CN110498832A - 一组ace抑制肽及其应用 - Google Patents
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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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
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Abstract
本发明提供一组ACE抑制肽,所述ACE抑制肽是C端为色氨酸的二肽,其氨基酸序列结构为X‑W,其中X为任意氨基酸;W为色氨酸。本发明的活性小分子二肽对ACE(血管紧张素转化酶)的活性具有明显的抑制作用,可用于高血压的防治。本发明的活性小分子二肽还具抗氧化与抗炎活性,能用于与氧化应激或(和)炎症相关疾病的防治。本发明所提供的活性小分子二肽兼具ACE抑制、抗氧化和抗炎等多种活性,能在体内产生协同降压效应,可用于制备辅助降压功能食品或药品,优点是结构简单、安全、兼具多种活性。
Description
技术领域
本发明属于食品医药技术领域,具体涉及一组具有ACE抑制活性的二肽及其应用。
背景技术
高血压是心脑血管病最主要的危险因素,也是我国心脑血管病死亡的主要原因。据统计我国成年高血压患者已达2.7亿,但其控制率仅为13.8%;还有一类介于正常和高血压间的正常高值血压人群,这类人群为高血压易患人群,其发生率也由1991年的23.9%增加到2011年的33.6%。因此,我国高血压防治工作正面临着严峻的挑战。目前高血压的治疗以药物为主,但长期服药会引起诸如口干、头痛、血管神经性水肿等不良反应;而正常高值血压因处于未病状态,其防治方法以生活方式干预为主如减少钠盐摄入、控制体重等,这种方式虽能有效降低罹患高血压的风险但人群依从性较低。因而迫切需要寻找更加安全、有效的方法用于高血压的防治,以降低高血压和心脑血管病的发生率。
血管紧张素转化酶(ACE)是人肾素-血管紧张素(RAS)系统中参与血压调节的一种羧肽酶,它能水解血管紧张素I将其转变成具有强效升压作用的血管紧张素II(AngII)而引起血管收缩、血压升高。因此ACE是治疗高血压的一个重要靶标。近年研究表明一些ACE抑制肽可抑制ACE酶活性,减少AngII的生成,从而降低血压,其已在小规模临床试验中对正常高值血压和轻度高血压人群表现出降压效果;而且相对于合成的降压药,具有对正常血压无影响,对人体无毒副作用等优点。特别是ACE抑制小肽(二肽和三肽),其与长链肽相比,通常具有活性高、易吸收和降解少等优点而备受关注。因而,ACE抑制小肽作为天然的降压活性成分可用于高血压的防治,尤其在正常高值血压干预方面具有广阔的应用前景。
尽管大量的ACE抑制肽已从各类食物蛋白的酶解物中被相继分离、鉴定,但这些ACE抑制肽的降压效果较弱,疗效有限。因此,寻找降压活性更强的ACE抑制小肽,并提升其降压作用,仍将是ACE抑制肽应用于高血压防治所急需解决的瓶颈。
虽然目前高血压的具体发病机制尚不明确,但研究表明其与氧化应激及炎症反应也存在密切联系。在RAS系统中Ang II与血管壁上特异的Ang II受体AT1R结合后,除了能直接引起血管收缩、升高血压外,还可以调节血管壁上存在的NADPH氧化酶,产生过量活性氧(ROS)如超氧阴离子(O2-)和过氧化氢(H2O2),引起氧化应激;进而活化血管内皮细胞转录因子NFκB,产生炎症因子引起炎症反应,从而引起内皮功能障碍,导致血压增加。因此,寻找一种既能抑制ACE活性又具备抗氧化性和减少炎症状态的小分子肽成为目前亟待解决的问题。
发明内容
本发明的第一个目的是提供一类多功能小肽,利用液相合成技术合成长度为2个氨基酸的短肽,并进一步验证ACE抑制、抗氧化和抗炎症活性。
本发明的ACE抑制肽是C端为色氨酸的二肽,其氨基酸序列结构为X-W,其中X为任意氨基酸;W为色氨酸。
