CN110590912A - 一种新型抗氧化活性多肽oa-vi12及其制备方法与应用 - Google Patents
一种新型抗氧化活性多肽oa-vi12及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种新型抗氧化活性多肽OA‑VI12及其制备方法与应用。所述的新型抗氧化活性多肽OA‑VI12的氨基酸序列为VIPFLACRPLGL。本发明从云南臭蛙皮肤分泌物中提取到一种抗氧化的活性多肽,该多肽具有来源天然、高活性等特点。纯化得到的新型抗氧化活性多肽OA‑VI12显示了较高的稳定性和抗氧化活性,在保护细胞免受紫外线照射且在过氧化氢刺激引起的氧化应激方面发挥了有益的作用,更重要的是,新型抗氧化活性多肽OA‑VI12还起到了保护皮肤免受紫外线照射引起的光损伤的作用,表明本发明新型抗氧化活性多肽OA‑VI12有较大的医疗应用前景。本发明所述的新型抗氧化活性多肽OA‑VI12可作为抗紫外线药物候选物的有益特点。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种新型抗氧化活性多肽OA-VI12及其制备方法与应用。
背景技术
在皮肤老化损伤过程的相关因素中,遗传和生理因素占3%,环境因素占97%,而在众多的环境因素中,太阳辐射是造成光老化损伤的重要原因之一。太阳光中有三种紫外线:长波紫外线UVA(315-400纳米)、中波紫外线UVB(280-315纳米)和短波紫外线UVC(100-280纳米)。几乎所有的UVC都被臭氧层吸收,UVB的生物活性又高于UVA,因此UVB是导致光老化损伤的主要原因。当紫外线照射强度大于每天3.7 mJ/cm2时,可破坏皮肤的氧化还原稳态,继而影响细胞膜脂质过氧化,乳酸脱氢酶(LDH)释放,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)等酶抗氧化剂和还原型谷胱甘肽(GSH)等抗氧化肽水平降低。当皮肤受到过量的紫外线照射时,还会导致活性氧(ROS)的积累,氧化与抗氧化系统的失衡,从而形成氧化应激。此外,ROS介导的氧化应激可引起脂质氧化和DNA损伤。这些氧化损伤会导致皮肤老化和光损伤,如红斑和黑色素沉积,并最终导致皮肤缺陷。
长期的辐射也影响两栖动物独特并且有效的抗氧化肽皮肤防御系统的进化,以应对氧化应激。其皮肤抗氧化防御可分为三类:1)由超氧化物歧化酶SOD组成的高分子量基因编码酶;2)由维生素C(VC)或内源性抗氧化剂如还原型谷胱甘肽和泛素-10组成的非基因编码的低分子量抗氧化剂;3)基因编码的小分子肽。这些抗氧化皮肤防御系统能迅速产生抗氧化肽,清除自由基,减少对皮肤的氧化损伤。自由基可引起脂质过氧化、代谢产物异常积累,最终导致细胞和组织的氧化应激,80%的自由基被认为是由UVA和UVB辐射引起的。因此,抗氧化剂经常通过清除自由基和抵抗UVB光损伤来保持平衡,从而保护身体免受氧化应激引起的疾病的伤害。
局部应用抗氧化剂已成为预防皮肤光老化损伤的重要措施之一。不同来源的抗氧化剂也提供不同的保护使细胞和组织免受自由基的影响。非基因编码的低分子量抗氧化剂,如维生素C,在保护细胞内部结构和含水量方面发挥重要作用;高分子量基因编码酶,如超氧化物歧化酶,则是保护线粒体和组织的防御系统;然而,小分子多肽由于其低能耗、高速度、高效率以及对目前对其鲜有认知,被认为具有广阔的发展前景。目前已知的小分子基因编码肽中的抗氧化肽主要有来自云南蛙的的AOP-P1、来自大绿臭蛙的RL等,但相对于其来源的丰富性来说还是远远不够的。因此,小分子基因编码肽的开发成为了近年来的研究热点。
我们从云南臭蛙皮肤分泌物提取的天然多肽(OA-VI12),序列为“VIPFLACRPLGL”,其具有强大的自由基清除能力,并发现OA-VI12在保护细胞免受紫外线照射和过氧化氢(H2O2)刺激引起的氧化应激方面发挥了有益的作用。更重要的是,OA-VI12还起到了保护皮肤免受紫外线照射引起的光损伤的作用。
