CN115669929A - 辅助改善记忆的功能性食品其制备方法 - Google Patents
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Abstract
本发明提供一种辅助改善记忆的功能性食品，其包含β‑烟酰胺单核苷酸(NMN)和L‑α‑甘油磷酸胆碱(α‑GPC)，其中，优选的含量是β‑烟酰胺单核苷酸的使用剂量为6～9mg/kg/d，L‑α‑甘油磷酸胆碱的使用剂量为12～20mg/kg/d。在经典的Morris水迷宫测试模型中，本发明组合物对人工造模后大鼠的的学习和记忆行为有着明显改善作用。本发明的组合物均为食品级原料，安全可靠、可长期服用。
Description
本申请是申请号为2021100525546，发明名称为“治疗阿尔茨海默症的组合物及其制备方法、应用”的分案申请，母案申请日为2021年1月15日。
技术领域
本发明涉及一种辅助改善记忆的功能性食品及其应用。
背景技术
阿尔茨海默症(以下也简称AD)是在1906年首次由德国精神病学家和神经发病学家Alzheimer Alois发现、并以其名字命名的老年痴呆，是一种慢性神经退行性疾病。阿尔茨海默症的主要临床表现为记忆力逐渐减退、认知功能发生障碍、行为异常和社交障碍等。随着人类平均寿命的增加，发病率逐年上升老年性痴呆症中有约70％为阿尔茨海默症，尚有血管性痴呆，两者并存的混合性痴呆。其主要病理特征是大脑萎缩、脑组织内老年斑、脑血管沉淀物和神经原纤维缠结。
目前的研究表明，阿尔茨海默症患者的主要病理学特征是β-淀粉样蛋白(Aβ)聚集成老年斑，细胞内Tau蛋白异常聚集形成神经元纤维缠结(NFT)和神经元死亡。近年来，针对阿尔茨海默症的发病机制，在早期的胆碱能神经元假说、Aβ毒性假说和Tau蛋白假说等基础上，研究相对较少的炎症假说、胰岛素假说、氧化不平衡假说和基因突变假说也越来越受关注。阿尔茨海默症的具体发病机制目前尚未完全研究透彻，只是存在多种假说，包括胆碱能神经元假说、Aβ毒性假说、Tau蛋白假说、胰岛素假说、自由基损伤假说等。然而，阿尔茨海默症是由遗传因素和环境因素共同引发的一种复杂性疾病，单一的假说并不能解释阿尔茨海默症的全部发病特征。由于阿尔茨海默症的致病因素是非常复杂的。文中综述的所有假说可能并未完全将其概括，一些还处于初步研究阶段或未被发现的机制，如线粒体动力学失衡假说、Ca浓度失衡假说、小胶质细胞影响假说等也许能够丰富对阿尔茨海默症致病因素的描述。在阿尔茨海默症发病机制尚未完全研究清楚之前，针对阿尔茨海默症的治疗只能是对症治疗，这并不能从根本上治愈阿尔茨海默症患者。所以，找到可逆转疾病进程的药物才是攻克阿尔茨海默症这一疾病的关键。众多临床试验失败的教训提示单一靶点的治疗对于阿尔茨海默症这种复杂疾病很难奏效。
至今阿尔茨海默症的病因仍未得以阐明，无法研制出特效治疗药物，因此对阿尔茨海默症的治疗一直是一个十分棘手的问题。开发相关的药物一直是业界的追求方向。另外，由于阿尔茨海默症的发病率逐年上升，该病的预防性对策也非常受重视。如果能够开发可长期安全服用的具有阿尔茨海默症治疗效果的功能性食品，也存在巨大市场前景，
发明内容
本发明的目的是开发一款效果确切、安全性令人信服的治疗阿尔茨海默症的组合物，以满足可以适合长期服用并能够预防或改善阿尔茨海默症的需求。本发明的发明人经过深入研究，意外地发现将NMN和α-GPC组合使用，显示出的阿尔茨海默症的症状的治疗效果，从而完成了本发明。本发明的组合物，可以应用于制备用于治疗阿尔茨海默症的药物，或者以预防和治疗为目的的功能性食品。
具体而言，本发明提供一种治疗阿尔茨海默症的组合物，其特征在于，其包含β-烟酰胺单核苷酸(NMN)和L-α-甘油磷酸胆碱(α-GPC)，其中，β-烟酰胺单核苷酸的使用剂量为6～9mg/kg/d，L-α-甘油磷酸胆碱的使用剂量为12～20mg/kg/d。
作为本发明优选实施方式的药物制剂，可以为片剂、胶囊剂。
本发明还提供一种功能性食品，包含上述的治疗阿尔茨海默症的组合物以及可食用的辅料。
本发明的组合物，可以应用于制备用于治疗阿尔茨海默症的药物。
