CN111995659A - 一种来源于牡丹籽粕的ace抑制肽 - Google Patents
一种来源于牡丹籽粕的ace抑制肽 Download PDFInfo
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- CN111995659A CN111995659A CN202010941644.6A CN202010941644A CN111995659A CN 111995659 A CN111995659 A CN 111995659A CN 202010941644 A CN202010941644 A CN 202010941644A CN 111995659 A CN111995659 A CN 111995659A
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Abstract
本发明公开了一种牡丹籽粕ACE抑制肽及其制备方法。所述ACE抑制肽氨基酸序列为His‑Trp‑Ser（HWS），体外实验表明，该多肽具有良好的ACE抑制活性，IC50为0.64 mg/mL。本发明所涉及的ACE抑制肽具有结构简单、安全、活性强等特点，易于进行生产，可用于制备高血压预防药物，或者作为功能食品添加剂供高血压患者食用。
Description
技术领域
本发明涉及一种来源于牡丹籽粕的ACE抑制肽，属于食品生物技术领域。
背景技术
研究调查表明高血压影响到全世界约25%的成人人口，预计在2025年高血压患病率将增加29%，届时将有15.6亿人受到影响。高血压是一种心血管病综合征，也是危及人们心血管疾病的主要因素之一，其常伴随其他病症，如动脉粥样硬化、心肌梗死和中风等。血管紧张素转换酶（ACE）是一类含锌的二肽羧基肽酶，它主要存在于肺和一些器官中。血管紧张素转换酶在参与高血压调节起到重要的作用，参与肾素-血管紧张素系统（RAS）和激肽释放酶-激肽系统（KKS），使血压升高。ACE抑制剂能够通过抑制或降低ACE酶活性达到降低血管紧张素II的生成，并避免激肽、缓激肽的失活，起到调节血压的作用。常见的ACE 抑制剂药物有卡托普利、苯那普利、依那普利、培哚普利等，但是临床上显示长期服用降压药，会有咳嗽、血管性水肿、头痛等不良反应。因此，食源的降压肽因其具有天然、安全性高、易吸收、无副作用的特点而备受关注。
牡丹籽粕是牡丹籽榨油后副产物，大多丢弃或者作为动物饲料，生物利用率低。牡丹籽粕中蛋白含量为18%-35%，其中的必需氨基酸含量与大豆相似，具有很高的营养价值，可作为天然活性肽的良好来源。牡丹籽粕中蛋白质的高效利用对提高牡丹籽粕的附加值具有重要作用。牡丹籽粕中蛋白质的提取并酶解生产制备ACE抑制肽方面的研究开发，能够对牡丹籽粕进行有效地利用，提高牡丹籽的附加价值，实现资源的综合利用。
发明内容
本发明的目的是提供一种来源于牡丹籽粕的ACE抑制肽。
为实现上述目的，采用以下技术方案：
本发明提供一种ACE抑制肽，其氨基酸序列为His-Trp-Ser，缩写为HWS，即由组氨酸-色氨酸-丝氨酸3个基酸残基构成。
进一步，所述ACE抑制肽来自于牡丹籽天然提取或人工氨基酸合成。
本发明提供一种所述ACE抑制肽的制备方法，包括如下步骤，
1）牡丹籽粕蛋白酶解产物的制备：牡丹籽粕经粉碎后，经石油醚脱脂干燥后过筛备用；采用碱提酸沉法得到牡丹籽粕蛋白，采用中性蛋白酶将其在酶解条件：底物浓度2%（w/v）、pH 7.50、酶添加量7200 U/g、温度43 ℃，酶解时间2 h，酶解后沸水浴10 min灭活，冷却至室温后，调节pH至中性后于4℃10000 rpm离心20 min，取上清液冷冻干燥，即为牡丹籽粕蛋白质酶解产物。
2）牡丹籽粕蛋白的酶解产物利用Sephadex G-25凝胶色谱进行分离，以去离子水为洗脱液，流速0.3 mL/min，测量洗脱组分214nm波长处的吸光值；收集具有最佳ACE抑制活性的峰，利用反相高效液相色谱（RP-HPLC）进一步分离；RP-HPLC的洗脱梯度为0-3 min，5%（V/V）乙腈（含0.1%三氟乙酸）；3-43 min，5%-40%（V/V）乙腈（含0.1%三氟乙酸）；43-53 min，40%（V/V）乙腈（含0.1%三氟乙酸）；流速为2.0 mL/min，检测波长214nm，收集洗脱时间为19-21 min的洗脱峰，真空冷冻干燥即得所述ACE抑制肽。
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。
本发明的优点在于：
