WO2018006667A1 - 降血压肽和降血压蛋白质及其应用 - Google Patents

降血压肽和降血压蛋白质及其应用 Download PDF

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WO2018006667A1
WO2018006667A1 PCT/CN2017/086294 CN2017086294W WO2018006667A1 WO 2018006667 A1 WO2018006667 A1 WO 2018006667A1 CN 2017086294 W CN2017086294 W CN 2017086294W WO 2018006667 A1 WO2018006667 A1 WO 2018006667A1
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blood pressure
pressure lowering
peptide
antihypertensive
converting enzyme
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PCT/CN2017/086294
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English (en)
French (fr)
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谢静莉
陈绪军
朱巧莎
吴俊杰
周琰
魏东芝
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华东理工大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of food or medicine, and relates to a blood pressure lowering peptide and a blood pressure lowering protein and an application thereof.
  • Angiotensin converting enzyme is an enzyme that causes an increase in blood pressure and has the following two regulatory pathways.
  • angiotensin converting enzyme can excise two amino acids (His-Leu) at the end of angiotensin I, thereby converting the angiotensin I into angiotensin II.
  • amino acid sequence of angiotensin I is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, abbreviated as DRVYIHPFHL.
  • the amino acid sequence of angiotensin II is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, abbreviated as DRVYIHPF.
  • Angiotensin II is a vasoconstrictor that promotes contraction of blood vessels, thereby raising blood pressure.
  • angiotensin converting enzyme is capable of excising two amino acids (Phe-Arg) at the end of bradykinin to inactivate it.
  • the amino acid sequence of the bradykinin is Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
  • the amino acid sequence of the inactivated bradykinin is Arg-Pro-Pro-Gly-Phe-Ser-Pro .
  • the bradykinin has a function of expanding blood vessels, but when the bradykinin is inactivated, the ability to dilate blood vessels is lost, and blood pressure is increased.
  • ACE is a metal peptidase that contains a site that binds to Zn 2+ , which is the "obligatory binding site" that binds to the substrate.
  • the Zn 2+ binding site is the site of the active group of the ACE catalytic reaction.
  • the combined action of various ACE inhibitors is to bind to the Zn 2+ binding site of ACE and inactivate it.
  • angiotensin-converting enzyme If angiotensin-converting enzyme is inactivated, angiotensin-converting enzyme cannot convert angiotensin I to angiotensin II, and blood vessels will not contract. Meanwhile, angiotensin-converting enzyme cannot make bradykinin Inactivated, the blood vessels will expand. The combined effect of the two will lower blood pressure. Therefore, the search for a polypeptide or protein capable of inactivating angiotensin converting enzyme has become a technical problem to be solved.
  • One of the objects of the present invention is to provide a blood pressure lowering peptide capable of inhibiting angiotensin converting enzyme.
  • Another object of the present invention is to provide an application of the above blood pressure lowering peptide.
  • the solution of the present invention is:
  • a hypotensive peptide having an amino acid sequence of VHW and referred to as a second antihypertensive peptide is referred to as a second antihypertensive peptide.
  • a blood pressure lowering peptide having an amino acid sequence as shown in SEQ ID NO: 1, is referred to as a third blood pressure lowering peptide.
  • Any of the above blood pressure lowering peptides can be used as an angiotensin converting enzyme inhibitor.
  • Any of the above blood pressure lowering peptides can be used for the preparation of a blood pressure lowering drug.
  • Any of the above-mentioned blood pressure lowering peptides can be used for preparing a blood pressure lowering health care product.
  • a blood pressure lowering protein comprising one or more of TTW, VHW and an amino acid sequence as shown in SEQ ID NO: 1.
  • the above blood pressure lowering protein can be used as an angiotensin converting enzyme inhibitor.
  • the above blood pressure lowering protein can be used to prepare a blood pressure lowering drug.
  • the above blood pressure lowering protein can be used for preparing a blood pressure lowering health care product.
  • the antihypertensive peptide of the invention is screened from the hydrolysate of natural food, has obvious inhibitory activity on angiotensin converting enzyme, and can be used alone for preparing blood pressure lowering medicine or blood pressure lowering health care product, and can also be present A technical antihypertensive drug is used in combination to achieve a better synergistic blood pressure lowering effect.
