CN110372778A - 一种ace抑制肽、制备方法及用途 - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C—CHEMISTRY; METALLURGY
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
本发明公开了一种ACE抑制肽、制备方法及用途。所述ACE抑制肽的氨基酸序列为GPPGP,来源于天然动物蛋白,其IC50为190.43μmol/L,分子量小,具有稳定、安全、人体易吸收等特点,同时能进行产业化生产。氨基酸序列为GPPGP的ACE抑制肽,可用于制备高血压治疗/预防药物,或作为功能食品添加剂,供高血压患者长期治疗保健使用。
Description
技术领域
本发明涉及生物活性肽技术领域,具体涉及ACE抑制肽、制备方法及用途。
背景技术
随着我国经济的快速发展和人们生活习惯的改变,慢性非传染性疾病已成为影响人民健康的公共卫生问题。高血压是一种非传染性的慢性疾病,对于中风、心力衰竭和冠状动脉疾病的患者,高血压是一个非常重要的风险因素。据来自195个国家和地区的数据估计,2015年,有8.74亿成年人的收缩压(SBP)为140毫米汞柱或更高。预计,到2025年全世界将有约15.6亿人患有高血压。高血压现已成为全球主要的健康问题。
血管紧张素转换酶(angiotensin I-converting enzyme,ACE)是哺乳动物组织中广泛存在的羧基肽酶(EC 3.4.15.1),在肾素-血管紧张素系统(RAS)和激肽系统(KKS)中扮演着重要的血压调节功能。ACE不仅可以将血管紧张素I转化为具有血管收缩作用的血管紧张素II,还可将血管舒张作用的缓激肽转变为没有活力的缓释肽片段。ACE活力升高破坏了正常体液中升压和降压体系的平衡,导致人体血压的升高。可见抑制ACE活性是血压降低的关键。现市场流通的降血压药,如卡托普利,依那普利,赖诺普利等,通过直接抑制ACE或阻断血管紧张素II受体起作用。尽管这些药物在控制血压方面是有效的,但它们会产生许多副作用,如:咳嗽,味觉障碍,皮疹,肾衰竭等。因此,研究学者一直致力于寻找高效的食源性ACE抑制肽。
发明内容
本发明的目的在于提供一种ACE抑制肽。
为实现上述目的,本发明提供一种ACE抑制肽,其特征在于,所述ACE抑制肽的氨基酸序列为GPPGP。
进一步,所述ACE抑制肽来自于海参天然提取或人工氨基酸合成。
本发明还提供一种所述ACE抑制肽的制备方法,包括如下步骤,
海参蛋白的提取:将海参溶于NaOH溶液中,组织均浆后,离心收集沉淀,可重复此步骤1-5次,再用蒸馏水反复搅拌冲洗至中性后再溶于乙酸中,搅拌后离心,所得上清即为海参蛋白溶液;
海参蛋白酶解:将所得海参蛋白溶液预热,加入胃蛋白酶酶解后,再加入胰蛋白酶及胰凝乳蛋白酶的混合酶进行酶解,再加入脯氨酸内肽酶酶解得到酶解液;
ACE抑制肽的制备:酶解液经超滤膜超滤浓缩后得到透过液,将透过液进行冷冻干燥浓缩,得到冻干粉;将所得到的冻干粉溶于纯水,采用SuperdexTM Peptide凝胶柱进行洗脱,收集ACE抑制活性较高的组分,将收集组分冷冻干燥进行浓缩,获得海参蛋白ACE抑制肽组分;再用纯水溶解该海参蛋白ACE抑制肽组分后上样于高效液相色谱ZORBAX SB-C18柱进行分离,收集ACE抑制活性较高的活性峰即为高纯度的海参蛋白ACE抑制肽;
ACE抑制肽的筛选:利用质谱检测活性较高的组分,得到具有ACE抑制活性的肽段的氨基酸序列为DEGHKGPPGP;利用在线软件Biopep模拟胃蛋白酶、胰蛋白酶及胰凝乳蛋白酶对得到的肽段进行酶切;利用分子对接技术对原序列及酶解后得到的肽序列进行高活性筛选,得到ACE抑制活性肽序列GPPGP。
所述海参蛋白提取的作用是富集海参中的蛋白,去除海参多糖、脂质及皂苷;
所述ACE抑制肽的分离纯化是以ACE抑制活性为指标,利用超滤(3kDa超滤膜)、SuperdexTM Peptide凝胶柱及反相高效液相色谱(RP-HPLC)对ACE抑制肽组分进行分离纯化。
所述ACE抑制肽鉴定是通过LC-MS/MS对活性肽进行氨基酸序列鉴定。
本发明还提供一种所述ACE抑制肽在制备ACE抑制药物及辅助降压食品中的应用。
所述ACE抑制肽在ACE抑制药物及辅助降压食品中的应用为将ACE抑制肽作为活性成份添加于药物或降压食品中。
