Antihypertensive peptide or Chinese clam antihypertensive peptide extract and its application

Abstract

The present invention discloses a Chinese clam antihypertensive peptide and its application in the preparation of antihypertensive drugs and health foods. The Chinese clam antihypertensive extract is obtained by enzymatic hydrolysis, and the extract is further separated and purified to obtain a polypeptide of a specific sequence, and the amino acid sequence of the antihypertensive peptide is Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val (KDEYKHLNFGEV). The Chinese clam antihypertensive peptide disclosed in the present invention has significant angiotensin converting enzyme (ACE) inhibitory activity. In vitro, it is manifested as temperature, pH, simulated gastrointestinal digestion and angiotensin converting enzyme (ACE) degradation stability. It shows good antihypertensive effect both in vitro and in non-spontaneous hypertensive rats (SHRs), and can be used in health products and medicines related to hypertension treatment.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, and relates to a blood pressure lowering peptide extract of Chinese clams and a blood pressure lowering peptide with a specific sequence and applications thereof in blood pressure lowering medicines and health products. Background Art

[0002] Hypertension is characterized by elevated blood pressure in human arteries (systolic ≥ 140 mmHg, diastolic ≥ 90 mmHg). Hypertension is associated with increased mortality from cardiovascular disease and diabetes. The World Health Organization (WHO) reports that more than 17.5 million people die from cardiovascular disease each year. Hypertension has become the leading cause of death worldwide. The renin-angiotensin system (RAS) and the kallikreo-bradykinin system (KKS) can play an important role in blood pressure regulation. Angiotensin-converting enzyme (ACE) is a Zn-containing enzyme that is ubiquitous in mammalian tissues and can regulate the renin-angiotensin system (RAS) and the kallikreo-bradykinin system (KKS). 2+ Therefore, ACE is considered an important target for the treatment of hypertension.

[0003] Most of the common ACE inhibitors on the market are "prils", such as captopril, lisinopril, enalapril, fosinopril, etc. Although these drugs can play a good antihypertensive effect, they also produce a series of side effects, such as cough, rash, nausea, acute renal failure, proteinuria, etc. These side effects will reduce patient compliance and increase additional medical expenses. Therefore, more and more research has begun to focus on the development of low-toxic natural ACE inhibitory peptides as an auxiliary and substitute for antihypertensive drugs. Food-derived peptides can not only play the same anti-stress role, but also have safety and low toxicity due to their natural origin, so the development of antihypertensive peptides from natural foods is meaningful and promising.

[0004] Chinese clams, commonly known as "yellow clams", "yellow clams" and "flying clams", are scientifically named Mactra chinensis. They belong to the phylum Mollusca, class Bivalvia, order Heterostriatum, family Mactra, and genus Mactra. They are mainly distributed along the Yellow Sea and Bohai Sea coasts of Liaoning and Shandong in my country. They can be harvested all year round when cultivated artificially, especially in late autumn and spring. Chinese clams are suitable for living in the fine sand beach of the intertidal zone to the shallow sea area with a depth of 60m. They are a practical and economic tidal shellfish with rich nutrition and delicious meat. At present, many peptides with ACE inhibitory effects have been screened from marine shellfish, such as oysters, scallops, and Manila clams. However, there are few reports on the isolation of ACE inhibitory peptides from Chinese clams. Summary of the invention

[0005] In view of this, the purpose of the present invention is to select Chinese clams as raw materials, and identify an ACE inhibitory peptide with an amino acid sequence of Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val (KDEYKHLNFGEV) through proteinase K enzymolysis and liquid-mass spectrometry technology.

[0006] The Chinese clam antihypertensive peptide of the present invention:

[0007] Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val(KDEYKHLNFGEV) exhibits high angiotensin-converting enzyme (ACE) inhibitory activity in vitro and exerts a good antihypertensive effect in spontaneously hypertensive rats (SHRs) in vivo.

[0008] Another object of the present invention is to provide a Chinese clam antihypertensive peptide extract, which is prepared according to the following method: crushing Chinese clams into a homogenate, adding purified water 0.5-20 times the weight of the Chinese clams, adding proteinase K for enzymolysis, taking the supernatant after centrifugal filtration, desalting by nanofiltration with a molecular weight cutoff of 180 Daltons, concentrating, and freeze-drying or spray-drying the concentrate to obtain the Chinese clam enzymolysis antihypertensive peptide extract. The amount of proteinase K added is 0.2%-5% of the weight of the Chinese clam, the enzymolysis temperature is controlled at 35°C-55°C, the enzymolysis pH is controlled at 7.0-9.0, the enzymolysis time is 2h-5h, and after the enzymolysis is completed, the enzyme is inactivated by boiling water for 10-60min.

