Jeong Ji Hee, Kim Hyeon Ju, Park Seon Kyeong, Jin Dong Eun, Kwon O-Jun, Kim Hyun-Jin, Heo Ho Jin
Division of Applied Life Science, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, South Korea.
BMC Complement Altern Med. 2014 Dec 13;14:482. doi: 10.1186/1472-6882-14-482.
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.
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.
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.
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.
研究黑豆种皮提取物中的非花青素组分（NAF）对Aβ诱导的氧化应激的生理影响，以确认其神经保护作用。此外，我们还研究了NAF对脑室内注射Aβ诱导的认知缺陷的预防作用。
使用2',7'-二氯荧光素二乙酸酯（DCF-DA）测量细胞氧化应激水平。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和乳酸脱氢酶（LDH）测定法研究神经元细胞活力。为了通过Y迷宫和被动回避试验研究NAF的体内抗遗忘作用，用Aβ诱导小鼠的学习和记忆障碍。在体内试验后，测定小鼠脑中乙酰胆碱酯酶（AChE）活性和丙二醛（MDA）水平，以确认认知效果。使用超高效液相色谱（UPLC）精确质量四极杆飞行时间（Q-TOF）UPLC/MS对NAF中的单个酚类进行定性分析。
NAF对氧化应激诱导的细胞毒性具有细胞保护作用。与仅用Aβ1-40处理的细胞相比，通过Aβ1-40处理积累的细胞内活性氧显著减少。在MTT和LDH测定中，NAF对Aβ1-40处理的细胞毒性也具有神经保护作用。最后，在体内行为试验中，给小鼠施用这种NAF可显著逆转Aβ1-40诱导的认知缺陷。行为试验后，收集小鼠大脑以检查脂质过氧化和AChE活性。NAF以剂量依赖性方式抑制AChE活性。用NAF处理的小鼠脑匀浆中MDA的生成减少。Q-TOF UPLC/MS分析从非花青素组分中鉴定出三种主要酚类；表儿茶素、原花青素B1和原花青素B2。
结果表明，黑豆种皮提取物中的NAF可能通过降低氧化应激来改善Aβ对PC12细胞的细胞毒性，并且对Aβ引起的体内学习和记忆缺陷具有抗遗忘作用。Q-TOF UPLC/MS分析显示三种主要酚类；（-）-表儿茶素、原花青素B1和原花青素B2。上述结果表明，（-）-表儿茶素是主要成分，也是黑豆种皮NAF抗遗忘作用的贡献者。