  • Fig. 1 is a schematic view showing the hydrogen bond formation of the first antihypertensive peptide of the present invention and an angiotensin converting enzyme active center.
  • FIG. 2 is a schematic view showing the hydrogen bond formation of the second antihypertensive peptide and the angiotensin converting enzyme active center of the present invention.
  • Fig. 3 is a schematic view showing the hydrogen bond formation of the third antihypertensive peptide and the angiotensin converting enzyme active center of the present invention.
  • Figure 4 is a graph showing the hypotensive effect of a hypotensive peptide.
  • the present invention provides a plurality of blood pressure lowering peptides and uses thereof.
  • the present invention provides a blood pressure lowering peptide composed of three amino acids, the amino acid sequence of which is Thr-Thr-Trp, abbreviated as TTW.
  • TTW blood pressure lowering peptide
  • the amino acids in the present invention are all arranged from the N-terminus to the C-segment.
  • the formation of these seven hydrogen bonds is shown in Figure 1.
  • the amino acids in the circle represent the amino acid sequence in the ACE, and the middle chain is the configuration in which the sequence of the blood pressure lowering peptide is formed in the ACE.
  • amino acid residues are important amino acid residues in the active center of angiotensin converting enzyme (ACE) and have an important influence on the activity of ACE.
  • ACE angiotensin converting enzyme
  • the active center of angiotensin converting enzyme (ACE) can no longer bind to angiotensin, thereby vascular
  • the angiotensin converting enzyme (ACE) is inactivated, and therefore, the first hypotensive peptide can act as an angiotensin converting enzyme inhibitor.
  • the first antihypertensive peptide can be used to prepare a blood pressure lowering drug.
  • the first antihypertensive peptide of the present invention can be used alone as a blood pressure lowering drug.
  • the first antihypertensive peptide needs to be used together with an auxiliary material to prepare a pharmaceutical granule, a capsule, a tablet (such as a sugar-coated tablet or a film-coated tablet), Pills, oral liquids or injections.
  • the excipient may be maltodextrin and/or vanillin.
  • the adjuvant may be starch and/or magnesium stearate.
  • the excipient may be sodium benzoate, aspartame, acesulfame, essence, and purified water.
  • the excipient may be mannitol, sodium dihydrogen phosphate, and/or disodium hydrogen phosphate.
  • the injection can be configured as a 0.9% sodium chloride injection or a 5% glucose injection followed by intravenous drip or intramuscular injection.
  • the first antihypertensive peptide of the present invention can also be used in combination with a blood pressure lowering drug of the prior art in order to achieve a better synergistic blood pressure lowering effect.
  • Prior art blood pressure lowering drugs include beta blockers, calcium antagonists, ACE inhibitors, and diuretics.
  • the beta blocker is selected from the group consisting of nebivolol, pradolol, arololol, atenolol, celrilol, carvedilol, labetalol, bisoprolol.
  • the calcium antagonist is selected from the group consisting of nifedipine, amlodipine (including levamlodipine), laccapine, felodipine, nilvadipine, lacidipine, and nisoldipine.
  • ACE inhibitors are selected from the group consisting of alapril, benazepril, captopril, cilostril, cilazapril, delaipril, enalapril, enalapril, fosinopril, Lisinopril, ramipril, ramipril, perindopril, quinapril, spironolide, temocapril, trandolapril.
  • the diuretic is selected from the group consisting of hydrochlorothiazide and trichlorothiazide.
  • the first blood pressure lowering peptide can be used for preparing a blood pressure lowering health care product.
  • the first antihypertensive peptide of the invention together with the auxiliary material can be used as a blood pressure lowering health care product, and the auxiliary materials can include: starch, gelatin, titanium dioxide, whey protein, sucrose, vitamin E, glutathione, arginine, melon Acid, dietary fiber, collagen, cephalin, cellulose, glucose, xylose, honey, lecithin, carotenoid, vitamin B1, vitamin B2, vitamin C, iron, calcium, bee pollen, black fungus powder ( 200-500 mesh), mushroom powder (300-500 mesh), ganoderma powder (300-500 mesh), konjac flour (300-500 mesh), astragalus powder (200-500 mesh), tannin powder (200-500 mesh) And one or more of ginseng powder (500-1000 mesh) and the like.
