CN116602998B - 香榧籽油用于制备改善认知和/或神经保护药物的应用 - Google Patents
香榧籽油用于制备改善认知和/或神经保护药物的应用 Download PDFInfo
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Abstract
本发明公开了香榧籽油用于制备改善认知和/或神经保护食品的应用，动物实验和行为学实验验证了香榧籽油的干预能改善东莨宕碱诱导的小鼠认知记忆混乱，进一步的qPCR，免疫荧光染色与生化指标检测表明，香榧籽油可以通过增强PSD‑95的表达，降低小胶质细胞的表达水平以及上调脑内乙酰胆碱水平，下调乙酰胆碱酯酶水平来实现神经保护，从而改善认知障碍。
Description
技术领域
本发明属于生物医药中的天然药物领域，具体涉及香榧籽油用于制备改善认知和/或神经保护药物的应用。
背景技术
慢性神经退行性疾病包括阿尔茨海默症(AD)，帕金森(PD)等，主要特征为运动障碍，认知功能与记忆的丧失等。目前，大部分慢性神经退行性疾病的致病因素尚不明确，现有报道表明其发病可能与氧化应激，神经炎症以及胆碱能假说等有关。大量报道与实验表明，胆碱能系统在神经退行性疾病的发病机制中起重要作用，胆碱能系统往往伴随着神经炎症，脑部升高的神经炎症诱导神经元细胞的死亡，从而导致记忆障碍。因此，发掘能够调节胆碱能紊乱的治疗方案对于防治神经退行性疾病具有重要的意义。
香榧(TorreyagrandisFort.ex Lindl.)是我国特有树种，主要分布在我国的江苏、浙江、福建、江西、安徽、湖南等地，具有极高的利用价值，木材为建筑、造船、家具等的优良木材；种子为干果香榧，亦可榨食用籽油；其假种皮可提炼芳香籽油(香榧壳籽油)。种子入药，杀虫，消积，润燥。主治肠道寄生虫病，小儿疳积，肺燥咳嗽，肠燥便秘，痔疮。研究表明，不同产地的香榧籽油也已经被证实了有抗炎和抗氧化活性(Chen et al.2006,Cui etal.2018,Ni et al.2014,Shi et al.2009,Shi et al.2018,Zhou et al.2019,Zhu etal.2019)以及降血脂(Xiao et al.2022)。
Chen BQ,Cui XY,Zhao X,Zhang YH,Piao HS,Kim JH,Lee BC,Pyo HB,YunYP.Antioxidative and acute antiinflammatory effects ofTorreyagrandis.Fitoterapia.2006；77(4):262-7.
Cui HX,Duan FF,Jia SS,Cheng FR,Yuan K.Antioxidant and TyrosinaseInhibitory Activities of Seed Oils from Torreyagrandis Fort.ex Lindl.BiomedRes Int.2018；2018:5314320.
Ni L,Shi WY.Composition and Free Radical Scavenging Activity ofKernel Oil from Torreyagrandis,CaryaCathayensis,and Myrica R ubra.Iran JPharm Res.2014；13(1):221-6.
Shi H,Wang H,Wang M,Li X.Antioxidant activity and chemicalcomposition of Torreyagrandis cv.Merrillii seed.Nat Prod Commun.2009；4(11):1565-70.
Shi LK；Mao JH；Zheng L；Zhao CW；Jin QZ；Wang XG.Chemicalcharacterization and free radical scavenging capacity of oils obtained fromTorreyagrandis Fort.ex.Lindl.and Torreyagrandis Fort.var.Merrillii:Acomparative study using chemometrics.Industrial Crops and Products,2018,115,250-260.
Xiao M,Huang M,Huan W,Dong J,Xiao J,Wu J,Wang D,Song L.Effects ofTorreyagrandis Kernel Oilon Lipid Metabolism and Intestinal Flora in C57BL/6JMice.Oxid Med Cell Longev.2022；
2022:4472751.
Zhou X,Shang J,Qin M,Wang J,Jiang B,Yang H,Zhang Y.FractionatedAntioxidant andAnti-inflammatory Kernel Oil fromTorreyafargesii.Molecules.2019；24(18):3402.
Zhu MF,Tu ZC,Zhang L,Liao H.Antioxidant,metabolic enzymes inhibitoryability of Torreyagrandiskernels,and phytochemical profiling identified byHPLC-QTOF-MS/MS.J Food Biochem.
