WO2022156190A1 - 一种降压肽、长效降压肽及其制备方法 - Google Patents

一种降压肽、长效降压肽及其制备方法 Download PDF

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WO2022156190A1
WO2022156190A1 PCT/CN2021/111448 CN2021111448W WO2022156190A1 WO 2022156190 A1 WO2022156190 A1 WO 2022156190A1 CN 2021111448 W CN2021111448 W CN 2021111448W WO 2022156190 A1 WO2022156190 A1 WO 2022156190A1
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polyethylene glycol
seq
peptide
antihypertensive
antihypertensive peptide
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French (fr)
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吕应年
戚怡
罗辉
唐晓宁
林芊杏
刘欢
黄细香
蔡晓璇
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南方海洋科学与工程广东省实验室(湛江)
广东医科大学
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    • A—HUMAN NECESSITIES
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    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

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  • the invention relates to the technical field of antihypertensive drugs, in particular to an antihypertensive peptide, a long-acting antihypertensive peptide and a preparation method thereof.
  • Angiotensin converting enzyme inhibitory peptide is a polypeptide substance that inhibits the activity of angiotensin converting enzyme (ACE).
  • Angiotensin-converting enzyme inhibitory peptide is generally composed of 2 to 20 amino acid residues, and is usually obtained by enzymatic hydrolysis of protein. The decomposition of peptides to achieve the effect of lowering blood pressure. Since the cleavage site of the protease is not fixed, the polypeptides obtained after cleavage are also diverse, and the later screening and separation and purification have become technical difficulties.
  • a blood pressure lowering peptide which is a small molecule biological peptide with blood pressure lowering effect, which can be used for the prevention and treatment of hypertension, and has less side effects and good blood pressure lowering effect.
  • the antihypertensive peptide is the polypeptide sequence shown in SEQ ID NO.5.
  • the peptide of this sequence has the best antihypertensive effect among the 6 peptides.
  • the above preparation method can stably and accurately cut the enzyme to obtain the above antihypertensive peptide.
  • the above antihypertensive peptide is obtained by separation and purification in sandworms for the first time, and experiments have proved that the polypeptide has the effect of reducing blood pressure.
  • angiotensin-converting enzyme inhibitory peptides depends on the amino acid type, sequence and spatial structure of the polypeptide.
  • protein macromolecules are firstly cleaved into polypeptide fragments by protease, and as the degree of enzymatic hydrolysis deepens, polypeptides are further enzymatically cleaved to form free amino acids. Therefore, controlling the enzymatic hydrolysis process parameters (including enzyme type, dosage, pH value, temperature, enzymatic hydrolysis time) can control the cleavage site and enzymatic hydrolysis degree, which has a decisive effect on the size and structure of the polypeptide fragment.
  • the step S1 is specifically as follows: wash the sandworms, grind them into a homogenate, add 10-15 times the volume of water, mix well, and add lye to adjust the pH to 7.5-8.5.
  • the step S2 is specifically: adding the enzyme powder slurry according to 0.5%-1% of the weight of the sandworm, the enzymolysis temperature is 45 ⁇ 5°C, the enzymolysis time is 4-6h, and after the enzymolysis is completed, The temperature was raised to 95 ⁇ 5°C, and the temperature was maintained for 3-8 minutes to inactivate the enzyme, thereby obtaining an enzymatic hydrolysis mixture.
  • the enzyme powder slurry contains trypsin and neutral protease, and the weight ratio of trypsin and neutral protease is (2-4):1.
  • the step S3 is specifically: centrifuging the enzymatic hydrolysis mixture, discarding the precipitate, and retaining the supernatant to obtain an enzymatic hydrolysis solution.
  • the present invention also provides a method for preparing a polyethylene glycol-modified antihypertensive peptide, comprising the following steps:
  • the step 2) is specifically: mixing polyethylene glycol and amino acid, reacting and dehydrating under acidic conditions to obtain activated polyethylene glycol, dissolving it in an acetonitrile-water solution, and dissolving the activated polyethylene glycol in an acetonitrile-water solution.
  • concentration of ethylene glycol is 50-150 mg/ml, and the temperature is lowered to 2-8 °C for use.
  • the amino acid is glycine.
  • Glycine has a small molecular weight and good stability, and the activation yield is as high as 70%, while the activation yield of other amino acids is about 30%-50%. Activation with glycine yields higher yields with fewer by-products.
