CN116284301B - 一种抗氧化多肽及其应用 - Google Patents
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Abstract
本发明提供了一种抗氧化多肽及其应用，属于生物医药技术领域。本发明所述的抗氧化多肽来源于大豆，具有较强的抗氧化作用，可显著清除自由基，可作为抗氧化剂用于食品、药品、化妆品及添加剂等领域中。此外，通过实验发现，本发明所述的抗氧化多肽对于氧化损伤的细胞具有较好的保护作用，对于神经退行性疾病的治疗具有重要意义。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种抗氧化多肽及其应用。
背景技术
氧化还原反应是机体生命活动的基础，该反应在产生能量的同时也产生了各种自由基(FR)，如1,1-二苯基-2-三硝基苯肼(DPPH·)、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS+·)、活性氧簇(ROS)等。FR是带有未配对电子的原子或基团，是机体的正常代谢产物，具有强氧化性。机体内FR与抗氧化剂(ATO)处于动态平衡，各种因素导致机体产生过多FR，则FR的强氧化性会导致生物膜脂质过氧化，DNA及蛋白质被破坏，导致机体损伤、肿瘤、免疫性损伤、加速人体衰老等。
抗氧化剂(ATO)可捕获并中和机体内的FR，从而消除其对机体的伤害。例如，ATO可保护细胞组织免受氧化作用的损害，起到保护心脑血管系统，抗癌及抗衰老的作用，如在年龄相关性黄斑病变中，补充维生素型的抗氧化剂可减缓疾病的进程。ATO类型众多，如按来源可分为天然ATO和人工合成ATO；按溶解性可分为脂溶性ATO和水溶性ATO；还有酶类和非酶类抗氧化剂；机体内源产生的ATO和外源性ATO等。例如：(1)过氧化氢酶(CAT)是存在于细胞内的过氧化物体，是以H2O2为电子受体催化底物氧化的酶，可清除H2O2，是过氧化物酶体的标志酶；(2)超氧化物歧化酶(SOD)是生物体内重要的抗氧化酶，广泛分布于各种生物体内；(3)谷胱甘肽过氧化物酶(GPX)催化H2O2还原型谷胱甘肽(GSH)还原H2O2，GSH则变为氧化型谷胱甘肽(GSSG)，GSSG又可被还原为GSH，继续清除H2O2；(4)肌肽则是一种由β丙氨酸和L组氨酸两种氨基酸组成的天然二肽，大量存在于骨骼肌中，肌肽能抑制由金属离子、血红蛋白、脂酶和活性氧引起的脂质过氧化；(5)非酶类抗氧化剂，主要是一些具有还原性的维生素，如维生素A、维生素C、维生素E、类胡萝卜素等。
来源于动物或植物的生物活性抗氧化多肽是蛋白质经特定蛋白水解酶酶解或发酵后形成的从二肽到具有复杂线性、环状结构的不同肽类的总称。例如，专利CN202110511611.2公开了一种来自于花生的抗氧化多肽，其氨基酸序列为PGCPST。专利CN201711446377.X则公开了一种从蚕蛹蛋白酶解液中分离制备得到的抗氧化多肽，其氨基酸序列为SVLGTGC。
因此，开发新的抗氧化多肽对于新的功能食品、药品、化妆品以及添加剂领域具有重要意义。
发明内容
针对上述不足，本发明提供了一种大豆来源的抗氧化多肽。所述的抗氧化多肽具有较强的抗氧化作用，可显著清除自由基，可作为抗氧化剂用于食品、药品、化妆品及添加剂等领域中。此外，通过实验发现，本发明所述的抗氧化多肽对于氧化损伤的细胞具有较好的保护作用，对于神经退行性疾病的治疗具有重要意义。
为了实现上述发明目的，本发明的技术方案如下：
一方面，本发明提供了一种抗氧化多肽，所述的抗氧化多肽的氨基酸序列为YAVRHFLMFQTTMNRWKHAHWFY(SEQ ID NO:1)所示的序列。
另一方面，本发明提供了一系列编码所述的抗氧化多肽的核酸分子。
具体地，所述的核酸分子包含一种或多种经密码子优化的核酸分子。
