CN111944009A - 一种海蓬子降血压肽的制备方法及应用 - Google Patents
一种海蓬子降血压肽的制备方法及应用 Download PDFInfo
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- CN111944009A CN111944009A CN202010820052.9A CN202010820052A CN111944009A CN 111944009 A CN111944009 A CN 111944009A CN 202010820052 A CN202010820052 A CN 202010820052A CN 111944009 A CN111944009 A CN 111944009A
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Abstract
本发明公开了一种海蓬子降血压肽的制备方法及应用，步骤如下：首先对海蓬子植物进行预处理，并切段备用；再将海蓬子茎段与NaCl和EDTA混合进行匀浆破碎，过滤除去固体残渣，再冷冻离心得到混合蛋白液体，调节pH，继续100℃水浴，离心得到海蓬子粗蛋白；最后在水解反应体系中加入海蓬子粗蛋白，再加入菠萝蛋白酶进行酶解，酶解产物即为所述海蓬子降血压肽。该制备方法简便易行，生产成本低。降血压肽来源于药食两用植物资源，使用方便、安全，同时提高了盐生植物海蓬子的高附加值利用，具有广阔的开发前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种海蓬子降血压肽的制备方法及应用。
背景技术
高血压是我国最常见的心血管疾病，患病人数众多，已经成为威胁广大人民群众生命安全的一种疾病。高血压的发病率逐年升高且呈年轻化趋势，据统计，目前我国高血压患病人数已经达到2.6亿，其中45-75岁人群中50％以上人群患有高血压，另外尚有部分患者为察觉，因此尚未进行干预治疗。通过几十年的研究，高血压已经不再被认为是局限于心脑血管系统的疾病，而是多种心血管危险因素聚集、遗传的综合征，因此也被称为高血压综合征，是代谢综合征的一种特殊形式。目前，临床上有多种药物用于高血压疾病的治疗，但这些药物都存在一些问题，如长期服用一种药物会导致用药者对该药物敏感性降低，单纯增加药量不能达到控制血压的效果，其他一些问题如引发血管水肿、咳嗽、高钾血症、肾功能减退等风险。因此，科学家开始寻找一些更加有效、更加安全的高血压防治手段。血管紧张素转换酶(angiotensin-converting enzyme，ACE)抑制剂目前仍是使用广泛且效果显著的抗高血压药物，但该类药物仍然存在一些不可忽视的问题。研究者发现人体在食用食物蛋白后，经过体内蛋白酶的水解作用形成寡肽和氨基酸等被机体利用，寡肽具有抗肿瘤、抗衰老、降血脂、降血压等多种功效，且安全性高、不良反应小且易被人体吸收等多种优势，因此食源性降血压肽因其安全性高、来源广、不良反应小、效果显著等成为高血压研究领域的热点。20世纪80年代研究者已经开始研究食源性ACE抑制肽。随后研究者展开了乳源性、发酵来源、植物、动物、海洋生物来源食源性ACE抑制肽的研究，获得了大量的食源性ACE抑制肽。我国植物种类丰富，且有着以植物性食材为主的饮食传统，研究植物性来源的ACE抑制肽具有重要意义。目前，已经从植物中获得了较多的抗高血压活性肽，部分多肽获得了动物实验数据，具有明显的降压效果。Zenezini Chiozzi等分别采用胰蛋白酶、胃蛋白酶水解消化花椰菜后进行活性筛选，得到6种具有ACE抑制活性的多肽，其中3种为新发现的ACE活性肽，对6种肽体外活性实验测定发现仅FFA-PYAPNFPFK的活性较高，其IC50＝0.461μmol/L，有效浓度较低具有商业开发潜力。Mkinen等将土豆溶解后分离纯化得到土豆蛋白肽冻干粉，对油菜籽碱裂解后进行分类纯化获得油菜籽蛋白肽冻干粉，在自发性高血压小鼠模型中喂食600mg/kg的土豆蛋白肽及油菜籽蛋白肽与喂食20mg/kg的卡托普利阳性对照，降压效果相当，在预防高血压方面效果良好。
