高血压作为一种以动脉血压持续升高为特征的慢性心血管疾病,已成为全球公共卫生领域的重大挑战。归因于全球死亡的首要危险因素是血压升高,每年导致1 080万人死亡[1]。据世界卫生组织2025年统计数据,全球约有12.8亿成年人受到高血压的困扰,这一数字预计在未来十年将持续增长[2]。我国人群高血压患病率持续升高,知晓率、治疗率和控制率仍处较低水平。目前我国有2.7亿高血压患者,成人高血压控制率仅16.8%[2]。高血压不仅直接导致心脑血管疾病发病率的上升,更因其长期性、隐匿性的特点,给患者生活质量和社会医疗体系带来沉重负担。传统降血压药物虽能有效控制血压,但普遍存在患者依从性低、需频繁给药以及长期服用伴有副作用等问题,促使医学界不断探索更安全、有效的治疗方案[3]。

近年来,食物来源的生物活性肽由于其分子质量小、易于吸收且安全无害等特点备受关注。目前已有大量食源性活性肽被报道可通过抑制血管紧张素转化酶(angiotensin-converting enzyme, ACE)的活性、上调内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)表达及调节氧化应激平衡等多途径机制实现持续降血压效果。在降血压肽的研究中,抑制ACE是最常用的机制。ACE抑制肽可通过特异性阻断ACE的活性,从而有效扩张血管、降低外周阻力。例如,从发酵牛乳中分离得到的降血压肽VPP和IPP作为典型ACE抑制肽,通过抑制ACE活性在降低收缩压(systolic blood pressure, SBP)和舒张压(diastolic blood pressure, DBP)方面具有显著作用[4]。来自沙丁鱼的ACE抑制肽VY也已被证明可降低轻度高血压或血压正常受试者的血压,并且未观察到不良反应[5-6]。这种基于膳食的干预方式因其安全性高、作用温和以及依从性好等特点正成为高血压非药物防治的重要方向。因此,本研究归纳了食源性降血压肽的制备方法、构效关系及作用机制,以期为其在功能性食品或药品中的应用提供理论依据。

1 食源性降血压肽的作用机制

药物是高血压治疗的主要手段,临床常用降血压药物主要包括利尿剂、β受体拮抗剂、钙通道阻滞剂、ACE抑制剂和血管紧张素Ⅱ受体拮抗剂5大类,其作用机制围绕减少心输出量(利尿剂、β受体拮抗剂)和降低外周阻力(钙通道阻滞剂、ACE抑制剂和血管紧张素Ⅱ受体拮抗剂)两大核心途径(图1)。现代研究证实,肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosterone system, RAAS)的过度激活是高血压发生的关键驱动因素,该系统通过级联反应生成血管紧张素Ⅱ(angiotensin Ⅱ, Ang Ⅱ),后者通过血管紧张素Ⅱ 1型受体(angiotensin Ⅱ type 1 receptor, AT1R)介导血管收缩、醛固酮分泌及交感神经兴奋,最终导致外周血管阻力与循环血量双重升高[7]。基于抑制RAAS的作用机制,开发了肾素抑制剂、ACE抑制剂、Ang Ⅱ受体拮抗剂和盐皮质激素受体拮抗剂等降血压药物,其作用主要针对减少Ang Ⅱ和醛固酮的形成,以减少血管损伤和血管收缩等。研究表明,食源性降血压肽可通过多维度调控机制发挥降血压作用,涉及多种分子和信号通路。例如,研究人员发现带鱼肌球蛋白水解物对自发性高血压大鼠(spontaneously hypertensive rats, SHRs)具有降低血压的作用,可以显著降低SHRs体内Ang Ⅱ水平,提高缓激肽和一氧化氮(nitric oxide, NO)水平。水解物还可以下调细胞间黏附分子-1(intercellular adhesion molecule-1, ICAM-1)和血管细胞黏附分子-1(vascular cell adhesion molecule-1, VCAM-1)的表达,表明具有较强的抗炎作用。此外,硝基酪氨酸和Ⅰ型胶原蛋白的表达也降低,提示带鱼肌球蛋白水解物具有显著的抗氧化作用,这反映了其涉及多种降血压机制[8]。表1总结了研究中报道的食源性降血压肽的作用机制[9-20]。可以发现,食源性降血压肽的作用机制并不局限于对ACE的抑制,还可以调节参与高血压发生和发展的相关分子的表达,最终导致血压下降。