  • the step 4) is specifically as follows: using a dialysis membrane with a molecular weight cut-off of 3000 Da to dialyze the product of the step 3), the dialysis membrane is covered with solid PEG6000 for dehydration, concentrated, and subjected to gel column chromatography, using Acetate buffer elution to obtain polyethylene glycol-modified antihypertensive peptide.
  • the present invention also provides a polyethylene glycol-modified antihypertensive peptide obtained by the above preparation method.
  • the present invention also provides the use of the above antihypertensive peptide in the preparation of antihypertensive drugs.
  • the dosing frequency can be extended to once a week or once a month to improve the drug efficacy and treatment compliance and reduce the pain of the patient.
  • the present invention also provides the use of the above-mentioned polyethylene glycol-modified antihypertensive peptide in preparing an antihypertensive drug.
  • polyethylene glycol to modify small molecule peptides can prolong the duration of efficacy of antihypertensive peptides.
  • Fig. 1 is the MS spectrum of the polypeptide shown in SEQ ID NO.1;
  • Fig. 7 is the MS spectrum of the polypeptide shown in SEQ ID NO.4;
  • Fig. 8 is the HPLC spectrum of the polypeptide shown in SEQ ID NO.4;
  • Fig. 10 is the HPLC spectrum of the polypeptide shown in SEQ ID NO.5;
  • Figure 13 is the ACE inhibition rate of 6 kinds of polypeptides (10mmol/L);
  • Example 1 The small molecule peptide obtained in Example 1 was dissolved in a Tris-HC buffer with a pH of 9.0, prepared into a peptide solution with a concentration of 20 mg/ml, and cooled to 4°C;
  • step (3) the product of step (3) is bagged for dialysis with a 3000Da dialysis membrane to remove unreacted small molecular components, the dialysis membrane is put into an aluminum box, stored in a refrigerator, covered with solid PEG6000 for dehydration, and concentrated to 1/5 of the volume;
  • Example 1 Take the six kinds of polypeptides isolated in Example 1 and carry out the ACE inhibitory activity experiment, and the test method is as follows:
  • test results are shown in Figure 13. It can be seen from the results that the above polypeptides all have ACE inhibitory activity, and S4525 has the best inhibitory effect.

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Abstract

本发明提供一种降压肽、经聚乙二醇修饰的降压肽及其制备方法。本发明的降压肽包含至少一种如SEQ ID NO.1-SEQ ID NO.6所示的多肽序列。本发明的多肽是首次从沙虫中分离纯化得到，实验结果表明该多肽具有降压效果，可用于制备降压药物。

Description

一种降压肽、长效降压肽及其制备方法 技术领域
本发明涉及降压药物技术领域，特别是涉及一种降压肽、长效降压肽及其制备方法。
背景技术
血管紧张素转化酶抑制肽(angiotensin converting enzymeinhibitory peptides)是一种对血管紧张素转换酶(ACE)活性具有抑制作用的多肽类物质。血管紧张素转化酶抑制肽一般由2～20个氨基酸残基组成，通常是由蛋白质酶解而得，其通过抑制血管紧张素转换酶的活性，阻碍血管紧张素Ⅱ的生成以及抑制血管舒缓激肽的分解而达到降血压的作用。由于蛋白酶的酶切位点不固定，酶切后得到的多肽也是多种多样的，后期的筛选及分离纯化成为技术难点所在。
发明内容
基于此，有必要针对上述问题，提供一种降压肽，该降压肽为具有降血压作用的小分子生物肽，可用于高血压的防治，其副作用小、降压效果好。
一种降压肽，包括至少一种如下多肽序列：
Gly-Phe-Ala-Gly-Asp-Asp-Ala-pro-Arg(SEQ ID NO.1)，
Gly-Lue-Gly-Gly-Lue-Ser-Pro-Glu-Lys(SEQ ID NO.2)，
Lue-Pro-Lys(SEQ ID NO.3)，
Pro-Arg-Pro(SEQ ID NO.4)，
Ser-Arg-Pro(SEQ ID NO.5)，
Arg-Pro-Ala(SEQ ID NO.6)。
上述降压肽，是首次从海洋生物沙虫中分离纯化得到的，实验证明其具有较好的降压的效果，可用于高血压的防治。
在其中一个实施例中，所述降压肽为SEQ ID NO.5所示的多肽序列。该序列的多肽在6种多肽中降压效果最佳。
本发明还提供一种上述降压肽的制备方法，包括以下步骤：
S1、选取沙虫，加碱液调节pH值为7.5-8.5；
S2、加酶进行酶解，酶解完成，升温使酶失活，得到酶解混合物；
S3、将酶解混合物进行固液分离，保留液体，得酶解液；
S4、将酶解液进行超滤透析，保留分子量≤100KDa的超滤液；
S5、对所述超滤液进行分离提纯，即得如SEQ ID NO.1-SEQ ID NO.6所示的多肽。
上述制备方法，可以稳定、准确地对酶进行切割，得到上述降压肽，上述降压肽是首次在沙虫中分离纯化得到的，且实验证明该多肽具有降压的效果。
血管紧张素转化酶抑制肽的活性，取决于多肽的氨基酸种类、序列及其空间结构。在上述酶解过程中，蛋白质大分子首先被蛋白酶切割成多肽片段，随着酶解程度加深，多肽被进一步酶解切割形成游离氨基酸。因此，控制好酶解工艺参数(包括酶的种类、用量、pH值、温度、酶解时间)，可控制切割位点及酶解程度，这对于多肽片段的大小及结构具有决定作用。
在其中一个实施例中，所述步骤S1具体为：将沙虫洗净，绞碎匀浆，加入10-15倍体积的水，混匀，加碱液调节pH值为7.5-8.5。
在其中一个实施例中，所述步骤S2具体为：按沙虫重量的0.5％-1％加入酶粉浆，酶解温度为45±5℃，酶解时间4-6h，酶解完成后，升温至95±5℃，保温3～8min，使酶失活，得到酶解混合物。
在其中一个实施例中，所述酶粉浆中含有胰蛋白酶和中性蛋白酶，胰蛋白酶和中性蛋白酶的重量比为(2-4):1。
在其中一个实施例中，所述沙虫和酶的用量比为100g：0.5-1g。在沙虫(方格星虫)匀浆液的酶解过程中，若酶解不足，则星虫蛋白以大分子状态存在，不能切割得到具有ACE抑制活性的肽段；若酶解过度，则肽段被进一步切割形成游离氨基酸，不具有活性结构，从而失去了ACE抑制活性。
在其中一个实施例中，所述步骤S3具体为：对所述酶解混合物进行离心，弃去沉淀，保留上清液，得到酶解液。
在其中一个实施例中，所述步骤S5具体为：将聚乙二醇修饰铜离子螯合琼脂糖装柱，水洗，磷酸盐缓冲液洗脱至平衡，再将所述超滤液过柱层析，得到降压肽。
本发明还提供一种聚乙二醇修饰的降压肽的制备方法，包括以下步骤：
1)将权利要求1-2任一项所述的降压肽溶于缓冲液中，得到多肽溶液；
2)将聚乙二醇与甘氨酸混合，经反应得到活化的聚乙二醇；
3)将多肽溶液与活化的聚乙二醇混合，反应完成后加酸调节pH值为2-4，终止反应；