本发明还提供一种ACE抑制药物,其特征在于,含有所述ACE抑制肽作为活性成份。
所述ACE抑制五肽来源于天然动物蛋白,其IC50为190.43μmol/L分子量小,具有稳定、安全、人体易吸收等特点,同时能进行产业化生产。该ACE抑制肽可用于制备高血压治疗/预防药物,或作为功能食品添加剂,供高血压患者长期治疗保健使用。
附图说明
图1是3kDa超滤膜透过液的ACE抑制活性测定图;
图2是GPPGP的ACE抑制活性测定图;
图3是GPPGP体外模拟胃肠液消化产物稳定性分析图;
图4是ACE-GPPGP分子对接图谱;
图5是GPPGP细胞毒性检测图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:天然ACE抑制肽的制备
1、海参蛋白的提取:取200g海参,溶于2000mL已预冷的0.1mol/LNaOH溶液中,组织均浆后,离心(12000g,4℃,20min),所得沉淀继续溶于NaOH缓冲液中,搅拌过夜后离心(12000g,4℃,20min),收集沉淀。反复4次后,将最后一次离心后收集的沉淀用蒸馏水反复漂洗至洗出液pH为中性。水洗后蛋白溶于10倍体积0.5mol/L乙酸中,搅拌过夜后离心(12000g,4℃,20min),所得上清即为海参蛋白溶液。
2、海参蛋白酶解:海参蛋白溶液置于37℃水浴中预热5min,加入重量百分比0.5%的胃蛋白酶置于37℃下进行酶解,酶解1h后终止反应,再加入重量百分比0.5%的T+C酶(胰蛋白酶及胰凝乳蛋白酶质量比为1:1的混合酶)于37℃下酶解2h后终止反应,最后加入重量百分比0.5%的脯氨酸内肽酶,于25℃条件下酶解12h后终止反应,得到酶解液。
3、ACE抑制肽的分离纯化:所得酶解液经3kDa超滤膜超滤分离后得到透过液,将透过液进行冷冻干燥浓缩,得到冻干粉。将所得到的冻干粉溶于纯水,上样于SuperdexTMPeptide凝胶柱,流速为0.3mL/min,以含0.1%三氟乙酸的30%乙腈为洗脱液,在波长220nm下进行洗脱,测定各收集组分的ACE抑制率,收集ACE抑制活性较高组分冷冻干燥进行浓缩,获得海参蛋白ACE抑制肽组分。用纯水溶解该海参蛋白ACE抑制肽组分后上样于高效液相色谱ZORBAX SB-C18柱进行分离,收集主峰进行ACE抑制活性测定,以检测海参蛋白ACE抑制肽组分中ACE抑制肽段的分布情况,而后收集ACE抑制活性较高的活性峰。所收集的活性峰即为高纯度的海参蛋白ACE抑制肽。色谱条件为:流速1mL/min,柱温25℃,检测波长220nm,流动相为含0.1%三氟乙酸的乙腈和含0.1%三氟乙酸的纯水不同比例的混合液;洗脱过程中乙腈比例为:0~40min,乙腈0~20%。
4、ACE抑制肽的鉴定:利用LC-MS/MS质谱检测活性较高的组分,得到具有ACE抑制活性的肽段的氨基酸序列为GPPGP。
实施例2:人工合成ACE抑制肽的制备
1、称量取代度为0.35mmol/g的Fmoc-Pro-Wang Resin(for P1900520)或Fmoc-Arg(Pbf)-Wang Resin(for P1900521)0.3mmol,放置于中号反应柱中,DMF浸泡120min;
2、抽干DMF,加树脂体积3倍量的20%Pip/DMF,鼓氮气30min,用以脱除Fmoc,DMF洗5遍,茚三酮检测现深蓝色;
3、按比例投入原料(AA:HBTU:NMM=3:2.85:6),加入适量DMF,鼓氮气反应,直到茚三酮检测透明;
4、重复步骤2-3,完成序列的合成;
5、抽干反应器内树脂,转移至切割管中,加入40ml F液,摇床控温1.5h;
6、抽滤,将切割滤液收集至离心管中,加入6倍体积冰乙醚,低速离心机沉淀;
7、将沉淀的粗品用乙醚洗3遍,得最后粗品;
8、将粗品放置干燥锅中,真空干燥过夜,送纯化部门纯化,得精品。
实施例3:天然ACE抑制活性检测
将酶解液经3kDa超滤膜超滤分离后得到的透过液,将进行冷冻干燥浓缩,得到冻干粉。用水稀释成不同浓度后检测其ACE抑制活性,结果如图1所示。从图中可以看出:透过液的ACE抑制活性IC50为81.37μg/mL。天然制备所得ACE抑制肽是经胃肠蛋白酶酶解后所得,可避免该活性肽经人体摄入后被胃肠道消化液降解而失活。
实施例4:合成ACE抑制活性检测