所述牡丹籽粕ACE抑制肽HWS具有较强的ACE抑制活性，其IC50值为0.64 mg/mL。该ACE抑制肽在细胞水平上无毒副作用，能够提高细胞NO的释放量，且具有分子量小、安全、稳定和容易吸收等特点。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，供高血压患者长期保健使用。
附图说明
图1：牡丹籽粗蛋白酶解产物Sephedex G-25凝胶过滤色谱图。
图2：凝胶色谱洗脱组分F4反相高效液相色谱图。
图3：ACE抑制肽鉴定的质谱图。
图4：ACE抑制肽的活性测定。
图5：ACE抑制肽分子对接图。
图6：ACE抑制肽细胞毒性结果图。
图7：ACE抑制肽对HUVEC细胞NO释放量的影响结果图。
图8：ACE抑制肽对HUVEC细胞ROS水平的影响结果图。
具体实施方式
本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和过程，但本发明的保护范围不仅限于下述的实施实例。
实施例1
本发明所述ACE抑制肽的分离纯化包括Sephadex G-25凝胶过滤色谱和反相高效液相色谱（RP-HPLC）两个步骤。
牡丹籽粕蛋白酶解产物的制备：牡丹籽粕经粉碎后，经石油醚脱脂干燥后过筛备用。采用碱提酸沉法，加入NaOH溶液从脱脂处理后的牡丹籽粕中提取蛋白，提取工艺条件为：料液比1：25（w/v），浸提pH 9.25，提取温度53℃，提取时间70 min。将提取液4℃条件下10000 r/min离心15 min，取上清并调节pH为4.0进行使蛋白酸沉，在4℃条件下10000 r/min离心15 min后取沉淀，沉淀用水复溶，调节pH为中性，冷冻干燥备用。采用中性蛋白酶将其在酶解条件：底物浓度2%（w/v）、pH 7.50、酶添加量7200 U/g、温度43 ℃，酶解时间2 h，酶解后沸水浴10 min灭活，冷却至室温后，调节pH至中性后于4℃ 10000 rpm离心20 min，取上清液冷冻干燥，即为牡丹籽粕蛋白质酶解产物。
Sephadex G-25凝胶过滤色谱：将牡丹籽粕蛋白质酶解产物冻干粉，溶解于去离子水中，于4℃下，10000 rpm离心10 min。取上清液用0.22 μm微滤膜过滤除杂后上样。Sephadex G-25凝胶柱（1.6cm×100 cm）用去离子水平衡，将已过滤的样品上柱。用去离子水洗脱，流速0.3 mL/min，于214nm波长处检测洗脱液吸光值，绘制洗脱曲线，如图1所示。收集各组分进行ACE抑制活性测定，以检测ACE抑制肽不同分子量的组分的ACE抑制活性情况。收集洗脱组分F4，真空冷冻干燥，-20 ℃低温保存备用。
高效液相色谱：去离子水溶解上述组分F4干粉，采用高效液相色谱柱Gemini 5μC18 （250mm×10mm）（Phenomenex，UK）进行进一步分离纯化，用水和乙腈（含0.1%的三氟乙酸）构成的洗脱系统进行梯度洗脱。RP-HPLC的洗脱梯度为0-3 min，5%（V/V）乙腈；3-43min，5%-40%（V/V）乙腈；43-53 min，40%（V/V）乙腈。流速为2.0 mL/min，检测波长214nm，洗脱曲线如图2所示，收集洗脱时间为19-21 min的洗脱峰，真空冷冻干燥。
将收集到的ACE抑制肽组分冷冻干燥，采用高效液相色谱进行二次纯化，检验组分纯度。经检测，该抗氧化肽组分纯度达到95%。
用液相色谱与质谱连用（LC-MS/MS）方法测定氨基酸序列（图3），得到该ACE抑制肽的氨基酸序列HWS。
实施例2
对分离纯化获得的ACE抑制肽HWS进行活性检测。在96孔板上添加0.1 U/mL的ACE酶10μL，1 mmol/L底物FAPGG 50 μL，一定浓度的样品40 μL，用酶标仪在340 nm下测吸光值，记为A1。在37℃下继续反应30 min，再次测定吸光值，记为A2，平行实验3次，结果取平均值。其中FAPGG溶解于100 mmol/L的硼酸缓冲液中（pH 8.3，含300 mmol/L NaCl），以去离子水作为空白对照。∆A （∆A=A1-A2）表示单位时间内ACE酶活性的变化，ACE抑制活性计算公式如下：
式中，∆As为加入样品时30 min内ACE活性的变化，∆Ab为空白组30 min内吸光值的变化。
从图4可以看出， HWS的ACE抑制活性IC50为0.64mg/mL。
实施例3
ACE抑制肽与ACE的分子对接。从RCSB蛋白数据库中(http://www.rcsb.org/pdb/home/home.do)下载ACE蛋白的三维结构文件(PDB ID：1O8A)，用AutoDock Tool 4.0与AutoDockVina软件进行蛋白及ACE抑制肽的准备及对接，根据预测的结合能，选择与ACE的最佳结合方式，并确定了ACE抑制肽与ACE之间的相互作用方式。
