CN117503812A - 千里光的萃取方法及其在预防和治疗神经退行性疾病和/或抗衰药品中的应用 - Google Patents
千里光的萃取方法及其在预防和治疗神经退行性疾病和/或抗衰药品中的应用 Download PDFInfo
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Abstract
本发明属于天然药物技术领域,具体涉及一种千里光的萃取方法及其在预防和治疗神经退行性疾病和/或抗衰药品中的应用。通过提取分离得到千里光萃取物,其中,乙酸乙酯萃取物可显著提高对神经损伤细胞的保护作用,明显延长野生型秀丽隐杆线虫的平均寿命,对秀丽隐杆线虫阿尔茨海默症和帕金森病病理模型具有显著的治疗作用,显著改善阿尔茨海默症模型小鼠的学习记忆障碍,提示具有预防和治疗神经退行性疾病和/或延缓衰老的潜力,且无毒副作用,能够用于制备防治神经退行性疾病和/或抗衰的药品。
Description
技术领域
本发明属于天然药物技术领域,具体涉及一种千里光的萃取方法及其在预防和治疗神经退行性疾病和/或抗衰药品中的应用。
背景技术
神经退行性疾病(NDDs)是中枢神经系统(CNS)或周围神经系统(PNS)神经元进行性丢失的一组神经系统疾病,影响着全球数百万人的生活。神经网络结构和功能的崩溃和神经元的丢失,由于其终末分化的本质而不能有效地自我更新,导致核心沟通回路的崩溃,最终导致记忆、认知、行为、感觉或运动功能受损。常见的主要有阿尔茨海默病(Alzheimer's disease,AD)、帕金森病(Parkinson'sdisease,PD)、亨廷顿病(Huntington's disease,HD)等多种类型。除了少数家族性神经退行性疾病有明确的致病基因外,大多数疾病的发病机制尚不清楚,因此缺乏有效的治疗手段。
AD是痴呆症的主要病因,并正迅速成为本世纪最昂贵、最致命和负担最重的疾病之一。现代医学研究表明,AD发病机制与氧化应激、胆碱能损伤、神经炎症等相关。关于AD发病机制科学家提出了几种假说,分别为Aβ级联假说、Tau蛋白假说、胆碱能假说和氨基酸假说等。Tau蛋白的病理变化与AD患者认知能力下降有较大的相关性。Tau蛋白的正常生理功能是与微管蛋白结合,以维持细胞骨架的稳定性。过度磷酸化的Tau蛋白与微管脱离后,可积累形成神经元纤维缠结,从而造成突触结构的可塑性损伤以及神经元的变性和凋亡,进而引发AD。现今获FDA批准上市的AD药物主要为胆碱酯酶抑制剂与NMDA受体阻断剂,靶点单一且副作用大。
PD是仅次于AD第二高发的神经退行性疾病,主要症状为震颤麻痹。帕金森病的发病特点为黑质致密部多巴胺能(DA)神经元的减少,以及多巴胺神经元中以α-突触蛋白(α-Syn)聚集为主的路易小体的形成。正常情况下α-Syn是一种由140种氨基酸组成的蛋白质,大量存在于突触前神经元中,在调控神经递质的释放中发挥重要作用。病理情况下神经元中的α-Syn异常聚集和积累,α-Syn具有高度的细胞毒性,能够导致黑质DA神经元的神经退化以及由纹状体的DA神经元减少从而产生一系列的临床症状。目前PD的主要治疗手段为药物治疗,其次为手术治疗,但是这两种治疗方法都只能改善症状,并不能根治疾病,也不能阻止疾病的发展。
