CN114478698A - 抗氧化短肽m5、其制备方法及应用 - Google Patents
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Abstract
本发明公开了抗氧化短肽M5、其制备方法及应用，属于生物医药技术领域，抗氧化短肽M5，抗氧化短肽M5为直链小肽，含有9个氨基酸残基，全序列一级结构为：苯丙氨酸‑亮氨酸‑脯氨酸‑甘氨酸‑酪氨酸‑谷氨酸‑半胱氨酸‑缬氨酸‑色氨酸。抗氧化短肽M5的制备方法，包括以下步骤：S1：根据短肽的氨基酸序列合成得到粗多肽，S2：将合成得到的粗多肽进行层析脱盐纯化，得到纯化后的多肽，S3：通过质谱确定纯化后的多肽的分子量，S4：测定纯化后的多肽的氨基酸序列结构。本发明的抗氧化短肽M5、其制备方法及应用，抗氧化短肽M5具有分子量小、结构简单、溶血活性低、细胞毒性极低的优点，具有极强的抗氧化活性，且制备方法简单。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及一种抗氧化短肽M5、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、保健品等领域中亟待开发。
发明内容
本发明的目的在于提出一种抗氧化短肽M5、其制备方法及应用，抗氧化短肽M5具有分子量小、结构简单、溶血活性低、细胞毒性极低的优点，具有极强的抗氧化活性，且制备方法简单。
为达此目的，本发明采用以下技术方案：
本发明提供的抗氧化短肽M5，抗氧化短肽M5为直链小肽，含有9个氨基酸残基，全序列一级结构为：苯丙氨酸-亮氨酸-脯氨酸-甘氨酸-酪氨酸-谷氨酸-半胱氨酸-缬氨酸-色氨酸。
优选地，抗氧化短肽M5的分子量为1113.3Da，等电点为4.0。
本发明还提供一种抗氧化短肽M5的制备方法，包括以下步骤：
S1：根据短肽的氨基酸序列合成得到粗多肽；
S2：将合成得到的粗多肽进行层析脱盐纯化，得到纯化后的多肽；
S3：通过质谱确定纯化后的多肽的分子量；
S4：测定纯化后的多肽的氨基酸序列结构。
优选地，步骤S2中，采用RP-HPLC C18柱进行层析脱盐纯化，得到纯化后的多肽后，通过RP-HPLC鉴定纯化后的多肽的纯度，纯度≥95％。
优选地，步骤S3具体包括：使用基质辅助激光解析电离飞行时间质谱确定纯化后的多肽的分子量。
优选地，步骤S4具体包括：通过等电聚焦电泳测定多肽的等电点，并采用自动氨基酸测序仪测定纯化后的多肽的氨基酸序列结构。
本发明还提供一种抗氧化短肽M5的制备方法制备的抗氧化短肽M5在制备抗氧化物质的应用。
优选地，抗氧化物质为抗氧化药物、食品添加剂、抗氧化化妆品或保健品。
本发明的有益效果为：
1、抗氧化短肽M5具有极强的抗氧化活性，在体外实验中在浓度为10μg/ml时，对ABTS·+正离子自由基清除率I％可以达到74.21％，且抗氧化活性随着浓度升高表现出浓度依赖性增加，优于临床常使用的抗氧化制剂谷胱甘肽和母本肽Macro-A1。
2、抗氧化短肽M5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。
3、抗氧化短肽M5应用在抗氧化药物、食品添加剂、抗氧化化妆品或保健品中，可以提高抗氧化药物、食品添加剂、抗氧化化妆品或保健品的抗氧化效果，具有很好的应用前景。
附图说明
图1是本发明的多肽ABTS·+自由基清除活性图。
具体实施方式
现结合附图和具体实施方式对本发明进一步说明。
本实施例中提供的抗氧化短肽M5，抗氧化短肽M5为直链小肽，含有9个氨基酸残基，全序列一级结构为：苯丙氨酸-亮氨酸-脯氨酸-甘氨酸-酪氨酸-谷氨酸-半胱氨酸-缬氨酸-色氨酸。
其中，抗氧化短肽M5的分子量为1113.3Da，等电点为4.0。
实施例还提供一种抗氧化短肽M5的制备方法，包括以下步骤：
S1：根据短肽的氨基酸序列：苯丙氨酸-亮氨酸-脯氨酸-甘氨酸-酪氨酸-谷氨酸-半胱氨酸-缬氨酸-色氨酸，用自动多肽合成仪合成得到粗多肽。
S2：将粗多肽通过RP-HPLC C18柱层析脱盐纯化，得到纯化后的多肽，并通过RP-HPLC鉴定纯化后的多肽的纯度，纯度≥95％。
S3：使用基质辅助激光解析电离飞行时间质谱确定纯化后的多肽的分子量。
S4：通过等电聚焦电泳测定多肽的等电点，并采用自动氨基酸测序仪测定纯化后的多肽的氨基酸序列结构。
本实施例还提供一种抗氧化短肽M5的制备方法制备的抗氧化短肽M5在制备抗氧化物质的应用。其中，抗氧化物质为抗氧化药物、食品添加剂、抗氧化化妆品或保健品。
抗氧化短肽M5的抗氧化活性测定：
抗氧化短肽M5的抗氧化活性用ABTS·+自由基正离子清除活性检测进行确定。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。将不同浓度的4μl多肽样品和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)。
