanidin B2. Above results suggest that (-)-epicatechins are the major components, and contributors to the anti-amnesic effect of the NAF from black soybean seed coat.