1.1 ACE活性抑制机制

抑制ACE活性是食源性降血压肽调节RAAS功能并发挥降血压作用的核心机制。第一个ACE抑制肽早在1971年从蛇毒中被鉴定出来[21],自此引发了对降血压肽的广泛研究。ACE的催化位点主要包括C端和N端两个功能结构域,包含3个催化活性位点S1、S1′和S2[22]。多肽主要通过与ACE活性位点的催化残基(如Ala 354、Gln 281、His 513、Tyr 520、Lys 511和Glu 162等)形成氢键来抑制其活性[23]。ACE抑制肽作为一种酶活性抑制剂,按照其抑制机理可以分为4类:竞争性抑制、非竞争性抑制、反竞争性抑制和混合性抑制。大多数已知的ACE抑制肽的抑制类型以竞争性抑制为主[24],通过螯合锌离子或竞争性结合ACE的活性位点有效阻断Ang Ⅰ向Ang Ⅱ的转化,进而实现血管舒张与血压调控[25]。Yu等[26]在体外实验中证实了蛋清蛋白来源的五肽(QIGLF和RVPSL)具有良好的ACE抑制活性,而体内研究显示二者分别使SHRs收缩压显著降低48 mmHg和46 mmHg。分子对接结果表明,QIGLF和RVPSL与ACE的3个活性位点建立了相互作用,通过竞争性抑制ACE活性而发挥降血压作用。此外,抑制ACE还能通过激肽释放酶-激肽系统(kallikreinkinin system,KKS)调控血压,该系统通过影响血管舒张因子缓激肽的降解参与血压调节。

1.2 ACE2激活和上调机制

随着基因组学技术的快速发展,人们对RAAS的认知不断深化,特别是血管紧张素转化酶2(angiotensin-converting enzyme 2, ACE2)的发现为食源性降血压肽的作用机制研究开辟了新的视角。ACE2是ACE的同源酶,在2000年被鉴定出来[27]。ACE2作为RAAS中的关键酶,可以将Ang Ⅱ降解为代谢产物血管紧张素-(1-7)[angiotensin-(1-7), Ang-(1-7)],Ang-(1-7)进而通过Mas受体(Mas/receptor, MasR)发挥舒张血管、抗炎、抗增殖的作用,以平衡ACE的影响[28]。多项研究表明,ACE2在心血管功能中发挥着诸多有益作用,因此被认为是心血管治疗的新靶点[29]。食源性降血压肽可通过上调ACE2的基因表达和激活其酶活性发挥降低血压和心血管保护作用。Majumder等[30]通过转录组学研究发现,灌胃蛋清蛋白源三肽IRW可显著增加SHRs肠系膜动脉ACE2 mRNA的表达,同时使SBP和DBP显著降低,这揭示了降血压肽的体内新机制。Ehlers等[31]发现,酪蛋白三肽IPP在离体主动脉实验中可以通过调节ACE2对MasR的结合来促进Ang-(1-7)的生成,表明其通过激活ACE2的活性来增强血管弹性效应的作用机制。Liao等[32]发现,豌豆蛋白肽AKSLSDRFSY对大鼠主动脉平滑肌细胞中ACE2的表达有显著上调作用,但其在体内的降血压作用需要进一步研究。类似地,Zhou等[33]发现,大豆蛋白水解物显著降低了人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)中Ang Ⅱ诱导的细胞过度迁移,降低活性氧(reactive oxygen species, ROS)水平并增加NO水平,从而改善了Ang Ⅱ诱导的内皮功能障碍。然而,ACE2抑制剂MLN-4760部分逆转了这些作用,这表明大豆蛋白水解物对ACE2具有潜在的调节作用。进一步的研究表明,从大豆蛋白水解物中鉴定得到的四肽IVPQ和五肽IAVPT能增强HUVECs中ACE2的活性,且IVPQ还能上调ACE2蛋白在细胞中的表达。

1.3 血管内皮功能调控机制

内皮功能障碍的发生、发展与血管收缩因子内皮素-1(endothelin-1, ET-1)和促炎因子超氧化物的过量产生,以及血管舒张因子NO的降低密切相关。食源性降血压肽可通过多途径改善内皮功能:促进血管舒张因子生成、抑制血管收缩因子分泌以及发挥抗氧化作用清除超氧化物。研究表明,酪蛋白来源的三肽VPP和IPP能通过上调SHRs主动脉组织中eNOS的mRNA表达,显著增强NO的合成与释放,从而实现血管舒张和血压调节[34]。此外,VPP和IPP还能有效改善SHRs的血管内皮功能,特别是有助于恢复肠系膜动脉的内皮依赖性舒张功能[35]。临床观察发现,对于轻度高血压患者而言,摄入含VPP和IPP的酪蛋白水解物虽未显著改变全身血压,却能明显改善血管内皮功能,表明VPP和IPP对血管功能具有保护作用[36]。