  • the auxiliary materials can include: starch, gelatin, titanium dioxide, whey protein, sucrose, vitamin E, glutathione, arginine, melon Acid, dietary fiber, collagen
  • the invention provides a blood pressure lowering peptide composed of three amino acids, the amino acid sequence of which is Val-His-Trp, abbreviated as VHW.
  • VHW Val-His-Trp
  • the following are collectively referred to as a second blood pressure lowering peptide.
  • V in the second antihypertensive peptide forms two hydrogen bonds with Glu411 of the angiotensin converting enzyme (ACE) active center; H in the second antihypertensive peptide and Ala354, His353, Glu384 A hydrogen bond is formed; W in the second blood pressure lowering peptide forms a hydrogen bond with Tyr520, so the second blood pressure lowering peptide forms a total of 7 hydrogen bonds with angiotensin converting enzyme (ACE).
  • ACE angiotensin converting enzyme
  • amino acid residues are also important amino acid residues in the active center of angiotensin converting enzyme (ACE) and have an important influence on the activity of ACE.
  • ACE angiotensin converting enzyme
  • the active center of angiotensin converting enzyme (ACE) can no longer bind to angiotensin, thereby vascular
  • the angiotensin converting enzyme (ACE) is inactivated, and therefore, the second antihypertensive peptide can also act as an angiotensin converting enzyme inhibitor.
  • the second antihypertensive peptide can be used to prepare a blood pressure lowering drug.
  • the second blood pressure lowering peptide can be used for preparing a blood pressure lowering health care product.
  • the auxiliary substance which can be used together with the second blood pressure lowering peptide can refer to the first blood pressure lowering peptide.
  • the present invention provides a blood pressure lowering peptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • a blood pressure lowering peptide having an amino acid sequence as shown in SEQ ID NO: 1.
  • the third antihypertensive peptide consists of four amino acids, and its amino acid sequence is Lys-Ala-Lys-Trp, abbreviated as KAKW.
  • amino acid residues are important amino acid residues in the active center of angiotensin converting enzyme (ACE) and have an important influence on the activity of ACE.
  • ACE angiotensin converting enzyme
  • the active center of angiotensin converting enzyme (ACE) can no longer bind to angiotensin, thereby vascular
  • the angiotensin converting enzyme (ACE) is inactivated, and therefore, the third antihypertensive peptide can act as an angiotensin converting enzyme inhibitor.
  • the third antihypertensive peptide can be used to prepare a blood pressure lowering drug.
  • the third blood pressure lowering peptide can be used for preparing blood pressure lowering health care products.
  • the auxiliary compound which can be used together with the third blood pressure lowering peptide can refer to the first blood pressure lowering peptide.
  • a blood pressure lowering protein comprising one or more of TTW, VHW and an amino acid sequence as shown in SEQ ID NO: 1.
  • polypeptides and proteins can be synthesized by artificial methods, so the synthesis method will not be described again.
  • blood pressure lowering protein can be used to prepare blood pressure lowering drugs.
  • blood pressure lowering protein can be used to prepare blood pressure lowering health products.
  • the auxiliary substance which can be used together with the blood pressure lowering protein can refer to the first blood pressure lowering peptide.
  • the various antihypertensive peptides of the present invention all belong to competitive inhibitors with strong affinity to the active region of angiotensin converting enzyme, and have higher affinity with the active region than angiotensin I or bradykinin, and once combined It is also not easily released from the binding region, thereby reducing the activity of the active region of angiotensin converting enzyme and even inactivating it, thereby preventing angiotensin converting enzyme from converting angiotensin I into angiotensin II and obstructing blood vessels.
  • Angiotensin-converting enzyme inactivates soothing kinin, which in turn lowers blood pressure.
  • the ability of various hypotensive peptides to inhibit angiotensin converting enzyme was verified by experiments below.
  • Hip-His-Leu is a substrate for angiotensin-converting enzyme (ACE) and is hydrolyzed to hippuric acid and His-Leu.