4)将步骤3)的产物进行透析，经分离纯化，得到聚乙二醇修饰的降压肽。
在其中一个实施例中，所述步骤1)具体为：将所述降压肽溶于Tris-HCl缓冲液中，配制成浓度5-50mg/ml的肽溶液，降温到2-8℃备用。
在其中一个实施例中，所述步骤2)具体为：将聚乙二醇与氨基酸混合，在酸性条件下反应脱水得到活化的聚乙二醇，将其溶于乙腈-水溶液中，活化的聚乙二醇的浓度为50-150mg/ml，降温至2-8℃备用。
优选地，所述聚乙二醇分子量为3000-30000。包括但不限于支链或直链的聚乙二醇。
优选地，所述氨基酸选用甘氨酸。甘氨酸分子量小，稳定性好，活化的收率高达70％，而其他氨基酸活化收率约为30％-50％。采用甘氨酸进行活化产率更高，副产物更少。
在其中一个实施例中，所述步骤3)具体为：将上述肽溶液和活化的聚乙二醇溶液按照1:(10-50)的比例混合，2-8℃下搅拌36-72h；反应完成，加酸调节pH值为2-4，终止反应。
聚乙二醇的羟基和单个氨基酸如甘氨酸的羧基结合，在酸性条件下形成酯键，得到带氨基NH 2末端的活化聚乙二醇。活化的聚乙二醇和小分子肽的羧基发生脱水缩合反应生成酰胺 键，即可将聚乙二醇加长到肽分子上，得到经聚乙二醇修饰的降压肽。
在其中一个实施例中，所述步骤4)具体为：用截留分子量为3000Da的透析膜对步骤3)的产物进行透析，透析膜加固体PEG6000覆盖脱水，浓缩，过凝胶柱层析，用醋酸盐缓冲液洗脱，得到聚乙二醇修饰的降压肽。
本发明还提供一种采用上述制备方法得到的聚乙二醇修饰的降压肽。
本发明还提供一种上述降压肽在制备降压药中的用途。
由于多肽类的生物制品口服给药容易在消化道被破坏，降压肽被降解活性消失，因此可采用制备为注射剂，进行注射给药。给药频率可延长至每周一次或每月一次，提高药物的药效和治疗依从性，减轻病人的痛苦。
本发明还提供一种上述经聚乙二醇修饰的降压肽在制备降压药中的用途。
采用聚乙二醇修饰小分子肽，可延长降压肽的药效持续时间。
与现有技术相比，本发明具有以下有益效果：
本发明的降压肽，是首次从海洋生物沙虫中分离纯化得到的，实验证明其具有较好的降压的效果，可用于高血压的防治。采用本发明制备方法，可分离得到上述降压肽，制备结果稳定可靠，分离提纯效率高。本发明的经聚乙二醇修饰的降压肽可延长多肽的药效时间。
附图说明
图1为SEQ ID NO.1所示多肽的MS图谱；
图2为SEQ ID NO.1所示多肽的HPLC图谱；
图3为SEQ ID NO.2所示多肽的MS图谱；
图4为SEQ ID NO.2所示多肽的HPLC图谱；
图5为SEQ ID NO.3所示多肽的MS图谱；
图6为SEQ ID NO.3所示多肽的HPLC图谱；
图7为SEQ ID NO.4所示多肽的MS图谱；
图8为SEQ ID NO.4所示多肽的HPLC图谱；
图9为SEQ ID NO.5所示多肽的MS图谱；
图10为SEQ ID NO.5所示多肽的HPLC图谱；
图11为SEQ ID NO.6所示多肽的MS图谱；
图12为SEQ ID NO.6所示多肽的HPLC图谱；
图13为6种多肽(10mmol/L)的ACE抑制率；
图14为6种多肽的ACE抑制活性IC 50值测试结果。
具体实施方式
为了便于理解本发明，以下将给出较佳实施例对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
实施例1
降压肽的酶解提取纯化，包括以下步骤：
(1)沙虫洗净至无沙，绞碎匀浆；
(2)加入沙虫12倍重量的纯水，混匀，加1％的氢氧化钠溶液调节为pH7.5；
(3)胰蛋白酶和中性蛋白酶以重量比3:1混合，加水溶解成浆，按沙虫重量的0.8％加入酶粉浆，沙虫与酶的用量比为100g：0.7g；
(4)将酶粉浆缓慢加入沙虫液中，边加边搅拌混匀，设置温度为45℃，酶解5h；
(5)酶解完毕，升温至95℃，保温5min杀酶；
(6)使用低温高速离心机进行离心处理，分离固液，弃去沉淀，上清备用；
(7)将上清液进行超滤透析，保留分子量≤100KDa的超滤液；
(8)将聚乙二醇修饰金属鳌合亲和层析介质(聚乙二醇修饰铜离子螯合琼脂糖Chelating Sepharose Fast Flow，mPEG-CSFF-Cu)装柱，采用自然沉降法装柱，柱体积约为20ml，用蒸馏水洗脱以除去未结合的金属离子，用pH6.8磷酸钠缓冲液(内含0.5mol/L氯化钠)洗脱至平衡；上样，上样缓冲液为PH6.8磷酸钠缓冲液(内含0.5mol/L氯化钠)，洗脱缓冲液为pH5.0的柠檬酸缓冲液，经金属螯合色谱柱层析，得到以下多肽：
表1.多肽序列
序列号 序列 序列简写 名称
SEQ ID NO.1 Gly-Phe-Ala-Gly-Asp-Asp-Ala-pro-Arg GFAGDDAPR S-4521
SEQ ID NO.2 Gly-Lue-Gly-Gly-Lue-Ser-Pro-Glu-Lys GLGGLSPEK S-4522