1.4 其他作用机制

食源性降血压肽可通过多种机制发挥降血压作用(图2),能与肾素、ACE、ACE2和血管紧张素受体等RAAS关键组分相互作用,并能通过调控精氨酸-NO途径、钙离子通道、内皮功能、血管重构和交感神经系统等生理过程发挥作用。以交感神经调控为例,交感神经兴奋性升高可以引起血压升高,释放去甲肾上腺素等神经递质,导致心率加快,心肌收缩力增强,从而引起心排血量增多。而Miyazaki等[37]在SHRs模型中发现,摄入酪蛋白三肽IPP和VPP后,大鼠皮肤动脉交感神经活动与平均动脉压均呈现显著下降趋势。此外,Ang Ⅱ诱导的血管平滑肌细胞(vascular smooth muscle cell, VSMCs)过度增殖、炎症、氧化应激和迁移是引发血管重构的主要机制,具体表现为血管中层膜厚度增加、弹性下降以及舒张收缩功能障碍,最终促进高血压病程的进展。鸡肉蛋白来源的八肽VVHPKESF通过显著降低循环Ang Ⅱ水平,同时增加ACE2和Ang-(1-7)水平,并增强主动脉ACE2与MasR的表达,有效抑制血管炎症并减轻氧化应激,以控制血管重构的发生并降低SHRs的血压,展现出防治高血压及心血管疾病的综合潜力[38]。值得注意的是,某些活性肽虽然不具备直接的降血压活性,但是可以辅助药物的降血压效果,例如油菜籽肽可提高卡托普利的降血压能力、延长其作用时间,虽然在SHRs的器官中并没有观察到ACE的活性降低,但血清中NO水平升高,提示了油菜籽肽和卡托普利可能通过非RAAS途径产生协同降血压作用[39]。这些发现为研究食源性降血压肽的作用机制提供了新的思路。

2 食源性降血压肽的制备及活性评价

2.1 食源性降血压肽的原料来源

近年来,全球范围内已有大量研究团队系统性地从多种多样的食源性蛋白质中挖掘得到具有降血压特性的活性肽,主要包括动物、植物、微生物以及一些加工副产物等(图3)。动物来源的降血压肽主要来自乳制品、肉类和蛋类等。乳制品是制备食源性降血压肽的优质蛋白来源,其含有丰富的蛋白质,通过酶解或其他方法可以从中分离得到具有降血压活性的肽[40-42]。蛋清蛋白来源的肽如ITKPNDVYS、IRW和TNGIIR已被证明对SHRs具有体内降血压作用[43-45]。丰富的海产品如比目鱼[46]、牡蛎[47]和罗非鱼[48]也是降血压肽的良好来源。此外,胶原蛋白是一种广泛存在于动物体内的蛋白质,通过酶解等方法可以从胶原蛋白中提取得到具有降血压活性的肽[49]。植物来源的降血压肽主要来自谷物[50]和豆类[51]等。例如,大豆来源的四肽FGSF具有体内降血压活性,口服给药SHRs后SBP最大降低21.95 mmHg[52]。相比之下,微生物来源的降血压肽较少,主要来自海洋微藻和发酵微生物。Mirzaei等[53]从酵母提取物中提取了十肽YGKPVAVPAR和九肽LPESVHLDK,它们被证实具有体外抑制ACE的能力。总之,食源性降血压肽的原料来源非常广泛,这为降血压肽的研究与开发提供了丰富的资源。同时,不同来源的降血压肽在结构和功能上可能存在差异,这为开发具有不同特点和优势的食源性降血压肽产品提供了可能。

2.2 食源性降血压肽的制备方法

食源性降血压肽的制备方法主要包括酶解法,微生物发酵法和化学水解法,其中酶解法的使用频率最高。由于不同蛋白酶的特异性不同,利用一种或多种蛋白酶可以获得一些具有生物活性的小分子肽。常用的蛋白酶包括嗜热菌蛋白酶、碱性蛋白酶、胃蛋白酶、胰蛋白酶、胰凝乳蛋白酶和风味蛋白酶等。研究表明,碱性蛋白酶和嗜热菌蛋白酶相比其他蛋白酶,能够产生具有相对更高ACE抑制活性的肽段[54-55]。由于碱性蛋白酶的切割偏好性,其产生的肽段C末端残基通常富含疏水氨基酸,如Ala、Val、Leu、Ile、Phe、Pro、Trp和Met[56]。而嗜热菌蛋白酶产生的具有疏水性N末端残基(如Trp、Tyr、Phe、Ile、Leu、Val、Ala或Met)的肽段对ACE抑制效力至关重要[57]。但是由于单一蛋白酶水解缺乏酶切位点的多样性,使用两种或两种以上的蛋白酶组合进行蛋白质水解可以提高潜在降血压肽的产量[58]。此外,胃蛋白酶、α-胰凝乳蛋白酶和胰蛋白酶常组合使用以模拟人类食物蛋白质胃肠道消化。研究表明,仅用嗜热菌蛋白酶或嗜热菌蛋白酶-胃蛋白酶组合水解大豆蛋白时,其ACE抑制活性相近,半数抑制浓度(half maximal inhibitory concentration,IC50)值分别为51.8 μg/mL和53.6 μg/mL。然而,当采用嗜热菌蛋白酶、胃蛋白酶和胰蛋白酶的三重组合时,IC50值升高至115.6 μg/mL,表明胰蛋白酶的进一步消化可能降低大豆蛋白的ACE抑制活性[59]。