2019；43(12):e13043.
有关香榧籽油在改善认知作用与神经保护方面的研究未见报道。我们首次在细胞水平，动物实验层面研究了香榧籽油的神经保护及改善认知功能。
发明内容
本发明描述了香榧籽油的神经保护活性以及改善记忆作用，通过动物行为学实验以及后续体内组织的mRNA水平检测首次验证了香榧籽油对于小鼠的记忆改善作用。
具体包括：
香榧籽油用于制备改善认知和/或神经保护的药物的应用。
香榧籽油用于制备治疗阿尔茨海默病药物的应用。
可选的，按哺乳动物体重计，香榧籽油的用量为1000mg/kg·day。
可选的，按哺乳动物体重计，香榧籽油的用量为3000mg/kg·day。
一种治疗阿尔茨海默病的药物，该药物中含有香榧籽油，按哺乳动物体重计，香榧籽油的用量为1000mg/kg·day或3000mg/kg·day。
一种改善认知的药物，该药物中含有香榧籽油，按哺乳动物体重计，香榧籽油的用量为1000mg/kg·day或3000mg/kg·day。
一种神经保护的药物，该药物中含有香榧籽油，按哺乳动物体重计，香榧籽油的用量为1000mg/kg·day或3000mg/kg·day。
本发明的香榧籽油作为神经保护类药物，在神经退行性疾病的防治方面具有开发应用潜力。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1为香榧籽油神经保护活性的测定及应用的实验方法流程图；
图2为在香榧籽油干预21天后小鼠在新物体实验中表现的认知作用改善影响；
图3为在香榧籽油干预21天后小鼠在巴恩斯迷宫实验中表现得认知作用改善影响；
图4为在香榧籽油干预21天后小鼠在Y迷宫实验中表现得认知作用改善影响；
图5为香榧籽油干预21天后对小鼠脑皮层区小胶质细胞激活表达的影响；
图6为香榧籽油干预21天后对小鼠脑皮层区PSD-95的mRNA表达的影响；
图7为香榧籽油干预21天后对小鼠脑皮层中Ach，Ach-E表达量的影响。
具体实施方式
以下将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，以下所描述的实施例仅是本发明的一部分实施例，并非全部实施例，也并未对本发明做任何形式上的限制，凡是利用本实施例的技术方案，包括对本实施例做了简单的变化，均属于本发明保护的范围。
本发明的香榧籽油通过超临界提取采自安徽岳西县的干香榧果(由安徽康馨祥香榧农业科技有限公司提供)。
本发明中，东莨宕碱诱导的4月龄小鼠为老年认知混乱模型；对小鼠具有改善认知作用的最低浓度为1000mg/kg。
一、本发明的香榧籽油神经保护活性的测定及应用：
1、实验材料
香榧籽油：去壳干香榧子经CO2超临界提取而得。
生物材料：Scoplamine(纯度≥98％)、多奈哌齐(纯度≥98％)购自大连美伦生物科技有限公司(中国大连)。C57BL/6小鼠购买于16-20周龄(中国西安)。实验前，这些小鼠进行了一周的适应性喂养。所有实验小鼠置于相对湿度为50±15％、温度为22±2℃、光照/暗周期为12/12h的可控环境下，提供实验室标准食物(AIN-93M)和水。在接下来的实验中，将小鼠随机分为5组:对照组(Con)、东莨菪碱组(SCOP)、多奈哌齐组(DNP)、香榧籽油低剂量组(TGOL)和香榧籽油高剂量组(TGOH)。香榧籽油低剂量组和香榧籽油高剂量组分别以1000mg/kg和3000mg/kg BW灌胃，每日1次，连续30d；多奈哌齐组以多奈哌齐(3mg/kg)灌胃，连续30d。在第21天至第30天注射SCOP(3mg/kg)30min后进行行为学测试。实验过程如图1所示。SCOP和多奈哌齐的剂量根据既往报道确定。所有的动物实验程序都遵循《实验动物护理和使用指南:第八版》(ISBN-10:0-309-15396-4)。动物行为试验等研究方案已通过西北农林科技大学批准。ACh，AChE试剂盒购自南京建成。