SEQ ID NO.3 Lue-Pro-Lys LPK S-4523
SEQ ID NO.4 Pro-Arg-Pro PRP S-4524
SEQ ID NO.5 Ser-Arg-Pro SRP S-4525
SEQ ID NO.6 Arg-Pro-Ala RPA S-4526
(9)小分子肽液经透析脱盐浓缩，真空冻干，保存。
本实施例中得到的多肽的质谱和高效液相色谱结果如图1-12所示。多肽S-4521～S-4526的HPLC测试结果分别如表2-7所示。
表2.S-4521 HPLC检测结果
Figure PCTCN2021111448-appb-000001
表3.S-4522 HPLC检测结果
Figure PCTCN2021111448-appb-000002
表4.S-4523 HPLC检测结果
Figure PCTCN2021111448-appb-000003
表5.S-4524 HPLC检测结果
Figure PCTCN2021111448-appb-000004
表6.S-4525 HPLC检测结果
Figure PCTCN2021111448-appb-000005
表7.S-4526 HPLC检测结果
Figure PCTCN2021111448-appb-000006
实施例2
聚乙二醇修饰降压肽的制备。
(1)将实施例1中得到的小分子肽溶解于pH为9.0的Tris-HC缓冲液中，配制成浓度20mg/ml的肽溶液，降温到4℃；
(2)将聚乙二醇(分子量4000)与甘氨酸按摩尔比1:1混合，在酸性条件下，经反应得到活化的聚乙二醇，将其溶解于乙腈-水溶液(乙腈与水的体积比为98:2)中，得到活化的聚乙二醇溶液，活化的聚乙二醇的浓度为100mg/ml，降温到4℃；
(3)将肽溶液和活化的聚乙二醇溶液按体积比1:40混合，4℃避光缓慢搅拌40h，反应完成加醋酸调节pH为3，终止反应；
(4)步骤(3)的产物用3000Da的透析膜装袋透析，除去未反应小分子成分，透析膜放入铝盒，冰箱保存，加固体PEG6000覆盖脱水，浓缩至体积为1/5；
(5)将浓缩的样品上样凝胶柱层析，用醋酸盐缓冲液洗脱，根据紫外吸收分段收集洗脱峰，电泳检测确定纯度大于90％的样品。
实验例1
一、ACE抑制活性测试。
取实施例1中分离得到的六种多肽进行ACE抑制活性实验，测试方法如下：
将上述六种多肽分别配制为10mmol/L的肽溶液，采用经典马尿酸含量法测定ACE抑制活性。
测试结果如图13所示。从结果可以看出，上述多肽均具有ACE抑制活性，其中S4525抑制效果最佳。
二、ACE抑制活性IC 50值测试。
将上述六种多肽分别配制成不同浓度，采用上述相同方法测试各种多肽的ACE抑制活性IC 50值。
结果如图14和表8所示。从结果可以看出，S4525对应的IC 50值最低(30.5μmol/L)。
表8.多肽抑制ACE的IC 50值
多肽 IC 50(mmol/L)
S-4521 0.858
S-4522 0.906
S-4523 13.34
S-4524 0.465
S-4525 0.0305
S-4526 0.961
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
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. 一种降压肽，其特征在于，包括至少一种如下多肽序列：
    Gly-Phe-Ala-Gly-Asp-Asp-Ala-pro-Arg(SEQ ID NO.1)，
    Gly-Lue-Gly-Gly-Lue-Ser-Pro-Glu-Lys(SEQ ID NO.2)，
    Lue-Pro-Lys(SEQ ID NO.3)，
    Pro-Arg-Pro(SEQ ID NO.4)，
    Ser-Arg-Pro(SEQ ID NO.5)，
    Arg-Pro-Ala(SEQ ID NO.6)。
  2. 根据权利要求1所述的降压肽，其特征在于，所述降压肽为SEQ ID NO.5所示的多肽序列。
  3. 一种如权利要求1或2所述的降压肽的制备方法，其特征在于，包括以下步骤：
    S1、选取沙虫，加碱液调节pH值为7.5-8.5；
    S2、加酶进行酶解，酶解完成，升温使酶失活，得到酶解混合物；
    S3、将酶解混合物进行固液分离，保留液体，得酶解液；
    S4、将酶解液进行超滤透析，保留分子量≤100KDa的超滤液；
    S5、对所述超滤液进行分离提纯，即得如SEQ ID NO.1-SEQ ID NO.6所示的多肽。
  4. 根据权利要求3所述的制备方法，其特征在于，所述步骤S1具体为：将沙虫洗净，绞碎匀浆，加入10-15倍体积的水，混匀，加碱液调节pH值为7.5-8.5；
    所述步骤S2具体为：按沙虫重量的0.5％-1％加入酶粉浆，酶解温度为45±5℃，酶解时间为4-6h，酶解完成后，升温至95±5℃，保温3-8min，使酶失活，得到酶解混合物。