肽段的分离纯化能够显著增强其ACE抑制活性[60]。蛋白质水解产物中的多肽在链长、疏水性、净电荷和活性方面的不同为选择适当的分离技术提供了科学依据。常见的技术包括凝胶过滤色谱、离子交换色谱、反相色谱、超滤法、电泳和固定化金属亲和层析等[61-62]。由于ACE抑制肽通常含有疏水氨基酸,其在疏水色谱柱中表现出较高的保留值[63]。近年来,反相高效液相色谱和快速蛋白液相色谱等改进技术被广泛应用于提高肽的纯度和产量。例如,蘑菇ACE抑制肽经反相高效液相色谱技术纯化后,IC50值从初始水提取物的310 μg/mL显著降低至40 μg/mL,表明纯化过程可有效提升蘑菇ACE抑制肽的浓度和活性[64]。此外,可通过聚丙烯凝胶电泳法、反相高效液相色谱-串联质谱法、四级杆飞行时间质谱法、中红外光谱法和毛细管电泳-质谱法等方法进行肽段的鉴定和表征。然而,从酶解到分离纯化的过程耗时长,设备昂贵,未来需要通过工艺参数优化和技术革新来进一步提升效率与成本效益。

从蛋白水解产物中筛选降血压肽是一项复杂而富有挑战性的工作,计算机辅助技术的应用可以显著提升筛选效率。与传统的制备方法相比,计算机技术能够通过预测酶切位点和活性肽结构,快速锁定潜在候选肽段。例如,基于蛋白酶的切割特异性,可利用BIOPEP和Expasy-Peptide Cutter数据库等生物信息学工具精准预测食物来源蛋白质底物释放的生物活性肽特性[65]。ACE是一种含锌离子的二肽羧肽酶,其活性中心对底物的选择具有一定的特征。因此,研究人员可以利用这些特征来建立定量结构-活性关系(quantitative structure-activity relationship, QSAR)模型,以预测和评估肽的降血压活性。研究表明,通过液相色谱串联质谱法(liquid chromatographic-tandem mass spectrometry, LC-MS/MS)鉴定的大豆蛋白肽序列,其QSAR模型预测的IC50值与实验值高度吻合[59],验证了该方法的可靠性。为了优化水解条件、减少实验工作量并最大化生物活性肽的产量与功效,机器学习被广泛应用于生物活性肽的预测和筛选。Jiang等[66]采用大语言模型(large language models, LLMs)模拟乳清蛋白酶解以确定制备降血压肽的最佳条件,最终确定了最佳的多酶组合体系(中性蛋白酶-木瓜蛋白酶-胃蛋白酶),其产物的ACE抑制率达89.08%(1 mg/mL),活性较单酶处理产物显著提升。Zhang等[67]使用了一种名为ProtBERT的深度学习模型来筛选降血压肽,从大豆分离蛋白中高效鉴定出三条高活性ACE抑制肽:LVPFGW(IC50=20.63 μmol/L)、VSFPVL(IC50=2.57 μmol/L)和VLPF(IC50=5.78 μmol/L)。总之,食源性降血压肽的制备涉及多个领域的技术和方法。通过综合应用这些方法,研究人员可以更有效地挖掘和优化具有降血压活性的肽序列,为高血压的预防和治疗提供新的候选。

2.3 食源性降血压肽的活性评价

2.3.1 体外活性评价

ACE抑制作为食源性降血压肽发挥功效的核心机制,其体外活性的测定是评估降血压肽功能的关键环节,目前ACE抑制活性测定主要采用以下方法:经典的分光光度法通过测定马尿酰-组氨酰-亮氨酸(hippuryl-His-Leu-OH, HHL)的水解产物马尿酸(hippuric acid, HA)的浓度来评估ACE抑制活性[68],但存在未水解HHL的波长干扰和复杂样品基质中的背景干扰问题[69]。新型底物如N-(3-(2-呋喃基)丙烯酰基)-L-苯丙氨酰甘氨酰甘氨酸(FAPGG)和3-羟基丁酰甘氨酰-甘氨酰-甘氨酸(3HB-GGG)通过优化其发色团结构可显著提升检测的特异性[70-71]。现代分析技术中,荧光检测体系采用355 nm/405 nm波长对,以30 s间隔连续监测60 min,可达到纳摩尔级的灵敏度,适合于高通量筛选[72];而色谱分析体系通过高效液相色谱梯度洗脱以分离HHL和HA,可实现高分辨率定量[73]。