本发明中的NMN(Nicotinamide mononucleotide)是β-烟酰胺单核苷酸的简称，天然存在于活细胞中，是辅酶1NAD+(烟酰胺腺嘌呤二核苷酸)的前体。可适当延长寿命。真正发挥抗衰老作用的是一种称为烟酰胺腺嘌呤二核苷酸(NAD+)的重要能量代谢物。NMN是一种营养物质，市场上可以获得商品。NMN会在细胞中转化为NAD+，它归类为一种全新的维生素B3，且超越了普通维生素的功效，被认为是预防衰老和恢复年轻的补充剂。NMN本身是人体内含有的物质，存在于母乳及食物中，例如毛豆、西兰花、黄瓜、圆白菜、鳄梨、番茄等，但含量非常少(每100克中含有0.25-1.88毫克)。生牛肉和虾等也含有非常少量的(每100克中含有0.06-0.42毫克)NMN成分。所以想要提高NMN，还要从外界直接补充。哈佛遗传教授大卫·辛克莱(David Sinclair)提出NMN有逆转衰老的功效：通过维持细胞内充足的NAD+，可以保持DNA的自我修复能力，使年龄增长带来的DNA损伤得以有效修复，从而抑制衰老。而NMN原料是NAD+的前体，服用NMN可以提高体内NAD+水平。但是迄今为止，尚无直接证据证实NMN的摄入能够治疗或者改善阿尔茨海默症的症状。
本发明中的α-GPC是L-α-甘油磷酸胆碱的简称，其是具有由构成细胞膜的主要磷脂PC(磷脂酰胆碱)脱去2个脂肪酸后的水溶性物质，原本在生物体内广泛存在，是存在于人的母乳及体液肿的身体成分之一，2009年的食品药品分类修订中，将L-α-甘油磷酸胆碱(有时也可以称为sn-甘油-3-磷酸胆碱)定义为食品。α-GPC为可以通过血脑屏障少数几种营养素之一，因此可以迅速的从胆碱转换为乙酰胆碱，被认为是防止胆碱缺乏、增加神经递质、促进促生长激素的分泌等的营养素。而且，由于乙酰胆碱酯酶抑制剂(AChEI)是到目前为止，临床上使用最为广泛的AD治疗药物，FDA批准用于治疗AD的5种药物中除了美金刚属于NMDA受体拮抗药外，其余4种[他克林(Tacrine)、多奈哌齐(Donepezil)、加兰他敏(Galanthamine)和卡巴拉汀(Rivastigmine)]均属于AChEI。然而，可能是由于单纯的摄入α-GPC营养品由于机体的调控作用，不会增多乙酰胆碱含量，因此目前为止尚没有α-GPC的摄入能够治疗或者改善阿尔茨海默症的症状的报道。
然而本发明的发明人意外地发现，将NMN和α-GPC这两种内源性的营养成分组合使用，在动物模型上显示了对阿尔茨海默症的症状改善作用，提示了NMN和α-GPC作为预防或治疗阿尔茨海默症的前景，具体可以参见实施例中的具体实施例。
作为本发明的优选的实施方式，本发明还提供一种治疗阿尔茨海默症的药物制剂，其包含上述组合物，还包含药学上可用接受的辅料。
本发明的组合物在使用时，优选制成口服给药的固体剂型来给药。作为用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，将组合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，组合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了本发明组合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了本发明组合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
从服用方便角度考虑，本发明的组合物优选制成片剂或者胶囊剂。为制备本发明组合物的粉剂的片剂或胶囊，可将本发明组合物的粉剂与制剂载体混合，该载体为常用的制片剂组分，如玉米淀粉、蔗糖、山梨醇、脂肪酸、硬脂酸镁和其它药用稀释剂。
本发明的治疗阿尔茨海默症的组合物，可以与其他类的治疗阿尔茨海默症的药物合用。作为其他的疗阿尔茨海默症的药物，可举出他克林、多奈哌齐、卡巴拉汀、氢溴酸加兰他敏、美金刚、九期一等临床常见的药物。
本发明还提供一种功能性食品，包含上述的治疗阿尔茨海默症的组合物以及可食用的辅料。
由于本发明的组合物由于原料完全可食用，非常适合开发成功能食品，例如可制备成口含片、糖果、固体饮料、液体饮料形式的功能性食品。本发明的功能性食品，包含本发明的上述组合物和可食用的辅料，作为可以使用的符合相关标准的食品添加剂，例如符合中华人民共和国GB2760-2014标准的食品添加剂