1.1香榧籽油在新物体识别试验中对小鼠认知记忆的改善作用
采用新物体识别试验(New object recognition test，NOR)评价实验动物的短期非空间识别记忆(Lu et al.2018a)。试验装置为40cm×40cm×40cm的箱子和数据自动采集处理系统组成。试验可分为适应阶段、熟悉阶段和测试阶段。在适应阶段(第1天)，在训练日，让小鼠在无物体的试验装置中自由探索5分钟，以减少动物对新环境的恐惧。在第2天的熟悉阶段，在试验装置的对角线上放置2个相同的物品，小鼠被放置在测试箱的中心，然后将小鼠从测试箱中以相同的距离放置，自由探索5分钟，记录小鼠在每个物体上的探索时间。在第二天测试阶段，其中一个物体被一个新的物体所取代，位置保持不动，并记录小鼠在花费在新物体和旧物体上的探索时间。试验结束后，采用新物体识别指数(NOR Index)评估小鼠的短期非空间识别记忆。注意每只小鼠测试完成后，用酒精喷洒在物体，待酒精挥发后再放入小鼠，以消除小鼠停留在物体上的气味。
如附图2所示，模型组(SCOP)中小鼠的新物体识别指数较低；而在香榧籽油干预的组别中，1000mg/kg与3000mg/kg BW每日干预的小鼠(TGOL组与TGOH组)均可以使新物体识别指数上升且效果高于阳性药(DNP组)，意味着小鼠在新物体识别实验中认知能力的改善，其中1000mg/kg即低剂量的香榧籽油对于新物体识别指数的干预效果高于3000mg/kg剂量香榧籽油的干预效果。综上，香榧籽油干预能够显著改善小鼠的认知记忆混乱(p<0.01)，且最佳干预剂量为1000mg/kg。
1.2香榧籽油在巴恩斯迷宫实验中对小鼠记忆认知的影响
巴恩斯迷宫利用小鼠的避光以及恐高习性，试验装置位于地板上方140cm处，20个孔均匀地位于表面周边，直径为5cm。目标洞是这些孔中的1个，连接到暗室允许小鼠逃离强光的刺激。试验分为4天的适应期以及最后1天的测试天，适应期允许小鼠在熟悉暗室1min后让小鼠在丧失方向感的前提下从装置中间开始探索，为时3min，3min或找到目标洞后结束适应，记录小鼠找到目标洞的潜伏时间；测试天将小鼠在丧失方向感的前提下直接置于装置中央，允许其进行自由探索，为时90s，记录小鼠找到目标洞的潜伏时间来判断小鼠记忆能力。
如图3所示，从适应期的第3天开始，东莨宕碱诱导记忆混乱的模型组小鼠相对空白组潜伏时间显著更长(p<0.01)，而阳性对照组，高低剂量组小鼠潜伏时间则显著更短(p<0.01)。在测试天的结果中，与模型组(SCOP组)相比，香榧籽油干预组的逃逸潜伏期有显著的降低，且效果优于阳性药组，在剂量方面，低剂量组的逃逸潜伏期也略低于高剂量组的表现，可以认为低剂量组对于小鼠的长期记忆改善能力更强。综上，香榧籽油的干预可以改善小鼠的长期记忆能力，且最佳剂量为1000mg/kg·day。
1.3香榧籽油在Y迷宫实验中对小鼠记忆认知的影响
Y迷宫。Y迷宫的设计是为了测试老鼠的工作记忆。在测试中，小鼠被放置在设备的中心，并有3分钟的时间自由探索三个手臂(40厘米×20厘米*4厘米)。工作记忆采用交替百分比进行评估。
交替百分比＝[N/(t1)]×100％(N＝三个臂的不重复输入总数；T＝总臂项数)。
如图4所示，与空白组小鼠相比，东莨宕碱诱导的模型组小鼠在Y迷宫中的交替百分比明显下调，意味东莨宕碱的诱导损伤了小鼠的工作记忆及短期记忆能力即建模成功，与给药组(DNP组，TGOL组以及TGOH组)对比，给药组的交替百分比显著上升，其中两种剂量的香榧籽油的干预效果都优于阳性药组，进一步确定最佳剂量，我们发现TGOL组的交替百分比略高于TGOH组，可以认为低剂量的香榧籽油干预改善小鼠工作记忆和短期记忆障碍的能力更强。综上，香榧籽油具有改善小鼠工作记忆和短期记忆障碍的能力，其最佳剂量为1000mg/kg·day。
1.4香榧籽油在脑皮层中减少小胶质细胞激活的影响