高血压作为心血管疾病的主要风险因素,其发生发展与血管损伤密切相关。持续性的血压升高会导致身体的血管系统发生一系列病理改变,包括血管壁张力异常增高、内皮功能紊乱、慢性炎症反应、氧化应激失衡以及病理性血管重塑等。为模拟这一病理环境,研究常采用以下体外细胞模型:人脐静脉内皮细胞(HUVECs)、人主动脉内皮细胞(human aortic endothelial cells, HAECs)和血管平滑肌细胞(VSMCs)。例如,HUVECs可以分泌多种血管活性物质,如Ang Ⅱ、NO、eNOS、前列环素I2(prostaglandin I2, PGI2)以及血管收缩因子ET-1等,共同参与血管稳态的调节。通过检测细胞活力、ELISA定量血管活性因子分泌水平,以及Western blot分析信号通路蛋白表达,可系统评估肽段的细胞毒性和生理调节作用。研究显示,从南瓜籽粕酶解液中分离得到SNHANQLDFHP和PVQVLASAYR两种降血压肽,可通过减少ET-1的分泌,增加NO的释放,对EA.hy926细胞起到保护作用[74]。这些细胞模型与生化检测方法相结合,构成了降血压肽体外活性评估的完整体系,为作用机制解析和临床应用转化提供了重要支撑。

2.3.2 体内活性评价

降血压肽的体外活性与体内效应往往存在差异,这是由于活性肽在进入循环系统,到达靶器官前通常会被体内各种酶水解,产生无活性的片段,使肽活性下降甚至失去降血压活性。尽管许多肽段在体外实验中表现出显著的ACE抑制活性,但其在生物体内的降血压效果可能有限。因此,在高血压动物模型和人体试验中进行体内验证成为不可或缺的研究环节。目前,自发性高血压大鼠(SHRs)因其遗传性高血压特征与人类原发性高血压的高度相似性,是最常用于原发性高血压及其并发症的实验动物。通过灌胃或胃插管给药后,研究者会系统监测收缩压变化,并以合成药物(如卡托普利)和生理盐水分别作为阳性和阴性对照进行对比分析[75]。

Zou等[50]在体外评估了麦麸蛋白水解物对ACE和肾素的抑制作用后,进一步在SHRs体内验证了它们的降低血压能力。同样地,油菜籽蛋白来源的肽LY、RALP和GHS在体外被鉴定为有效的ACE和肾素抑制肽,经过5周的灌胃干预后,分别使SHRs收缩压降低41 mmHg(5.47 kPa)、31 mmHg(4.13 kPa)和28 mmHg(3.73 kPa),并伴随ACE和肾素mRNA及蛋白表达的显著下调[76]。这些结果不仅验证了肽段的体内活性,也揭示了其多靶点调控的潜在机制。然而必须注意的是,体外ACE抑制活性与体内降血压效果之间往往缺乏线性相关性[77],这是由于体内研究能完整反映生理转化与代谢互作对肽活性的影响,而体外实验则更纯粹地评估其分子作用能力。因此,在开发新型降血压肽时,需建立多维评估体系,除评估活性外,还应评估其生物利用度,借助动物模型验证综合效应,最终实现从分子机制到临床应用的系统转化。

3 食源性降血压肽的构效关系

原料的选择以及制备工艺的差异都会显著影响肽段的组成,进而导致其降血压活性的差异。因此,明确食源性降血压肽的构效关系是开发靶向制备策略的关键基础,可为高活性肽段的定向设计提供理论依据。作为食源性降血压肽中研究最广泛的类别,ACE抑制肽自在血压调节领域展现出巨大应用潜力后,其构效关系研究已成为该类肽功能解析与结构优化的核心科学问题,且目前相关构效关系已相对明晰。ACE抑制肽的活性与其分子量大小、氨基酸序列、两亲性和空间构象等特征密切相关。如图4所示,本研究系统统计了67条经文献报道对SHRs具有显著体内降血压作用的食源性降血压肽,并对其肽序列的构效特征进行了分析,现结合分析结果与现有文献报道共同讨论食源性降血压肽构效关系的研究现状。

3.1 分子质量的影响

现有研究表明,ACE抑制肽通常是短链肽,主要由2～12个氨基酸组成[78-80]。Mirzapour等[81]发现,分子质量小于3 kDa的苦杏仁苷水解物,其ACE抑制活性显著优于高分子质量组分。Sonklin等[82]通过超滤膜分离技术对绿豆蛋白菠萝蛋白酶水解液进行分析,发现不同分子质量区间的组分IC50值存在显著差异:分子质量大于10 kDa的组分为(0.68±0.02) mg/mL,5～10 kDa的组分为(0.58±0.02) mg/mL,1～5 kDa的组分为(0.64±0.01) mg/mL,而小于1 kDa的组分为(0.50±0.01) mg/mL。这些研究从侧面揭示了ACE抑制肽分子量与活性的潜在关联,分子量较小的组分可能具有较高的ACE抑制活性。然而,不同分子量分级组分中的肽组成复杂,分子量并非唯一决定因素,并不足以建立肽段分子量大小与其ACE抑制能力之间的直接联系。Ding等[83]对454条ACE抑制肽的统计显示,仅9条ACE抑制肽的氨基酸数量超过12个,分子质量低于400 Da的二肽或三肽占比达42.9%,而分子质量在1 000 Da及以上的肽仅占9.4%,表明食源性降血压肽多为含2～5个氨基酸的短肽。类似地,本文对经SHRs体内验证的67条食源性降血压肽的肽段长度进行分析,其中仅7条肽链长度超过10个氨基酸,而二肽和三肽的占比达43.3%[图4(a)]。分子质量较小的肽通常表现出更强的ACE抑制活性,这可能源于多肽分子量增加时,空间折叠程度显著增加,导致其难以进入ACE的活性口袋[84]。