结果如图1及表1所示，抗氧化短肽M5具有极强的抗氧化活性。在样品浓度为10μg/ml时，M5对ABTS·+正离子自由基清除率I％即可达到73.4％，显著优于母本肽Macro-A1和临床药物谷胱甘肽。且M5的抗氧化活性表现出明显的浓度依赖性，随着浓度升高其对ABTS·+正离子自由基清除率也显著升高。
表1.抗氧化短肽自由基清除率I％
抗氧化短肽M5溶血活性与细胞毒性测定：
(1)溶血活性检测。
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短肽M5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％。
溶血活性结果如表2所示：
表2.抗氧化短肽M5溶血活性
结果表明抗氧化短肽M5和母本肽Macro-A1以及谷胱甘肽均表现出较低的溶血活性。M5在浓度高达160μg/ml时的溶血率只有5.77％。说明M5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、化妆品添加剂或保健品领域进一步的开发应用。
(2)抗氧化短肽M5细胞毒性检测。
用MTT法检测抗氧化短肽M5对人皮肤成纤维细胞HFF-1细胞(购于上海细胞库)的毒性。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。加入抗氧化短肽样品，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。
细胞毒性结果如表3所示：
表3.抗氧化短肽M5细胞毒性
可以看出抗氧化短肽M5浓度为160μg/ml时的细胞毒性只有12.54％，说明抗氧化短肽M5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，因此十分利于其在抗氧化药物、食品或、化妆品添加剂或保健品领域进一步的开发应用。
以上实施例仅用以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；其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，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精神和范围。
SEQUENCE LISTING
<110> 宜肌坊（厦门）生物科技有限公司
<120> 抗氧化短肽M5、其制备方法及应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> 人工序列
<400> 1
Phe Leu Pro Gly Tyr Glu Cys Val Trp
1 5
Claims (8)
1.抗氧化短肽M5，其特征在于：
所述抗氧化短肽M5为直链小肽，含有9个氨基酸残基，全序列一级结构为：苯丙氨酸-亮氨酸-脯氨酸-甘氨酸-酪氨酸-谷氨酸-半胱氨酸-缬氨酸-色氨酸。
2.根据权利要求1所述的抗氧化短肽M5，其特征在于：
所述抗氧化短肽M5的分子量为1113.3Da，等电点为4.0。
3.抗氧化短肽M5的制备方法，其特征在于，包括以下步骤：
S1：根据短肽的氨基酸序列合成得到粗多肽；
S2：将合成得到的粗多肽进行层析脱盐纯化，得到纯化后的多肽；
S3：通过质谱确定纯化后的多肽的分子量；
S4：测定纯化后的多肽的氨基酸序列结构。
4.根据权利要求1所述的抗氧化短肽M5的制备方法，其特征在于：
所述步骤S2中，采用RP-HPLC C18柱进行层析脱盐纯化，得到纯化后的多肽后，通过RP-HPLC鉴定纯化后的多肽的纯度，纯度≥95％。
5.根据权利要求1所述的抗氧化短肽M5的制备方法，其特征在于：
所述步骤S3具体包括：使用基质辅助激光解析电离飞行时间质谱确定纯化后的多肽的分子量。
6.根据权利要求1所述的抗氧化短肽M5的制备方法，其特征在于：
所述步骤S4具体包括：通过等电聚焦电泳测定多肽的等电点，并采用自动氨基酸测序仪测定纯化后的多肽的氨基酸序列结构。
7.根据权利要求3-6任一项所述的抗氧化短肽M5的制备方法制备的抗氧化短肽M5在制备抗氧化物质的应用。
8.根据权利要求7所述的应用，其特征在于：
所述抗氧化物质为抗氧化药物、食品添加剂、抗氧化化妆品或保健品。
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| WO2015200656A1 (en) * | 2014-06-26 | 2015-12-30 | L'oreal | Short peptides and a method of use as an antioxidant |
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