优选地,所述二肽为GW、AW、VW、LW、IW、FW、WW、YW、DW、NW、EW、KW、QW、MW、SW、TW、CW、PW、HW或RW中的任意一种。
本发明的第二个目的是提供上述ACE抑制肽在制备ACE抑制药物中的应用。
一种ACE抑制药物,其含有上述ACE抑制肽作为活性成分。
优选地,所述ACE抑制药物包含上所述的ACE抑制肽其中两种或多种任意比例的混合物。
本发明的第三个目的是提供上述ACE抑制肽在制备抗氧化药物中的应用。
一种抗氧化药物,其含有上述ACE抑制肽作为活性成分。
优选地,所述抗氧化药物包含上所述的ACE抑制肽其中两种或多种任意比例的混合物。
本发明的第四个目的是提供上述ACE抑制肽在制备抗炎药物中的应用。
一种抗炎药物,其含有上述ACE抑制肽作为活性成分。
优选地,所述抗炎药物包含上所述的ACE抑制肽其中两种或多种任意比例的混合物。
本发明的第五个目的是提供上述ACE抑制肽在制备辅助降压保健食品中的应用。
一种保健食品,其含有上述ACE抑制肽作为活性成分。
优选地,所述保健食品包含上所述的ACE抑制肽其中两种或多种任意比例的混合物。
本发明的有益效果:本发明的活性小分子二肽对ACE具有明显抑制作用,具有降压潜力。本发明的活性小分子二肽还具备抗氧化与抗炎症活性,能防治因氧化应激引起的疾病(或)和炎症引起的疾病。另外因其兼具ACE抑制、抗氧化和抗炎症多种活性,可用于制备辅助降压功能食品或药品,通过作用于高血压的多种靶标,产生协同降压效应,有利于高血压的防治。
附图说明
图1为本发明二肽对DPPH自由基清除活性测定结果示意图。
图2为本发明二肽抗氧化能力测定结果示意图。
图3为本发明二肽对RAW264.7细胞生长的影响检测结果示意图。
图4为本发明二肽对脂多糖诱导RAW264.7细胞产生NO的抑制影响检测结果示意图。
图5为本发明二肽灌胃给药后各组大鼠尾动脉收缩压(SBP)的变化示意图。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例及其附图对本发明做进一步的详细描述。
实施例1合成二肽
叔丁氧羰基(BOC)-X-OH(本实施例X为Gly)与色氨酸的甲酯盐酸盐(Trp-OME.HCl),采用N,N’-二环己基碳二亚胺(DCC)/N-羟基苯并三唑酯(HOBt)法,在碱性条件下,过夜缩合反应得中间体BOC-Ala-Trp-OME,随后通过皂化反应得到BOC-Ala-Trp-OH,随后通过盐酸气脱去氨基保护基BOC,得到二肽的粗品Gly-Trp。粗品经过C18色谱柱,流速为1mL/min,采用溶剂A:含有0.1%三氟乙酸的乙腈,B含有0.1%三氟乙酸的水,洗脱梯度过程中A初始比例为20%,在0.01min到25min内,A的比例上升到45%,在25min到25.1min内,A的比例上升为100%,保持100%运行至30min停止,检测波长220nm收集多肽溶液,液氮速冷,然后冻干。得到纯度为95%以上的产品,并经ESI-MS鉴定结构。通过前述方法制得如表1所示的二肽以及纯度与分子量。
表1:20种合成二肽以及纯度与分子量
实施例2测定ACE酶抑制率
1、检测方法:
将实施例1制备的二肽用蒸馏水配置成不同浓度的溶液后,从中吸取20μL,与225μL浓度为25mU/mL的血管紧张素转化酶(ACE)溶液加入混合并置于37℃的水浴锅中放置10min,之后加入50μL浓度为2.5mg/mL的马尿酸-组氨酸-亮氨酸(HHL)溶液,于37℃的水浴锅中反应30min;反应结束后加入75μL 3M的HCl溶液终止反应。反应物于10,000rpm离心15min后,取上清液使用液相色谱法测定溶液中马尿酸(HA)的含量。空白对照组使用相同体积的蒸馏水代替二肽溶液按照上述步骤进行。
高效液相色谱法检测条件:使用安捷伦1260液相色谱仪及色谱柱Waters XbridgeC18(4.6×250mm,5μm)进行检测,进样量为20μL,检测波长为228nm,流速为1min/mL。移动相为0.1%三氟乙酸(TFA)/乙腈(A)及0.1%三氟乙酸(TFA)/蒸馏水(DW),0min,5%A;10min,60%A;12min,60%A;14min,5%A;20min,5%A。