发明内容
本发明的第一目的在于提供一种新型抗氧化活性多肽OA-VI12;第二目的在于提供所述的新型抗氧化活性多肽OA-VI12的制备方法;第三目的在于提供所述的新型抗氧化活性多肽OA-VI12的应用。
本发明的第一目的是这样实现的,所述的新型抗氧化活性多肽OA-VI12的氨基酸序列为VIPFLACRPLGL。
本发明的第二目的是这样实现的,包括以下步骤:
A、用电刺激法取云南臭蛙的皮肤分泌物,溶解于Tris-HCl,真空冷冻干燥后得到物料a于-80℃保存备用;
B、将物料a溶解于水中得到物料b,物料b经洗脱、分馏得到样品c;
C、将样品c进行高效液相色谱反相层析得到目标物新型抗氧化活性多肽OA-VI12。
本发明的第三目的是这样实现的,所述的新型抗氧化活性多肽OA-VI12在制备抗氧化保健食品、化妆品或药品中的应用。
本发明从云南臭蛙皮肤分泌物中提取到一种抗氧化的活性多肽,该多肽具有来源天然、高活性等特点。纯化得到的新型抗氧化活性多肽OA-VI12显示了较高的稳定性和抗氧化活性,在保护细胞免受紫外线照射且在过氧化氢刺激引起的氧化应激方面发挥了有益的作用,更重要的是,新型抗氧化活性多肽OA-VI12还起到了保护皮肤免受紫外线照射引起的光损伤的作用,表明本发明新型抗氧化活性多肽OA-VI12”有较大的医疗应用前景。本发明所述的新型抗氧化活性多肽OA-VI12可作为抗紫外线药物候选物的有益特点。
附图说明
图1为本发明抗氧化多肽OA-VI12的HPLC反相C18柱层析图;
图2为本发明抗氧化多肽OA-VI12的序列与质谱图;
图3为本发明抗氧化多肽OA-VI12的体外抗氧化活性图;
图4为本发明抗氧化多肽OA-VI12对HaCaT细胞活力影响图;
图5为本发明抗氧化多肽OA-VI12在细胞水平的活性氧表达图;
图6为 本发明抗氧化多肽OA-VI12在细胞水平乳酸脱氢酶和过氧化氢酶表达图;
图7为本发明抗氧化多肽OA-VI12对动物UVB损伤模型的保护作用;
图8为本发明抗氧化多肽OA-VI12对动物模型真皮与表皮的保护作用;
图9为本发明抗氧化多肽OA-VI12在组织中超氧化物歧化酶与还原性谷胱甘肽水平表达图。
具体实施方式
下面结合实施例和附图对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。
本发明所述的新型抗氧化活性多肽OA-VI12的氨基酸序列为VIPFLACRPLGL。
本发明所述的新型抗氧化活性多肽OA-VI12的制备方法,包括以下步骤:
A、用电刺激法取云南臭蛙的皮肤分泌物,溶解于Tris-HCl,真空冷冻干燥后得到物料a于-80℃保存备用;
B、将物料a溶解于水中得到物料b,物料b经洗脱、分馏得到样品c;
C、将样品c进行高效液相色谱反相层析得到目标物新型抗氧化活性多肽OA-VI12。
B步骤中所述的水为去离子水。
B步骤中所述的洗脱是于Sephadex G-75凝胶过滤柱用TriS-HCl(pH 7.8)与NaCl混合作为洗脱缓冲液洗脱。
B步骤中所述的分馏是使用自动分流收集器每8~12min收集一次分馏物,吸光度监测波长为280nm。
所述的高效液相色谱反相层析是将物料c上样到预先用含0.1%三氟乙酸的超纯水平衡好的Hypersil bds c1柱子,在流速为1ml/min的条件下,用乙腈在线性梯度条件下进行洗脱,检测波长为220nm。
本发明所述的新型抗氧化活性多肽OA-VI12的应用为所述的新型抗氧化活性多肽OA-VI12在制备抗氧化保健食品、化妆品或药品中的应用。
下面以具体实施案例对本发明做进一步说明:
实施例1
1. 样品收集和动物护理