  • ACE angiotensin-converting enzyme
  • the inhibitor can inhibit the ability of angiotensin converting enzyme to catalyze the hydrolysis of equine glutamic acid leucine, and reduce the content of hippuric acid in the product. Therefore, the inhibitor pair can be calculated by detecting the content of hippuric acid in the product. The inhibition rate of angiotensin converting enzyme.
  • reaction liquid in each EP tube is separately filtered with a 0.22 ⁇ m filter, and the filtered liquid is stored in a corresponding liquid phase vial;
  • the inhibition rate-sample concentration curve is obtained, and the concentration of the sample to be tested when the inhibition rate reaches 50% is obtained by the curve, that is, the test is to be tested.
  • the IC 50 of the sample is obtained.
  • the sample to be tested is a sample containing a first antihypertensive peptide, a second antihypertensive peptide, a third antihypertensive peptide or a blood pressure lowering protein.
  • the IC 50 of the sample to be tested is shown in Table 2.
  • Antihypertensive peptide IC 50 ( ⁇ M) First hypotensive peptide (TTW) 0.61 Second blood pressure lowering peptide (VHW) 0.91 Third blood pressure lowering peptide (KAKW) 2.02
  • This experiment mainly verified the effects of various antihypertensive peptides on blood pressure in hypertensive rats, including the following steps:
  • the antihypertensive peptide not only has a good antihypertensive activity in vitro, but also has good in vivo activity after digestion and absorption through the gastrointestinal tract, and its blood pressure lowering function is not lost.
  • the blood pressure lowering drug tablet of the present embodiment contains 5 wt% of a blood pressure lowering peptide and 95 wt% of an excipient.
  • the antihypertensive peptide is the first antihypertensive peptide, and the auxiliary materials are starch and magnesium stearate.
  • the antihypertensive peptide may also be any one or more of the second antihypertensive peptide, the third antihypertensive peptide, and the hypotensive protein.