值得注意的是,ACE抑制肽的抑制能力与分子质量之间并不是线性相关的。尽管短链肽的抑制能力通常随分子质量减小而增强,但部分长链肽段仍表现出较强ACE抑制活性,表明其抑制能力并非仅仅取决于分子质量大小。此外,对于分子质量低于一定范围的肽,氨基酸序列等因素的影响可能更为关键[85]。

3.2 氨基酸序列的影响

大量研究表明,食源性降血压肽对ACE的抑制作用与其氨基酸序列密切相关。早在1977年,Ondetti等[86]的研究指出,ACE抑制肽C端3个氨基酸残基序列对其活性有着关键的影响,特别是当C端序列为FGP时,该肽与ACE表现出较强的亲和力。随着研究的深入,不同团队从不同角度对ACE抑制肽的构效关系进行了更为细致的探索。Qin等[87]的研究发现,当肽序列C末端为疏水性氨基酸时,能够显著促进ACE与肽的结合,增强ACE抑制能力。具体而言,在序列长度为2～5个氨基酸的ACE抑制肽中,C末端一般为F(苯丙氨酸)、Y(酪氨酸)、P(脯氨酸);而对于长度大于5个氨基酸的ACE抑制肽,C末端则以P为主。这些疏水残基可通过疏水作用锚定在ACE的S1/S2亚位点,成为ACE抑制肽与活性中心结合的重要机制。其中P的作用尤为突出,当多肽的C末端为P时,其咪唑环与ACE活性中心氨基酸残基具有高亲和性,是增强其结合能力的核心[83]。Ding等[83]对454条ACE抑制肽的统计数据也印证了这一点:C末端氨基酸中P的出现频率高达23.21%,其余15种氨基酸总占比不足40%。Olalere等[22]对173条ACE抑制肽的系统性分析也表明,P在ACE与长肽的结合中不可或缺。Mizuno等[88]发现,从酪蛋白水解物中获得的高活性ACE抑制肽主要由XP型二肽和XPP型三肽组成,这些肽在动物实验中表现出了较强的降血压作用,进一步凸显了C末端P的关键地位。

不仅如此,N末端氨基酸的种类同样影响肽的ACE抑制活性。例如,两种ACE抑制肽(GQY和VQY)具有相同的肽链长度和C末端,但其ACE抑制活性存在显著差异(IC50值分别为52.3和9.8 μM)[89-90]。研究表明,当N末端为脂肪族氨基酸时,可促进肽与ACE之间的相互作用,从而增强ACE抑制肽的活性[91]。特别是,当脂肪族氨基酸在肽段N端重复出现时,其ACE抑制活性会进一步提高,如VVLYK[92]和AAATP[93]。Ding等[83]对454条ACE抑制肽的N末端氨基酸进行统计,发现出现频率较高的分别是亮氨酸(L)、缬氨酸(V)、异亮氨酸(I)、丙氨酸(A)、甘氨酸(G)、酪氨酸(Y)和苯丙氨酸(F),其中L出现频率最高(14.70%)。Olalere等[22]对173条ACE抑制肽的统计数据也强调了,N末端支链氨基酸L和I对活性至关重要。

对经SHRs体内验证的67条食源性降血压肽的氨基酸组成进行分析:末端氨基酸组成中,C末端为P的肽占25.4%,N末端为疏水性氨基酸的肽占79.1%;肽段序列中P的出现频次最高,其次为L、V和Y[图4(b)]。这些结果共同揭示了肽链两端氨基酸的性质和特定类型(C末端为P、N末端为L等),可通过影响肽与ACE活性中心的结合亲和力,进而共同影响其ACE抑制活性。

3.3 两亲性的影响

ACE抑制肽的疏水性和亲水性也与其活性密切相关[94]。其中,C末端疏水氨基酸含量能显著增强肽的ACE抑制活性[83]。Bougatef等[95]采用高效液相色谱法对白唇鱼皮蛋白水解物进行分级,在ACE抑制活性最强的两个级分中,C末端含疏水氨基酸的肽占比分别高达42.8%和71%。Huang等[96]的研究进一步证实,在已报道的ACE抑制肽序列中,疏水氨基酸占比普遍较高,部分序列甚至达到100%。在经SHRs体内验证的67条降血压肽中,疏水氨基酸占比达50.1%,C末端为疏水氨基酸或疏水氨基酸在肽段中占比大于60%的肽段达59.7%,这进一步凸显了疏水氨基酸在ACE抑制肽中的重要作用。

