CN107325155B - 一种抗氧化超短肽Mapin-WH2、其制备方法及应用 - Google Patents
一种抗氧化超短肽Mapin-WH2、其制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种抗氧化超短肽Mapin‑WH2、其制备方法及应用，属于生物医学技术领域。所述抗氧化超短肽Mapin‑WH2是直链多肽，仅含有5个氨基酸残基，全序列一级结构为：缬氨酸‑亮氨酸‑赖氨酸‑赖氨酸‑半胱氨酸；所述抗氧化超短肽Mapin‑WH2是通过化学合成的方法得到的；所述抗氧化超短肽Mapin‑WH2具有极强的抗氧化活性，相比于临床常用的抗氧化剂谷胱甘肽，清除自由基效果更优，且无溶血性及细胞毒性。本发明中的抗氧化超短肽Mapin‑WH2可以用于抗氧化药物、食品添加剂、抗氧化化妆品原料的制备，并且它的作用可能还不止局限于此。抗氧化超短肽Mapin‑WH2分子量小、结构简单、采用化学合成方法的成本极低，相比临床常用的抗氧化谷胱甘肽具有突出有益的特点。
Description
技术领域
本发明涉及生物医药领域，尤其涉及一种抗氧化超短肽Mapin-WH2、其制备方法及应用。
背景技术
自由基是具有强氧化活性的一组物质，极易发生反应。活性氧自由基不仅会使人体内的脂质与蛋白质发生链式氧化反应导致细胞膜、组织、酶和基因受损，对肌体造成了损害和衰老，而且会促使有关的基因突变，诱发相关的疾病，如动脉硬化症、高血压、类风湿性关节炎、白内障、老年痴呆症、帕金森氏病以及神经退行性病变等。越来越多的医学研究及临床试验证据显示，体内自由基含量越高寿命越短，自由基可谓“万病之源”。
近年来抗氧化在化妆品领域的应用中很广泛。由于皮肤的光老化作用是由于紫外线诱导皮肤细胞产生大量的氧自由基堆积而造成的。现在许多护肤品中加入抗氧化剂，有减少雀斑、防辐射、延缓衰老、调节免疫和提高皮肤自我保护的作用。此外食品中营养成分的氧化反应会产生过氧化物。过氧化物影响食品的营养价值，甚至严重还可导致疾病发生，寻找安全的抗氧化剂以抑制过氧化物产生一直是生化营养学的研究热点。
目前，化工领域常用抗氧化剂多为化学合成物，如BHA、BHT等，虽抗氧化效果良好，但对人体肝、脾、肺有蓄积性致癌作用。临床上常见的抗氧化剂如维生素C、维生素E与谷胱甘肽等。维生素C主要以清除·OH为主，但需大剂量使用才能达到目的，但同时，大剂量维生素C使用容易导致酸中毒；维生素E主要通过抑制细胞膜上的脂质过氧化来达到抗氧化的目的，但长期是用则有潜在的致突变风险。而临床还原型谷胱甘肽的产业化壁垒较高，已被日本协和发酵工业株式会社几乎垄断全球谷胱甘肽的供应，每公斤高达8000美金。同时，现在国内制药企业所用的谷胱甘肽制药原料还不能摆脱进口，谷胱甘肽价格一直居高不下。可见，活性佳、易合成、产量高的新型抗氧化肽已在医疗、食品、化妆品等领域中亟待开发。
发明内容
基于背景技术存在的技术问题，本发明提出了一种抗氧化超短肽Mapin-WH2、其制备方法及应用旨在解决上述技术问题。
本发明采用的技术方案为：一种抗氧化超短肽Mapin-WH2，所述抗氧化超短肽Mapin-WH2为一种直链小肽，含有5个氨基酸残基，全序列一级结构为：缬氨酸-亮氨酸-赖氨酸-赖氨酸-半胱氨酸。
优选的，所述抗氧化超短肽Mapin-WH2的分子量为589.79Da，等电点是9.31。
优选的，所述抗氧化超短肽Mapin-WH2的制备方法，具体步骤如下：
1)多肽肽链的合成；
2)对多肽进行纯化；
3)测定纯化后多肽的分子量；
4)测定多肽的等电点及序列。
优选的，所述抗氧化超短肽Mapin-WH2可用于抗氧化药物、食品添加剂、抗氧化化妆品原料的制备。
本发明的有益效果在于：所述抗氧化超短肽Mapin-WH2具有极强的抗氧化活性，在体外实验中对多种自由基的清除率均在90％以上，且浓度为3mM时，对DPPH自由基、ABTS·+正离子自由基清除率I％可以达到96.75％、95.84％，优于临床常使用的抗氧化制剂——谷胱甘肽。