  • the excipient may be any one or more of starch, sucrose, and maltodextrin.
  • the content of the blood pressure lowering peptide may be 1 to 20% by weight and the content of the auxiliary material may be 80 to 99% by weight.
  • Example 2 Blood pressure lowering health care product
  • the blood pressure lowering drug tablet of this example contained 15% by weight of a blood pressure lowering peptide and 85% by weight of an excipient.
  • the blood pressure lowering peptide is the third blood pressure lowering peptide, and the auxiliary materials are starch, honey, dietary fiber, arginine and glutathione.
  • the blood pressure lowering peptide may also be any one or more of the first blood pressure lowering peptide, the second blood pressure lowering peptide, and the blood pressure lowering protein.
  • the content of the blood pressure lowering peptide may be 1 to 20% by weight and the content of the auxiliary material may be 80 to 99% by weight.

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Abstract

降血压肽和降血压蛋白质及其应用；这些降血压肽的氨基酸序列分别为TTW、VHW和KAKW，降血压蛋白质的氨基酸序列含有TTW、VHW和KAKW的一种以上；上述的降血压肽和降血压蛋白质能够抑制血管紧张素转化酶的活性，从而起到降血压的作用，能够被用于制成各种降血压药品或保健品。

Description

降血压肽和降血压蛋白质及其应用 技术领域
本发明属于食品或药品技术领域，涉及降血压肽和降血压蛋白质及其应用。
背景技术
血管紧张素转化酶(Angiotensin converting enzyme，简称ACE)是一种能够导致血压升高的酶，其具有以下两种调控途径。
其一、血管紧张素转化酶能够切除血管紧张素I(AngiotensinI)末端的两个氨基酸(His-Leu)，从而将该血管紧张素I转化为血管紧张素II(AngiotensinII)。其中，血管紧张素I的氨基酸序列为Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu，简称为DRVYIHPFHL。血管紧张素II的氨基酸序列为Asp-Arg-Val-Tyr-Ile-His-Pro-Phe，简称为DRVYIHPF。血管紧张素II是一种血管收缩剂，其能够促进血管的收缩，从而使血压升高。
其二、血管紧张素转化酶能够切除舒缓激肽(Bradykinin)末端的两个氨基酸(Phe-Arg)，从而使其失活。其中，舒缓激肽的氨基酸序列为Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg，失活的舒缓激肽的氨基酸序列为Arg-Pro-Pro-Gly-Phe-Ser-Pro。舒缓激肽具有使血管扩张的功能，但舒缓激肽出于失活状态时，便失去了使血管扩张的能力，从而使血压升高。
ACE是一种金属肽酶，含有一个结合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的Zn2+结合位点结合，使之失活。
若使血管紧张素转化酶失活，则血管紧张素转化酶就不能将血管紧张素I转化为血管紧张素II，血管就不会收缩；同时，血管紧张素转化酶也就不能使舒缓激肽失活，血管就会扩张。二者的综合作用就会使血压降低。因此，寻找能够使血管紧张素转化酶失活的多肽或蛋白质就成为亟待解决的技术问题。