HWS与ACE的最低结合能为-9.1 kcal/mol，两者之间的相互作用力包括范德华力、氢键结合作用，同时还与金属离子作用有关。从图5可以看出，HWS与ACE的3个残基Ala354、Glu384、Glu411形成5个氢键，其中Ala354与Glu384是ACE蛋白的S1活性口袋。
实施例4
ACE抑制肽对HUVEC细胞毒性试验。将HUVEC细胞用胰酶消化成细胞悬液，以1×105个/mL的密度接种于96孔板中，培养箱培养1天贴壁后，弃培养基，加入配制好的不同浓度ACE抑制肽（0.125、0.25、0.5 mg/mL）继续培养，对照组不添加多肽。培养24 h后，弃培养基，加入新的DMEM培养液和10%的CCK-8试剂，于37℃条件下继续孵育1 h，用酶标仪测定其在450 nm处的吸光值，计算细胞存活率。
由图6可知，随着ACE抑制肽浓度的增加，细胞活力并未发生显著性变化，说明HWS无细胞毒性，可用于制备高血压的治理或预防药物，或作为功能食品添加剂，供高血压患者治疗保健使用。
实施例5
ACE抑制肽对HUVEC细胞毒性试验。将HUVEC细胞用消化为细胞悬液，使细胞以1×105个/mL的密度接种于96孔板中，培养1天贴壁后，弃培养基，加入新鲜的培养基。实验设置为3组，分别为对照组、Ang Ⅱ刺激组和多肽+ Ang Ⅱ组，其中Ang Ⅱ+多肽组先加入含有不同浓度的多肽（0.125、0.25和0.5 mg/mL）预处理2 h后，在Ang Ⅱ刺激组和多肽+ Ang Ⅱ组中加入0.01 mg/mL的Ang Ⅱ，对照组不做任何处理。培养24 h后取培养液测定NO含量。
从图7可以看出，HWS浓度达到0.5mg/mL时，对比AngII刺激组，NO释放量显著升高（P<0.05），说明在高剂量下多肽HWS能够促进内皮细胞NO的释放，保护细胞减少血管收缩剂Ang II对细胞的影响，起到一定的降血压的作用。
实施例6
ACE抑制肽对细胞ROS水平的影响。细胞内活性氧（ROS）水平过高与很多心血管疾病的发病机制有关，图8可得，Ang II高压刺激下，细胞的ROS水平显著提高（P<0.05），说明在AngII高压刺激下使得细胞发生氧化应激反应。在有ACE抑制肽HWS存在的情况下，细胞的ROS水平显著降低（P<0.001）。说明多肽HWS还能够抑制Ang II诱导的细胞氧化应激，对于内皮细胞有保护作用，进而可能保护心血管，减少高血压病症的发生。
尽管上面已经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。
SEQUENCE LISTING
<110> 福州大学
<120> 一种来源于牡丹籽粕的ACE抑制肽
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 3
<212> PRT
<213> 人工序列
<400> 1
His Trp Ser
1
Claims (4)
1.一种牡丹籽粕ACE抑制肽，其特征在于：所述ACE抑制肽的氨基酸序列为His-Trp-Ser。
2.根据权利要求1所述一种牡丹籽粕ACE抑制肽，其特征在于，所述ACE抑制肽来源于牡丹籽粕天然提取或人工合成。
3.一种如权利要求1所述的一种牡丹籽粕ACE抑制肽的制备方法，其特征在于：从脱脂后的牡丹籽粕中分离纯化，具体步骤包括如下：
（1）牡丹籽粕蛋白酶解产物的制备：牡丹籽粕经粉碎后，经石油醚脱脂干燥后过筛备用；采用碱提酸沉法得到牡丹籽粕蛋白，然后再采用中性蛋白酶将其在酶解条件：底物浓度2%（w/v）、pH 7.50、酶添加量7200 U/g、温度43 ℃，酶解时间2 h，酶解后沸水浴10 min灭活，冷却至室温后，调节pH至中性后于4℃ 10000 rpm离心20 min，取上清液冷冻干燥，即为牡丹籽粕蛋白质酶解产物；
（2）牡丹籽粕蛋白的酶解产物利用Sephadex G-25凝胶色谱进行分离，以去离子水为洗脱液，流速0.3 mL/min，测量洗脱组分214nm波长处的吸光值；收集具有最佳ACE抑制活性的峰，利用反相高效液相色谱RP-HPLC进一步分离；RP-HPLC的洗脱梯度为0-3 min，5%（V/V）乙腈；3-43 min，5%-40%（V/V）乙腈；43-53 min，40%（V/V）乙腈；流速为2.0 mL/min，检测波长214nm，收集洗脱时间为19-21 min的洗脱峰，真空冷冻干燥即得所述ACE抑制肽。
4.一种如权利要求1所述的牡丹籽粕ACE抑制肽在ACE抑制药物及辅助降压保健食品中的应用。
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