CN115043906A - 一种抗氧化活性多肽Cos-16及其应用 - Google Patents
一种抗氧化活性多肽Cos-16及其应用 Download PDFInfo
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Abstract
本发明公开了一种抗氧化活性多肽Cos‑16及其应用,其氨基酸序列如SEQ ID NO.01所示。本发明活性高、无细胞毒性,具有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。本发明能够保护皮肤免受紫外线照射引起的光损伤的作用,具有较大的医疗应用前景。
Description
技术领域
本发明属于功能多肽技术领域,具体涉及一种抗氧化活性多肽Cos-16及其应用。
背景技术
光老化的特征是由紫外线照射引起的皮肤随时间变化的复杂过程。它会导致皮肤过早老化,并叠加在由时间老化引起的变化上。光老化临床症状包括皱纹、痣、斑驳的色素沉着过度、半透明性丧失和弹性下降。这些特征进一步受到个体种族和遗传的影响。
紫外线是皮肤光老化产生皱纹的元凶,它穿透皮肤后,成纤维细胞会大量生成基质金属蛋白酶(MMPs),它不能促进合成胶原蛋白,相反却会破坏真皮中的胶原蛋白和弹性纤维。
抗衰老一直是化妆品行业中长期关注的焦点,延缓皮肤衰老是大部分人的追求目标之一。一直以来都有很多人热衷于抗衰老实验的研究,并开发出很多有关抗衰老的化妆品。
目前,在抗衰老化妆品中,多肽是最热门的几种抗衰老成分之一。具有抗衰老活性的多肽的研究因此也是层出不穷。
发明内容
本发明目的在于提供一种抗氧化活性多肽Cos-16。
本发明的另一目的在于提供上述抗氧化活性多肽Cos-16的应用。
本发明的技术方案如下
一种抗氧化活性多肽Cos-16,:其氨基酸序列如SEQ ID NO.01所示。
上述抗氧化活性多肽Cos-16在制备抗氧化保健组合物中的应用。
一种抗氧化保健组合物,其有效成分包括上述抗氧化活性多肽Cos-16。
在本发明的一个优选实施方案中,其有效成分为上述抗氧化活性多肽Cos-16。
上述抗氧化活性多肽Cos-16在制备化妆组合物中的应用。
一种化妆组合物,其有效成分包括上述抗氧化活性多肽Cos-16。
在本发明的一个优选实施方案中,其有效成分为上述抗氧化活性多肽Cos-16。
上述抗氧化活性多肽Cos-16在抗紫外线组合物中的应用。
一种抗紫外线组合物,其有效成分包括上述抗氧化活性多肽Cos-16。
在本发明的一个优选实施方案中,其有效成分为上述抗氧化活性多肽Cos-16。
本发明的有益效果是:
1、本发明活性高、无细胞毒性,具有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。
2、本发明能够保护皮肤免受紫外线照射引起的光损伤的作用,具有较大的医疗应用前景。
附图说明
图1为本发明实施例1制得的抗氧化活性多肽Cos-16的HPLC反相C18柱层析图。
图2为本发明实施例1制得的抗氧化活性多肽Cos-16的质谱图。
图3为本发明实施例2中抗氧化活性多肽Cos-16的体外自由基清除活性图之一。
图4为本发明实施例2中抗氧化活性多肽Cos-16的体外自由基清除活性图之二。
图5为本发明实施例3中抗氧化活性多肽Cos-16保护HaCaT细胞活力免受UVB诱导损伤影响图。
图6为本发明实施例4中抗氧化活性多肽Cos-16抑制UVB诱导的HaCaT细胞中的ROS积累。
图7为本发明实施例5中抗氧化活性多肽Cos-16对HaCaT细胞过氧化氢酶的活性调控图。
