摘要
【目的】
制定大豆异黄酮苷元制备工艺及建立质量控制方法;从细胞水平研究其对过氧化氢和β-淀粉样蛋白诱导的PC12细胞氧化损伤和神经毒性的保护作用;利用阿尔茨海默病模型小鼠(AD小鼠),从体内实验研究其对AD小鼠学习记忆能力、抗氧化能力、神经递质水平、Aβ神经毒性和海马组织损伤的影响,探讨大豆异黄酮苷元抗阿尔茨海默病(AD)的作用及机理,为开发治疗AD的新药提供依据。
【方法】
(1)从豆粕中提取总大豆异黄酮;用酸水解法将大豆异黄酮苷转化成苷元,并纯化精制;建立高效液相色谱方法测定异黄酮苷元的含量的方法。
(2)建立过氧化氢和Aβ25-35诱导的PC12细胞损伤模型。显微镜观察细胞形态变化,并利用MTT法检测细胞存活率及LDH法检测细胞乳酸脱氢酶活力,研究大豆异黄酮苷元对PC12细胞的氧化损伤和Aβ神经毒性损伤的保护作用。
(3)对AD模型小鼠用不同剂量的异黄酮苷元治疗,通过Y-迷宫实验和避暗试验观察AD小鼠学习记忆能力的改善;通过测定血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性、丙二醛(MDA)水平,研究AD小鼠抗氧化能力的改变;③通过测定乙酰胆碱酯酶活性,研究AD小鼠神经递质水平的变化;④通过测定β-分泌酶活力,研究AD小鼠抗Aβ神经毒性的作用;⑤通过脑组织切片,HE染色,观察对AD小鼠海马组织的保护情况。
【结果】
(1)本实验确定了一套大豆异黄酮苷元的制备工艺及质量控制方法,异黄酮苷元总含量为70.98%。其中3种苷元含量分别为:染料木素46.59%、大豆苷元21.215%、黄豆黄素3.175%。
(2)用终浓度为150umol/L的H2O2和20umol/Aβ25-35处理正常培养的PC12细胞显微镜下观察,发现细胞受损较严重、细胞形态异常、数量显著减少,PC12细胞存活率为74.10%,与正常对照组相比明显降低(P<0.05),培养液中LDH漏出率明显增大(P<0.01);异黄酮苷元保护组(5ug/ml、10ug/ml、20ug/ml)细胞贴壁良好,形态正常,活细胞较多,接近正常对照组,培养液中LDH漏出率比损伤组明显减小(P<0.05)。
(3)对AD模型小鼠用异黄酮苷元治疗后,与模型组相比,用异黄酮苷元组的学习记忆能力提高(P<0.05);血清中SOD、GSH-Px活力增加(P<0.05);MDA含量下降(P<0.05);AchE活力下降(P<0.05)、β-分泌酶活力活力下降(P<0.05)。
【结论】
(1)本实验中提取制备工艺可获得高纯度的大豆异黄酮苷元,工艺稳定可控,适合规模生产;
(2)异黄酮苷元可显著改善H2O2和Aβ25-35诱导引起的PC12细胞形态改变,减轻细胞膜的损伤,提高神经细胞存活率,有较好的神经保护作用;
(3)异黄酮苷元能改善AD模型小鼠学习记忆能力,增强抗氧化能力,降低AchE活力以保护神经递质、减轻Aβ神经毒,有良好的抗AD活性。
【Objective】
To formulate the technology of Soybean Isoflavone Aglycones , and establish the method for the content determination of isoflavone aglycone. Study its protection of PC12 cell against oxidative damage induced by H2O2 and neurotoxicity by Aβon cell level. In order to study mechanism that Soybean Isoflavone Aglycones anti- Alzheimer’s disease (AD) and providing proofs for developing new clinical anti-AD medicine ,we observe the effect of the Aglycones on AD model mice,s learning and memory, effect on levating serum anti—oxidative , effect on adjusting cholinergic transmitter in cortexs tissue , relievimg neurotoxicity, damage In hippocamps of the mouse were be obversed.
【Methods】
(1)T o extracte Soybean Isoflavones from defatted Soybean, then use acid hydrolysis to transformat glucoside of Soybean Isoflavones into Aglycones ,refine Aglycones . Establish an HPLC method for the content determination of isoflavone aglycones.
(2)We built PC12 cell impaired model induced by H2O2 and Aβ.To study Soybean Isoflavone Activity Aglycones protection of PC12 cell against oxidative damage induced by H2O2 and neurotoxicity by Aβ, We Observe under the microscope , use MTT assay to evaluate the survival rate and LDH to check the LDH activity.
(3)After treating with Soybean Isoflavone Aglycones, Y-maze and step experiments were used to observe the effect of on Alzheimer’s disease model mice,s learning and memory.To study the effect on serum anti—oxidative, we determinate SOD,GSH-P activity, MDA content. To study the effect on adjustting cholinergic transmitter in cortexs tissue we determinate AchE activity. To study the effect on relievimg neurotoxicity, we determinateβ-Secretase Activity. damage In hippocamps of the mouse were be obversed too to study protection of the Aglycones.
【Results】
(1)We get the preparing techniques of Soybean Isoflavone Aglycones and establish the method for the content determination.The contents of three Activity Aglycone are 70.98%, Genistein、Daidzein and Glycitein were 46.59%, 21.215 % and 3.175%.
(2)After being treated byH2O2 (150umol/L) and Aβ25-35(20umol/L) ,PC12 cell is strongly damaged . The PC12 cell form is abnormaland the quantity of cell is notably decreased.Compare to the normal control ,the the survival rate obviously reduces(P <0.05) and the LDH activity rise.The form of PC12 cells, which were protected by Soybean Isoflavone Activity Aglycone Component ,is normal ; the the survival rate of cell is near normal control and the LDH activity is obviously lower than damaged group (P <0.05).
(3)The memory of Alzheimer’s disease model mice was decreased significantly,and was improved by Soybean Isoflavone Aglycones treatment.Meanwhile,serum SOD activity, GSH-Px activity, AchE activity in cortexs tissue andβ-Secretase Activity in hippocampus were increased(P <0.05) by Soybean Isoflavone Aglycones treatment,whereas the contents of serum MDA were decreased(P <0.05) .
【Conclution】
(1)The preparing technique of Soybean Isoflavone Aglycones from defatted Soybean is stable and can be controlled.
(2) Our studies show that Soybean Isoflavone Aglycones can obviously improve the abnormal form of PC12 cell induced by H2O2 and Aβand show a good protection of PC12 cell against oxidative damage induced by H2O2 and neurotoxicity by Aβ.
(3)Soy isoflavones can improve learning and memory of Alzheimer’s disease model mice, elevate the level of serum anti—oxidative, adjust cholinergic transmitter in cortexs tissue , relieve neurotoxicity in central nerve system.
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