[0009] The Chinese clam antihypertensive peptide extract and the Chinese clam antihypertensive peptide of the invention are widely used in angiotensin inhibitors, antihypertensive drugs and health products.

[0010] The Chinese clam antihypertensive peptide disclosed in the present invention has significant angiotensin converting enzyme (ACE) inhibitory activity. It is manifested in vitro as temperature, pH, simulated gastrointestinal digestion and angiotensin converting enzyme (ACE) degradation stability. It shows good antihypertensive effect both in vitro and in vivo in non-spontaneous hypertensive rats (SHRs), and can be used for health products and medicines related to hypertension treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Liquid chromatography (A) and mass spectrometry identification (B) of the antihypertensive peptide KDEYKHLNFGEV.

[0012] Figure 2 ACE inhibitory activity of peptide KDEYKHLNFGEV (0.5 mg / ml) under different temperature treatments.

[0013] Figure 3 ACE inhibitory activity of peptide KDEYKHLNFGEV (0.5 mg / ml) under different pH treatments.

[0014] Figure 4 ACE inhibitory activity of the peptide KDEYKHLNFGEV (0.1 mg / ml and 0.5 mg / ml) after treatment in simulated gastrointestinal fluid. GC treated with simulated gastric fluid without pepsin; G treated with simulated gastric fluid containing pepsin; G+IC treated with simulated gastrointestinal fluid without pepsin and pancreatin; G+I treated with simulated gastrointestinal fluid containing pepsin and pancreatin.

[0015] Figure 5 Study on the binding stability of peptide KDEYKHLNFGEV (0.1 mg / ml) with ACE.

[0016] Figure 6 Effect of polypeptide KDEYKHLNFGEV on reducing hypertension in spontaneously hypertensive rats (SHRs); oral dose 10 mg / kg, A: systolic blood pressure; B: diastolic blood pressure, ● control group, ▉ captopril, ◆ polypeptide KDEYKHLNFGEV DETAILED DESCRIPTION

[0017] The present invention is further described in detail by the following specific examples:

[0018] Example 1

[0019] The preparation method of the Chinese clam antihypertensive peptide extract comprises the following steps:

[0020] (1) 500 g of fresh Chinese clam meat was homogenized, and 1 times its weight (500 g) of purified water was added and mixed.

[0021] (2) 500 mg of proteinase K was added for enzymatic hydrolysis, the enzymatic hydrolysis temperature was 42° C., the enzymatic hydrolysis pH was controlled at 8.0 (adjusted by 1 M sodium hydroxide), the enzymatic hydrolysis time was 3 h, and after the enzymatic hydrolysis was completed, the enzyme was inactivated at 100° C. for 10 min, and impurities were removed by centrifugation.

[0022] (3) The supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 180 Daltons, concentrated to 100 mL, and lyophilized to obtain the antihypertensive peptide extract.

[0023] (4) The ACE inhibitory activity of the Chinese clam antihypertensive peptide extract was evaluated using the following high performance liquid chromatography (HPLC) method. The IC50 value of the ACE inhibitory activity was 1.02±0.31 mg / ml.

[0024] Example 2

[0025] The preparation method of the Chinese clam antihypertensive peptide extract comprises the following steps:

[0026] (1) Chinese clam meat was dried and crushed, 20 g of dry powder was added to 400 ml of distilled water, stirred, and swollen at room temperature for 4 h.

[0027] (2) 400 mg of proteinase K was added and stirred for enzymolysis. The enzymolysis temperature was controlled at 42° C., the enzymolysis pH was controlled (adjusted by 1 M sodium hydroxide) at 7.8, the enzymolysis time was 2 h, and after the enzymolysis was completed, the enzyme was inactivated by boiling water at 100° C. for 10 min, and impurities were removed by filtration.

[0028] (3) The supernatant was desalted using a nanofiltration membrane with a molecular weight cutoff of 180 Daltons, concentrated to 20 mL, and spray-dried to obtain the Chinese clam antihypertensive peptide extract.

[0029] (4) The ACE inhibitory activity of the Chinese clam antihypertensive peptide extract was evaluated using the following high performance liquid chromatography (HPLC) method. The IC50 value of the antihypertensive peptide for inhibiting ACE was 0.93±0.24 mg / ml.