对于QW、PW、RW三种肽,由于其保留时间与马尿酸(HA)相似,故其流动相梯度变为0.1%三氟乙酸(TFA)/乙腈(A)及0.1%三氟乙酸(TFA)/蒸馏水(DW),0min,5%A;10min,20%A;17min,60%A;19min,5%A;25min,5%A。
ACE抑制活性计算:通过上述方法得到反应组和空白对照组反应产物HA的峰面积,肽ACE抑制活性为(AC-AS)×100/AC;其中AC为空白对照组中HA的峰面积。As为反应组中HA的峰面积。
2、实验结果
通过以上对20种肽ACE酶抑制活性的检测,检测结果如表2所示,其中IC50表示肽抑制ACE酶50%活性时所需浓度,其所需浓度越低表明该肽ACE酶抑制活性越高,从表2可知,二肽VW、CW、IW的ACE酶抑制活性最高,所需浓度低于1μM。
表2:20种二肽ACE酶抑制活性检测结果
实施例3检测抗氧化性
1、检测方法
1.1二肽DPPH自由基清除活性测定
将实施例1制备的肽分别用蒸馏水配置成5mM的溶液后,从中吸取25μL,与175μL浓度为0.4mM的DPPH混合于96孔板中,放于暗处反应30min后用酶标仪在517nm处测定其吸光值;同时设置空白对照组,用等体积的蒸馏水替代样品按照上述步骤进行。DPPH自由基清除活性为(ABSC-ABSS)×100/ABSC;其中ABSC为空白对照组的吸光值。ABSS为样品组反应后的吸光值。
1.2二肽铁离子还原力/抗氧化能力(FRAP)测定
醋酸缓冲液(300mM pH 3.6),10mM TPTZ与20mM三氯化铁(FeCl3)按照10:1:1的体积比混合配置成反应液。取配置好的肽溶液20μL,与180μL上述配置的反应液混合于96孔板中,放于暗处反应30min后用酶标仪在593nm处测定其吸光值;同时设置空白对照组,用等体积的蒸馏水替代样品按照上述步骤进行。FRAP还原能力为ABSs-ABSc,其中ABSc为空白对照组的吸光值。ABSs为样品组反应后的吸光值。
2、检测结果
检测结果如图1和图2所示,20种二肽对DPPH均呈现不同程度的抑制作用,其中CW在5mM的浓度下,对DPPH自由基的清除率超过80%以上,在4mM的浓度下总抗氧化能力的FRAP值大于1.0,具有超强的抗氧化作用;其次具有较强清除DPPH自由基的肽是LW和MW,对DPPH自由基的清除率超过40%;其次YW的总抗氧化能力的FRAP值大于0.5,也具有较强的抗氧化作用。而其他二肽也有不同程度的的抗氧化活性。
实施例4检测抗炎性
1、检测方法
1.1二肽对RAW264.7细胞生长的影响
使用0.25%胰酶对处于对数生长期的RAW264.7细胞消化3min后,弃胰酶,用含10%牛血清(FBS)的DMEM完全培养基中和胰酶,并使用移液管将细胞轻轻吹打至成单细胞悬液后离心弃上清,然后使用完全培养基重悬细胞并计数,将细胞浓度调整至5×105cell/mL,然后按每孔100μL,将细胞铺与96孔板中,并以蒸馏水作为对照组,每组设4个复孔,然后将细胞培养于37℃质量分数为5%的CO2的培养箱中培养24h后,每孔加入10μLCCK-8试剂,1h后用酶标仪在490nm处检测吸光度值。将空白对照组的吸光值设为100%,空白对照组为参照,将样品吸光值与空白对照组的比值的百分数作为细胞的存活率。
1.2二肽对脂多糖(LPS)诱导RAW264.7细胞产生NO的抑制影响
使用0.25%胰酶对处于对数生长期的RAW264.7细胞消化3min后,弃胰酶,用含10%牛血清(FBS)的DMEM完全培养基中和胰酶,并使用移液管将细胞轻轻吹打至成单细胞悬液后离心弃上清,然后使用完全培养基重悬细胞并计数,将细胞浓度调整至5×105cell/mL,然后按每孔500μL,将细胞铺与6孔板中,然后将细胞培养于37℃质量分数为5%的CO2的培养箱中培养24h后,加入LPS(1μg/mL)和样品,同时设置不加LPS的阴性对照组,加LPS(1μg/mL)的阳性对照组,和同时加LPS(1μg/mL)和实施例1肽的样品处理组(设置两则处理组,其中一组肽的浓度为1mM,另一组肽的浓度为10mM),每组3个复孔;再同样的条件下培养24h后,取上清液,使用Griess reagent(Promega Co.WI,USA)试剂盒NO试剂盒对各组中NO含量进行检测。将LPS处理的样品对照组NO的含量设为100%,以此为参照,将阴性对照组与样品处理组中NO的含量与阳性对照组中NO含量的比值作为其他组NO的产生量。