从云南省丽江市采集成年云南臭蛙并安全转运至实验室。臭蛙被集体安置在有面包虫以便臭蛙随时食用的一个50厘米×60厘米的容器里。在让其适应环境一周后,用一个6伏交流电的电子按摩器刺激云南臭蛙皮肤。而后用25 mM的Tis-HCl缓冲液(pH7.8)冲洗臭蛙的皮肤分泌物。将获得的分泌物在4000 g下离心15分钟(4度),收集上清液并冻干。所有样品在-80°C下保存至进一步分析。
2. 纯化程序
将冻干皮肤分泌物样品(500μL,OD280=50)溶解在去离子水中,并应用于Sephadex G-75凝胶过滤柱,用25 mM TriS-HCl(pH 7.8)与0.1 M NaCl混合作为洗脱缓冲液在0.1毫升/分钟流速洗脱。使用BSA-30A自动分馏收集器每10分钟收集一次分馏物,并在280nm处测量吸光度。然后将分离出的样品进行ABST+抗氧化活性测定,并使用hypersil bds c18反相高效液相色谱柱在水中用0.1%(v/v)三氟乙酸(tfa)平衡后,收集和纯化显示活性的组分。用含有0.1%(v/v)TFA的线性梯度(60分钟内0-60%乙腈)在乙腈中以1 ml/min的流速进行洗脱,并在220 nm处进行监测。显示ABTS+抗氧化活性的部分被收集并冻干以进行第二轮hplc纯化。
3. 肽一级结构测定
用质谱仪与α-氰基-4-羟基肉桂酸线性模式测定天然OA-VI12的分子量和纯度。根据制造商的标准程序和软件,执行所有协议并分析所有数据。采用日本岛津PPSQ-31A蛋白测序仪确定完整的氨基酸序列。最后,根据制造商提供的程序测定肽的半胱氨酸残基。
4. OA-VI12的cDNA基因克隆
基于我们先前建立的云南臭蛙皮肤cDNA文库,我们筛选了编码成熟肽的cDNA。所使用的引物,即5’PCR引物(5’-CCAA(G/C)ATGTTCACC(T/A)TGAAAA-3’)和3’PCR引物(5’-ATTCAGGCCGAGGCCG ACATG-3’),由BGI公司生产。聚合酶链反应(PCR)进行(94°C 2分钟,92°C 10秒,50°C 30秒,72°C 40秒)后所得产物用BioTekes DNA凝胶提取试剂盒(中国)检索,并连接到PMD19-T载体。PCR产物随后被克隆到大肠杆菌中,独立克隆用于ABI 3730XL DNA测序仪上的DNA测序。
5. 肽的合成
成熟的OA-VI12肽(VIPFLACRPLGL)由中国武汉生物技术有限公司合成。用质谱法分析了与天然OA-VI12共洗脱的市售OA-VI12,证实其与天然OA-VI12相同。合成的样品粉末在-20°C下储存。实验前,样品溶解在去离子水中,用质谱法测定样品中不存在二聚体,然后立即使用。
6. 抗氧化活性检测
自由基清除活性由2,2-叠氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)测定。ABTS原液是通过将2.8 M过硫酸钾与7 mM ABTS在水中在黑暗中孵育6 h来制备的。原液用去离子水稀释后立即使用。将溶于50 μL水的样品与50 μL稀释的储备溶液混合,在黑暗中室温保存30分钟,以样品溶剂(相同体积)作为阴性对照。最后在415 nm处吸光度的降低表明抗氧化活性。ABTS+清除活性用(Ablank-Asample)×100/Ablank计算。
2,2-二苯基-1-苦草酰(DPPH)自由基清除试验是将190 μL样品溶于乙醇的5×10-5M DPPH和10μl样品溶液组成的分析混合物,在室温下加入96孔微量滴定板30 min,样品溶剂为阴性对照。在517 nm处测定吸光度,并通过(Ablank-Asample)×100/Ablank计算DPPH清除活性(%)。
Fe3+清除研究:将不同浓度的样品与2.5 ml 1% K3Fe(CN)6和2.5 ml 0.2 M磷酸盐缓冲液(pH 6.6)在50℃下孵育20分钟,然后添加2.5 ml 10%TCA。然后在室温下将溶液在3000 g下离心10分钟,上层(2.5 mL)然后与蒸馏水(2.5 mL)和FeCl3(0.5 mL;0.1%)混合。在700纳米的空白处读取吸收率。以维生素C为阳性对照,根据反应混合物吸光度的增加测定还原力。
