CN107226836B - 一种具有改善记忆功效的多肽及其分离制备方法和应用 - Google Patents
一种具有改善记忆功效的多肽及其分离制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有改善记忆功效的多肽及其分离制备方法和应用，该多肽的氨基酸序列为Tyr‑Asn‑Glu，其分离制备方法包括如下步骤：将凤尾鱼加混合酶酶解得到酶解液，酶解液上Sephadex G‑25凝胶柱洗脱，测定各洗脱峰对应组分的乙酰胆碱酯酶抑制活性，将活性最高的组分采用反相高效液相色谱进一步纯化，各洗脱峰中乙酰胆碱酯酶抑制活性最高的组分即为本发明的多肽。本发明采用生物酶解技术及色谱分离技术制备出一种具有明显改善记忆作用的蛋白肽，制备方法简单可行，制得的产品纯度高。
Description
技术领域
本发明属于蛋白质精深加工领域，具体涉及一种具有改善记忆功效的多肽及其分离制备方法和应用。
背景技术
研究表明，记忆的下降或退化与大脑海马区病变、神经递质减少以及突触病变等有关。此外，发生在中枢神经系统(Central nervous system，CNS)的氧化应激会随着年龄的增加而增加，从而导致许多与年龄相关的神经退行性疾病的产生，如阿尔茨海默病、帕金森病、痴呆、多发性硬化症等疾病。预计，在2050年前患者数将增加到1.15亿。
人类大脑内神经递质-乙酰胆碱的缺失是导致记忆损伤的重要因素之一。乙酰胆碱是人类神经细胞中重要的神经递质，它能够在突触间进行神经信号的传递。但乙酰胆碱会被胆碱酯酶(Cholinesterase,ChE)降解，过度的乙酰胆碱降解会导致乙酰胆碱含量降低，从而造成神经信号传递的失败，导致记忆损伤。
目前，已经有许多乙酰胆碱酯酶抑制剂被列为研究对象。但某些抑制剂中，毒扁豆碱(Physostigmine)及塔克林(Tacrine)因货架期短及肝毒性等副作用，而逐渐从市场上消失。因而安全无副作用的乙酰胆碱酯酶抑制剂的研究吸引了人们的注意。对于天然、安全无副作用的乙酰胆碱酯酶抑制剂的筛选逐渐吸引了许多研究者的目光。
学者们对多种天然物质的酰胆碱酯酶抑制活性进行了系统研究，其中包括天然产物提取物、卵磷脂、多不饱和脂肪酸以及肽类物质等。天然产物提取物因成分复杂，可能存在试剂残留等危害；卵磷脂、多不饱和脂肪酸因价格昂贵、提取工艺复杂而难以大范围推广。而肽类物质，具有来源广泛、制备工艺简单、无副作用等优点，逐渐成为研究热点。
蛋白质和肽类物质具有维持脑细胞代谢，保持大脑的各种运动正常进行的功能。其中天然存在于哺乳动物中枢神经系统中的内源多肽约有200种，例如生长抑素(Somatostatin，SS)、促皮质激素释放因子(Corticotropin Releasing Factor，CRF)、加压素(Vasopressin，VP)、催乳素(Prolactin，PRL)、脑啡肽(Enkephalin)、内啡肽(Eindorphin)等已被证实对记忆具有调节作用。
目前也有很多学者采用生物酶解技术从植物或者动物组织中制备得到具有改善记忆的多肽。脑活素则是由猪脑水解后得到的含有85％游离氨基酸和15％低分子多肽的复合酶解产物，研究表明其具有改善记忆、保护神经细胞的作用，可以改善脑内神经递质和相关酶的活性。
目前的研究多集中于混合物的记忆功效增强或者合成改善记忆肽序列的公开，评价手段多采用动物行为学，改善记忆多肽纯品制备困难，评价指标过程繁琐，且很少采用乙酰胆碱酯酶活性抑制能力来评价多肽。
凤鲚(Coilia mystus(1inaeus))，属鲱形目，鲲科，鲚属，称为凤尾鱼、肉鲚等。凤尾鱼多用于制作罐头产品。其鱼肉中含15～20％的蛋白质，氨基酸组成合理，脂肪含量低，但局限于罐头产品等附加值低，鉴于其产量稳定、价格低廉，适宜开发新方向，因此如何充分开发利用凤尾鱼蛋白质资源，提高其附加值，是鱼肉蛋白资源综合利用的重要环节。
发明内容
为了克服现有改善记忆多肽纯品制备困难的问题，本发明的首要目的在于提供一种具有改善记忆功效的多肽，其氨基酸序列为Tyr-Asn-Glu。
本发明的另一目的在于提供上述多肽的分离制备方法。
本发明的再一目的在于提供上述多肽的应用。
本发明的目的通过下述技术方案实现：
一种多肽，其氨基酸序列为Tyr-Asn-Glu,分子量为424.41Da。
上述多肽的分离制备方法，包括如下步骤：
(1)将凤尾鱼去头去内脏，绞成肉糜，加水混合，加入占肉糜质量0.5～1.2％的混合酶，在45～60℃下酶解6～10h后，灭酶，冷却至室温后，离心，过滤收集滤液，得到凤尾鱼酶解液；