对化学合成小肽GPPGP进行体外ACE抑制活性进行检测,如图2所示。从图中可以看出:GPPGP小肽的ACE抑制活性IC50为190.43μmol/L(也相当于80.64μg/mL),与纯化过程中3kDa透过液作用相当。
对GPPGP进行模拟胃肠液消化,消化后的产物利用反向高效液相色谱及圆二色谱进行结构检测。如图3所示,从图中可以看出:模拟胃肠液消化后小肽的结构基本保持不变。GPPGP具有消化稳定性,能避免该活性肽经人体摄入后被胃肠道消化液降解而失活。
实施例5 ACE抑制肽与ACE的虚拟对接分析
使用AutoDock软件,将GPPGP与ACE(PDB ID:1O86)进行对接,阐明五肽与ACE酶特定的抑制性位点相互作用形式。
GPPGP与ACE(PDB ID:1O86)之间的相互作用主要包括范德华力、氢键结合作用力两种形式,从图4可以看出,ACE-GPPGP相互作用的活性口袋氨基酸残基分别为Gln281,His353,Lys511和His513;锌离子配位键氨基酸残基为His383。除此之外,还包括以氢键相互作用的其它氨基酸残基Leu375,Lys454。ACE-GPPGP相互作用的氨基酸中含有ACE活性口袋的关键氨基酸,锌离子配位键氨基酸残基,阐述了GPPGP具有ACE抑制活性的机理。
实施例6 ACE抑制肽细胞毒性试验
配制不同浓度的ACE抑制肽(合成或上述制备得到)溶液,加入至含有Caco-2细胞的96孔培养板中共培养,使肽终浓度达到0、0.05、0.1、0.5、1和2mmol/L,在CO2培养箱中放置24h。用PBS溶解MTT,使其浓度为2mg/mL,每孔加入100μL MTT溶液,于37℃中孵育4h,每孔加入200μL DMSO终止反应且溶解MTT晶体,使用酶标仪在波长570nm测定吸光值。
结果表明,合成或上述制备得到的ACE抑制肽GPPGP对Caco-2无明显毒性,结果见图5。其中横坐标为肽浓度,纵坐标为细胞相对存活率,从图中可以看出:即使该肽浓度达到2mmol/L时,细胞的存活率仍在90%以上。GPPGP无细胞毒性,可用于制备高血压治疗/预防药物,或作为功能食品添加剂,供高血压患者长期治疗保健使用。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (6)
1.一种ACE抑制肽,其特征在于,所述ACE抑制肽的氨基酸序列为GPPGP。
2.根据权利要求1所述ACE抑制肽,其特征在于,所述ACE抑制肽来自于海参天然提取或人工氨基酸合成。
3.一种权利要求1所述ACE抑制肽的制备方法,其特征在于,包括如下步骤,
海参蛋白的提取:将海参溶于NaOH溶液中,组织均浆后,离心收集沉淀,可重复此步骤1-5次,再用蒸馏水反复搅拌冲洗至中性后再溶于乙酸中,搅拌后离心,所得上清即为海参蛋白溶液;
海参蛋白酶解:将所得海参蛋白溶液预热,加入胃蛋白酶酶解后,再加入胰蛋白酶及胰凝乳蛋白酶的混合酶进行酶解,再加入脯氨酸内肽酶酶解得到酶解液;
ACE抑制肽的制备:酶解液经超滤膜超滤浓缩后得到透过液,将透过液进行冷冻干燥浓缩,得到冻干粉;将所得到的冻干粉溶于纯水,采用SuperdexTMPeptide凝胶柱进行洗脱,收集ACE抑制活性较高的组分,将收集组分冷冻干燥进行浓缩,获得海参蛋白ACE抑制肽组分;再用纯水溶解该海参蛋白ACE抑制肽组分后上样于高效液相色谱ZORBAX SB-C18柱进行分离,收集ACE抑制活性较高的活性峰即为高纯度的海参蛋白ACE抑制肽;
ACE抑制肽的筛选:利用质谱检测活性较高的组分,得到具有ACE抑制活性的肽段的氨基酸序列为DEGHKGPPGP;利用在线软件Biopep模拟胃蛋白酶、胰蛋白酶及胰凝乳蛋白酶对得到的肽段进行酶切;利用分子对接技术对原序列及酶解后得到的肽序列进行高活性筛选,得到ACE抑制活性肽序列GPPGP。
4.一种权利要求1所述ACE抑制肽在ACE抑制药物及辅助降压食品中的应用。
5.根据权利要求4所述ACE抑制肽在ACE抑制药物及辅助降压食品中的应用,其特征在于,含有权利要求1所述ACE抑制肽作为活性成份添加于药物或降压食品中。
6.一种ACE抑制药物,其特征在于,含有权利要求1所述ACE抑制肽作为活性成份。
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