千里光(Senecio scandens Buch-.Ham.)为菊科千里光属多年生草本植物,在我国西藏、陕西及南方多省份均有广泛分布,是一味传统中药材,临床上多用于急性炎性疾病的治疗。对千里光现代药理学研究表明,其主要含有鞣质、黄酮、酚酸、挥发油、生物碱及萜类等生物活性成分,并主要具有抗氧化、抗炎、抑菌、抗癌及抗病毒等多种药理学作用,其在神经损伤保护方面未有报道。
秀丽隐杆线虫(以下简称“线虫”)是研究分子遗传学、神经科学以及细胞生物学等科学的杰出模式动物,它是第一个基因组经过完全测序的动物,有超过70%的基因与人类相似或具有同源性。线虫因其生命周期短、神经系统结构简单、遗传信息清晰和易于获得大量个体模型等优点,常被用于衰老及神经退行性疾病的相关研究。
发明内容
本发明公开了一种千里光的萃取方法及其在预防和治疗神经退行性疾病和/或抗衰药品中的应用。
本发明提供的千里光的萃取方法步骤如下:
(1)取千里光饮片100g装进三颈烧瓶中,加入1L乙醇-水溶液(70%的乙醇),加热回流3次,趁热抽滤,合并滤液,减压浓缩,剩余100mL的醇提液后停止旋蒸;取出5mL冷冻干燥作为千里光醇萃取物;
加热回流3次的时间依次是2h、1.5h、1h。
(2)取出分液漏斗,检查气密性后开始萃取;向分液漏斗中加入剩余醇提液和100mL的石油醚,萃取三次收集上层溶液,将上层溶液旋干得到千里光石油醚萃取物;
(3)接着向下层溶液(加入石油醚提取后的下层溶液)中加入100mL二氯甲烷,萃取三次收集下层溶液,将下层溶液旋干得到千里光二氯甲烷萃取物;
(4)接着向上层溶液(加入二氯甲烷提取后的上层溶液)中加入100mL乙酸乙酯,萃取三次收集上层乙酸乙酯层,将上层溶液旋干得到千里光乙酸乙酯萃取物;
(5)接着向下层溶液(加入乙酸乙酯提取后的下层溶液)中加入100mL饱和正丁醇,萃取三次收集上层溶液,将上层溶液旋干得到千里光饱和正丁醇萃取物;
(6)保留下层水层(加入正丁醇提取后的下层水层),同样旋干得到千里光水层萃取物。
千里光萃取物,尤其是乙酸乙酯萃取物可显著提高对神经损伤细胞的保护作用,明显延长野生型线虫的寿命并对线虫AD病理模型Tau蛋白的异常磷酸化引起的运动障碍具有抑制作用、明显减少PD病理模型线虫的α-Syn荧光聚集,提示千里光乙酸乙酯萃取物具有预防和治疗神经退行性疾病及延缓衰老的潜力。
千里光萃取物,尤其是乙酸乙酯萃取物具有显著的抗AD作用,几乎无毒副作用,可制备成胶囊剂、片剂、颗粒剂、散剂、口服液、丸剂中任一种剂型,用于制备抗神经退行性疾病及延缓衰老的药物。
有益效果
本发明提供了活性成分为千里光萃取物的新应用。通过研究发现:该萃取物能够显著提高对神经损伤细胞的保护作用和明显抑制线虫AD病理模型Tau蛋白的异常磷酸化引起的运动障碍而防治神经退行性疾病,能够用于制备防治神经退行性疾病的药品或保健食品,为防治神经退行性疾病提供了新的途径。相比较千里光其他的提取物,乙酸乙酯萃取层具有较为明显的指向性,为后续的分离组分和人工合成提供便易。
附图说明:
图1千里光的提取流程图。
图2EESS不同浓度对PC12细胞毒性的影响。
图3H2O2不同浓度对PC12细胞毒性的影响。
图4千里光不同层对PC12细胞活性的影响。
图5EESS对N2线虫、AD病理模型及PD病理模型的影响。
图6EESS对AD小鼠学习及记忆功能的影响。
具体实施方式
本发明通过以下实施例进一步详细说明,但应注意本发明的范围并不受这些实施例的任何限制。
实施例1
(1)取100g千里光饮片装进三颈烧瓶中,加入1L 70%乙醇水溶液,然后放入电热套中80℃加热回流冷凝,等微沸后将温度控制在60℃左右,2h后收集第一批滤液,趁热抽滤;