步骤(1)所述的加水混合，水与肉糜的质量比为(1～3):1；
步骤(1)所述的混合酶，由木瓜蛋白酶和碱性蛋白酶组成；其中木瓜蛋白酶占肉糜质量的0.2～0.5％，碱性蛋白酶占肉糜质量的0.3～0.7％；所述的碱性蛋白酶优选Alcalase 2.4L；
步骤(1)所述的灭酶是将反应物在95℃下加热15min；
步骤(1)所述的离心优选在3500r/min转速下离心10min；
(2)将凤尾鱼酶解液添加至Sephadex G-25凝胶柱，用去离子水以0.5～1.5mL/min的流速进行洗脱，检测波长为220nm，收集各洗脱峰并测定其乙酰胆碱酯酶抑制活性，选取乙酰胆碱酯酶抑制活性最高的组分进行下一步分离；
(3)采用反相高效液相色谱对步骤(2)选取的目标组分进一步纯化，收集各洗脱峰并测定其乙酰胆碱酯酶抑制活性，乙酰胆碱酯酶抑制活性最高的组分即为本发明的多肽Tyr-Asn-Glu；
步骤(3)所述的反相高效液相色谱优选以下参数：Waters e2695HPLC，2998PDA检测器，色谱柱为XBridgeTM Prep BEH130 C18柱(10×150mm,5μm,Waters，USA)，流动相为A相和B相；A相是质量分数0.1％的三氟乙酸超纯水溶液，B相为甲醇；
洗脱程序为：0-1min内采用的流动相中A与B体积比为(95:5)-(90:10)；1-35min内采用的流动相中A与B体积比为(95:5)-(60:40)或(90:10)-(70:30)；35-36min内采用的流动相中A与B体积比为60:40；36-40min内采用的流动相中A与B体积比为(60:40)-(95:5)或(60:40)-(90:10)，流速为1mL/min，检测波长为220nm。
步骤(2)和(3)中所述的乙酰胆碱酯酶抑制活性的测定方法如下：
将25μL含有7.5mM的硫代碘代乙酰胆碱溶液、50μL HEPES溶液(0.75mM)、125μL含有0.5％牛血清蛋白的二硫代二硝基苯甲酸(3Mm，pH 7.4)和50μL样品(即各洗脱峰对应组分)混合，37℃下孵育15min；然后向混合物中加入25μL乙酰胆碱酯酶(0.11U)，412nm处用酶标仪检测(15min)各组吸光值。AchE抑制率计算公式如下：
AchE抑制率(％)＝[1-(A样品-A样品空白)/(A对照-A对照空白)]×100％
其中，对照组为不加样品加AchE组，对照空白组为不加样品不加AchE组。
本发明的多肽可用于制备具有改善记忆功效的药品、保健品或食品。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明所用原料为凤尾鱼，来源广泛，价格低廉，目前凤尾鱼多用于制作鱼罐头，现在将其作为蛋白原料，开发改善记忆肽，可显著提高凤尾鱼原料的附加值。
(2)本发明采用生物酶解技术及色谱分离技术制备出一种具有明显改善记忆作用的蛋白肽，制备方法简单可行，制得的产品纯度高。
(3)本发明提供的改善记忆肽活性优良，具有良好的乙酰胆碱酯酶抑制能力，可以作为功能性成分，用于保健品中。
(4)本发明提供的改善记忆肽为三肽产品，其肽分子量小，可被人体直接吸收。
(5)本发明通过化学合成对应的改善记忆肽，经实验验证效果与分离纯化得到的新肽效果接近。
附图说明
图1为凤尾鱼酶解产物的Sephadex G-25凝胶色谱分离洗脱曲线。
图2为凤尾鱼酶解产物Sephadex G-25凝胶色谱洗脱组分的乙酰胆碱酯酶抑制活性测定结果。
图3为凝胶色谱目标收集组分的反相高效液相色谱分离洗脱曲线。
图4反相高效液相色谱分离洗脱组分的乙酰胆碱酯酶抑制活性测定结果。
图5为改善记忆肽的氨基酸序列质谱分析图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细描述，但本发明的实施方式不限于此。
实施例
本发明多肽的分离制备方法，包括如下步骤：
(1)凤尾鱼去头去内脏，清洗干净后，放入搅拌机中制成肉糜；加入肉糜质量2倍的水，按肉糜质量的0.5％添加蛋白酶(以鱼肉肉糜的质量为计算基准，木瓜蛋白酶的加入量占0.25％，Alcalase 2.4L的加入量占0.25％)，在45℃下保温酶解6h，然后在95℃温度下加热15min灭酶，冷却至室温后，在3500r/min下离心10min，收集上清液，得到凤尾鱼蛋白酶解液；
