CN116284229B - 一种抗氧化珍珠贝活性肽及其应用 - Google Patents
一种抗氧化珍珠贝活性肽及其应用 Download PDFInfo
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Abstract
本发明涉及一种抗氧化珍珠贝活性肽。该抗氧化珍珠贝活性肽的氨基酸序列如下:GGSIT(Gly‑Gly‑Ser‑Ile‑Thr)。该抗氧化珍珠贝活性肽无毒性且具有良好的抗氧化活性和细胞氧化损伤保护作用,其可作为抗氧化剂或抗氧化添加剂,广泛应用于制备功能性食品、化妆品或制备治疗由活性氧或细胞氧化损伤引起的疾病的药品中。
Description
技术领域
本发明涉及抗氧化肽技术领域,更具体地,涉及一种抗氧化珍珠贝活性肽及其应用。
背景技术
马氏珠母贝(Pinctada martensii)又称珍珠贝,是中国海水珍珠养殖的主要品种。自古以来,人们认为珍珠具有良好的保健价值和药用价值。在珍珠养殖产业中,取珠后的贝肉、分泌物等利用附加值很低,经常被作为废弃物处理。
抗氧化肽具有安全性高,环境友好等优势,成为近年来研究的热点。抗氧化肽可以清除活性氧,从而避免过量的活性氧导致氧化损伤而引起机体失调(如,提前衰老、癌症、动脉粥样硬化、糖尿病或心血管疾病等)。抗氧化肽的活性与氨基酸组成、顺序、数量都有关系,对结构-活性关系研究有助于阐明作用机制及开发新型抗氧化肽。
文献(黄潘钿等,珍珠贝肉抗氧化肽制备工艺优化及其对酪氨酸酶的抑制活性)以探讨了珍珠贝肉抗氧化酶解物制备的最优工艺,但并未研究酶解物中何种活性成分起到较好的抗氧化活性。
对此,需研究发现珍珠贝肉酶解物中具有抗氧化活性的肽段。
发明内容
本发明的首要目的是克服上述现有珍珠贝肉酶解物的抗氧化活性成分研究不足的问题,提供一种抗氧化珍珠贝活性肽。该抗氧化珍珠贝活性肽无毒性且具有良好的抗氧化活性和细胞氧化损伤保护作用,其可作为抗氧化剂或抗氧化添加剂,广泛应用于制备功能性食品、化妆品或制备预防和/或治疗由活性氧或细胞氧化损伤引起的疾病的药品中。
本发明的进一步目的是提供上述抗氧化珍珠贝活性肽在制备抗氧化剂中的应用。
本发明的进一步目的是提供上述抗氧化珍珠贝活性肽在制备抗氧化添加剂中的应用。
本发明的上述目的通过以下技术方案实现:
一种抗氧化珍珠贝活性肽,具有如SEQ ID NO:1所示序列。
本发明的发明人从珍珠贝肉中提取出抗氧化珍珠贝活性肽,该抗氧化珍珠贝活性肽的氨基酸序列如下:GGSIT(Gly-Gly-Ser-Ile-Thr)。该抗氧化珍珠贝活性肽无毒性且具有良好的抗氧化活性和胞氧化损伤保护作用,其可作为抗氧化剂或抗氧化添加剂,广泛应用于制备功能性食品、化妆品或制备预防和/或治疗由活性氧或细胞氧化损伤引起的疾病的药品中。
上述抗氧化珍珠贝活性肽在制备抗氧化剂中的应用。
优选地,所述抗氧化剂为口服制剂或外用制剂中的一种。
优选地,所述抗氧化剂为用于清除DPPH自由基和/或ABTS自由基的抗氧化剂。
优选地,所述抗氧化剂为用于保护细胞免受氧化损伤的抗氧化剂。
经研究发现,本发明的抗氧化珍珠贝活性肽具有突出的细胞氧化损伤保护作用。
上述抗氧化珍珠贝活性肽在制备抗氧化添加剂中的应用。
优选地,所述抗氧化添加剂用于制备功能性食品、化妆品或药品。
优选地,所述药品为用于预防和/或治疗癌症、动脉粥样硬化、糖尿病或心血管疾病的药品。
优选地,所述护肤品为用于抗皮肤衰老或具有皮肤修复功能的化妆品。
优选地,所述抗氧化添加剂在功能性食品、化妆品或药品的添加量为0.005~1mg/mL。
在该添加量下,抗氧化珍珠贝活性肽无毒性且具有良好的抗氧化活性。
与现有技术相比,本发明的有益效果是:
本发明的抗氧化珍珠贝活性肽(GGSIT)无毒性且具有良好的抗氧化活性和胞氧化损伤保护作用,其可作为抗氧化剂或抗氧化添加剂,广泛应用于制备功能性食品、化妆品或药品或制备预防和/或治疗由活性氧或细胞氧化损伤引起的疾病的药品中。
附图说明
图1为实施例1的珍珠贝肉酶解物、<3kDa组分和≥3kDa组分的体外抗氧化能力评价测试结果图。
图2为实施例1的<3kDa组分两步纯化的得到的各组分的体外抗氧化能力评价结果图。
图3为本发明的抗氧化珍珠贝活性肽的细胞内抗氧化活性评价结果图。