然而值得注意的是,并非所有由疏水氨基酸构成的肽都必然表现出更强的活性。例如,含亲水氨基酸的ACE抑制肽GYD和LSL的IC50值都低于由100%疏水残基构成的ACE抑制肽LLY的IC50值[81]。这可能是由于酸性氨基酸的存在导致肽段整体呈现负电荷,带负电荷的肽段与ACE相互作用时,会螯合酶活性所必需的锌离子,从而促进肽与ACE的结合[97]。此外,Ding等[83]对不同IC50水平的ACE抑制肽中疏水氨基酸的占比进行统计,发现疏水氨基酸占比100%的ACE抑制肽,其平均IC50值要高于疏水氨基酸占比为50%～100%的ACE抑制肽,这提示了ACE抑制肽的疏水性需控制在适宜区间。

3.4 空间构象的影响

除了一级结构的特征外,肽的二级结构也是影响其ACE抑制活性的重要因素[98]。Cian等[99]发现ACE抑制肽的活性与其α-螺旋结构直接相关。基于ACE明晰的三维结构,可通过分子对接来研究肽段与ACE作用的空间结构,以预测肽段的活性强度并分析相互作用力。Xu等[100]通过分子对接研究揭示了从大豆分离蛋白中鉴定得到的ACE抑制肽与ACE的相互作用:这些肽能与ACE的C结构域形成多种氢键,其中ACE抑制活性最强的肽段IY展现出多位点结合特性,能与ACE的S2活性口袋中的His 353和His 513、S1活性口袋中的Ala 354以及其他活性口袋中的His 383等残基形成氢键;除氢键结合外,WMY、YVVF和LVLL还可以与ACE活性位点中的锌离子相互作用。

随着食源性降血压肽研究的深入,目前已制备和鉴定了大量的食源性ACE抑制肽,其构效关系研究也取得显著进展。近年来,随着分子模拟技术的进步和人工智能算法的发展,为食源性ACE抑制肽构效关系的研究提供了新工具。例如,与传统构效关系研究相比,通过优化人工智能算法模型,可以更准确且高效地预测新肽段的ACE抑制活性,甚至实现新型ACE抑制肽的设计[101]。Du等[102]基于蛋白质语言模型(protein language model, pLM)开发了ACE抑制肽活性分类器pLM4ACE,通过嵌入ESM-2 (evolutionary scale modeling v2)技术实现ACE抑制肽活性的高效预测。Liao等[103]采用LSTM (long short-term memory)算法构建的深度学习模型,能够预测肽段抑制ACE活性的IC50值。研究人员随后采用54条具有体外ACE抑制IC50值和体内降血压活性的肽段进行验证,结果显示模型对ACE抑制肽的活性具有较高的预测准确性。这些人工智能算法通常基于序列信息构建预测模型,通过不同的特征表示和机器学习算法提取和学习肽序列的特征向量,从而对新肽序列进行分类或回归分析。然而,人工智能算法仍存在局限性,其高度依赖高质量数据集可能导致过拟合,且当前模型多基于体外数据,缺乏对体内药代动力学特性的考量,影响了预测的生物学相关性。此外,由于不同来源的ACE抑制肽可能具有独特的构效特征,非活性位点氨基酸可能存在协同机制。因此,未来研究需整合分子动力学模拟等技术,从分子尺度深入解析肽-ACE相互作用的动态机制,并融合既往ACE抑制肽的多维度研究数据与人工智能驱动的预测算法,系统性建立其构效关系模型,从而实现ACE抑制肽的高效设计与开发。

4 总结与展望

高血压作为心血管疾病的主要风险因素,其发生发展与血管损伤密切相关。现有降血压药物虽疗效明确,但存在长期服用伴有副作用等问题,促使医学界不断探索更安全、有效的治疗方案。食源性降血压肽因其天然安全、多靶点调控的特性,在血压管理及心血管保护方面具有巨大潜力。这些来源于动物、植物和微生物的降血压肽,其制备方法既有传统的分离纯化方法,也有新兴的计算机辅助技术(生物信息学工具、QSAR模型和机器学习等),可通过抑制ACE、激活和上调ACE2等多种途径与RAAS相互作用,并且在调控血管内皮功能、交感神经、减缓血管重构等其他作用机制上发挥作用。在降血压肽中,ACE抑制肽是最受关注的一类。构效关系分析表明,ACE抑制肽的分子质量、疏水性氨基酸残基、氨基酸组成和序列(尤其是C末端为脯氨酸、N末端为亮氨酸等)以及空间构象等均对其ACE抑制活性有显著的影响。尽管这些肽在临床试验中展现出了积极的效果,但在实际应用中仍面临以下关键挑战与突破方向。