(2)保留药渣并继续向三颈烧瓶中加入1L 70%乙醇-水溶液,回流冷凝1.5h后收集第二批滤液,趁热抽滤;
(3)保留药渣并继续向三颈烧瓶中加入1L 70%乙醇-水溶液,回流冷凝1h后收集第三批滤液,弃去药渣,趁热抽滤,合并三批滤液;
(4)使用旋转蒸发仪减压浓缩,剩余100mL的醇提液后停止旋蒸,取出5mL冷冻干燥作为千里光醇提取物(EtSS);
(5)取出分液漏斗,检查气密性后开始萃取;向分液漏斗中加入剩余醇提液,量筒量取100ml石油醚加入其中,萃取三次,取上层,三次萃取液再用旋转蒸发仪浓缩得到千里光石油醚萃取物(PESS);
(6)接着向下层溶液(石油醚萃取后)中加入100mL二氯甲烷,萃取三次收集下层溶液,将下层溶液旋干得到千里光二氯甲烷萃取物(DESS);
(7)接着向上层溶液(二氯甲烷萃取后)中加入100mL乙酸乙酯,萃取三次收集上层乙酸乙酯层,将上层溶液旋干得到千里光乙酸乙酯萃取物(EESS);
(8)接着向下层溶液(乙酸乙酯萃取后)中加入100mL饱和正丁醇,萃取三次收集上层溶液,将上层溶液旋干得到千里光饱和正丁醇萃取物(BESS);
(9)保留下层水层(正丁醇萃取后),同样旋干得到千里光水层萃取物。
提取过程见图1。
实施例2
EESS对神经损伤细胞的保护作用
1.生物材料
PC12细胞来源于成年大鼠肾上腺髓质嗜铬细胞瘤,经过神经生长因子阻止增殖并促进分化后会变成类似交感神经节神经元的表型并且可以无限传代培养,因此常用于神经性疾病的研究。
2.试剂
(1)胎牛血清(FBS)的灭活
首先将-20℃冷冻的胎牛血清取出至室温解冻,取出一空瓶加入相同量的水并插入温度计,待血清彻底融化后把两样都放进水浴锅里,等到温度计示数显示56℃时开始计时30min。
(2)抗生素
本实验使用青霉素链霉素配置双抗,以配置100mL为例,首先用天平分别准确称取1g青霉素和1g链霉素放入250mL烧杯中,接着加入100mLDDW,在超净工作台中使用玻璃棒轻轻搅拌至完全溶解,尽量不要搅出气泡,过滤除菌,分装在无菌合适大小的离心管中,冻存于-20℃中。
(3)完全培养基(DMEM+胎牛血清+双抗)
将瓶装DMEM、灭活后的胎牛血清、双抗(青霉素链霉素)取出放入超净工作台内,取一根灭过菌的50mL离心管,按照10%FBS以及1%双抗的比例,分别加入4mLFBS以及400μL双抗,后用DMEM定容到40mL,用于后续实验。
(4)H2O2溶液
从超净工作台中取出灭好菌的1.5mL棕色离心管,放入天平中称重并记录,接着用微量移液器量取100μL30%H2O2原液,称重,用DDW稀释,配置成100mM的储备液,分装在棕色1.5mL离心管中(鉴于过氧化氢易分解,不宜过多冻存,故每管分装200μL),冻存于-20℃冰箱中,下次自行解冻使用。
(5)1×PBS缓冲液
1000mL的1×PBS溶液配置需要分别称取8.0g NaCl、0.2g KCl、1.4gNa2HPO4和0.2gKH2PO4粉末,并将以上粉末放于干净的1L烧杯中。首先加入900mL的DDW搅拌溶解,并使用pH计调节溶液的pH值在7.2-7.4之间,之后定容到1L,并再次使用pH计以确保溶液的pH值在7.2-7.4范围内,经高压蒸汽灭菌后使用,4℃保存。
(6)0.25%胰酶