图8为本发明实施例5中抗氧化活性多肽Cos-16对HaCaT细胞谷胱甘肽过氧化物酶活性调控图。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1多肽合成、纯化与鉴定
称取适量2-cl-Trt树脂,用二氯甲烷溶胀20min。设置取代度(SD),目标序列比较短设置SD为0.3,按定量称取1.3eq(eq=树脂量×取代度)的第一个氨基酸Fmoc-L-oh。树脂溶胀后用二氯甲烷反复清洗2-3次,用二氯甲烷作为反应溶剂DIEA催化;加入第一个氨基酸反应1-2H,二氯甲烷易挥发,过程适当补充二氯甲烷。反应结束后按体积1∶1加入适量的甲醇封头30min。封头结束洗涤3次后用20%pip脱除fmoc 10min。脱保护结束后洗涤5-6次检测阳性,表明第一个氨基酸已经成功连接在树脂上,就可以开始后面的反应了,后面的反应的3eq投量。反应方式HOBT+DIC或者HBTU+DIEA都可,反应时长1H。结束后检测阴性就进入下一步骤,阳性重复上一步骤即可,直至完全结束。抽干树脂,使用TFA∶EDTt∶TIS∶H2O=95∶2∶2∶1按1g树脂配比10mL切割液标准放入摇床裂解2h。过滤树脂,放入冰***析出离心得到粗品多肽。通过HPLC-ESI-MS对粗品多肽提纯鉴定,获得如SEQ ID NO.01和图2所示的抗氧化活性多肽Cos-16,氨基酸序列为ACFLEGL。
上述HPLC条件:inertsil ODS-SPC18柱(4.6mm×250mm,5um),柱温为室温,进样量30μL。流动相为0.1%三氟乙酸水溶液,等度冲洗,流速1mL/min。HPLC纯化鉴定结果如图1和表1所示,可知4.2min出峰为Cos-16,经提纯后Cos-16纯度达到95.75%。质谱条件:电喷雾电离源正离子模式ESI,选择监测模式Scan和SIR,全扫分子量区间100~500,特征扫描分子量297,喷雾电压5.5KV,雾化气流量15Lh,离子源温度500℃,锥孔电压15V,碰撞气体为氮气,数据采集扫描时间20s。
表1
实施例2 DPPH检测
使用DPPH自由基清除能力检测试剂盒(碧云天,货号:BC4750)检测实施例1制得的抗氧化活性多肽Cos-16清除自由基的活性。取500μL试剂二溶液,冰上解冻后,加入试剂一溶液(无水乙醇)2625μL,充分混匀,置于冰上待用。取1mL试剂盒提供的提取液加入到试剂三的棕色玻璃管中,充分混匀后,分装保存50μL每管,置于-20℃;从-20℃冰箱中取出多肽母液,加入DMSO稀释,置于冰上待用同时准备溶剂备用。分光光度计预热30min以上,调节波长至515nm,无水乙醇调零。将10mg/mL维生素C溶液(试剂三)用提取液配制成大于0.3mg/mL的维生素C溶液待用(向储存离心管加入1.2mL的提取液,充分混匀)。在1.5mL EP管中分别加入抗氧化活性多肽Cos-16溶液、DMSO、提取液以及工作液,使多肽终浓度为1.25uM、2.5uM、5uM。混匀后室温避光静置30min,于515nm处的吸光度。空白管、阳性对照管、对照管和测定管的吸光值分别记为A空白、A阳性对照、A对照和A测定。空白管只需测1-2次。仪器开机预热,调试参数。使用仪器进行检测,记录检测数据并分析得出结果如图3、图4所示,图3表明抗氧化活性多肽Cos-16具有较强的自由基清除活性,并且具有浓度依赖性,图4表明抗氧化活性多肽Cos-16浓度为5uM时,与对照组相比,具有显著的自由基清除活性。
实施例3抗氧化活性多肽Cos-16对HaCaT细胞活力影响
