CN104447948B - 一种从荠菜中分离获得的降血压多肽 - Google Patents
一种从荠菜中分离获得的降血压多肽 Download PDFInfo
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Abstract
本发明公开了11种降血压多肽，氨基酸序列具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11中任一项所述的序列。本发明提供的降血压多肽具有明显的血管紧张素转化酶的抑制活性，为治疗高血压的食品、药物或保健品的开发利用提供了新的解决途径，具有较好的应用前景。
Description
技术领域
本发明属于生物蛋白领域，具体地说，是从荠菜中水解得到的多肽作为血管紧张素转化酶(Angiotensin Converting Enzyme，ACE)抑制剂的应用。
背景技术
ACE(血管紧张素转化酶，Angiotensin Converting Enzyme)在人体内的作用是把血管紧张素I(Angiotensin I Converting Enzyme)转化成血紧张素II(Angiotensin IIConverting Enzyme)，同时还会使血管舒缓激肽失活，而血管紧张素II为强烈的血管收缩物质，从而使体内血压升高。ACE可催化血管紧张素I(十肽)水解成八肽的血管紧张素II，使血管进一步收缩，血压升高。也可作用于肾上腺皮质，促进醛固酮的分泌。因此，ACE是肾素-血管紧张素-醛固酮的重要成分。ACE还催化具有降压作用的缓激肽水解而失去活性。ACE广泛分布于人体各组织，以附睾、睾丸及肺的含量较丰富，其中肺毛细血管内皮细胞ACE活性最高。
ACE是一种金属肽酶，含有1个结合Zn2+的位点，这就是与底物结合的“必须结合位点(Obligatory Binding Site)”。Zn2+结合位点是ACE催化反应的活性基团所在部位。各种ACE抑制物的共同作用是与ACE活性部位的Zn2+结合，使之失活。降血压肽是由数个氨基酸组成的肽，是对ACE活性区域亲和力较强的竞争性抑制剂，它们与ACE的亲和力比血管紧张素转化酶I(AngI)或舒缓激肽更强，而且也较不容易从ACE结合区释放，降低ACE活性或使其失去活性，从而阻碍ACE催化血管紧张素I转化成血管紧张素II，使血管不收缩，以及催化水解舒缓激肽成为失活片段的两种生化反应过程，起到降血压的作用。
目前我国临床常用的一线抗高血压药主要包括利尿药、肾上腺素受体阻断药、钙通道阻滞药和血管紧张素转化酶抑制药等。虽然这些药具有很好的降压效果，但都具有一定的副作用或不良反应。而来源于食品的降血压肽 (Angiotensin-eonverting EnzymeInhibitory Peptides，ACEIPs)是一类能够降低人体血压的小分子多肽的总称。由于其降血压效果明显且对正常血压无影响、无副作用等优点，已成为目前研究的热点。
发明内容
本申请的发明人在研究降血压多肽的过程中发现，荠菜作用常见的降血压食物，其必然具有相应的降血压物质能够起到相应的作用。申请人从荠菜的水解产物中得到了一系列的多肽有些具有明显的抑制血管紧张素转化酶(ACE)的活性；经过大量的筛选和测序，获得了多个具有抑制ACE活性的多肽序列。
本发明的第一个目的在于提供降血压多肽，所述降血压多肽的氨基酸序列具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11中任一项所述的序列。序列如下：
SEQ ID NO：1 DHJXY-004 WPF
SEQ ID NO：2 DHJXY-013 RKGWAREPSR
SEQ ID NO：3 DHJXY-025 KGSRWPSGSSGE
SEQ ID NO：4 DHJXY-047 GSSEPWVFG
SEQ ID NO：5 DHJXY-086 GEPSRSSGWPPRK
SEQ ID NO：6 DHJXY-097 GSYASGRGSS
SEQ ID NO：7 DHJXY-131 YNGAWLSRVVY
SEQ ID NO：8 DHJXY-156 KEHLRYTSQVSGG
SEQ ID NO：9 DHJXY-176 GNGRYYAGSSWTA
SEQ ID NO：10 DHJXY-192 YRGNAWLSRRVEKVSS
SEQ ID NO：11 DHJXY-205 KGGGNARLSVRVGKQQPPG
本发明的第二个目的在于提供所述降血压多肽在制备降血压药物中的应用。
本发明的第三个目的在于提供所述降血压多肽在制备食品中的应用。
本发明的第四个目的在于提供所述降血压多肽在制备保健品中的应用。