所述抗氧化超短肽Mapin-WH2具有分子量小、结构简单、溶血活性低、细胞毒性极低、制备方法简单、稳定性高、无蛋白修饰、产量高、纯度高等特点，对比临床用谷胱甘肽成本更低。
所述抗氧化超短肽Mapin-WH2活性强、效价高，因此可将其应用在抗氧化药物、食品或者化妆品添加剂中，可以提高药物、食品或化妆品添加剂的抗氧化效果，具有很好的应用前景。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
一种抗氧化超短肽Mapin-WH2，所述抗氧化超短肽Mapin-WH2为一种直链小肽，含有5个氨基酸残基，全序列一级结构为：缬氨酸-亮氨酸-赖氨酸-赖氨酸-半胱氨酸。
优选的，所述抗氧化超短肽Mapin-WH2的制备方法，具体步骤如下：
1、根据超短肽的氨基酸序列：缬氨酸-亮氨酸-赖氨酸-赖氨酸-半胱氨酸，用自动多肽合成仪(型号：433A，Applied Biosystems)合成得到粗多肽；
2、将粗多肽通过HPLC反相柱层析脱盐纯化，通过多次分离纯化、并鉴定其纯度，直到多肽的纯度不低于95％；
HPLC纯化及鉴定方法：将1mg待测样品溶于1mL含有0.1％TFA的超纯水中，若有不溶解的杂质，用0.22μm滤膜过滤，流动相A为0.1％TFA-H2O，流动相B为0.1％TFA-CH3CN(所用洗脱溶剂均需过滤并超声处理，防止色谱柱中产生气泡)，待基线平稳后开始上样，上样量为10μL；色谱柱为硅胶烷基键合相C18柱(4.6mm id×250mm，胶粒大小5μm id，孔径大小为300目)，采用二元流动相梯度洗脱系统，进行梯度洗脱，即在30min内，流动相B在洗脱剂中的含量从0％-80％按线性关系增长，流速1mL/min，检测波长215nm，25℃下测定。
3、使用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)确定其分子量为589.79Da；
测定方法：将纯化后的多肽溶于去离子水中，配置成2μmol/mL的溶液，取1μL与等体积的饱和基质溶液(将α-氰基-4-羟基肉桂酸溶于含0.1％TFA的50％ACN溶液中，制成饱和溶液，离心，取上清液)混合后测定。
4、通过等电聚焦电泳测定纯化后多肽的等电点为9.31，并采用自动氨基酸测序仪测定纯化后多肽的氨基酸序列结构，确定为：缬氨酸-亮氨酸-赖氨酸-赖氨酸-半胱氨酸。
优选的，所述抗氧化超短肽Mapin-WH2可用于抗氧化药物、食品添加剂、抗氧化化妆品原料的制备。
(一)、抗氧化超短肽Mapin-WH2的抗氧化实验：
1.1 DPPH自由基清除活性(DPPH radical scavenging assay)
称取一定量的DPPH(2，2-diphenyl-1-picrylhydrazyl hydrate，Sigma，美国)，用甲醇溶解，配成6×10-5M的溶液，现配现用。将48μl DPPH溶液和2μl样品混合(最终样品与DPPH的质量比为3∶1)，室温下避光静置30min，于517nm处测定吸光值。空白对照组以样品溶解介质代替待测样品。实验做三个平行，紫外分光光度计调零时使用甲醇。
DPPH·清除率(％)＝(AB-AA)/AB×100(AB：空白对照组吸光值；AA：样品组吸光值)。
1.2 ABTS·+自由基正离子清除活性
ABTS(3-ethylbezothiazoline-6-sulfonic acid)(3-乙基苯并噻唑啉-6-磺酸)用PBS缓冲液(pH7.4)配成2mM的ABTS储存液。将ABTS储存液和70mM过硫酸钾(K2S2O8)水溶液按体积比250∶1混合，于室温避光放置15-16h。试验开始前，将ABTS·+释至734nm波长处的吸光值为0.80±0.03。将4μl样品和96μl上述校正过的ABTS·+溶液混合，室温放置10min后，于734nm波长处检测反应液的吸光值。空白对照组为溶解样品所用灭菌去离子水。实验做三个平行。
ABTS·+清除率I(％)＝(AB-AA)/AB×100(AB：空白对照组吸光值；AA：样品组吸光值)。