作为本发明的功能性食品的优选形式，可以举出口含片。将本发明的组合物制成口含片的方式没有特殊限制，可以使用公知的方法，例如，将本发明的组合物的造粒，添加可食用的辅料等混合，进行压片的过程。作为可食用的辅料，可以举出淀粉、蔗糖、甲基纤维素低聚果糖、茶粉、食盐、甜菊糖苷、山梨糖醇、维生素C等，但不限于这些，只要是符合食品标准(例如中华人民共和国GB2760-2014)的辅料均可以适当添加。
本发明的原料本身的口味无刺激性，制成口含片时，不会有不良的味道，无须包衣。但如果为了利于食用、利于防止吸潮等目的，也可以包衣。从食品角度出发，优选使用海藻酸钠、黄原胶、阿拉伯胶等完全符合食品标准的辅料进行包衣。
作为本发明的功能性食品的优选形式，从食品化观感强，接受度高的角度考虑，本发明的组合物，还可以制成食品口服液的形式。作为食品口服的液体制剂的剂型有溶液、糖浆或混悬液。这些液体制剂的制备方法中，分散媒一般为水，非水溶性的赋形剂如杏仁油、油脂；作为药剂学上允许使用的填加剂包括如山梨醇、氢化食用油、甲基纤维素；还包括防腐剂如甲基或丙基P-酚；乳化剂如卵磷脂、阿拉伯橡胶：人工色素或甜味剂。
作为给药的剂量，应根据病人的年龄、体重、健康状况及剂型的不同而定。推荐的使用剂量为β-烟酰胺单核苷酸的使用剂量为6～9mg/kg/d，L-α-甘油磷酸胆碱的使用剂量为12～20mg/kg/d。但是由于本发明的原料成分均有着很强的安全性，本领域的技术人员可以根据个体需要，适当增加每一组分有效用量。
本发明的发明人通过实验动物模型，验证了本发明的组合物的治疗阿尔茨海默症的药效。具体而言，本研究中采用Morris水迷宫实验，该测试是强迫实验动物游泳，学习寻找隐藏在水中平台的实验，主要用于测试实验动物对空间位置感和方向感(空间定位)的学习记忆能力，被广泛应用于阿尔茨海默症的药效评价研究中。本发明中，采用经典的Aβ1-40对大鼠的海马CA1区定向注射制备阿尔茨海默症动物模型，结合经典的Morris水迷宫测试模型大鼠的学习和记忆行为，从而评价给药成分的效果。结果显示，单独利用NMN灌胃大鼠没有表现出改善阿尔茨海默症的症状的现象，同样地，单独利用α-GPC灌胃大鼠也未表现出善阿尔茨海默症的症状的现象。然而当组合使用NMN和α-GPC进行灌胃时，对于模型大鼠产生了认知改善作用，提示了组合使用NMN和α-GPC具有预防或治疗阿尔茨海默症的应用前景，
本发明的组合物与现有技术相比，存在以下明显的优势：本发明所开发的组合物的原料均为食品，是效果明确、安全性令人信服的治疗阿尔茨海默症的组合物，本发明的组合物并采用科学的动物表型验证了确切的效果，虽然其效果比照对照品的西药的药效弱，但是考虑到其特别适合以满足可以适合长期服用，可以用于预防阿尔茨海默症症状，因此有着广阔的市场前景。
附图说明
图1是实施例2中各组大鼠的逃避潜伏期结果的统计图表。
图2是实施例2中各组大鼠的穿台测试结果的统计图表。
具体实施方式
以下集合具体的实施例对本发明进行进一步详细的描述，实施例仅仅是为了说明本发明技术方案的实例，并非为了对本发明的保护范围进行任何限定。
实施例1AD实验动物模型的建立
模式动物品系选择SPF级SD大鼠，健康，雄性，体重为250g-300g，由上海南方模式生物科技发展有限公司提供。实验分组分为正常对照组、AD模型组、阳性药组、受试药组1、受试药组2、受试药组3，总共六组，每组8至11只。
除了正常对照组，采用β-淀粉样蛋白(具体为Aβ1-40，购于Sigma公司)注射至海马诱导大鼠为AD大鼠的建模方法进行建模方式。具体为：大鼠腹腔注射10％水合氯醛溶液麻醉，用量为4.5ml/kg。SD大鼠均采用1％戊巴比妥钠40μg/g腹腔内注射麻醉，麻醉后继而固定于脑立体定位仪上。按照大鼠脑立体定位图谱，以前囟为零起点，前囟后3.5mm处为穿刺点，中线右侧旁开2mm，而后牙科钻钻开颅骨，采用微量注射器自脑表面垂直进针3mm，双侧海马CA1区缓慢匀速注射Aβ1-40各10μg(1μL)，留针5min，退针后牙科水泥封闭颅骨钻孔处，缝合伤口，皮针+4.0手术缝合线缝合伤口，大腿肌肉注射庆大霉素1mg/kg。造模后恢复7d，开始灌胃喂养，正常对照组、AD模型组灌胃生理盐水，阳性药组灌胃盐酸美金刚3mg/kg/d，受试药组1灌胃β-烟酰胺单核苷酸8mg/kg/d，受试药组2灌胃L-α-甘油磷酸胆碱10mg/kg/d，受试药组3灌胃β-烟酰胺单核苷酸8mg/kg/d以及L-α-甘油磷酸胆碱10mg/kg/d，连续给药21天后，立即进行以下四天的行为学测试。