根据本发明，所述降血压多肽用于抑制血管紧张素转化酶的活性。
本发明的有益效果：经过大量筛选和测序，本发明从荠菜的水解物中筛选到11种降血压多肽，均具有明显的血管紧张素转化酶的抑制活性，为治疗高血压 的食品、药物或保健品的开发利用提供了新的解决途径，具有非常广泛的应用前景。
具体实施方式
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例1降血压多肽的获得
取10g荠菜叶片及根茎，研钵捣碎，加入2M的NaOH溶液处理，通过滤膜出去纤维素杂质。由于酶解的顺序对最终的结果影响很大，因此，需要分别依次加入适量的蛋白酶E、胰凝乳蛋白酶、胶原蛋白酶、胰蛋白酶和碱性蛋白酶进行彻底酶解，将得到的酶解产物超滤，超滤后的产物采用Tricine-SDSP-AGE电泳进行分析，根据不同条带大小，分别进行割胶回收，测序。
实施例2降血压多肽活性检测
采取体外模拟测定方法测定上述降血压多肽对ACE的抑制活性，得到了具有较好结果的11个多肽，结果如表1所示。ACE抑制活性的测定原理及方法步骤如下：
多肽的ACE抑制活性测定采用体外模拟测定，体外ACE抑制率测试原理为：底物马尿酰组氨酰亮氨酸(HHL)在ACE的催化作用下，可以生产马尿酸(HA)与一个组氨酸和亮氨酸结合的二肽，通过高效液相色谱的检测方法测定马尿酸的含量，进而可以间接测定ACE活性；而降血压多肽存在时可以抑制ACE的活性，致使HA的生成减少，从而间接反应了降血压多肽的活性。
测试方法步骤如下：选取eppendof(EP)管作为反应器，编号1、2、3、4等，1号管加入20μL去离子水作为对照，2、3、4等管加入20μL的ACE抑制剂，同时各管内加入10μL的底物HHL，37℃温浴5min，然后加入10μL的ACE启动反应，在37℃水浴中反应40min，反应结束后，加入100μL1M HCl终止反应。反应液进行高速离心，过0.45μm的滤头，然后利用高效液相色谱(HPLC)测定。HPLC条件是：15％乙腈：85％水(0.1％TFA)，检测波长228nm，柱温30℃，上样量10μL。
ACE抑制率(X)按下式计算：
X＝(A对照-A1)/A对照×100％
式中：X--ACE抑制率(％)
A对照--对照组生成HA的峰面积
A1--实验组生成HA的峰面积。
实施例3多肽稳定性检测
为了使得多肽能够更加适合药用，验证多肽在血清中的稳定性。将实施例1得到的多肽，采用常规的辅料制备成为注射制剂，注入BALB/c小白鼠体内，注射量为10μg/只(每个多肽设置三个重复)，每间隔3d抽取血样，检测所述多肽时候存在，结果如表1所示。
表1、降血压多肽的ACE抑制活性的测定结果
| 多肽名称 | 序列号 | IC50(μM) | 多肽残留时间(d) |
| DHJXY-004 | SEQ ID NO：1 | 52.6 | 3 |
| DHJXY-013 | SEQ ID NO：2 | 8.7 | 9 |
| DHJXY-025 | SEQ ID NO：3 | 6.9 | 12 |
| DHJXY-047 | SEQ ID NO：4 | 8.2 | 12 |
| DHJXY-086 | SEQ ID NO：5 | 7.3 | 9 |
| DHJXY-097 | SEQ ID NO：6 | 6.9 | 12 |
| DHJXY-131 | SEQ ID NO：7 | 5.9 | 12 |
| DHJXY-156 | SEQ ID NO：8 | 7.6 | 12 |
| DHJXY-176 | SEQ ID NO：9 | 8.1 | 9 |
| DHJXY-192 | SEQ ID NO：10 | 8.8 | 12 |
| DHJXY-205 | SEQ ID NO：11 | 9.1 | 12 |
从表1的结果可以看出，分离的11个多肽均具有较好的抑制ACE酶的活性，并且在体内具有较长的存活时间，能够适用于制造药物，用于相应的高血压的治疗。
另外，本申请的多肽可以用于制造相应的食物或者保健品，可以同样达到相应的效果。
Claims (5)
1.降血压多肽，其特征在于：从荠菜中提取获得，所述降血压多肽的氨基酸序列为SEQID NO：3所述的序列。
2.如权利要求1所述的降血压多肽在制备降血压药物中的应用。
3.如权利要求1所述的降血压多肽在制备食品中的应用。
4.如权利要求1所述的降血压多肽在制备保健品中的应用。
5.如权利要求2～4中任一项所述的应用，其特征在于，所述降血压多肽用于抑制血管紧张素转化酶的活性。
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