图4为本发明的抗氧化珍珠贝活性肽的AAPH诱导的细胞损伤的保护作用结果图。
具体实施方式
为了更清楚、完整的描述本发明的技术方案,以下通过具体实施例进一步详细说明本发明,应当理解,此处所描述的具体实施例仅用于解释本发明,并不用于限定本发明,可以在本发明权利限定的范围内进行各种改变。
实施例1珍珠贝肉的酶解、分离和纯化
1.1珍珠贝肉的酶解
将珍珠贝(也称马氏珠母贝,购自北海黑珍珠海洋生物科技有限公司)肉搅碎成肉糜,加超纯水(料液比1:1,w/w)并调整pH为7.25,加入中性蛋白酶(酶底比0.4%,w/w,中性蛋白酶(10kU/g)购自南宁庞博生物工程有限公司),在50℃下酶解反应3h。酶解结束后立即在90℃下灭酶15min,冷却室温后在4000r/min下离心15min获得珍珠贝肉酶解物,并保存于-20℃用于进一步分析。
1.2珍珠贝肉酶解物的分离
在室温下,采用截留分子质量为3kDa的超滤膜分离第1.1步得到的珍珠贝肉酶解物,得到两个不同相对分子质量的组分:<3kDa组分和≥3kDa组分,并将两个组分分别收集和冷冻干燥,保存于-20℃用于进一步分析。
将珍珠贝肉酶解物、<3kDa组分和≥3kDa组分作为样品,进行体外抗氧化能力评价(DPPH自由基清除能力评价和ABTS自由基清除能力评价)。
DPPH自由基清除能力评价的方法如下:
取100μL样品溶液(0.25~2mg/mL)和100μL DPPH(1,1-二苯基-2-三硝基苯肼,购自上海源叶生物科技有限公司)溶液(0.2mM,95%乙醇)混合,室温避光反应30min。在酶标仪(Enspire Xenon Light Module,200Perkin–Elmer,Beaconsfield,U.K.)波长517nm下吸光值At。同时以乙醇替代DPPH溶液的吸光值Ac,以超纯水替代样品溶液的吸光值A0。根据公式(1)计算DPPH自由基清除率。
DPPH自由基清除率/%=[1-(At-Ac)/Ao]×100%(1)
式中:At为样品溶液和DPPH溶液体系的吸光值;Ac为样品溶液和乙醇体系的吸光值;A0为超纯水和DPPH溶液体系的吸光值。
ABTS自由基清除能力评价的方法如下:
首先以5mL ABTS(2,2-气联氮-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐,购自上海源叶生物科技有限公司)溶液(7mM)和88μL过硫酸钾溶液(140mM)混匀并在室温下避光反应12h,稀释得到吸光值为0.7±0.02(波长734nm)的ABTS·+工作液。取100μL ABTS·+工作液和100μL样品溶液(0.25~2mg/mL)混合,在室温下避光反应10min后,波长734nm测吸光值At'。同时以超纯水替代ABTS·+工作液的吸光值Ac',以超纯水替代样品溶液的吸光值A0'。根据公式(2)计算ABTS自由基清除率。
ABTS自由基清除率/%=[1-(At'-Ac')/Ao']×100%(2)
式中:At'为样品溶液和ABTS·+工作液体系的吸光值;Ac'为样品溶液和超纯水体系的吸光值;A0'为超纯水和ABTS·+工作液体系的吸光值。
珍珠贝肉酶解物、<3kDa组分和≥3kDa组分的体外抗氧化能力评价测试结果如图1所示。图1(A)是DPPH自由基清除能力评价结果,图1(B)是ABTS自由基清除能力评价结果,可知,珍珠贝肉酶解物、<3kDa组分和≥3kDa组分都可以有效清除DPPH和ABTS自由基;在浓度1mg/mL下,<3kDa组分的DPPH和ABTS自由基率分别为49.97%和71.93%;同一浓度的<3kDa组分的自由基清除能力显著优于珍珠贝肉酶解物和≥3kDa组分(p<0.05)。可知,<3kDa组分具有更强的体外抗氧化活性,对<3kDa组分做进一步的纯化。
1.3<3kDa组分的纯化
采用制备型高效液相(LC-8,Shimadzu,Japan)***对<3kDa组分进行第一次纯化,具体的过程和色谱条件为:将<3kDa组分加载到平衡良好的反向C18玻璃柱(20mm×450mm,10μm,Shimadzu)上;收集分离组分,冻干后分别测定其体外抗氧化能力(DPPH自由基清除能力评价和ABTS自由基清除能力评价),筛选活性最强组分;流动相(A液:双蒸水+0.1%TFA;B液:甲醇+0.1%TFA);梯度洗脱:B液0~45min:5%~10%,流速:10mL/min;检测波长:214nm。