以配置100mL为例,用天平准确称取0.25g胰蛋白酶放入250mL烧杯中,加入100mL1×PBS溶液,用灭过菌的玻璃棒缓慢搅拌不要出气泡,溶解后密封把他放入4℃过夜,第二天将其除菌过滤后分装在无菌大小合适的离心管中,冻存于-20℃中。
(7)MTT溶液
以配置100mL为例,用天平准确称取0.5g MTT粉末至250mL烧杯中,加入100mL的DDW,不断搅拌,等到完全溶解后分装1mL在1.5mL棕色离心管中,冻存于-20℃中备用。
3.实施步骤
(1)细胞复苏
将水浴锅调至37℃,预热DMEM、血清、抗生素或完全培养基。取出PC12细胞冻存管37℃融化,随后置于超净工作台中操作。将冻存管中的细胞液转移至含有3mL完全培养基的15mL离心管后,使用台式离心机(800rpm,3min)对细胞悬液进行离心。准备一个60mm×60mm的小皿,向小皿中加入4mL完全培养基,待离心好后使用真空泵将离心管上层溶液吸弃,加入1mL完全培养基混匀底部的细胞沉淀并转移至小皿中。摇匀后置于37℃细胞培养箱中培养。
(2)细胞传代
首先使用光学显微镜观察培养皿内细胞形态及密度,当确认细胞状态良好并且细胞铺满底部90%左右时可进行传代。接着与细胞复苏前期准备一致,预热完培养基后从二氧化碳培养箱中取出需传代的皿,去除原有培养基后用1×PBS清洗细胞,洗两次每次2mL,最后一次吸弃后向皿中滴加600μL 0.25%胰酶,摇晃铺匀,大约20s后先加入2mL完全培养液,吹打收集细胞悬液至无菌的15mL离心管中,再加入2mL培养基继续吹打收集细胞悬液至无菌的15mL离心管中,800×g离心3min。最后除去培养基,加入4mL完全培养基混匀底部的细胞沉淀,取出一个10mm培养皿,向其中加入9mL完全培养基,按照所需的传代比例取细胞悬液加入皿中,手动摇匀,37℃,5%CO2条件下继续培养。
(3)细胞冻存
首先观察培养皿内细胞情况,当皿内细胞密度达90%以上且细胞状态良好时可进行冻存,一般冻存选用代数靠前的细胞以达到保存优秀种子的目的。预先在37℃水浴锅中预热完全培养基、胰酶消化液和1×PBS溶液,表面喷75%酒精后放入超净工作台中。首先吸弃小皿中的细胞培养液,沿壁加入1mL的1×PBS溶液,轻轻晃动后吸弃,重复两次以确保皿内没有残余的血清。加入300μL的胰酶消化液,待细胞液中出现密密麻麻的小白点时说明消化完成,大约20s左右,此时再向皿中加入1mL完全培养基将细胞吹下来到无菌的15mL离心管中,再加入2mL培养基继续吹打收集细胞悬液至无菌的15mL离心管中,800×g离心3min。待离心后,吸除离心管上层培养液,加入8:1:1的由血清、完全培养基和DMSO组成的总体积为1mL的冻存混合液,加入冻存管中,液氮保存。
(4)MTT细胞存活率检测
①细胞种板:参照细胞传代中的方法获得细胞悬浮液,800×g离心3min。除去培养基,加入4mL完全培养基混匀底部的细胞沉淀,取出细胞计数板和台酚蓝溶液,按照特定的稀释比例进行稀释计数。以PC12细胞为例:按照细胞悬液:台酚蓝溶液=1:3的比例,分别取20μL细胞悬液和60μL的台酚蓝溶液至1.5mL离心管中对细胞进行染色,反复吹匀后取10μL涂布在细胞计数板上进行计数。按所需的种板密度,将细胞种在96孔板中,PC12细胞每孔的接种密度是1.2×105个/mL,体积为80μL。
②加药:细胞培养过夜后观察皿内情况,以本课题为例,给药时皿内细胞有60%属最佳,根据实验要求使用完全培养基配制不同浓度的H2O2和EESS,为避免偶然性,所有实验样本需设置三个复孔用以后续计算偏差。以给药结束为时间节点开始计时,继续培养24h。
