CN110590913A - 一种抗氧化损伤皮肤保护活性多肽aop-p1及其制备方法与应用 - Google Patents
一种抗氧化损伤皮肤保护活性多肽aop-p1及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种抗氧化损伤皮肤保护活性多肽AOP‑P1及其制备方法与应用。所述的抗氧化损伤皮肤保护活性多肽AOP‑P1的氨基酸序列为FLPGLECVW。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。纯化得到的抗氧化损伤皮肤保护活性多肽AOP‑P1显示了较高的稳定性和抗氧化活性,可以有效保护皮肤免受紫外线照射引起的光损伤,表明本发明抗氧化损伤皮肤保护活性多肽AOP‑P1”有较大的医疗应用前景。本发明所述的抗氧化损伤皮肤保护活性多肽AOP‑P1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,具体涉及一种抗氧化损伤皮肤保护活性多肽AOP-P1及其制备方法与应用。
背景技术
在过去的几年中,随着环境污染的增加和地球臭氧层的消耗,紫外线(UV)照射的水平,特别是UVA和UVB已经严重增加。紫外线照射(280-320纳米)是对人体皮肤最有害的紫外线,它可以在体内造成氧化应激。当紫外线照射强度大于3.7mJ/cm2/天时,会破坏皮肤氧化还原平衡。体内抗氧化剂会减少,过氧化产物的积累也将增加。这些氧化损伤使皮肤光损伤的发生,并最终导致皮肤癌的发展。目前使用最广泛抗氧化剂都存在不同方面的缺陷,如活性低、稳定性差,难以大规模合成更甚者存在潜在毒性和致癌性。因此,开发新的抗氧化剂对于处理氧化损伤是非常重要的。肽抗氧化剂由于具有活性高、毒性代谢产物少、与其它药物相互作用少、易于大规模合成等特点而引起人们的广泛关注。
两栖动物作为一种皮肤裸露且栖息地复杂的物种,很容易受到外部伤害,因而,它们形成了一种保护性的皮肤防御系统。考虑到两栖动物皮肤的复杂功能、在水生和陆地环境中保持皮肤完整性的需要以及表皮的脆弱性,两栖动物(与其他脊椎动物相比)必须有效地保护其皮肤免受氧化应激。之前关于两栖动物分泌物的研究已经确定了多种功能多样的生物活性肽,包括抗菌肽、抗氧化肽、伤口愈合肽、细胞因子、神经调节肽和神经毒素等。而生长在高海拔强且受紫外线辐射的云南臭蛙,由于其独特的生理特点被认为是应对光损伤的抗氧化应激肽的主要来源之一。
我们从云南臭蛙的皮肤分泌物中分离出一种新型肽AOP-P1,它可以有效保护皮肤免受紫外线照射引起的光损伤,且具有较高的稳定性及抗氧化活性。因此,天然产生的抗氧化肽AOP-P1可作为抗紫外线药物的候选物。
发明内容
本发明的第一目的在于提供一种抗氧化损伤皮肤保护活性多肽AOP-P1;第二目的在于提供所述的抗氧化损伤皮肤保护活性多肽AOP-P1的制备方法;第三目的在于提供所述的抗氧化损伤皮肤保护活性多肽AOP-P1的应用。
本发明的第一目的是这样实现的,所述的抗氧化损伤皮肤保护活性多肽AOP-P1的氨基酸序列为FLPGLECVW。
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抗氧化损伤皮肤保护活性多肽AOP-P1。
本发明的第三目的是这样实现的,所述的抗氧化损伤皮肤保护活性多肽AOP-P1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。纯化得到的抗氧化损伤皮肤保护活性多肽AOP-P1显示了较高的稳定性和抗氧化活性,可以有效保护皮肤免受紫外线照射引起的光损伤,表明本发明抗氧化损伤皮肤保护活性多肽AOP-P1”有较大的医疗应用前景。本发明所述的抗氧化损伤皮肤保护活性多肽AOP-P1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AOP-P1的HPLC反相C18柱层析图;
图2为本发明抗氧化多肽AOP-P1对HaCaT细胞活力影响图;
图3为本发明抗氧化多肽AOP-P1在细胞水平丙二醛,乳酸脱氢酶和过氧化氢酶表达图;
图4为本发明抗氧化多肽AOP-P1对动物UVB损伤模型的保护作用;
图5为本发明抗氧化多肽AOP-P1对动物模型真皮与表皮的保护作用;
图6为本发明抗氧化多肽AOP-P1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。
本发明所述的抗氧化损伤皮肤保护活性多肽AOP-P1的氨基酸序列为FLPGLECVW。
本发明所述的抗氧化损伤皮肤保护活性多肽AOP-P1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抗氧化损伤皮肤保护活性多肽AOP-P1。
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 ODS2 5 mm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。
本发明所述的抗氧化损伤皮肤保护活性多肽AOP-P1的应用为所述的抗氧化损伤皮肤保护活性多肽AOP-P1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Tris-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℃),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溶解在去离子水中,并应用于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。使用自动分馏收集器每10分钟收集一次分馏物,并在280 nm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的线性梯度在乙腈中进行洗脱,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AOP-P1的分子量和纯度。采用蛋白测序仪确定完整的氨基酸序列,所述保护肽的氨基酸序列为“FLPGLECVW”。