本实验通过LPS诱导小鼠巨噬细胞RAW264.7建立炎症反应模型,检测本发明20种二肽的抗炎作用。
2、检测结果
检测结果如图3所示,20种肽浓度为1mM时对RAW264.7细胞生长无任何影响,增加浓度至10mM本发明的20种肽仍然对细胞生长无任何影响,其细胞活性均在100%左右,表明本发明的20种肽对RAW264.7没有毒性作用,不影响细胞的生长。由图4可知,当肽浓度为2.5mM时,20种肽在不同程度上均对NO的产生有抑制作用,随着肽浓度的增加20种肽对NO产生抑制活性也随之增加,其中QW的抑制活性最强,LW次之。由于本发明20种肽能抑制LPS诱导的RAW264.7细胞产生NO,因此能发挥抗炎作用。
实施例5动物实验验证本发明二肽的降压效果
1、实验方法
12只雄性SPF级自发性高血压大鼠(SHR)购自北京市维通利华实验动物技术有限公司。所有大鼠购回后饲养于中山大学SPF级动物实验中心。所有大鼠均可自由饮食、饮水。在16周龄时(体重240-295g),大鼠被随机分为三组,空白组(蒸馏水Control,N=4)、阳性对照组(Captopril,30mg/kg BW,N=4)和肽处理组(Peptide:VW,CW和QW的1:1:1混合物,其中每种肽的药量为30mg/kg BW,N=4)。每组大鼠在一次灌胃给药后,使用DP-128型鼠尾动脉无创血压系统监测给药1、3、6、9、24h后大鼠尾动脉收缩压(systolic blood pressure,SBP)的变化。在进行血压测量时,先将大鼠固定于鼠网保温套中于39℃下恒温预热10-15min,使尾动脉扩张,血流畅通后进行测量,每次测量3次,取平均值。
2、实验结果
结果如图5所示,在给药后阳性对照组大鼠尾动脉收缩压(SBP)明显下降,在给药6h后达到最低值(-41.3mmHg),随后逐渐上升。肽处理组的大鼠尾SBP变化与阳性对照组相似,其在给药3h后达到最低值(-30.2mmHg)。可见本发明的二肽与抗高血压药相似,具有显著的降压活性。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一组ACE抑制肽,其特征在于,所述ACE抑制肽是C端为色氨酸的二肽,其氨基酸序列结构为X-W,其中X为任意氨基酸;W为色氨酸。
2.如权利要求1所述的ACE抑制肽,其特征在于,所述二肽为GW、AW、VW、LW、IW、FW、WW、YW、DW、NW、EW、KW、QW、MW、SW、TW、CW、PW、HW或RW中的任意一种。
3.一种ACE抑制药物,其特征在于,以如权利要求1或2所述的ACE抑制肽作为活性成分。
4.如权利要求3所述的ACE抑制药物,其特征在于,包含如权利要求2所述的ACE抑制肽其中两种或多种任意比例的混合物。
5.一种抗氧化药物,其特征在于,以如权利要求1或2所述的ACE抑制肽作为活性成分。
6.如权利要求5所述的抗氧化药物,其特征在于,包含如权利要求2所述的ACE抑制肽其中两种或多种任意比例的混合物。
7.一种抗炎药物,其特征在于,以如权利要求1或2所述的ACE抑制肽作为活性成分。
8.如权利要求7所述的抗炎药物,其特征在于,包含如权利要求2所述的ACE抑制肽其中两种或多种任意比例的混合物。
9.一种保健食品,其特征在于,包含如权利要求1或2所述的ACE抑制肽作为活性成分。
10.如权利要求9所述的保健食品,其特征在于,包含如权利要求2所述的ACE抑制肽其中两种或多种任意比例的混合物。
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