表1.超短肽Mapin-WH2抗氧化活性
结果如表1所示，抗氧化超短肽Mapin-WH2具有极强的抗氧化活性。在样品浓度为3mM时，Mapin-WH2对DPPH和ABTS·+正离子自由基清除率I％分别可以达到96.75％、95.84％。与临床药物谷胱甘肽具备相当抗氧化活性。故抗氧化超短肽Mapin-WH2将在抗氧化药品、食品等领域具有优秀的开发潜力。
(二)、抗氧化超短肽Mapin-WH2溶血性与细胞毒性测定
2.1溶血性检测。
将采集的健康人血与阿氏液混合抗凝，生理盐水洗涤2次并重悬成107-108cell/ml的悬浮液。上述稀释好的红细胞悬液与溶解于生理盐水的抗氧化超短肽Mapin-WH2样品混合，37℃保温30min，再于1000rpm离心5min，上清液于540nm测吸收值。阴性对照使用生理盐水，阳性对照使用Triton X-100，溶血百分比按以下公式计算：溶血百分比H％＝A样品-A阴性对照/A阳性对照×100％。
表2.抗氧化超短肽Mapin-WH2溶血性
溶血性结果如表2所示，结果表明抗氧化超短肽Mapin-WH2浓度高达160μg/ml时的溶血率只有0.77％。说明Mapin-WH2不具有溶血性，不易引起人体红细胞破裂溶解而对人体产生伤害，因此十分利于其在抗氧化药物、食品或者化妆品添加剂领域进一步的开发应用。
2.2抗氧化超短肽Mapin-WH2细胞毒性检测。
用MTT法检测该组抗氧化超短肽对人皮肤成纤维细胞HFF-1细胞(购于上海细胞库)毒性。先将成纤维细胞于含有15％胎牛血清以及双抗(青霉素和链霉素各100U/ml)的DMEM中培养，待细胞长满后，用0.25％的胰蛋白酶消化下来，用上述培养基洗两次，重新悬浮细胞，细胞计数后将细胞悬浮液100μl加入到96孔细胞培养板中，使每孔细胞数达到105个。加入样品，对照组加入相同体积的灭菌超纯水，置37℃，5％CO2培养箱内培养24h。培养结束后，96孔细胞培养板每孔加入20μl 5mg/ml MTT溶液(用细胞培养PBS缓冲液配制)，继续培养5h，用注射器吸出孔中液体，每孔加入100μl DMSO，用移液枪吹打几次使紫色结晶完全溶解。酶标仪检测光吸收，测定波长490nm，参考波长630nm。
表3.抗氧化超短肽Mapin-WH2对HFF-1细胞的毒性
细胞毒性结果如表3所示，可以看出抗氧化超短肽Mapin-WH2浓度为160μg/ml时的细胞毒性只有14.65％，说明抗氧化超短肽Mapin-WH2对人皮肤成纤维细胞细胞毒性极低，对人体正常皮肤细胞不会产生伤害，因此十分利于其在在抗氧化药物、食品或者化妆品添加剂领域进一步的开发应用。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
序列表
<110>于海宁王慧王义鹏
<120>一种抗氧化超短肽Mapin-WH2、其制备方法及应用
<160> 1
<210> 1
<211>5
<212> PRT
<213>人工序列
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Claims (4)
1.一种抗氧化超短肽Mapin-WH2，其特征在于：所述抗氧化超短肽Mapin-WH2为一种直链小肽，含有5个氨基酸残基，全序列一级结构为：缬氨酸-亮氨酸-赖氨酸-赖氨酸-半胱氨酸。
2.如权利要求1所述的一种抗氧化超短肽Mapin-WH2，其特征在于：所述抗氧化超短肽Mapin-WH2的分子量为589.79Da，等电点是9.31。
3.如权利要求1-2任一项所述的一种抗氧化超短肽Mapin-WH2的制备方法，具体步骤如下：
1)多肽肽链的合成；
2)对多肽进行纯化；
3)测定纯化后多肽的分子量；
4)测定多肽的等电点及序列。
4.如权利要求1-2任一项所述的一种抗氧化超短肽Mapin-WH2在抗氧化药物、食品添加剂、抗氧化化妆品原料的制备中的应用。
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