4. AOP-P1对紫外线照射与过氧化氢处理下HaCaT细胞活性影响
将生长状态良好的细胞用磷酸盐缓冲液(PBS)洗涤三次,分为四组(UVB辐照模型组、过氧化氢处理组、空白组和样品组),接种于96孔板(3000个细胞/孔)。在暴露于9 w/01 UVB灯或200 μM过氧化氢前,样品组中的细胞在37°C下用不同浓度的AOP-P1(0.5、1、5和10μM)或10 μM VC预处理2 h。UVB辐照强度为30 mJ/cm2。而后将20 μL MTS加入到培养细胞中再培养2-4小时,在490 nm的测试波长下,检测AOP-P1和VC对Hacat细胞活力的影响。
5. AOP-P1对过氧化氢处理或UVB照射下HaCaT细胞中乳酸脱氢酶,过氧化氢酶和丙二醛水平的影响
HaCaT细胞用PBS洗涤三次。用0.25%胰蛋白酶(Bi,Israel)消化4分钟,在37°C以1000g离心10分钟后,用0.5 ml冷PBS悬浮细胞,在冰盒中用超声波细胞粉碎机粉碎。然后在4°C条件下,以12000 g离心分离15分钟,收集上清液使用试剂盒测定丙二醛和过氧化氢酶的水平。HaCaT细胞培养基上清液使用试剂盒测定乳酸脱氢酶的水平。
6. AOP-P1对皮肤光损伤的保护活性
选取6周龄雌性昆明小鼠(20±2g)进行试验。实验前一周驯养,然后随机分为4组(每组6只):对照组、模型组(UVB)、样品组(UVB+AOP-P1)、阳性对照组(UVB+VC)。
将小鼠背部皮肤脱毛,然后暴露于UVB照射(UVB灯,TL20 W/12,飞利浦,荷兰)中,在最初的7天内每天150 mJ/cm2,在接下来的2周内每隔一天300 mJ/cm2。抗氧化肽AOP-P1(10 μM)或VC(10 μM)(均为液体)在小鼠皮肤的辐照部位每日局部施用一次。最后一次紫外线照射后,处死小鼠,迅速取出背部无毛皮肤,进行以下步骤。最后一天拍摄了照片损伤小鼠皮肤的图像。
将一部分皮肤组织在4%多聚甲醛中固定24-48小时,再由70%到100%的无水乙醇脱水,二甲苯透明,然后于石蜡中包埋。将组织样本切成6 μm厚的切片,并用H&E和Masson染色液染色。结果通过正置显微镜观察。
将另一部分皮肤组织进行匀浆(0.1g皮肤组织在1ml冷PBS中匀浆3min),然后在4°C条件下10000g离心15min,应用试剂盒进行超氧化物歧化酶与谷胱甘肽水平检测。
结果如图1~图6,具体说明如下:图2结果表明:AOP-P1(0.5~10 μM)对HaCaT的细胞活力无明显影响。相反,当暴露于UVB照射或用H2O2处理时,HaCaT的活性显著降低 (图2A)。并且,AOP-P1的预处理可以显著地逆转因UVB照射和H2O2处理导致的细胞活力降低(图2B)。当AOP-P1预处理组浓度为10 μM时,细胞活力与VC(阳性对照)相当,表明AOP-P1对细胞活力有显著的保护作用。如图3所示,在HaCaT中的UVB暴露和H2O2刺激可导致丙二醛水平(图3A、B)和乳酸脱氢酶活性(图3C、D)显著增加与过氧化氢酶活性(图3E、F)降低。而AOP-P1(10μM)预处理能显著降低丙二醛水平和乳酸脱氢酶活性(图3A,B,C,D),并且增加过氧化氢酶的活性(图3E,F)。在紫外线照射21天后,老鼠背部皮肤的发生病理改变:皮肤损伤,红斑出现和表皮增厚。从宏观上看,抗氧化剂AOP-P1能有效抑制上述光损伤现象,如图4。与正常皮肤相比,紫外线照射后表皮厚度(图5A,C)和真皮厚度(图5B,D)显著增加,而AOP-P1明显减少这种增厚。Masson的三色染色显示,紫外线照射可以降低胶原纤维的含量,而AOP-P能抑制胶原纤维的减少(图5B,D)。在UVB照射的小鼠皮肤中,超氧化物歧化酶活性(图6A)和谷胱甘肽水平(图6B)均显著降低,同样的抗氧化剂AOP-P1可分别提高其表达水平,以此进行光保护与抗氧化应激作用(图6)。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种抗氧化损伤皮肤保护活性多肽AOP-P1及其制备方法与应用
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> 抗氧化损伤皮肤保护活性多肽AOP-P1的氨基酸序列
<400> 1
Phe Leu Pro Gly Leu Glu Cys Val Trp
1 5
Claims (7)
1.一种抗氧化损伤皮肤保护活性多肽AOP-P1,其特征在于所述的抗氧化损伤皮肤保护活性多肽AOP-P1的氨基酸序列为FLPGLECVW。
2.一种权利要求1所述的抗氧化损伤皮肤保护活性多肽AOP-P1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抗氧化损伤皮肤保护活性多肽AOP-P1。
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 ODS2 5 mm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所述的抗氧化损伤皮肤保护活性多肽AOP-P1的应用,其特征在于所述的抗氧化损伤皮肤保护活性多肽AOP-P1在制备抗氧化保健食品、化妆品或药品中的应用。
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