又一方面，本发明提供了一系列载体，所述的载体包含本申请所述的一个或多个核酸分子。
具体地，所述的载体包括但不限于质粒、病毒、噬菌体。
又一方面，本发明提供了一系列包含上述核酸分子或上述载体的宿主细胞。
具体地，所述的宿主细胞包括但不限于微生物、植物或动物细胞，可通过本领域技术人员已知的方法将本发明所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等方法。
又一方面，本发明提供了上述抗氧化多肽、核酸分子、载体或宿主细胞在制备抗氧化剂中的应用。
又一方面，本发明提供了一种抗氧化剂，所述的抗氧化剂包含上述抗氧化多肽、核酸分子、载体或宿主细胞。
又一方面，本发明提供了上述抗氧化多肽、核酸分子、载体或宿主细胞在制备食品、药品或化妆品中的应用。
又一方面，本发明提供了一种食品、药品或化妆品，所述的食品、药品或化妆品包含上述抗氧化多肽、核酸分子、载体或宿主细胞。
具体地，所述的药品还包含任选的药学上可接受的载体。
进一步具体地，所述的药学上可接受的载体包括但不限于：稀释剂、赋形剂、填充剂、润湿剂、崩解剂、矫味剂和粘合剂。
具体地，所述的药品包括但不限于胶囊、片剂、锭剂、丸剂、滴丸、栓剂、喷雾、乳膏、贴剂等形式。
在某些实施例中，本发明所述的抗氧化多肽可通过胰酶酶解大豆蛋白获得；也可通过微生物或者宿主细胞内表达所述多肽片段，分离提纯获得；也可通过化学合成获得。所述的微生物和宿主细胞为公众所知且常规使用的，例如大肠杆菌(E.coli)，放线菌(Actinomycetes)，芽孢杠菌(Bacillus)，链霉菌(Streptomyces)等宿主细胞。本发明所述的多肽分离提纯方法为本领域技术人员公知的方法技术，例如可通过常规的液相色谱法进行分离和纯化。
与现有技术相比，本发明的积极和有益效果在于：
本发明提供了一种大豆蛋白来源的抗氧化多肽(SEQ ID NO:1)，所述的抗氧化多肽的氨基酸序列为YAVRHFLMFQTTMNRWKHAHWFY所示的序列。本发明所述的抗氧化多肽具有较强的抗氧化作用，能够显著清除自由基，且其对于氧化损伤的细胞也具有较好的保护作用。本发明所述的抗氧化多肽可应用于食品、药品、化妆品及添加剂领域，具有十分重要的意义。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1.抗氧化多肽的合成
委托南京金斯瑞生物科技有限公司通过化学合成如下SEQ ID NO:1所示的抗氧化多肽序列：YAVRHFLMFQTTMNRWKHAHWFY。
本发明所述的抗氧化多肽可通过胰酶酶解大豆蛋白获得(酶解条件：pH为7.0-8.0，45℃恒温水浴4h，底物浓度为2.5％，胰酶的添加量为1％)；也可通过微生物或者宿主细胞内表达所述多肽片段，分离提纯获得；也可通过化学合成获得。所述的微生物和宿主细胞为公众所知且常规使用的，例如大肠杆菌(E.coli)，放线菌(Actinomycetes)，芽孢杠菌(Bacillus)，链霉菌(Streptomyces)等宿主细胞。本发明所述的多肽分离提纯方法为本领域技术人员公知的方法技术，例如可通过常规的液相色谱法进行分离和纯化。
实验例1.抗氧化多肽活性测定
1.DPPH自由基清除活性测定
取DPPH溶于95％乙醇，配制终浓度为0.1mmol/L的DPPH溶液。将2mL实施例1制备的抗氧化多肽稀释液或GSH与2mL DPPH溶液混合均匀，室温避光放置30min，于517nm处检测其吸光值，记为Ai。以2mL 95％乙醇+2mL DPPH溶液混合均匀后于517nm处测定的吸光值作为对照，记为Ac。以2mL95％乙醇+2mL抗氧化多肽稀释液混合均匀后于517nm处测定的吸光值记为Aj。
抗氧化多肽对DPPH自由基的清除率根据以下公式进行计算：
DPPH自由基清除率(％)＝[1-(Ai-Aj)/Ac]×100％。