海蓬子又称海虫草、海芦笋、海鹿茸、富贵菜，是藜科(Chenopodiaceae)海蓬子属(Salicornia)的无叶茎肉质化的真盐生植物。主要品种有欧洲海蓬子(Salicorniaeuropaea L.)和北美海蓬子(Salicornia bigelovii Torr.)。海蓬子有很强的耐盐能力，耐旱不耐涝，非常适合在亚热带滨海地区生长。其耐盐度可高达5％NaCl浓度，超过海水盐度的20％～40％，内茎含有的可溶性盐分的量达37％(干质量百分比)，因此可以使用海水直接进行灌溉。海蓬子富含多种活性物质，如黄酮类、生物碱类、有机酸类、多糖和三萜皂苷类成分，具有抗氧化、抗肿瘤、抗炎、免疫调节、降血脂等多种作用。《中华本草》记载海蓬子(Salicornia europaea L.)具有平肝、利尿、降压的功效，主治高血压和头痛。同时，海蓬子具有海鲜的风味和清爽嫩脆的口感，且富含维生素A、C和多种矿物质元素及微量元素，是公认的绿色有机蔬菜，素有“植物海鲜”和“海人参”的美誉，在欧美地区有着悠久的食用历史。海蓬子籽实可作为提炼高级食用油的原料；作为西方传统减肥草药，其枝梗可用来做减肥茶，具有清热解毒、降脂、减肥的功效；而作为具有极强的集盐功能的天然盐生植物，可用来提炼低钠、富含矿物质和有机碘的生物盐；海蓬子全草可作利尿、抗坏血病的中药。课题组前期研究表明海蓬子茎中含有16种氨基酸，其中必需氨基酸8种，各种氨基酸比例均衡。海蓬子茎部位蛋白质含量高达到16.53％，必需氨基酸含量58.04mg/g。必需氨基酸与非必需氨基酸比值0.59，氨基酸比值系数分65.72，符合WHO/FAO的推荐标准，表明海蓬子具有较高的营养价值和良好的开发前景，目前尚未检索到利用海蓬子制备降血压肽的报道。
我国患高血压的人群不断增加，且有年轻化趋势，防治形势严峻，且高血压需终身服药，服药周期长，存在引发血管水肿甚至致死的风险。食源性ACE抑制肽因安全性高将会成为研究的重要方向，如开发降血压功能食品等。海蓬子植物具有降血压的功效，且蛋白质含量丰富，是制备ACE抑制肽的新原料，海蓬子生长在沿海滩涂盐碱地，可直接用海水灌溉，大量种植海蓬子既能够改良滩涂盐碱地，海蓬子既不与农田争土地、不与淡水争资源，又可以改良盐碱地将沿海滩涂荒地变为宝地。因此开发海蓬子降血压肽具有重要科学意义和应用价值。
发明内容
针对现有技术的不足，本发明提供一种海蓬子降血压肽的制备方法及应用，采用酸沉法提取海蓬子中大分子蛋白，以菠萝蛋白酶酶解的方式得到小分子降血压肽。制备方法简便易行，生产成本低，降血压肽来源于药食两用植物资源，使用方便、安全，同时提高了盐生植物海蓬子的高附加值利用，具有广阔的开发前景。
本发明是通过以下技术方案实现的：
一种海蓬子降血压肽的制备方法，包括以下步骤：
步骤1)材料预处理：精选新鲜海蓬子植物，清水洗净去除泥沙，沥干水分；将处理好的材料切成段备用；
步骤2)海蓬子蛋白的制备：将海蓬子茎段放入破碎机后，加入NaCl和EDTA，进行匀浆破碎，过滤除去固体残渣，再冷冻离心得到混合蛋白液体，采用盐酸调整液体pH，继续100℃水浴，离心得到沉淀，即为海蓬子粗蛋白；