发明内容
本发明的其中一个目的在于提供能够抑制血管紧张素转化酶的降血压肽。
本发明的另外一个目的在于提供上述降血压肽的应用。
为达到上述目的，本发明的解决方案是：
一种降血压肽，其氨基酸序列为TTW，称为第一降血压肽。
一种降血压肽，其氨基酸序列为VHW，称为第二降血压肽。
一种降血压肽，其氨基酸序列如SEQ ID NO：1所示，称为第三降血压肽。
上述的任意一种降血压肽均可以用于作为血管紧张素转化酶抑制剂。
上述的任意一种降血压肽均可以用于制备降血压药物。
上述的任意一种降血压肽均可以用于制备降血压保健品。
一种降血压蛋白质，其含有TTW、VHW和如SEQ ID NO：1所示的氨基酸序列的一种以上。
上述的降血压蛋白质可以用于作为血管紧张素转化酶抑制剂。
上述的降血压蛋白质可以用于制备降血压药物。
上述的降血压蛋白质可以用于制备降血压保健品。
由于采用上述方案，本发明的有益效果是：
本发明的降血压肽是从天然食品的水解物中筛选得到的，对血管紧张素转化酶均具有明显的抑制活性，既能够单独用于制备降血压药物或降血压保健品，也能够与现有技术的降血压药物复配使用，以便取得更好的协同降血压效果。
附图说明
图1为本发明的第一降血压肽与血管紧张素转化酶活性中心的氢键形成示意图。
图2为本发明的第二降血压肽与血管紧张素转化酶活性中心的氢键形成示意图。
图3为本发明的第三降血压肽与血管紧张素转化酶活性中心的氢键形成示意图。
图4为降血压肽的降压效果图。
具体实施方式
本发明提供了多个降血压肽及其应用。
<第一降血压肽>
本发明提供了一种降血压肽，该降血压肽由三个氨基酸组成，其氨基酸序列为Thr-Thr-Trp，简称为TTW。为便于说明起见，以下统称第一降血压肽。本发明中的氨基酸均从N端到C段排列。
通过分子对接实验表明，第一降血压肽中的第一个T与血管紧张素转化酶(ACE)活性中心的Ala354形成3个氢键；该第一降血压肽中的第二个T分别与His353、Glu384和His513各形成一个氢键；该第一降血压肽中的W与Glu403形成一个氢键，故第一降血压肽与血管紧张素转化酶(ACE)一共形成7个氢键。这7个氢键的形成情况如图1所示。圆圈中的氨基酸代表ACE中的氨基酸序列，中间的链是降血压肽的序列在ACE中形成的构型。
这些氨基酸残基均为血管紧张素转化酶(ACE)的活性中心中重要的氨基酸残基，对ACE的活性有重要的影响。当第一降血压肽与血管紧张素转化酶(ACE)的活性中心的这些氨基酸残基形成氢键后，血管紧张素转化酶(ACE)的活性中心就无法再结合血管紧张素，由此血管紧张素转化酶(ACE)便失活，因此，该第一降血压肽能够作为血管紧张素转化酶抑制剂。
<第一降血压肽的应用>
1、第一降血压肽可以用于制备降血压药物。
本发明的第一降血压肽可以单独作为降血压药物而使用，此时，第一降血压肽需与辅料配合使用以制成药品颗粒、胶囊、片剂(如糖衣片或薄膜衣片)、丸剂、口服液或注射剂等。
当第一降血压肽被制成药品颗粒而使用时，辅料可以为麦芽糊精和/或香兰素。
当第一降血压肽被制成糖衣片而使用时，辅料可以为淀粉和/或硬脂酸镁。
当第一降血压肽被制成口服液而使用时，辅料可以为苯甲酸钠、阿斯巴甜、安赛蜜、香精和纯化水。
当第一降血压肽被制成注射剂而使用时，辅料可以为甘露醇、磷酸二氢钠和/或磷酸氢二钠。可以将该注射剂配置成0.9％氯化钠注射液或5％葡萄糖注射液然后静脉滴注或肌肉注射。
本发明的第一降血压肽也可以与现有技术的降血压药物复配使用，以便取得更好的协同降血压效果。
现有技术的降血压药物包括β受体阻滞剂、钙拮抗剂、ACE抑制剂和利尿剂等。
其中，β受体阻滞剂选自奈必洛尔、普拉洛尔、阿罗洛尔、阿替洛尔、塞利洛尔、卡维地洛、拉贝洛尔、比索洛尔。
钙拮抗剂选自硝苯地平、氨氯地平(包括左旋氨氯地平)、拉卡地平、非洛地平、尼伐地平、拉西地平、尼索地平。
ACE抑制剂选自阿拉普利、贝那普利、卡托普利、西罗普利、西拉普利、地拉普利、依那普利、依那普利拉、福辛普利、赖诺普利、雷米普利、雷米普利拉、培哚普利、喹那普利、螺普利、替莫普利、群多普利。
利尿剂选自氢氯噻嗪、三氯噻嗪。
2、第一降血压肽可以用于制备降血压保健品。
本发明的第一降血压肽加上辅料即可制成降血压保健品，辅料可以包括：淀粉、明胶、钛白粉、乳清蛋白、蔗糖、维生素E、谷胱甘肽、精氨酸、瓜氨酸、膳食纤维、胶原蛋白、脑磷脂、纤维素、葡萄糖、木糖、蜂蜜、卵磷脂、胡萝卡素、维生素B1、维生素B2、维生素C、铁、钙、蜂花粉、黑木耳粉(200-500目)、香菇粉(300-500目)、灵芝粉(300-500目)、魔芋粉(300-500目)、黄芪粉(200-500目)、枸杞粉(200-500目)和人参粉(500-1000目)等中的任意一种或几种。