(2)将凤尾鱼蛋白酶解液用Sephadex G-25凝胶柱进行分离纯化，用去离子水以0.5mL/min的流速进行洗脱，检测波长为220nm，共收集到6个组分，洗脱曲线见图1，测定各洗脱峰对应组分的乙酰胆碱酯酶抑制活性，结果见图2，组分Fr.4活性最高，然后收集组分Fr.4，采用反相高效液相色谱法对改组分进一步纯化(Waters e2695HPLC，2998PDA检测器)，色谱柱为XBridgeTM Prep BEH130 C18柱(10×150mm,5μm,Waters,USA)，流动相为A相(0.1％的三氟乙酸超纯水溶液)和B相(乙腈)，洗脱程序为：0-1min内A:B为90:10(体积比,下同),1-35min内A:B为90:10-70:30，35-36min内A:B为70:30，36-40min内A:B为70:30-90:10，流速为1mL/min，检测波长为220nm，共收集到10个峰，洗脱曲线见图3，测定各洗脱峰对应组分的乙酰胆碱酯酶抑制活性，结果见图4，其中组分5活性最高，收集组分5，得到改善记忆肽。
(3)最后采用电喷雾串联质谱测定步骤(2)中获得的改善记忆肽的氨基酸序列。结果见图5，改善记忆肽的氨基酸序列为Tyr-Asn-Glu。
本发明制备的改善记忆肽与脑活素(Cerebrolysin)的乙酰胆碱酯酶抑制活性检测结果如表1，并采用脑活素(Cerebrolysin)作为对照，样品及脑活素浓度为10mg/mL，结果如表1。
表1改善记忆肽及脑活素的乙酰胆碱酯酶抑制能力
注：改善记忆肽和脑活素检测浓度为10mg/mL。
已有大量文献报道，脑力记忆下降受损多与胆碱能系统的紊乱有关。由表1可知本发明制备的改善记忆肽具有优秀的乙酰胆碱酯酶抑制活性，具有潜在改善大脑胆碱能系统中乙酰胆的动态平衡状态的作用，表明本发明提供的改善记忆肽具有较强的记忆改善功能，可用于药品、保健品和食品等行业中。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.一种多肽，其特征在于氨基酸序列为Tyr-Asn-Glu。
2.权利要求1所述多肽的分离制备方法，其特征在于包括如下步骤：
（1）将凤尾鱼去头去内脏，绞成肉糜，加水混合，加入占肉糜质量0.5~1.2%的混合酶，在45~60℃下酶解6~10 h后，灭酶，冷却至室温后，离心，过滤收集滤液，得凤尾鱼酶解液；
（2）将凤尾鱼酶解液添加至Sephadex G-25 凝胶柱，用去离子水以0.5~1.5 mL/min的流速进行洗脱，检测波长为220 nm，测定各洗脱峰对应组分的乙酰胆碱酯酶抑制活性，收集乙酰胆碱酯酶抑制活性最高的组分；
（3）步骤（2）收集的乙酰胆碱酯酶抑制活性最高的组分采用反相高效液相色谱进一步纯化，测定各洗脱峰对应组分的乙酰胆碱酯酶抑制活性，乙酰胆碱酯酶抑制活性最高的组分采用电喷雾串联质谱测定，得到多肽Tyr-Asn-Glu；
步骤（1）所述的混合酶，由木瓜蛋白酶和碱性蛋白酶组成；所述的木瓜蛋白酶占肉糜质量的0.2~0.5%；所述的碱性蛋白酶占肉糜质量的0.3~0.7%；
步骤（3）所述的反相高效液相色谱采用以下参数：Waters e2695 HPLC，2998 PDA 检测器，色谱柱为XBridgeTM Prep BEH130 C18柱，流动相为A 相和B 相；A相是质量分数0.1% 的三氟乙酸超纯水溶液，B相为甲醇；
洗脱程序为：0-1min 内采用的流动相中A与B 体积比为（95:5）-（90:10）；1-35 min 内采用的流动相中A与B 体积比为（95:5）-（60:40）或（90:10）-（70:30）；35-36 min 内采用的流动相中A 与B 体积比为60:40；36-40 min 内采用的流动相中A 与B 体积比为（60:40）-（95:5）或（60:40）-（90:10），流速为1 mL/min，检测波长为220 nm。
3.根据权利要求2所述的分离制备方法，其特征在于：步骤（1）所述的加水混合，水与肉糜的质量比为（1~ 3）:1。
4.根据权利要求2所述的分离制备方法，其特征在于：步骤（1）所述的灭酶是将反应物在95 ℃下加热15 min。
5.根据权利要求2所述的分离制备方法，其特征在于：步骤（1）所述的离心是在3500 r/min转速下离心10 min。
6.权利要求1所述的多肽在制备具有改善记忆功效的药品、保健品或食品中的应用。
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