步骤3)菠萝蛋白酶水解海蓬子蛋白制备降血压肽：取海蓬子粗蛋白用20mM PBS缓冲液中溶解，并稀释到蛋白浓度为10mg/mL，采用Bradford法测定蛋白质含量；水解反应体系中加入浓度10mg/mL的海蓬子粗蛋白，再加入菠萝蛋白酶进行酶解，酶解产物即为所述海蓬子降血压肽。
优选地，步骤2)所述NaCl的添加量为海蓬子重量份的0.3％，所述EDTA的浓度为5mmol/L。
优选地，步骤2)所述破碎的操作如下：在破碎机外侧加上冰袋，破碎时间为2min。
优选地，步骤2)所述采用盐酸调整混合蛋白液体的pH至4.0。
优选地，步骤2)所述离心的转速为4000rpm，时间为10min。
优选地，步骤3)所述菠萝蛋白酶的含量与底物比例为5％。
优选地，步骤3)所述酶解的时间为12h，pH为6.5。
上述方法制得的海蓬子降血压肽在制备具有降血压作用的保健食品或食品添加剂中的应用。
上述方法制得的海蓬子降血压肽在制备降血压药物中的应用。
一种降血压药物，含有上述方法制得的海蓬子降血压肽作为活性成分。
本发明的有益效果如下：
海蓬子原料为药食两用天然植物，广泛分布于滩涂盐碱地；产品制备过程简便易行，生产成本低，质量稳定可控，易于实现机械化操作，能够满足工业化生产需求。将海蓬子降血压肽喂食自发性高血压大鼠(SHR)发现该降血压肽能够显著降低SHR大鼠的血压，并且能显著降低SHR大鼠血清中血管紧张素Ⅱ水平，降血压效果显著，降血压肽来源于药食两用植物资源，使用方便、安全，同时提高了盐生植物海蓬子的高附加值利用，具有广阔的开发前景。
附图说明
图1为海蓬子酶解产物的SDS-PAGE结果；
图1中：1、Marker；2、海蓬子粗蛋白；3、菠萝蛋白酶酶解产物；
图2为海蓬子蛋白ACE抑制肽对大鼠血血浆AngⅡ的影响与对照组比较；
图2中：*：p<0.05，**：p<0.01。
具体实施方式
下面结合附图与具体实施例对本发明作进一步详细说明。
实施例1
一种海蓬子降血压肽的制备方法，具体步骤如下：
(1)材料预处理：精选新鲜海蓬子植物(自行采集)，清水洗净去除泥沙，沥干水分。先将处理好材料切成5cm左右小段，之后采用机械破碎机进行打浆处理。由于海蓬子植物中含水量非常高，因此在打浆过程中不需要另外添加水。
(2)海蓬子蛋白的提取：将海蓬子茎段放入破碎机后，加入0.3％的NaCl，5mmol/L的EDTA，进行匀浆破碎，破碎时间为2min，得到海蓬子浆。
①NaCl添加量对蛋白提取的影响：蛋白质提取过程中，NaCl通过盐溶作用促进蛋白的水解，并且盐离子和蛋白的结合可以保护蛋白质不发生变性。本实施例采用单因素实验对酶解过程中NaCl的添加量进行研究，结果发现在海蓬子提取时加盐量为0.3％时蛋白质提取率就已经达到12.65％，推测是因为海蓬子是盐生植物，体内本身就含有大量的盐分。
②EDTA添加量对蛋白提取的影响：EDTA作为蛋白酶抑制剂，能够保证提取蛋白不被水解，通过对不同浓度的EDTA作用下蛋白质的降解曲线分析发现，当EDTA浓度达到5mmol/L时，能够最大限度的降低蛋白质的水解。
③打浆时间对海蓬子蛋白提取的影响：本实施例采用机械式打浆机进行打浆，打浆时间过短会导致植物组织不能被搅碎，细胞破壁不完全，导致蛋白质不能流出；时间过长又会导致刀片产生过多热量使蛋白质发生变性，提前析出。通过实验摸索最终确定在破碎时间为2min。为保证破碎时温度，在破碎机外侧加上冰袋用来消除机器产生热量。
(3)海蓬子蛋白的析出：将得到的海蓬子浆采用纱布进行过滤除去固体残渣，之后采用冷冻离心机进行离心4000rpm离心10min得到混合蛋白液体，采用1mol/L的盐酸调整液体pH为4.0，再转移至100℃水浴锅中，待溶液中有明显的絮状物出现后继续保温1min，最后4000rpm离心10min，沉淀即为海蓬子粗蛋白。