第一次纯化分离得到3个峰,分别标记为组分F1、组分F2和组分F3,结果如图2(A)所示。从图2(A)可知,组分F1响应值最高,且对DPPH和ABTS自由基的清除率最好,分别为65.02%和73.06%(1mg/mL),因此选择富集组分F1进行第二次纯化。
第二次纯化的具体的过程和色谱条件为:将组分F1加载到平衡良好的反向C18钢柱(19mm×250mm,5μm,Waters)上,收集分离组分,冻干后分别测定其体外抗氧化能力(DPPH自由基清除能力评价和ABTS自由基清除能力评价),筛选活性最强组分进行质谱鉴定。色谱条件为:流动相(A液:双蒸水+0.1%TFA;B液:甲醇+0.1%TFA);梯度洗脱:B液0~60min:5%~10%,检测波长:214nm。
第二次纯化得到5个峰,分别标记为组分F1-1、组分F1-2、组分F1-3、组分F1-4和组分F1-5,结果如图2(B)所示。从图2(B)可知,组分F1-1具最高的DPPH和ABTS自由基清除率,分别为69.12%和75.34%(1mg/mL),因此富集组分F1-1进一步进行质谱鉴定,确定其肽谱组成。
实施例2肽段的质谱鉴定、生物信息学评价以及合成
对实施例1得到的组分F1-1进行质谱鉴定,具体过程为:将冻干粉溶解后加载到Easy NLC 1200***(ThermoFisher)上,经反相C18柱(2μm,75μm×25cm,AcclaimPepMap RSLC)分离;在装有Nano Flex离子源的ThermoFisher Q Exactive质谱仪(ThermoFisher,USA)采集MS数据。数据采集条件:离子喷雾电压(1.9KV),界面加热器温度(275℃)。采用PEAKS Studio8.5(Bioinformatics Solutions Inc.Waterloo,Canada)软件对原始raw图谱文件进行处理质谱数据,在Uniprot上的Pinctada martensi物种蛋白数据库中进行检索肽序列,确定组分F1-1中肽段的一级结构。
对鉴定后的肽段进行生物信息学评价,筛选出抗氧化珍珠贝活性肽,具体的评价方法为:通过Pepdraw(http://www.pepdraw.com/)模拟计算肽段的疏水性;通过ToxinPred(https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php)预测肽段的潜在毒性;通过在ExPASy peptide cutter(https://web.expasy.org/peptide_cutter/)选择胃蛋白酶(pH=1.3和>2.0)和胰蛋白酶进行模拟酶切(最低的概率为20%)分析肽段对胃肠道消化的抵抗能力。
经过质谱鉴定和生物信息学评价后,得到序列为GGSIT(Gly-Gly-Ser-Ile-Thr)的抗氧化珍珠贝活性肽,其氨基酸残基为5个,分子量为433.2173Da,鉴定结果及其肽段特征如表1。疏水性氨基酸是肽清除自由基能力关键因素,该抗氧化珍珠贝活性肽(GGSIT)含有疏水性氨基酸Gly和Ile,表明其具有良好的清除自由基(DPPH和ABTS自由基)的能力。此外,通过ToxinPred和ExPASy peptide cutter的模拟计算表明该抗氧化珍珠贝活性肽无毒性且能完全抵抗胃蛋白酶和胰蛋白酶的模拟酶切。
表1抗氧化珍珠贝活性肽(GGSIT)的鉴定结果及其肽段特征
抗氧化珍珠贝活性肽 | -10lgP | 等电点 | 毒性 | 模拟消化后肽段 | 疏水性(Kcal/mol) |
GGSIT | 20.65 | 5.36 | 无 | GGSIT | +9.79 |
为了更好的评估抗氧化珍珠贝活性肽(GGSIT)的抗氧化能力,通过固相合成法合成该氧化珍珠贝活性肽(纯度>98%),进一步研究其在细胞层面的抗氧化能力。
实施例3抗氧化珍珠贝活性肽细胞层面的抗氧化活性测定
3.1细胞培养