实施例2行为学检测——Morris水迷宫模型测试
前3天进行逃避潜伏期试验，第4天撤去平台进行穿台试验。
2.1逃避潜伏期实验
所谓潜伏期实验，是指每只大鼠测试中从同一个位置入水，须有实验人员辅助大鼠缓慢入水。为了保证大鼠游泳上台环境尽量相同，注意补水保持平台距离水面2cm，大鼠入水后开始计时，找到平台并停留2秒的情况，记录时间，所耗费时间为潜伏期，超过120秒仍未找到平台也记作120秒。时间越短表明记忆改善效果越好。
2.2穿台实验
所谓穿台实验，是指在前3天水迷宫潜伏期测试之后，撤去水迷宫中的平台，记录大鼠入水后的运动轨迹，与平台位置相交一次记为一次穿台，记录每只大鼠120秒内穿台总次数，次数越多说明记忆改善效果越好。
2.3结果及分析
每只大鼠在前3天在各个象限入水4次，总共落水12次，将最终将得到的时间值汇总取平均值并四舍五入，作为该只大鼠的最终潜伏期，第4天每只大鼠在各个象限入水4次，将得到的4个数字取平均值，为该只大鼠的穿台次数。
利用统计学分析，比较模型组与正常组的上台潜伏期和穿台次数。本发明中正常对照组和AD模型组在潜伏期和穿台次数均有统计学差异，认定造模成功(p<0.05认为有统计学差异，p<0.01则认为有显著统计学差异)。实验结果显示：正常对照组和AD模型组之间p<0.05，本发明的模型是可用的。
利用统计学分析，比较模型组与各给药分组上台潜伏期和穿台次数。
1.各组对模型大鼠逃避潜伏期的作用
各组大鼠的逃避潜伏期统计结果参见表1和图1。阳性对照组(美金刚组)显示了较明显的药效，其与正常对照组几乎达到同样的水平。受试药组1和受试药组2与AD模型组比较未表现出功效，但是受试药组3相对于AD模型明显显现了记忆改善效果，虽然没有阳性对照组效果明显，但是其药效具有显著性差异(p<0.05)。
表1大鼠逃避潜伏期的统计表
2.各组对大鼠穿台测试的作用
各组大鼠的穿台测试的统计结果参见表2和图2。与上述的逃避潜伏期类似，受试药组1和受试药组2与AD模型组比较未表现出功效，但是受试药组3相对于AD模型明显显现了记忆改善效果，但是其药效具有显著性差异(p<0.05)。阳性对照组(美金刚组)显示了的药效没有逃避潜伏期统计明显，但是其也显示了明确的药效。
表2大鼠穿台测试的统计表
上述动物实验显示，受试组3所用的组合物显示了在治疗阿尔茨海默病中具有良好的应用前景，即，本发明的组合物具有治疗或者预防阿尔茨海默症的活性，可以用于制备治疗和预防阿尔茨海默症的药物或者功能性食品。
最后应说明的是：以上所述的各实施例仅用于说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分或全部技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
本说明书中引用的所有出版物和专利文献引入本文作为参考，如同每个出版物或专利被分别明确指明引入本文作为参考。在不偏离本申请公开的实质和范围的情况下，可对本申请公开的各实施方案进行多种改变和用等同物替换。除非上下文中另有说明，否则本公开的实施方案的任何特征、步骤或实施方案都可以与任何其他特征、步骤或实施方案组合使用。
Claims (3)
1.一种辅助改善记忆的功能性食品，其特征在于，其包含β-烟酰胺单核苷酸NMN和L-α-甘油磷酸胆碱α-GPC，其中，β-烟酰胺单核苷酸的使用剂量为3～9mg/kg/d，L-α-甘油磷酸胆碱的使用剂量为8～20mg/kg/d。
2.根据权利要求1中所述的功能性食品，还包含可食用的辅料。
3.根据权利要求2所述的功能性食品，其为片剂、丸剂。
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