将HaCaT细胞铺至96孔板(每孔约1×104个细胞),24h细胞完全贴壁后,去除培养基,加入含不同浓度的抗氧化活性多肽Cos-16的100μL无血清培养基,孵育24h后,用PBS洗3遍,并在孔板底部留50μL PBS溶液,采用UVB紫外辐照仪对HaCaT细胞进行20mJ/em2UVB辐照剂量辐照。辐照后,立即加入新鲜培养基,继续培养24h后,在每孔中加入10ul的CCK-8试剂,孵育2h后,在450nm的波长下,检测OD值。结果如图5所示,可以看到,抗氧化活性多肽Cos-16对HaCaT细胞无毒性且当HaCaT细胞受到UVB诱导损伤时,能够随着Cos-16浓度的增加提高HaCaT细胞的活力。
实施例4细胞内ROS水平检测
使用荧光测定法(DCFH-DA测定法)测量细胞内ROS水平。接种在96孔板中的HaCaT细胞用指定浓度的5mM N-乙酰半胱氨酸(NAC)或Cos-16预处理1小时。然后将细胞在加10μMH2DCFDA的无血清培养基中孵育30分钟,并暴露于20mJ/cm2UVB,然后用0.1%Triton X-100裂解10分钟。在485nm的激发和528nm的发射下测量荧光强度。细胞内ROS水平表示为荧光强度的倍数变化,结果如图6所示,Cos-16能够抑制紫外线诱导的HaCaT细胞中的ROS积累。
实施例5 HaCaT细胞内GPx、CAT酶活性的测定
实验设置四个不同浓度梯度组,每组设置3个复孔。将处于对数生长期的HaCaT接种在24孔板中,每孔注入1mL细胞悬液,接种密度为以1×105/mL,然后将其置于5%CO2培养箱中贴壁培养,培养温度为37℃。培养24h后,弃去培养液,制备好不同浓度(2.5、5、10μM)的抗氧化活性多肽Cos-16溶液,然后将600μL不同浓度的抗氧化活性多肽Cos-16溶液分别添加到样品组的每孔细胞培养液中,继续培养2h后,以PBS洗涤细胞2次。将细胞在4℃裂解10min,收集裂解后的细胞,根据试剂盒说明书上操作获得上清液。按照试剂盒操作方法,分别测定其中GPx、CAT酶活性,以BCA蛋白浓度测定方法测定蛋白浓度。结果如图7、图8所示,可以看到,随着抗氧化活性多肽Cos-16浓度的增加,HaCaT细胞内GPx、CAT酶活性上调。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
序列表
<110> 厦门大学
<120> 一种抗氧化活性多肽Cos-16及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ala Cys Phe Leu Glu Gly Leu
1 5
Claims (10)
1.一种抗氧化活性多肽Cos-16,其特征在于:其氨基酸序列如SEQ ID NO.01所示。
2.权利要求1所述的抗氧化活性多肽Cos-16在制备抗氧化保健组合物中的应用。
3.一种抗氧化保健组合物,其特征在于:其有效成分包括权利要求1所述的抗氧化活性多肽Cos-16。
4.如权利要求3所述一种抗氧化保健组合物,其特征在于:其有效成分为权利要求1所述的抗氧化活性多肽Cos-16。
5.权利要求1所述的抗氧化活性多肽Cos-16在制备化妆组合物中的应用。
6.一种化妆组合物,其特征在于:其有效成分包括权利要求1所述的抗氧化活性多肽Cos-16。
7.如权利要求6所述一种化妆组合物,其特征在于:其有效成分为权利要求1所述的抗氧化活性多肽Cos-16。
8.权利要求1所述的抗氧化活性多肽Cos-16在抗紫外线组合物中的应用。
9.一种抗紫外线组合物,其特征在于:其有效成分包括权利要求1所述的抗氧化活性多肽Cos-16。
10.如权利要求6所述一种抗紫外线组合物,其特征在于:其有效成分为权利要求1所述的抗氧化活性多肽Cos-16。
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