1)食源性降血压肽的生产成本高昂:需要开发更高效、特异性更强的制备方法,提高降血压肽的产量和活性,降低生产成本。此外,改进分离纯化技术,提高肽段纯度和分离效率,简化操作流程,以实现大规模工业化生产。

2)食源性降血压肽的筛选效率低:先前的研究主要集中于食源性降血压肽的纯化和鉴定。然而,逐个确认具有不同氨基酸序列的肽的活性是烦琐且不现实的。同时,低含量但高活性的肽可能会被忽略。一些新兴的技术,如人工智能、微流控芯片和噬菌体展示可能提供解决方案。此外,由于蛋白质分子结构与生物活性密切相关,了解降血压肽的结构特征与其血压调节功能之间的关系将有助于这些肽的设计和开发。

3)食源性降血压肽到达目标部位的能力未知:虽然已经分离并鉴定了很多食源性降血压肽,但是经过临床研究证实对人体具有体内降血压功效且已在降血压功能性食品开发中获得实际应用的仅有极少数几种。口服摄入的降血压肽需要经过胃肠道消化、吸收,进入血液循环系统才能发挥作用,大多数研究忽视了这一关键方面。此外,还需要开展更多高质量的临床试验,验证降血压肽在人体中的安全性和有效性,建立科学的评价体系。

4)食源性降血压肽的作用机制仍有待探究:高血压的病理影响是多方面的,这意味着仅针对ACE这一靶点是不够的。虽然有一些证据表明部分降血压肽可以降低血压,但它们的具体作用机制仍不完全清楚。这限制了我们对这些肽如何发挥作用的理解,也限制了我们在实际应用中的灵活性。因此,需要进一步的研究来揭示降血压肽的作用机制,并研究降血压肽与其他药物或治疗手段联合使用的协同效应,为高血压的综合治疗提供新方案。

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Research Progress on Preparation, Structure-Activity Relationship and Mechanism of Action of Food-Derived Antihypertensive Peptides

ZHAO Mouming1, ZHOU Minzhi1, ZHENG Lin1, WANG Shuguang2

(1.School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China;2.School of Food Science and Engineering, Kunming University of Science and Technology, Kunming 650500, China)

Abstract：Hypertension, as the most prevalent cardiovascular disease worldwide, emerged as one of the major mortality risks threatening human health. Although traditional clinical antihypertensive drugs effectively controlled blood pressure, they required long-term administration and were accompanied by significant side effects. Food-derived antihypertensive peptides, as natural components with prominent biological activities, gained extensive attention due to their considerable potential in lowering blood pressure and preventing cardiovascular diseases. The mechanisms of action of food-derived antihypertensive peptides involved regulating key components of the renin-angiotensin-aldosterone system, such as angiotensin-converting enzyme (ACE), renin, ACE2, and angiotensin receptors, and exerted effects through multiple pathways including the arginine-nitric oxide pathway, calcium ion channels, endothelial function, vascular remodeling, and the sympathetic nervous system, among which ACE inhibition represented the most common antihypertensive mechanism. The antihypertensive properties of food-derived peptides from animal, plant, and microbial sources were extensively explored via in vitro ACE inhibitory activity assays, vascular cell models, and spontaneously hypertensive rat models. The ACE inhibitory activity of peptides was significantly influenced by their structural characteristics, including molecular weight, amino acid sequence, amphiphilicity, and spatial conformation. The study research progress on the preparation of food-derived antihypertensive peptides (including raw material sources and preparation methods), in vitro and in vivo activity evaluation approaches, the structure-activity relationship and their mechanisms of action was systematically summarized. Finally, the challenges faced by food-derived antihypertensive peptides in industrial production and stability enhancement, as well as their future development directions, were elaborated.

Keywords：bioactive peptides; antihypertensive activity; preparation methods; structure-activity relationship; mechanism of action

中图分类号：TS202.1

文献标志码：A

doi：10.12301/spxb202500588

文章编号：2095-6002(2025)06-0001-16

引用格式：赵谋明,周敏之,郑淋,等.食源性降血压肽的制备、构效关系及作用机制研究进展[J]. 食品科学技术学报,2025,43(6):1-16. ZHAO Mouming, ZHOU Minzhi, ZHENG Lin, et al. Research progress on preparation, structure-activity relationship and mechanism of action of food-derived antihypertensive peptides[J]. Journal of Food Science and Technology, 2025,43(6):1-16.

收稿日期：2025-11-10

基金项目：云南省科技人才和平台计划(院士专家工作站)项目(202405AF140067)。

Foundation: Science and Technology Talents and Platform Program of Yunnan Province (202405AF140067).

第一作者：赵谋明,男,教授,博士,主要从事食品生物技术方面的研究。

(责任编辑：李 宁)