[0030] Example 3

[0031] The Chinese clam antihypertensive peptide extract prepared in Example 2 was analyzed by liquid chromatography-mass spectrometry using UPLC-ESI-Q-Exactive Focus-MS / MS (eg Figure 1 The mass spectrometry data was compared with the Chinese clam transcriptome database to obtain a polypeptide with a specific sequence of KDEYKHLNFGEV. The polypeptide was synthesized by solid phase synthesis to obtain a polypeptide with a sequence of KDEYKHLNFGEV and a purity of ≥98%.

[0032] Information of SEQ ID No.1

[0033] (a) Sequence characteristics

[0034] *Length: 12 amino acid residues

[0035] * Type: Amino Acid

[0036] *Chain type: Single chain

[0037] *Topology: Linear

[0038] (b) Molecular type: protein

[0039] Sequence description: SEQ ID No.1

[0040] (I) Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val HPLC

[0041] Determination of ACE inhibitory activity of peptides in vitro by HPLC

[0042] Unless otherwise specified, the drugs and instruments used in this example can be obtained commercially.

[0043] The principle of high performance liquid chromatography (HPLC) to determine the activity of ACEI is: Hip-His-Leu (HHL, Sigma, USA) can be hydrolyzed by ACE (Sigma, USA) into hippuric acid and dipeptide products. Hippuric acid has a characteristic absorption peak at an ultraviolet wavelength of 228nm. When the inhibitor acts on angiotensin converting enzyme (ACE), the amount of hippuric acid generated decreases. By measuring the amount of hippuric acid generated before and after the addition of the inhibitor, the inhibitor's inhibition rate on ACE can be determined. The calculation formula is as follows:

[0044]

[0045] In the above formula, A 空白 is the peak area of ​​hippuric acid when no inhibitory peptide is added, A 抑制剂 IC50 is the peak area of ​​hippuric acid when inhibitory peptide is added; IC50 represents the concentration of inhibitory peptide that can inhibit half of the activity of ACE.

[0046] Results The IC50 value of the Chinese clam antihypertensive peptide KDEYKHLNFGEV was 7.59±0.36μM. Reference: Feng-Jun Wang, Xiao-Yan Yin, Joe M Regenstein, Jian-Zhong Wang. Separation and purification of angiotensin-I-converting enzyme (ACE) inhibitory peptides from walnuts (Juglans regia L.) meal. Eur Food Res Technol (2016) 242: 911–918

[0047] (II) Physicochemical properties of peptides

[0048] Peptide KDEYKHLNFGEV The molecular weight is 1479.60Da , non-toxic.

[0049] (III) Evaluation of thermal stability and pH stability

[0050] The polypeptide (0.5 mg / ml) was treated at a temperature range of 20°C-70°C for 2 hours. After the treatment, 20 μL of the polypeptide solution was taken and its ACE inhibitory activity was determined according to the above method.

[0051] Hydrochloric acid and sodium hydroxide were used to prepare aqueous solutions of pH 2-12, respectively. The polypeptide (0.5 mg / ml) was dissolved in a solution in the pH range of 2-12. After treatment for 2 hours under this condition, the polypeptide solution was neutralized with 1M HCL and 1M NaOH. 20 μL of the polypeptide solution was taken and its ACE inhibitory activity was determined according to the above method.

[0052] Attached Figure 2 and attached Figure 3 It was shown that the Chinese clam antihypertensive peptide KDEYKHLNFGEV had temperature and pH stability in the range of 20-70°C and pH 2-12.

[0053] 4. Evaluation of degradation stability in simulated gastrointestinal fluid

[0054] In vitro simulated gastrointestinal fluid degradation was used to evaluate the stability of the peptide in simulated gastrointestinal fluid.The peptide (KDEYKHLNFGEV) was dissolved in 0.1M potassium chloride-hydrochloric acid (KCl-HCl) (pH 2.0) buffer to a final concentration of 0.1mg / mL and 0.5mg / ml, respectively. Pepsin (≥250U / mg) was added to the polypeptide solution to a final concentration of 0.8mg / mL, and the solution was incubated at 37°C for 4h, after which the system was inactivated in 100°C boiling water for 10min, the pH of the solution was adjusted to 7.0 using 1M sodium hydroxide, and the solution was centrifuged at 12000rpm for 5min, and 20μL of the supernatant in the system was used to determine the ACE inhibitory activity according to the above-mentioned high performance liquid chromatography method; trypsin (8x USP) was added to the remaining supernatant to a final concentration of 10mg / mL, and the solution was incubated at 37°C for another 4h, inactivated in a 100°C boiling water bath for 10min, and centrifuged at 12000rpm for 5min, and 20μL of the supernatant was used to determine the ACE inhibitory activity according to the above-mentioned high performance liquid chromatography method.