(4)菠萝蛋白酶水解海蓬子蛋白制备降血压肽：取适量海蓬子粗蛋白在20mM PBS缓冲液中溶解，并稀释到蛋白浓度为10mg/mL，采用Bradford法测定蛋白质含量。水解反应体系中加入浓度10mg/mL的海蓬子粗蛋白，按照设定的酶含量与底物比例(5％)加入菠萝蛋白酶(购于上海源叶生物科技有限公司)进行酶解，酶解时间为12h，pH为6.5，酶解产物即为海蓬子降血压肽。
实施例2
(1)菠萝蛋白酶对海蓬子蛋白的酶解作用
采用实施例1的方法制备得到海蓬子降血压肽，将最优酶解条件下制得的酶解产物，10000rpm离心10min，取适量上清加等体积的2×Loading Buffer混匀，至沸水浴中10min。将处理好的样品采用SDS-PAGE凝胶电泳进行分析，先采用80V电压运行30min，之后转换成120V电压继续运行至指示剂到达分离胶底部后终止电泳，采用考马斯亮蓝G-250对凝胶进行染色，脱色液进行脱色后采集照片。SDS-PAGE结果如图1所示，菠萝蛋白酶将海蓬子蛋白中大分子酶解为均匀的小分子片段，该结果表明菠萝蛋白酶对海蓬子蛋白的酶解效果较好。
(2)海蓬子酶解产物对ACE抑制作用
ACE抑制作用反应体系：取酶解产物25μL，ACE酶液15μL，反应体系在37℃下保温15min后，加入10μL的底物，保持温度反应30min后，加入100μL1M的HCl中止反应。采用乙酸乙酯进行萃取，加入400μL乙酸乙酯充分涡旋混匀，10000rpm离心10min，充分吸取上清，氮气吹干，采用蒸馏水进行溶解，用于分析检测。采用安捷伦LC 1260液相色谱仪，色谱柱为ZORBAX SB-C18(4.6×250mm，5μm)，流动相A：0.1％甲酸水溶液，流动相B：乙腈溶液，流速：0.8mL/min(0～30min B液10％)，柱温为30℃，进样量为10μL，检测波长228nm。计算ACE抑制率。ACE抑制实验结果表明，海蓬子蛋白经菠萝蛋白酶酶解产物的IC50值为0.46mg/mL，优于文献中报道的采用碱性蛋白酶水解杏仁蛋白得到的酶解产物效果(IC50值为0.98mg/mL)。因此，采用该方法获得的海蓬子降血压肽有着较高的生物活性，以海蓬子为原料制备ACE抑制肽有着极大的开发前景。
实施例3
(1)海蓬子降血压肽对SHR大鼠的血压的影响
采用体内实验验证海蓬子降血压肽的降压作用及对血管紧张素的影响，实验设置空白对照组、阴性对照组、阳性对照组(卡托普利5mg/kg)、降压肽低剂量组(1g/kg)、中剂量组(3g/kg)、高剂量组(5g/kg)，其中空白对照组选用普通(Sprague-Dawley，SD)雄性大鼠，其余各组均选用SHR大鼠，所有大鼠均为8周龄大。空白对照组灌胃降压肽高剂量(5g/kg)，阴性对照组灌胃等量生理盐水，连续给药28天，每周测定一次血压值和体重。
结果如表1所示，海蓬子降血压肽对野生型SD大鼠血压基本无影响。阴性对照组SHR大鼠随着时间的生长，血压值逐渐上升。海蓬子降压肽组结果表明，海蓬子降血压肽剂量组均对SHR大鼠血压有着降低作用，降压肽低剂量组对SHR大鼠血压有显著降低作用p<0.05，特别是高剂量组，灌胃海蓬子降血压肽第四周后，SHR大鼠血压极显著降低，血压值降至147.31±6.37mmHg，p<0.01。表明5g/kg剂量的海蓬子蛋白降血压肽的降压活性可能与5mg/kg剂量的卡托普利降压效果接近。同时，海蓬子蛋白ACE抑制肽对SHR大鼠的降血压效果有一定的持续性，降压效果呈现剂量依赖性，给药剂量越大，降血压效果越显著。