③检测:药物与细胞共培养24h后,向每孔中加入10μLMTT,以加完MTT为时间节点继续培养4h,4h后吸除上清液,注意不要讲底部结晶吸走,每孔加100μL DMSO。然后检测570nm和630nm处的吸光度值。
(5)结果分析
如图2所示,EESS处理组在1-100μg/mL浓度区间的细胞存活率与对照组相比,没有显著性差异。这一结果说明,当该浓度区间的EESS单独作用于PC12细胞时不会对细胞活力产生影响,即无毒,可以此浓度进行后续实验。
使用400-700μM的H2O2处理PC12细胞24h。实验结果如图3所示,H2O2对PC12细胞有明显的细胞毒性,具有明显的浓度依赖性,经过500μM的H2O2处理后的细胞存活率与对照组相比约为69.2%,损伤程度适中,因而在后续的实验中我们选用500μM的H2O2作为刺激浓度建立体外阿尔兹海默氏病模型。
如图4所示,H2O2处理组的细胞存活率不足70%,有非常明显的损伤,EESS处理组在1-100μg/mL浓度区间的细胞存活率表现出了一定程度的增加,这一结果说明EESS有效减少了由H2O2造成的PC12细胞损伤。另外,千里光其余萃取层的活性均低于EESS层。
实施例3
EESS对秀丽隐杆线虫的治疗作用
1.生物材料
(1)秀丽隐杆线虫N2、BR5270、BR5271、NL5901均购自Caenorhabditis GeneticsCenter(CGC),为转基因品系。
(2)大肠杆菌OP50(尿嘧啶渗漏突变株),作为秀丽隐杆线虫的食物。
2.试剂
3.实施步骤
(1)大肠杆菌OP50的培养
在超净工作台中吸取10μL低温冻存的E.coli OP50菌液,加入到湿热灭菌后冷却至室温的100mL LB液体培养基中,置于37℃摇床中180rpm培养过夜。取出置于4℃保存,可使用一个月。
(2)线虫NGM生长培养基的制备
NGM冷却凝固后,点燃酒精灯,在酒精灯旁向每皿加入200μL OP50作为线虫的食物,均匀涂开。待OP50吹干后,将其置于37℃恒温培养箱中培养过夜,取出置于15℃以下恒温培养箱保存备用。
(3)含药线虫NGM培养基的制备
湿热灭菌后温度降至70℃左右的NGM,在超净工作台中加入对应终浓度为1mM的MgSO4溶液、CaCl2溶液和5μg/mL的胆固醇溶液,混匀,后加入适宜稀释度的EESS,充分混匀后倒入玻璃培养皿中。含药的NGM板EESS终浓度分别为0.75mg/mL、1.5mg/mL、3.0mg/mL(L、M、H)。静置待NGM冷却凝固后,向每皿加入450μL OP50作为线虫的食物,均匀涂开。待OP50吹干后,将其置于37℃恒温培养箱中培养过夜,取出置于15℃以下恒温培养箱保存备用。
(4)线虫的同步化
在超净工作台中,取成长至性成熟且正在产卵的线虫,标志为体视镜下观察NGM板菌苔表面随处可见线虫卵,用M9缓冲液将线虫洗至无菌15mL离心管中,静置3-5min。待成虫沉淀后,弃掉上清液,加入4mL现配的线虫裂解液,剧烈震荡至溶液澄清,后迅速将其转移至1.5mL离心管中,5000rpm离心1min。离心结束后倒掉上清,每管迅速加入1mLM9缓冲液,涡旋3s后5000rpm离心2min。离心结束后仔细弃上清,每管加入1mL M9缓冲液,涡旋3s后5000rpm离心3min。离心结束后仔细弃上清,将所有沉淀转移至1个1.5mL离心管中,加入1mLM9缓冲液,涡旋混匀,置于相应温度恒温培养箱中培养24-48h。