小胶质细胞作为中枢神经系统内的一部分，具有多突触，高可塑性的特点，他的活化一般被视作神经退行性疾病发生的主要特征之一。其中，IBA-1为小胶质细胞激活的标志物之一，IBA-1的高表达意味着小胶质细胞被激活，从而介导脑内的生理学变化，诱发神经疾病。
如图5所示，在脑内CA1区以及皮层区，无论在IBA-1信号激活细胞以及阳性信号面积上，模型组相较空白组都有了显著提高，而香榧籽油的干预可以逆转这一病理学变化，其中低剂量组与高剂量组的效果差异不显著，可以认为在神经保护层面上二者的功效相当。综上，香榧籽油的干预可以降低东莨宕碱诱导的小鼠脑皮层内IBA-1信号激活细胞的数量以及阳性信号面积，意味着可以预防小鼠脑内小胶质细胞的激活，起到神经保护的作用，在这部分内容中低剂量香榧籽油与高剂量组的表现差异不显著。
1.5香榧籽油对小鼠脑内Ach，Ach-E含量表达的改变
胆碱能假说作为AD的常见假说之一，在药物开发以及治疗方案扩展的研究方向上亦举足轻重。胆碱能系统的紊乱与否与神经疾病的发生密切联系在一起，其中ACh是胆碱能神经元中一种重要的神经递质，在中枢神经系统及胆碱能传递中都起重要的作用，AChE是合成和水解ACh的关键酶类。因此，针对这两项指标在脑内的表达水平可以作为判断脑内胆碱能系统是否紊乱的标准。
如图6所示，我们对脑皮层内Ach以及AChE的含量进行了测定。可以观察到，东莨宕碱的干预升高了小鼠脑皮层内的AChE含量，降低了ACh含量，诱使了小鼠脑内胆碱能紊乱，进一步导致了认知功能障碍；而香榧籽油的干预逆转了这一变化，升高了ACh水平，降低了AChE水平。在乙酰胆碱水平上，低剂量组小鼠脑内的乙酰胆碱水平高于高剂量组以及阳性药组，而在乙酰胆碱酯酶水平上，低剂量组小鼠脑内的乙酰胆碱酯酶水平亦低于高剂量组以及阳性药组。综上，香榧籽油的干预可以起到调控胆碱能系统紊乱的效果，且最佳干预剂量为1000mg/kg·day。
1.6香榧籽油对小鼠脑内PSD-95mRNA含量表达的改变
突触后致密物(postsynaptic density,PSD)是位于中枢神经系统突触后膜的特殊结构，是突触后信号转导和整合的关键物质，其中PSD-95是PSD的主要成分，通过不同结构域与相关受体及信号分子结合，形成信号复合物发挥作用，具有参与突触连接的形成，维持突触的可塑性等多种生物学功能。
如图7，PSD-95的mRNA表达量随着香榧籽油浓度的升高呈浓度依赖性增加，TNF-α和iNOS的mRNA表达量则随着香榧籽油浓度的升高而减少。荧光定量PCR分析结果表明，东莨宕碱诱导小鼠模型的脑部皮层区PSD-95的mRNA表达相较模型组有明显的下调(p<0.01)，模型组和低剂量组组之间无显著差异，阳性对照组，高剂量组与模型组相比，有着明显的上调(p<0.05)。
综上所述，在神经退行性疾病的防治方面，香榧籽油具有进一步开发的潜力。尤其是我们首次在体内证明了香榧籽油的神经保护活性，对胆碱能系统紊乱的改善作用活性，首次验证了香榧籽油具有改善记忆的作用。
以上结合附图详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。
Claims (1)
1.香榧籽油作为唯一活性成分用于制备治疗阿尔茨海默病药物的应用。
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| 7种木本油料油脂品质综合评价;陈振超;倪张林;莫润宏;钟冬莲;汤富彬;;中国油脂;20181120(11);第90页右栏最后一段、表1 * |
| 孙小红等人.超声波法提取香榧籽油工艺及其理化特性.食品工业.2019,第40卷(第6期),38-41. * |
| 超声波法提取香榧籽油工艺及其理化特性;孙小红等人;食品工业;20191231;第40卷(第6期);38-41 * |
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