使用的HepG2细胞代数在20~35代之间,使用添加了10%的胎牛血清(FBS,购自赛默飞世尔科技(中国)有限公司)和1%青霉素-链霉素-新霉素抗生素混合物(购自赛默飞世尔科技(中国)有限公司)的基础培养基(DMEM,购自赛默飞世尔科技(中国)有限公司)培养细胞。将细胞在37℃,5% CO2培养箱中培养,隔天换液,细胞长至80%~90%进行传代培养。
3.2细胞毒性测定
抗氧化珍珠贝活性肽对HepG2细胞存活率影响采用MTT法测定。在96孔板中将HepG2细胞以细胞密度为1×104个/孔接种,并在37℃,5% CO2条件下培养24h。在样品处理组中加入100μL含抗氧化珍珠贝活性肽(0.025、0.05、0.1、0.25、0.5、1mg/mL)的培养基,以不含抗氧化珍珠贝活性肽的新鲜培养基为空白组。继续培养24h后,弃去培养基,加入100μLMTT溶液(0.5mg/mL)避光孵育4h。弃去MTT溶液,加入100μL DMSO,震荡10min使蓝紫色结晶全部溶解。在酶标仪波长490nm测定并计算细胞存活率。
细胞毒性测定结果如图3(A)所示,可知,样品处理组的HepG2细胞存活率都高于90%,表明抗氧化珍珠贝活性肽无毒性。
3.3细胞内抗氧化活性测定
抗氧化珍珠贝活性肽的细胞内抗氧化活性通过采用CAA法进行表征。CAA法的原理是在HepG2细胞中的引入荧光探针DCFH-DA,细胞内脂肪酶将荧光探针DCFH-DA降解为DCFH,在AAPH诱导下被氧化为荧光DCF,而抗氧化珍珠贝活性肽可以保护探针不被氧化。具体过程为:在96孔板中,将HepG2细胞以细胞浓度6×104个/孔接种并在37℃,5% CO2条件下培养24h。在样品处理组中加入50μL DCFH-DA工作液(50μM)和50μL的抗氧化珍珠贝活性肽溶液(0.005、0.01、0.025、0.05和0.1mg/mL),在对照组和空白组中分别加入50μL的50μM DCFH-DA的溶液和50μL的无菌水。孵育1h后用100μL PBS(购自赛默飞世尔科技(中国)有限公司)洗涤,在样品处理组和对照组中加入100μL AAPH工作液(600μM),在空白组中加入新鲜培养基。在激发波长为485nm,测定波长528nm条件下测定荧光值,每5min测定一次,连续测定1h。同时以谷胱甘肽(GSH,购自上海源叶生物科技有限公司)作为阳性对照。通过荧光曲线面积(Area-under-the-curve,AUC)计算CAA,如公式(3)所示。同时计算CAA等于50时所对应的样品的半数有效浓度(EC50)。
式中:AUCt、AUCc和AUCo分别为样品处理组、对照组和空白组的荧光曲线面积。
抗氧化珍珠贝活性肽和谷胱甘肽的CAA值如图3(B)所示,可知,抗氧化珍珠贝活性肽具有细胞内抗氧化活性且呈浓度依赖性,通过计算后得到其EC50为0.085mg/mL,这与谷胱甘肽的EC50(0.021mg/mL)处于同一水平,低于松仁源抗氧化肽的EC50值0.13mg/mL(Liang,R.;Zhang,Z.;Lin,S.,Effects of pulsed electric field on intracellularantioxidant activity and antioxidant enzyme regulating capacities of pine nut(Pinus koraiensis)peptide QDHCH in HepG2 cells.Food Chem.2017,237,793-802.),以及明显低于玉米源抗氧化肽的EC50值2.85mg/mL(Wang,L.;Ding,L.;Yu,Z.;Zhang,T.;Ma,S.;Liu,J.,Intracellular ROS scavenging and antioxidant enzyme regulatingcapacities of corn gluten meal-derived antioxidant peptides in HepG2cells.Food Res.Int.2016,90,33-41.),这表明本发明的抗氧化珍珠贝活性肽具有良好的细胞内抗氧化活性。
实施例4抗氧化珍珠贝活性肽细胞氧化损伤保护作用测定
4.1 AAPH损伤模型的构建