(5)线虫的冻存
取三个培养皿中饥饿处理L1-L2线虫,用S Buffer洗下,分装在3个冻存管中,清洗三次,每个冻存管中终体积0.5mL,加等体积的冻存液,混合均匀。在冻存管上标记好秀丽线虫品系的名字和冻存日期,放入冻存盒,于-80℃放置最少24h,转入液氮保存。
(5)线虫的复苏培养
从液氮中取出目的线虫冻存管,迅速将其融化并倒在NGM固体培养基上,吹干后置于相应温度恒温培养箱中培养。待发现复苏线虫后,将其在无菌环境下挑入新的NGM固体培养基规模培养。
(6)野生型(N2)线虫的寿命试验
设空白组和EESS梯度浓度组,EESS浓度分别为0.75mg/mL、1.5mg/mL、3.0mg/mL。挑取同步化至L4期的N2线虫到各组培养基中,每组含虫60条,置于20℃培养箱中。从转移之日起开始统计线虫存活时间,转移当天记为寿命实验第0天。隔天检查线虫,记录已死亡线虫数目,剔除意外死亡、逃逸和袋样线虫。线虫死亡判别标准为铂丝轻触虫体而无任何反应。结果显示,与空白组相比,EESS以剂量依赖地方式延长了秀丽隐杆线虫的平均寿命,结果见图5A。
(7)AD品系线虫BR5270的基础慢反应实验
BR5270品系线虫由于Tau蛋白的聚集,表现为从成年第1天开始就运动不协调、运动神经元轴突缺损,因此,通过测定线虫的摆动次数,可探究EESS对BR5270品系线虫运动能力的影响。将BR5271、BR5270品系线虫进行同期化处理,然后于16℃条件下培养24h后,将BR5271品系线虫作为对照组,BR5270品系线虫分为空白组(不加药物)和EESS不同质量浓度组(0.75、1.5、3.0mg/mL),每组30条,并设3个平行。将线虫置于含不同药物的NGM培养基(含食物)上,于16℃条件下培养至成年(培养5天)后,测定各组线虫30s内的摆动次数。结果显示,与空白组比较,对照组与EESS 0.75mg/mL、1.5mg/mL、3.0mg/mL组线虫在30s内的摆动次数均显著增加,结果见图5B。
(8)PD线虫NL5901α-Syn::YFP聚集的荧光分析
NL5901品系线虫在unc-54启动子的控制下于体壁肌肉细胞中表达人类α-Syn融合YFP蛋白。α-Syn聚集在衰老的过程中逐渐增加,因此通过NL5901线虫α-Syn::YFP聚集的荧光析,研究了EESS对α-Syn聚集的影响。将同步化至L4期的NL5901线虫转至对照组和EESS不同浓度(0.75、1.5、3.0mg/ml)处理组,20℃培养2天后,用荧光倒置显微镜检测线虫肌肉组织绿色荧光的聚集情况,统计分析。如图5C和图5D所示,与对照组相比,EESS处理组的α-Syn积累显著降低。
(9)结果分析
本研究的AD品系线虫的基础慢反应实验结果显示,EESS可以明显增加BR5270品系线虫的摆动次数,这提示其可改善线虫因Tau蛋白聚集而导致的运动能力受损。寿命实验结果显示,EESS可显著延长野生型品系线虫的平均寿命。PD品系线虫NL5901荧光聚集分析实验结果显示,EESS明显减少了α-Syn的荧光聚集。
实施例4
EESS对AD模型小鼠的行为学实验
1.动物分组、模型建立及给药
(1)空白组。
(2)模型组:皮下注射的溶液量0.1-0.3ml/10g,每天一次D-gal(150mg/kg)+亚硝酸钠(70mg/kg)皮下注射,AlCl3(40mg/kg)按小鼠口服灌胃药液量0.1-0.3ml/10g配制液体,灌胃,持续8周。