<第二降血压肽>
本发明提供了一种降血压肽，该降血压肽由三个氨基酸组成，其氨基酸序列为Val-His-Trp，简称为VHW。为便于说明起见，以下统称第二降血压肽。
通过分子对接实验表明，第二降血压肽中的V与血管紧张素转化酶(ACE)活性中心的Glu411形成2个氢键；该第二条降血压肽中的H与Ala354、His353、Glu384各形成一个氢键；该第二条降血压肽中的W与Tyr520形成一个氢键，故第二降血压肽与血管紧张素转化酶(ACE)一共形成7个氢键。这7个氢键的形成情况如图2所示。
这些氨基酸残基也为血管紧张素转化酶(ACE)的活性中心中重要的氨基酸残基，，对ACE的活性有重要的影响。当第二降血压肽与血管紧张素转化酶(ACE)的活性中心的这些氨基酸残基形成氢键后，血管紧张素转化酶(ACE)的活性中心就无法再结合血管紧张素，由此血管紧张素转化酶(ACE)便失活，因此，该第二降血压肽也能作为血管紧张素转化酶抑制剂。
<第二降血压肽的应用>
1、第二降血压肽可以用于制备降血压药物。
2、第二降血压肽可以用于制备降血压保健品。
其中，在使用第二降血压肽制备降血压药物或降血压保健品时，能够与该第二降血压肽搭配使用的辅料可以参考第一降血压肽。
<第三降血压肽>
本发明提供了一种降血压肽，其氨基酸序列如SEQ ID NO：1所示。为便于说明起见，以下统称第三降血压肽。该第三降血压肽由四个氨基酸组成，其氨基酸序列为Lys-Ala-Lys-Trp，简称为KAKW。
通过分子对接表明，第三降血压肽中的第一个K与ACE活性中心的Ala356形成了两个氢键和与Glu411形成了一个氢键；该第三条降血压肽中的W与Gln281形成了2个氢键，与Tyr520、Asp415各形成了一个氢键；该第三降血压肽中的第二个K与His353、、Lys511各形成一个氢键，故第三降血压肽与血管紧张素转化酶(ACE)一共形成9个氢键。这9个氢键的形成情况如图3所示。这些氨基酸残基均为血管紧张素转化酶(ACE)的活性中心中重要的氨基酸残基，对ACE的活性有重要的影响。当第三降血压肽与血管紧张素转化酶(ACE)的活性中心的这些氨基酸残基形成氢键后，血管紧张素转化酶(ACE)的活性中心就无法再结合血管紧张素，由此血管紧张素转化酶(ACE)便失活，因此，该第三降血压肽能够作为血管紧张素转化酶抑制剂。
<第三降血压肽的应用>
1、第三降血压肽可以用于制备降血压药物。
2、第三降血压肽可以用于制备降血压保健品。
其中，在使用第三降血压肽制备降血压药物或降血压保健品时，能够与该第三降血压肽搭配使用的辅料可以参考第一降血压肽。
<降血压蛋白质>
一种降血压蛋白质，其特征在于：其含有TTW、VHW和如SEQ ID NO：1所示的氨基酸序列的一种以上。
上述的多肽和蛋白质均可以通过人工方法合成，故对其合成方法不再赘述。
<降血压蛋白质的应用>
1、降血压蛋白质可以用于制备降血压药物。
2、降血压蛋白质可以用于制备降血压保健品。
在使用降血压蛋白质制备降血压药物或降血压保健品时，能够与该降血压蛋白质搭配使用的辅料可以参考第一降血压肽。
以下结合实验对各种降血压肽等的降血压活性进行说明。
<实验1：各种降血压肽对血管紧张素转化酶的体外抑制实验>
本发明的各种降血压肽均属于与血管紧张素转化酶的活性区域的亲和力较强的竞争性抑制剂，其与该活性区域的亲和力高于血管紧张素I或者舒缓激肽，并且一旦结合也不容易从结合区域释放，从而能够降低血管紧张素转化酶的活性区域的活性，甚至使其失活，从而阻碍血管紧张素转化酶将血管紧张素I转化为血管紧张素II，并且阻碍血管紧张素转化酶失活舒缓激肽，进而起到降低血压的作用。以下通过实验验证各种降血压肽对血管紧张素转化酶的抑制能力。
本实验的测定原理如下：
马尿酰组氨酰亮氨酸(Hip-His-Leu，简称HHL)能够作为血管紧张素转化酶(ACE)的底物，并且被水解为马尿酸和His-Leu。抑制剂可以抑制血管紧张素转化酶催化水解马尿酰组氨酰亮氨酸的能力，使产物中的马尿酸的含量降低，因此，可以通过检测产物中马尿酸的含量来计算出抑制剂对血管紧张素转化酶的抑制率。
本实验的测定步骤如下：
(1)、取多个EP管作为反应容器，对照组的EP管加入20μL去离子水，实验组的EP管加入20μL不同浓度的待测样品；
(2)、向各个EP管中加入80μL5M的马尿酰组氨酰亮氨酸(HHL)，于37℃下水浴5min；
(3)、向各个EP管中加入10μL 310mU/mL的血管紧张素转化酶(ACE)，于37℃下水浴5min，启动反应；
(4)、待反应进行完全时，向各个EP管中加入400μL l M的HCL终止反应；