一氧化氮清除实验:将不同浓度的样品与5 mM硝普钠在蒸馏水中孵育。在25°C下培养120分钟后,去除0.5毫升溶液,后与0.5毫升Griess试剂混合。然后使用微板阅读器在550 nm处测量吸光度。NO清除活性用(Ablank-Asample)×100/Ablank计算。
7. 细胞培养
(DMEM/F12培养基中培养人角质形成细胞(HaCaT)细胞,辅以1% 双重抗生素(100单位/毫升青霉素,100单位/毫升链霉素)和10% 胎牛血清在37度,含 5%的二氧化碳加湿培养箱中培养。
8. OA-VI12对UVB照射的HaCaT细胞活力的影响
用磷酸盐缓冲液(PBS)洗涤HaCaT细胞三次后将细胞分成三组:UVB照射模型组、空白组和样品组。然后将细胞接种于96孔板(3×103细胞/孔)中,其中含有90 μL的DMEM/F12(无血清)。用不同浓度的OA-VI12(0.5、1、5和10 μM)或10 μM VC对样品组的细胞进行预处理2 h后讲其暴露于9W/01紫外线灯(30 mJ/c m2),在37°C培养箱中培养,而后添加20 μL MTS(Promega,Madison,WI,USA)。继续培养细胞2-4 h,在490nm的酶标仪上进行检测,以探求OA-VI12或VC对HaCaT细胞活力的影响。
9. OA-VI12对过氧化氢处理的HaCaT细胞活力的影响
按上述方法将细胞分组,接种于培养板上。用200 μM的H2O2处理细胞,在37°C下用不同浓度的OA-VI12或VC预处理2小时。MTS法检测细胞活力与上述方法相同。
10. OA-VI12对H2O2和UVB照射或过氧化氢刺激的HaCaT细胞ROS水平的影响
将细胞分为4组(过氧化氢处理组、UVB照射组、空白组和样品组)并接种于6孔板(1.2×106细胞/孔)中。用不同浓度的OA-VI12或VC在37℃下处理2小时后用UVB照射细胞或用过氧化氢处理细胞2小时。而后,将细胞重新悬浮在含有1 μM H2DCFDA荧光探针在37℃下孵育45分钟。用PBS洗涤三次,用0.25%胰蛋白酶消化4min,1000g离心5min,最后用1mL冷PBS悬浮细胞并用流式细胞术检测细胞内ROS的产生。
11. OA-VI12对过氧化氢或UVB刺激后的HaCaT的过氧化氢酶与乳酸脱氢酶活性的影响
用PBS洗涤HaCaT细胞三次,然后用0.25%胰蛋白酶消化4分钟,在37℃下用1 000 g离心5分钟,用0.5毫升冷PBS悬浮,然后用超声波细胞粉碎机破碎细胞。将破碎的细胞混合物在12 000 g下4℃离心15分钟,然后收集上清液采用商品化试剂盒对细胞上清液进行检测,并按标准操作。
12. 紫外线照射背部皮肤光损伤动物模型
在昆明医科大学实验动物室中,将小鼠置于单独的通风笼中。实验小鼠在22±2°c的空调室内和12/12h的明暗循环条件下,用食物和自由饮用水喂养。实验前小鼠进行7d驯化,然后随机分为4组(每组6只):对照组、模型组(UVB)、样品组(UVB+OA-VI12)和阳性对照组(UVB+VC)。用1%戊巴比妥钠(0.1毫升/20克体重)腹腔注射(I.P)麻醉小鼠,然后脱毛并暴露于紫外线照射中,在前7天每天照射150兆焦耳/平方厘米,接下来每隔一天照射300兆焦耳/平方厘米。用紫外辐射计监测辐射强度。OA-VI12(10 μM)或VC(10 μM)(均为液体)在受照射的小鼠皮肤上每日局部给药一次。最后一次UVB照射后,处死小鼠,取背部无毛皮肤,进行以下步骤。在最后一天拍摄了受损皮肤的照片。鉴于小鼠的个体差异,最具代表性的图片放在结果中。
13. 苏木精-伊红(H&E)和masson三色染色在UVB辐射小鼠皮肤中的应用