表1海蓬子降血压肽对SHR大鼠血压的影响(n＝6)
表1注：与空白对照组比较*p<0.05，**p<0.01。
如下表2所示，研究还发现，未灌胃海蓬子蛋白ACE抑制肽的大鼠体重增加量为28.38g，灌胃海蓬子蛋白ACE抑制肽4周后，大鼠体重增幅显著，高剂量组大鼠体重增幅达55.06g，结果表明海蓬子蛋白ACE抑制肽不仅具有降血压的效果，同时还能促进机体的生长。
表2海蓬子降血压肽对SHR大鼠体重的影响(n＝6)
(2)海蓬子降血压肽对SHR大鼠血浆中血管紧张素Ⅱ的影响
给药结束后，采用戊巴比妥对大鼠进行麻醉，从腹主动脉采集动脉血5mL于含有酶抑制剂的冰浴冷却抗凝管中，迅速颠倒混匀放回冰浴中。采用冷冻离心机，2500rpm离心5min，分离得到血浆。采用放射免疫分析法检测各组大鼠血浆中AngⅡ质量浓度，实验操作严格按照试剂盒说明进行，大鼠血管紧张素Ⅱ(Ang-Ⅱ)ELISA试剂盒(购于上海生工NO.D731188)。海蓬子降血压肽对大鼠血浆中AngⅡ质量浓度影响的结果如图2所示，海蓬子降血压肽低剂量组能降低血浆中AngⅡ质量浓度，中剂量组能够显著降低血浆中AngⅡ浓度(p<0.05)，高剂量组能够极显著降低血浆中AngⅡ浓度(p<0.01)。血压的平稳受到多种因素的影响，其中AngⅡ在高血压的发生中起到非常关键的作用，是目前已知最强的缩血管活性物质之一。本研究结果表明海蓬子蛋白降压肽对SHR大鼠血压有降低的作用，并且降压是通过降低大鼠血浆中AngⅡ水平来实现的。
Claims (10)
1.一种海蓬子降血压肽的制备方法，其特征在于，包括以下步骤：
步骤1)材料预处理：精选新鲜海蓬子植物，清水洗净去除泥沙，沥干水分；将处理好的材料切成段备用；
步骤2)海蓬子蛋白的制备：将海蓬子茎段放入破碎机后，加入NaCl和EDTA，进行匀浆破碎，过滤除去固体残渣，再冷冻离心得到混合蛋白液体，采用盐酸调整液体pH，继续100℃水浴，离心得到沉淀，即为海蓬子粗蛋白；
步骤3)菠萝蛋白酶水解海蓬子蛋白制备降血压肽：取海蓬子粗蛋白用20mM PBS缓冲液中溶解，并稀释到蛋白浓度为10mg/mL，采用Bradford法测定蛋白质含量；水解反应体系中加入浓度10mg/mL的海蓬子粗蛋白，再加入菠萝蛋白酶进行酶解，酶解产物即为所述海蓬子降血压肽。
2.根据权利要求1所述的一种海蓬子降血压肽的制备方法，其特征在于，步骤2)所述NaCl的添加量为海蓬子重量份的0.3％，所述EDTA的浓度为5mmol/L。
3.根据权利要求1所述的一种海蓬子降血压肽的制备方法，其特征在于，步骤2)所述破碎的操作如下：在破碎机外侧加上冰袋，破碎时间为2min。
4.根据权利要求1所述的一种海蓬子降血压肽的制备方法，其特征在于，步骤2)所述采用盐酸调整混合蛋白液体的pH至4.0。
5.根据权利要求1所述的一种海蓬子降血压肽的制备方法，其特征在于，步骤2)所述离心的转速为4000rpm，时间为10min。
6.根据权利要求1所述的一种海蓬子降血压肽的制备方法，其特征在于，步骤3)所述菠萝蛋白酶的含量与底物比例为5％。
7.根据权利要求1所述的一种海蓬子降血压肽的制备方法，其特征在于，步骤3)所述酶解的时间为12h，pH为6.5。
8.权利要求1～7任一项制得的海蓬子降血压肽在制备具有降血压作用的保健食品或食品添加剂中的应用。
9.权利要求1～7任一项制得的海蓬子降血压肽在制备降血压药物中的应用。
10.一种降血压药物，其特征在于，以权利要求1～7任一项制得的海蓬子降血压肽作为活性成分。
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