  5. 根据权利要求3所述的制备方法，其特征在于，所述酶粉浆中含有胰蛋白酶和中性蛋白酶，胰蛋白酶和中性蛋白酶的重量比为(2-4):1；
    所述沙虫和酶的用量比为100g：0.5-1g。
  6. 根据权利要求3所述的制备方法，其特征在于，所述步骤S3具体为：对所述酶解混合物进行离心，弃去沉淀，保留上清液，得到酶解液；
    所述步骤S5具体为：将聚乙二醇修饰铜离子螯合琼脂糖装柱，水洗，磷酸盐缓冲液洗脱至平衡，再将所述超滤液过柱层析，得到降压肽。
  7. 一种聚乙二醇修饰的降压肽的制备方法，其特征在于，包括以下步骤：
    1)将权利要求1-2任一项所述的降压肽溶于缓冲液中，得到多肽溶液；
    2)将聚乙二醇与氨基酸混合，经反应得到活化的聚乙二醇；
    3)将多肽溶液与活化的聚乙二醇混合，反应完成后加酸调节pH值为2-4，终止反应；
    4)将步骤3)的产物进行透析，经分离纯化，得到聚乙二醇修饰的降压肽。
  8. 根据权利要求7所述的制备方法，其特征在于，所述步骤1)具体为：将所述降压肽溶于Tris-HCl缓冲液中，配制成浓度5-50mg/ml的肽溶液，降温到2-8℃备用；
    所述步骤2)具体为：将聚乙二醇与氨基酸混合，在酸性条件下反应脱水得到活化的聚乙二醇，将其溶于乙腈-水溶液中，活化的聚乙二醇的浓度为50-150mg/ml，降温至2-8℃备用；
    所述步骤3)具体为：将上述肽溶液和活化的聚乙二醇溶液按照1:(10-50)的比例混合，2-8℃下搅拌36-72h；反应完成，加酸调节pH值为2-4，终止反应；
    所述步骤4)具体为：用截留分子量为3000Da的透析膜对步骤3)的产物进行透析，透析膜加固体PEG6000覆盖脱水，浓缩，过凝胶柱层析，用醋酸盐缓冲液洗脱，得到聚乙二醇修饰的降压肽。
  9. 一种采用权利要求7或8的制备方法得到的聚乙二醇修饰的降压肽。
  10. 权利要求1～2任一项所述的降压肽或权利要求9所述的聚乙二醇修饰降压肽在制备降压药中的用途。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101240016A (zh) * 2008-02-28 2008-08-13 山东大学 鲨鱼蛋白降压肽及其制备方法与应用
CN102231981A (zh) * 2008-02-08 2011-11-02 昌达生物科技公司 蛋白质或肽的缓释给药组合物
CN105777865A (zh) * 2015-12-25 2016-07-20 滨州万嘉生物科技有限公司 具有抗氧化功能的鱼肉耐消化肽、其制备方法和用途
CN112646003A (zh) * 2020-12-22 2021-04-13 广东湛江海洋医药研究院 一种方格星虫ace抑制肽及其制备方法和应用
CN112661811A (zh) * 2021-01-21 2021-04-16 南方海洋科学与工程广东省实验室(湛江) 一种降压肽、长效降压肽及其制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2618163A1 (en) * 2008-02-07 2009-08-07 K. W. Michael Siu Head and neck cancer biomarkers
CN102336806A (zh) * 2011-09-23 2012-02-01 华东理工大学 一种血管紧张素转化酶(ace)抑制物
CN104558114B (zh) * 2013-10-21 2018-06-08 中国科学院大连化学物理研究所 花粉中具有ace抑制活性的多肽及其应用
CN108358998B (zh) * 2018-04-25 2021-05-11 宁波大学 一种星虫肽及其在制备妊娠期高血压治疗药物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102231981A (zh) * 2008-02-08 2011-11-02 昌达生物科技公司 蛋白质或肽的缓释给药组合物
CN101240016A (zh) * 2008-02-28 2008-08-13 山东大学 鲨鱼蛋白降压肽及其制备方法与应用
CN105777865A (zh) * 2015-12-25 2016-07-20 滨州万嘉生物科技有限公司 具有抗氧化功能的鱼肉耐消化肽、其制备方法和用途
CN112646003A (zh) * 2020-12-22 2021-04-13 广东湛江海洋医药研究院 一种方格星虫ace抑制肽及其制备方法和应用
CN112661811A (zh) * 2021-01-21 2021-04-16 南方海洋科学与工程广东省实验室(湛江) 一种降压肽、长效降压肽及其制备方法

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