(3)模型组+阳性药多奈哌齐(Donepezil):另加多奈哌齐灌胃(3mg/kg)。
(4)模型组+EESS:另加EESS灌胃20mg/kg。
2.水迷宫实验
(1)水迷宫准备:在一个封闭的水池里将水注入水迷宫中,池中放入一个站台(平台),水的高度高过站台1cm左右。
(2)训练小鼠:训练实验共4天,每天于固定时间段训练。水迷宫分为4个象限,平台始终置于第一象限。训练第一天从池壁第一象限的起始点将小鼠面向池壁放入水中,记录小鼠找到平台的时间和游泳路径。第一象限训练结束后按象限顺序将小鼠分别从其他象限的起始点放入水中训练。第二天从第二象限开始按顺时针方向训练,第三天从第三象限开始按逆时针方向训练,第四天从第四象限按顺时针方向开始。若小鼠60s内成功爬上平台并停留10s,就将其放回笼中,若小鼠60s内未到达平台或停留平台时间不足10s,则将其放在平台上停留10s,然后放回笼中。
(3)测试小鼠:第五天撤掉平台,将所有小鼠从第三象限起始点面向池壁放入水中,每只计时60s。
3.实验结果
(1)各组小鼠在水迷宫实验的运动轨迹图及第5天穿台次数
小鼠的游行轨迹如图6A所示,模型组的游行轨迹呈现边缘式,而空白组和EESS组呈现趋向式。发现模型组小鼠穿台次数明显少于空白组图6C。
(2)各组小鼠在水迷宫实验1-4天的逃避潜伏期
水迷宫实验前4天考察小鼠的空间学习能力,如图6B所示,模型组的逃避潜伏期整体高于空白组,模型组小鼠学习能力下降,EESS和多奈哌齐的逃避潜伏期均高于模型组且EESS的较好。
(3)各组小鼠在水迷宫实验第5天在目标象限的运动路程百分比、活动时间百分比
模型组第一象限运动路程时间百分比(图6D)及活动时间百分比(图6E)均低于空白组,提示模型组小鼠空间记忆能力下降。
以上结果都说明了AD小鼠产生认知功能障碍,而用EESS处理后可改善AD小鼠的学习记忆能力。
Claims (6)
1.一种千里光的萃取方法,其特征在于,所述萃取方法步骤如下:
(1)将千里光饮片装进三颈烧瓶中,加入70%的乙醇-水溶液,加热回流3次,趁热抽滤,合并滤液,减压浓缩,取出部分冷冻干燥作为千里光醇萃取物;
(2)向分液漏斗中加入剩余醇提液和石油醚,萃取三次收集上层溶液,旋干得到千里光石油醚萃取物;
(3)向石油醚萃取后的下层溶液中加入二氯甲烷,萃取三次收集下层溶液,旋干得到千里光二氯甲烷萃取物;
(4)向二氯甲烷萃取后的上层溶液中加入乙酸乙酯,萃取三次收集上层乙酸乙酯层,旋干得到千里光乙酸乙酯萃取物;
(5)接着向乙酸乙酯萃取后的下层溶液中加入饱和正丁醇,萃取三次收集上层溶液,旋干得到千里光饱和正丁醇萃取物;
(6)保留正丁醇萃取后的下层水层,旋干得到千里光水层萃取物。
2.如权利要求1所述的千里光的萃取方法,其特征在于,加热回流3次的时间依次是2h、1.5h、1h。
3.一种如权利要求1所述的千里光萃取物。
4.如权利要求3所述的千里光萃取物,其特征在于,所述萃取物为千里光乙酸乙酯萃取物。
5.一种如权利要求1所述方法得到的千里光萃取物的应用,其特征在于,所述千里光的乙酸乙酯萃取物用于制备防治神经退行性疾病和/或延缓衰老的药品。
6.如权利要求5所述的千里光萃取物的应用,其特征在于,所述千里光的乙酸乙酯萃取物制备成胶囊剂、片剂、颗粒剂、散剂、口服液、丸剂中的任一种剂型。
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