将小鼠皮肤组织样本固定在4%多聚甲醛中24小时,然后按照先前的研究进行脱水和透明化。为了进行组织学分析,组织样本被切成6 μm厚的切片,并用H&E或Masson三色试剂染色。Image J软件用于估计表皮层和真皮层的厚度。
14.UVB照射小鼠皮肤中超氧化物歧化酶和谷胱甘肽的测定
取小鼠皮肤组织样品(0.1g)在1 mL冷pbs中匀浆3 min,然后在10 000g下在4℃离心15min。收集上清液用于测定超氧化物歧化酶(超氧化物歧化酶测定试剂盒)和谷胱甘肽(谷胱甘肽测定试剂盒)水平。
结果如图1~图9,具体说明如下:如图1所示,云南臭蛙皮肤分泌物在Sephadex G-75凝胶过滤后被分成约10个组分。以10min为间隔收集各组分,测定其ABTS+的清除活性。将具有清除ABTS+自由基活性的样品汇集在一起(图1A),并进行c18柱rp-hplc进一步纯化(图1B)。再次显示ABTS+清除活性的样品被收集用于第二轮c18 rp-hplc纯化(图1C),获得的一个峰显示出理想的形状和与前一个hplc程序相同的洗脱时间。纯化后的样品具有ABTS+自由基清除活性,以此初步建立了其结构。
如图2所示,测定显示抗氧化活性的纯化样品的主要结构并使用edman测序仪,从n-末端到c-末端的氨基酸序列为‘VIPFLACRPLGL’。
图3:在0.3 μM浓度下,OA-VI12对ABTS+的清除率为7.9%,而在5 μM浓度下,OA-VI12对ABTS+的清除率为66.6%,表现出剂量依赖性(图3A)。此外,OA-VI12(5 μM)在10秒内达到最大清除率(图3B)。在最大浓度为5 μM时,OA-VI12清除约10.1%的DPPH,但当浓度降至2.5 μM时活性消失了(图3D)。这些结果表明OA-VI12的DPPH清除活性弱于ABTS+清除活性。OA-VI12在最大浓度为5 μM时表现出对Fe3+的清除活性,但当浓度降至2.5 μM时,这种活性被消除(图3E)。这些结果表明OA-VI12表现出弱的Fe3+清除活性。OA-VI12通过抑制NO的供体SNP来抑制 NO释放。在浓度为1.25 μM时,OA-VI 12不具有清除活性,但在浓度为5 μM时清除率达到51.25%,说明OA-VI12具有剂量依赖性的清除自由基活性(图3C)。
图4:OA-VI12(0.5-10 μM)对HaCaT细胞的活力没有明显影响。当UVB照射(图4A)或用过氧化氢(图4B)刺激时,HaCaT细胞的活性显著降低;然而,用OA-VI12预处理后可以保护HaCaT细胞免受UVB照射(图4A)和过氧化氢(图4B)引起的活性降低。浓度为10 μM 的OA-VI12 预处理组的细胞活力与VC组(阳性对照组)相同,说明OA-VI12对细胞活力有明显的保护作用。
图5:OA-VI12在UVB和H2O2刺激的HaCaT细胞中表现出抗氧化活性。与对照组相比,随着UVB照射(图5A,B)和H2O2刺激(图5C,D),细胞内ROS荧光强度显著增加。OA-VI12的预处理显著降低UVB照射和H2O2刺激后细胞内ROS的释放。
图6:UVB辐射(图6A)和H2O2(图6B)对HaCaT细胞的攻击均能显著提高乳酸脱氢酶活性,降低过氧化氢酶活性。UVB照射后乳酸脱氢酶活性升高(117.63±8.52%),过氧化氢酶活性降低(77.31±4.34%);而OA-VI12(10 μM)预处理后乳酸脱氢酶活性显著降低(100.68±10.9%),过氧化氢酶活性显著升高(54.49±3.15%)(图6A,C)。同样的,H2O2刺激HaCaT细胞后,细胞释放的乳酸脱氢酶活性明显升高,过氧化氢酶活性降低;而用OA-VI12预处理组的乳酸脱氢酶活性下降92.12±4.78%,过氧化氢酶活性上升43.67±4.62%(图6B,D)。