2.ABTS自由基清除活性测定
用超纯水配制ABTS(7mmol/L)与过硫酸钾(2.45mmol/L)的混合溶液，置于室温避光孵育12-16h，制备ABTS自由基阳离子基液。用超纯水稀释ABTS自由基阳离子基液，使其在734nm处测定的吸光值为0.700±0.050，得到ABTS自由基阳离子工作液。取0.1mL实施例1制备的抗氧化多肽稀释液或GSH与3.9mL ABTS自由基阳离子工作液混合均匀后，室温避光放置6min，于734nm处测定其吸光值，记为Ai。以0.1mL超纯水+3.9mL ABTS自由基阳离子工作液混合均匀后于734nm处测定的吸光值作为对照，记为Ac。以0.1mL超纯水+3.9mL抗氧化多肽稀释液混合均匀后于734nm处测定的吸光值记为Aj。
抗氧化多肽对ABTS自由基的清除率根据以下公式进行计算：
ABTS自由基清除率(％)＝[1-(Ai-Aj)/Ac]×100％。
抗氧化多肽活性测定结果如下表1所示。
表1.抗氧化多肽活性
由表1可知，本发明所述的抗氧化多肽的活性较好，优于GSH。
实验例2.抗氧化多肽对氧化损伤细胞的保护作用
取对数生长期的PC12细胞，接种于96孔板中，每孔100μL，细胞浓度为5×104/mL，培养基为DMEM培养基。将其分为四组：空白组，对照组，实施例组，正常组。其中空白组细胞生长36h后，加入10μL PBS，孵育2h后，加入浓度为600mM的H2O2，在37℃、5％ CO2培养箱内孵育2h。对照组细胞生长36h后，加入10μL浓度为0.01mM的GSH，孵育2h后，加入浓度为600mM的H2O2，在37℃、5％ CO2培养箱内孵育2h。实施例组细胞生长36h后，加入10μL浓度为0.01mM的抗氧化多肽，孵育2h后，加入浓度为600mM的H2O2，在37℃、5％ CO2培养箱内孵育2h。正常组细胞生长36h后，加入10μL PBS，孵育2h后，加入等量PBS，在37℃、5％ CO2培养箱内孵育2h。采用MTT法检测细胞的存活率。
检测结果如下表2所示。
表2.细胞存活率
| 组别 | 细胞活率(％) |
| 正常组 | 100.0 |
| 空白组 | 23.4 |
| 对照组 | 56.9 |
| 实施例组 | 95.7 |
由表2可知，本发明所述的抗氧化多肽可显著保护氧化损伤的细胞。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种抗氧化多肽，其特征在于：所述的抗氧化多肽的氨基酸序列为SEQ ID NO:1：YAVRHFLMFQTTMNRWKHAHWFY所示的序列。
2.一种编码权利要求1所述的抗氧化多肽的核酸分子。
3.一种包含权利要求2所述核酸分子的载体。
4.根据权利要求3所述的载体，其特征在于：所述的载体包括质粒、病毒或噬菌体。
5.包含权利要求2所述的核酸分子或权利要求3所述的载体的宿主细胞。
6.根据权利要求5所述的宿主细胞，其特征在于：所述的宿主细胞包括微生物或动物细胞。
7.权利要求1所述的抗氧化多肽、权利要求2所述的核酸分子、权利要求3所述的载体或权利要求5所述的宿主细胞在制备抗氧化剂中的应用。
8.一种抗氧化剂，其特征在于：所述的抗氧化剂包含权利要求1所述的抗氧化多肽、权利要求2所述的核酸分子、权利要求3所述的载体或权利要求5所述的宿主细胞。
9.权利要求1所述的抗氧化多肽、权利要求2所述的核酸分子、权利要求3所述的载体或权利要求5所述的宿主细胞在制备食品、药品或化妆品中的应用。
10.一种食品、药品或化妆品，其特征在于：所述的食品、药品或化妆品包含权利要求1所述的抗氧化多肽、权利要求2所述的核酸分子、权利要求3所述的载体或权利要求5所述的宿主细胞。
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