AAPH常用于诱导细胞氧化损伤,通过不同浓度AAPH处理HepG2细胞,可建立氧化损伤模型。将细胞以1×104个/孔浓度接种到96孔板中并在37℃、含有5%CO2和95%空气的培养箱中培养48h。AAPH损伤组中加入150μL含有一系列不同浓度AAPH(浓度分别0.025、0.05、0.1、0.25、0.05、1、2、4、8、10、12、14、16、18、20、22和25mM)的新鲜培养基,对照组中加入等量不含AAPH的新鲜培养基,继续培养24h后采用MTT法测定细胞存活率,结果如图4(A)所示。从图4(A)可知,与对照组相比,AAPH损伤组细胞存活率降低,且具有量效关系。考虑到实际情况中的氧化损伤往往强度不同,因此采用浓度10mM和0.25mM的AAPH分别构建了细胞存活率为50%和80%的损伤模型,并在此基础上研究抗氧化珍珠贝活性肽对于氧化损伤程度细胞的保护作用。
4.2细胞氧化损伤保护作用测定
将细胞以1×104个/孔浓度接种到96孔板中培养24h。样品处理组加入100μL含样品(0.025、0.05、0.1和0.2mg/mL)的新鲜培养基,对照组和AAPH损伤组中加入等量新鲜培养基。处理24h后,样品处理组和AAPH损伤组中再添加50μL含AAPH(终浓度为10和0.25mM)的新鲜培养基,对照组添加等量新鲜培养基。继续培养24小时后,采用MTT法测定细胞存活率。
不同浓度抗氧化珍珠贝活性肽对10mM和0.25mM AAPH分别诱导的HepG2细胞氧化损伤的保护作用的结果如图4(B)和图4(C)所示,##表示AAPH损伤组与空白对照组比较p<0.01;#表示AAPH损伤组与空白对照组比较p<0.05;**表示样品处理组与AAPH损伤组比较p<0.01;*表示样品处理组与AAPH损伤组比较p<0.05。
从图4(B)可知,在10mM AAPH构造的细胞存活率为50%的细胞氧化损伤模型中,当抗氧化珍珠贝活性肽的浓度为0.05mg/mL,即可将细胞存活率从50.77%提升到62.06%,表现出显著性,随着抗氧化珍珠贝活性肽的浓度进一步提升(分别为0.1mg/mL和0.2mg/mL时),细胞存活率进一步提升(达到65.14%和66.35%),而谷胱甘肽(GSH)在相同的浓度下,对细胞存活率的提升都无显著性。
从图4(C)可知,在0.25mM AAPH构造的细胞存活率为80%的细胞氧化损伤模型中,当抗氧化珍珠贝活性肽的浓度为0.2mg/mL,细胞存活率从79.41%提升到96.06%,表现出显著性,而谷胱甘肽(GSH)在相同的浓度下,对细胞存活率的提升无显著性。
可知,本发明的抗氧化珍珠贝活性肽具有突出的细胞氧化损伤保护作用。
通过实施例1~4,表明本发明的抗氧化珍珠贝活性肽可清除自由基(DPPH自由基和ABTS自由基),具有良好的体外抗氧化活性。此外,通过进一步的研究和测定,该抗氧化珍珠贝活性肽还具有良好的细胞内抗氧化活性和细胞氧化损伤保护作用。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一种抗氧化珍珠贝活性肽,其特征在于,所述抗氧化珍珠贝活性肽为SEQ ID NO:1所示序列。
2.权利要求1所述抗氧化珍珠贝活性肽在制备抗氧化剂中的应用。
3.根据权利要求2所述应用,其特征在于,所述抗氧化剂为口服制剂或外用制剂中的一种。
4.根据权利要求2所述应用,其特征在于,所述抗氧化剂为用于清除DPPH自由基和/或ABTS自由基的抗氧化剂。
5.根据权利要求2所述应用,其特征在于,所述抗氧化剂为保护细胞免受氧化损伤的抗氧化剂。
6.权利要求1所述抗氧化珍珠贝活性肽在制备抗氧化添加剂中的应用。
7.根据权利要求6所述应用,其特征在于,所述抗氧化添加剂为用于功能性食品的抗氧化添加剂、用于化妆品的的抗氧化添加剂或用于药品的抗氧化添加剂。
8.根据权利要求7所述应用,其特征在于,所述药品为预防和/或治疗癌症、动脉粥样硬化、糖尿病或心血管疾病的药品。
9.根据权利要求7所述应用,其特征在于,所述化妆品为抗皮肤衰老或具有皮肤修复功能的化妆品。
10.根据权利要求7所述应用,其特征在于,所述抗氧化添加剂在功能性食品、化妆品或药品的添加量为0.005~1mg/mL。
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