[0055] Attached Figure 4 It shows that the Chinese clam antihypertensive peptide KDEYKHLNFGEV can resist degradation in simulated gastrointestinal fluid and maintain its structure and ACE inhibitory activity stability in simulated gastrointestinal fluid.

[0056] (V) Investigation of the stability of peptide binding to ACE

[0057] ACE is a carboxypeptidase with a wide range of specific substrates, which can produce the function of degrading antihypertensive peptides. 300 μL of polypeptide (0.1 mg / mL) was mixed with 300 μL of 0.1 U / mL ACE solution and incubated at 37°C for 4-24 hours, then inactivated by boiling water at 100°C for 10 minutes, and 20 μL of polypeptide solution was taken and the inhibitory activity of ACE was determined by the above-mentioned high-performance liquid chromatography method. Figure 5 It showed that the Chinese clam antihypertensive peptide KDEYKHLNFGEV still maintained ACE inhibitory activity within 24 hours of incubation with ACE.

[0058] (VI) Evaluation of antihypertensive effect after single administration in spontaneously hypertensive rats (SHRs)

[0059] Spontaneously hypertensive rats SHRs (male, 10 weeks old, 250-300g, no specific pathogens) were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd. SHRs were kept at 22±2℃ with a 12h light / 12h dark day and night cycle, and were fed with pure water and a standard diet (i.e., standard diet) at regular times every day. SHRs were divided into three groups (blank control group, positive control group, experimental group), each with 6 rats. When the high pressure was higher than 180mmHg, gavage could be started, and the gavage dose of KDEYKHLNFGEV was 10mg / kg.

[0060] Physiological saline was used as a blank control, and captopril was used as a positive control. The intragastric dose was 10 mg / kg, and the high and low pressures at 0, 1, 2, 4, 6, and 8 hours after intragastric administration were measured using the rat tail non-invasive blood pressure measurement system CODA. Each measurement was repeated 3 times.

[0061] Attached Figure 6 This indicates that KDEYKHLNFGEV can significantly reduce the blood pressure of SHRs 1 to 2 hours after oral administration (p<0.05). At 2 hours, the blood pressure can be reduced from 204 mmHg to 170 mmHg at most, and the blood pressure can be reduced by 34 mmHg at most. Then the blood pressure begins to recover slowly until 8 hours after oral administration, when the blood pressure returns to the previous level. KDEYKHLNFGEV reduces the blood pressure of SHRs from 145 mmHg to 118 mmHg at 2 hours (p<0.05), and then the blood pressure begins to recover slowly, and returns to the original level at 8 hours. In general, KDEYKHLNFGEV can produce a good antihypertensive effect in SHRs.

[0062] The above results show that the Chinese clam antihypertensive peptide KDEYKHLNFGEV has a strong inhibitory activity against ACE; the peptide can maintain activity in the range of 0℃-100℃ and pH2-12, and can also resist degradation by simulated gastrointestinal fluid and ACE; enzyme kinetics show that the peptide is a non-competitive inhibitor of ACE; and it can also show significant antihypertensive activity in spontaneously hypertensive rats (SHRs). The antihypertensive peptide of Chinese clam and the antihypertensive peptide KDEYKHLNFGEV with a specific sequence will be widely used in health products and drugs related to the treatment of hypertension.

[0063] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Liaoning Tianzengxiang Biotechnology Co., Ltd. <120> Antihypertensive peptide or Chinese clam antihypertensive peptide extract and its application <140> 2022107596392 <141> 2022-06-29 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 201 <212> AA <213> Artificial Sequence <220> <223> n=[a / g] <400> 1 Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val 12

Claims

1. A blood pressure lowering peptide, characterized in that : The antihypertensive peptide amino acid sequence is the amino acid sequence shown in SEQ ID No.1 in the sequence table; The amino acid sequence is KDEYKHLNFGEV.

2. A use of the antihypertensive peptide according to claim 1, characterized in that: The antihypertensive peptide is used in preparing medicines for treating hypertension.

3. The use according to claim 2, characterized in that: The antihypertensive peptide according to claim 1 is prepared by combining any carrier that is permitted or acceptable for food or drug production.

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