图7:采用紫外线致小鼠皮肤损伤模型,以此来评价OA-VI12的光保护作用。紫外线照射21d后,小鼠背部皮肤形态发生变化,出现皮肤损伤、红斑和表皮增厚。而OA-VI12能有效地抑制了光损伤引起的上述现象,表明OA-VI12具有相对有益的保护活性。与正常皮肤相比,UVB照射后表皮厚度显著增加160.81±6.93 μm,OA-VI12的治疗会使表皮厚度减轻到62.14±3.39 μm(图8A,C)。UVB照射后皮肤真皮厚度增加355.92±8.28 μm,而OA-VI12的治疗可以使皮肤厚度减少到266.67±6.36μm(图8B,D)。Masson三色染色显示UVB照射可以降低胶原纤维的含量,OA-VI12和VC处理都抑制了胶原纤维的含量(图8)。
图9:OA-VI12降低UVB照射小鼠皮肤的内源性氧化应激。在UVB照射的小鼠皮肤中,内源性抗氧化酶超氧化物歧化酶活性(图9A)和抗氧肽谷胱甘肽(图9B)水平分别显著降低了46.57±0.17%和91.02±6.25%。相反,应用抗氧化剂OA-VI12后,其水平分别升高了76.05±2.94%和68.09±6.01%。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种新型抗氧化活性多肽OA-VI12及其制备方法与应用
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213> 新型抗氧化活性多肽OA-VI12的氨基酸序列
<400> 1
Val Ile Pro Phe Leu Ala Cys Arg Pro Leu Gly Leu
1 5 10
Claims (7)
1.一种新型抗氧化活性多肽OA-VI12,其特征在于所述的新型抗氧化活性多肽OA-VI12的氨基酸序列为VIPFLACRPLGL。
2.一种权利要求1所述的新型抗氧化活性多肽OA-VI12的制备方法,其特征在于包括以下步骤:
A、用电刺激法取云南臭蛙的皮肤分泌物,溶解于Tris-HCl,真空冷冻干燥后得到物料a于-80℃保存备用;
B、将物料a溶解于水中得到物料b,物料b经洗脱、分馏得到样品c;
C、将样品c进行高效液相色谱反相层析得到目标物新型抗氧化活性多肽OA-VI12。
3.根据权利要求2所述的制备方法,其特征在于B步骤中所述的水为去离子水。
4.根据权利要求2所述的制备方法,其特征在于B步骤中所述的洗脱是于Sephadex G-75凝胶过滤柱用TriS-HCl(pH 7.8)与NaCl混合作为洗脱缓冲液洗脱。
5.根据权利要求2所述的制备方法,其特征在于B步骤中所述的分馏是使用自动分流收集器每8~12min收集一次分馏物,吸光度监测波长为280nm。
6.根据权利要求2所述的制备方法,其特征在于所述的高效液相色谱反相层析是将物料c上样到预先用含0.1%三氟乙酸的超纯水平衡好的Hypersil bds c1柱子,在流速为1ml/min的条件下,用乙腈在线性梯度条件下进行洗脱,检测波长为220nm。
7.一种权利要求1所述的新型抗氧化活性多肽OA-VI12的应用,其特征在于所述的新型抗氧化活性多肽OA-VI12在制备抗氧化保健食品、化妆品或药品中的应用。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112480209A (zh) * | 2020-11-16 | 2021-03-12 | 昆明学院 | 一种抗皮肤光损伤保护活性多肽rl-pl9及其应用 |
CN112574282A (zh) * | 2020-11-16 | 2021-03-30 | 昆明学院 | 一种抗氧化损伤皮肤保护活性多肽rl-pp10及其应用 |
CN115043906A (zh) * | 2022-05-27 | 2022-09-13 | 嘉文丽(福建)化妆品有限公司 | 一种抗氧化活性多肽Cos-16及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090093408A1 (en) * | 1999-05-17 | 2009-04-09 | Bridon Dominique P | Protection of exendin-4 peptides through conjugation |