(5)、将各个EP管中的反应液分别用0.22μm的滤膜过滤，将过滤后的液体保存于相对应的液相小瓶中；
(6)、对各个液相小瓶内的液体进行高效液相色谱检测，检测条件为：色谱柱：Thermo BDS HYPERSIL C18(250mm×3mm×5μm)；柱温：30℃；流速：0.8mL/min；检测波长：228nm；进样量：10μL；流动相A：超纯水(0.1％三氟乙酸TFA)；流动相B：乙腈；洗脱条件如表1所示。
表1高效液相色谱洗脱条件
Figure PCTCN2017086294-appb-000001
(7)、各个待测样品对血管紧张素转化酶的抑制率的计算方法如下：
X＝(A对照-A)/A对照
式中：X——ACE抑制率(％)；
A对照——对照组的峰面积；
A——实验组的峰面积。
(8)、待测样品的IC50的计算：
以待测样品的浓度为横坐标，ACE抑制率为纵坐标作图，得到抑制率-样品浓度关系曲线，通过该曲线得到抑制率达到50％时的待测样品的浓度，即为该待测样品的IC50。
其中，待测样品为含有第一降血压肽、第二降血压肽、第三降血压肽或降血压蛋白质的样品。
经检测得知，待测样品的IC50如表2所示。
表2待测样品的IC50
降血压肽 IC50(μM)
第一降血压肽(TTW) 0.61
第二降血压肽(VHW) 0.91
第三降血压肽(KAKW) 2.02
检测结果表明三条降血压肽在体外抑制血管紧张素转化酶实验中，具有明显的抑制血管紧张素转化酶的效果，其抑制效果接近治疗高血压的经典药物赖诺普利的体外抑制效果(IC50＝0.0214μM)。
<实验2：各种降血压肽对血管紧张素转化酶的体内抑制实验>
本实验主要验证各种降血压肽对高血压大鼠的血压的影响，其包括如下步骤：
(1)、获取自发性高血压大鼠(SHRs，10周大小，雄性，体重250–320g)，血压超过180mmHg，购自上海斯莱克动物实验公司；
(2)、高血压大鼠6只一组饲养，在22±2℃的温度下12小时循环光照，供给食物和饮水；
(3)、将不同的降血压肽溶解在生理盐水中，实验组按照每只高血压大鼠3μmol/Kg的量计算，生理盐水按照1ml/100g大鼠体重的量进行灌胃，对照组以相同剂量的生理盐水进行灌胃同时喂食降血压药赖诺普利；
(4)、灌胃后，分别测量这些高血压大鼠在0h、1h、2h、4h、6h和8h的血压。所有的结果都进行了三次重复测量。
结果如图4所示。从图4中可以看出，三条降血压肽相较于空白实验组(control)，血压都有明显下降，说明其在体内有降血压效果，同时与赖诺普利组相比，三条降血压肽组的降血压效果在前期可以认为是要比赖诺普利效果要好的。
综上所述，降血压肽不仅具有较好的体外降血压活性，在经过胃肠道的消化吸收之后同仍然具有良好的体内活性，其降血压功能并未丧失。
以下结合各个实施例对本发明作进一步的说明。
实施例1：降血压药品片剂
本实施例的降血压药品片剂含有5wt％的降血压肽和95wt％的辅料。降血压肽为第一降血压肽，辅料为淀粉和硬脂酸镁。
实际上，降血压肽也可以为第二降血压肽、第三降血压肽和降血压蛋白质中的任意一种或几种。辅料可以为淀粉、蔗糖、麦芽糊精中的任意一种或几种。降血压肽的含量可以为1-20wt％并且辅料的含量可以为80wt-99wt％。
实施例2：降血压保健品
本实施例的降血压药品片剂含有15wt％的降血压肽和85wt％的辅料。降血压肽为第三降血压肽，辅料为淀粉、蜂蜜、膳食纤维、精氨酸、谷胱甘肽。
其中，降血压肽也可以为第一降血压肽、第二降血压肽和降血压蛋白质中的任意一种或几种。降血压肽的含量可以为1-20wt％并且辅料的含量可以为80wt-99wt％。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (10)

  1. 一种降血压肽，其特征在于：其氨基酸序列为TTW。
  2. 一种降血压肽，其特征在于：其氨基酸序列为VHW。
  3. 一种降血压肽，其特征在于：其氨基酸序列如SEQ ID NO：1所示。
  4. 如权利要求1至3任一所述的降血压肽作为血管紧张素转化酶抑制剂的应用。
  5. 如权利要求1至3任一所述的降血压肽在制备降血压药物中的应用。
  6. 如权利要求1至3任一所述的降血压肽在制备降血压保健品中的应用。
  7. 一种降血压蛋白质，其特征在于：其含有TTW、VHW和如SEQ ID NO：1所示的氨基酸序列的一种以上。
  8. 如权利要求7所述的降血压蛋白质作为血管紧张素转化酶抑制剂的应用。
  9. 如权利要求7所述的降血压蛋白质在制备降血压药物中的应用。
  10. 如权利要求7所述的降血压蛋白质在制备降血压保健品中的应用。
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