CN101724632A (zh) * | 2009-07-15 | 2010-06-09 | 吉林大学 | 牛蛙皮肤活性肽及其基因和在制药中的应用 |
CN104530192A (zh) * | 2015-01-19 | 2015-04-22 | 云南民族大学 | 一种抗氧化多肽aop-om1及其制备方法与应用 |
CN107344959A (zh) * | 2017-06-29 | 2017-11-14 | 常州市千康生物科技有限公司 | 一种促皮肤修复超短肽Purin‑WH、其制备方法及应用 |
-
2019
- 2019-10-12 CN CN201910966155.3A patent/CN110590912A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090093408A1 (en) * | 1999-05-17 | 2009-04-09 | Bridon Dominique P | Protection of exendin-4 peptides through conjugation |
CN101724632A (zh) * | 2009-07-15 | 2010-06-09 | 吉林大学 | 牛蛙皮肤活性肽及其基因和在制药中的应用 |
CN104530192A (zh) * | 2015-01-19 | 2015-04-22 | 云南民族大学 | 一种抗氧化多肽aop-om1及其制备方法与应用 |
CN107344959A (zh) * | 2017-06-29 | 2017-11-14 | 常州市千康生物科技有限公司 | 一种促皮肤修复超短肽Purin‑WH、其制备方法及应用 |
Non-Patent Citations (4)
Title |
---|
SAIGE YIN等: "Potential skin protective effects after UVB irradiation afforded by an antioxidant peptide from Odorrana andersonii", 《BIOMEDICINE & PHARMACOTHERAPY》 * |
XIAOQING CAO等: "Identification and Characterization of a Novel Gene-encoded Antioxidant Peptide from Odorous Frog Skin", 《PROTEIN & PEPTIDE LETTERS》 * |
罗永康: "《生物活性肽功能与制备》", 31 March 2019 * |
陈刚: "《激光医疗技术》", 31 March 2018 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112480209A (zh) * | 2020-11-16 | 2021-03-12 | 昆明学院 | 一种抗皮肤光损伤保护活性多肽rl-pl9及其应用 |
CN112574282A (zh) * | 2020-11-16 | 2021-03-30 | 昆明学院 | 一种抗氧化损伤皮肤保护活性多肽rl-pp10及其应用 |
CN115043906A (zh) * | 2022-05-27 | 2022-09-13 | 嘉文丽(福建)化妆品有限公司 | 一种抗氧化活性多肽Cos-16及其应用 |
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