A polypeptide having ace inhibitory activity and use thereof

Abstract

This invention discloses a polypeptide with ACE inhibitory activity and its applications. The polypeptides LVSKP, LVAP, and VVAGP were obtained by solid-phase synthesis, and their IC50 values ​​for angiotensin-converting enzyme (ACE) inhibition were measured to be 22.3 µM, 26.3 µM, and 103.5 µM, respectively. In vivo antihypertensive effects were evaluated using a spontaneously hypertensive rat model. The results showed that LVSKP and VVAGP significantly reduced systolic blood pressure in spontaneously hypertensive rats. These polypeptides can be used to prepare ACE inhibitors, antihypertensive products, drugs, health foods, or functional foods, and have broad application prospects in industrial applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a polypeptide with ACE inhibitory activity and its applications. Background Technology

[0002] Hypertension is a significant risk factor for the development and progression of cardiovascular and cerebrovascular diseases, seriously threatening human health. Angiotensin-converting enzyme (ACE) plays a crucial role in the renin-angiotensin system, catalyzing the conversion of angiotensin I into angiotensin II, which has a strong vasoconstrictive effect, and simultaneously degrading bradykinin, which has a vasodilatory effect, thus leading to elevated blood pressure. Therefore, inhibiting ACE activity is considered an important approach to preventing and treating hypertension. Currently, commonly used ACE inhibitors such as captopril and enalapril have clear antihypertensive effects, but long-term use may be accompanied by adverse reactions such as cough, hypotension, taste abnormalities, and kidney damage. Therefore, developing safe, low-toxicity, and suitable natural ACE inhibitors for long-term intake, especially food-derived bioactive peptides with antihypertensive functions, has become an important research direction in the fields of functional foods and biomedicine.

[0003] In recent years, studies have reported that some peptides possess certain ACE inhibitory activities. However, existing studies on peptide screening have mostly focused on in vitro activity evaluation, lacking verification of in vivo antihypertensive effects. Since the in vivo activity of peptides is also affected by various factors such as digestive stability, absorption and transport, and in vivo metabolism, in vitro ACE inhibition results cannot be directly equated with in vivo antihypertensive effects. Therefore, there is an urgent need to obtain ACE inhibitory peptides with clearly defined application value that have been verified both in vitro and in vivo. This is of great significance for developing novel natural antihypertensive active ingredients, enriching ACE inhibitory peptide resources, and promoting the research and development of related drugs. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a polypeptide with ACE inhibitory activity.

[0005] Another object of the present invention is to provide the application of the above-mentioned polypeptide having ACE inhibitory activity.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A polypeptide with ACE inhibitory activity, comprising at least one of the following polypeptides:

[0008] LVSKP, with the amino acid sequence Leu-Val-Ser-Lys-Pro;

[0009] LVAP, with the amino acid sequence Leu-Val-Ala-Pro;

[0010] VVAGP has the amino acid sequence Val-Val-Ala-Gly-Pro.

[0011] The polypeptide with ACE inhibitory activity was prepared by chemical synthesis.

[0012] The above-mentioned peptides with ACE inhibitory activity are used in the preparation of ACE inhibitors.

[0013] The above-mentioned peptides with ACE inhibitory activity are used in the preparation of drugs for the treatment or prevention of cardiovascular diseases.

[0014] The above-mentioned peptides with ACE inhibitory activity are used in the preparation of drugs for the treatment or prevention of hypertension.

[0015] The above-mentioned peptides with ACE inhibitory activity are used in the preparation of health products with auxiliary blood pressure lowering function.

[0016] The above-mentioned peptides with ACE inhibitory activity are used in the preparation of health foods, functional foods or special medical purpose formula foods.

[0017] The present invention has the following advantages and effects compared with the prior art:

[0018] (1) This invention provides novel ACE-inhibiting peptides LVSKP, LVAP and VVAGP with well-defined sequences, and measures their IC50 values. 50 The values ​​were 22.3 µM, 26.3 µM and 103.5 µM, respectively, indicating strong ACE inhibitory activity;

[0019] (2) Validated by in vivo model, LVSKP and VVAGP have good antihypertensive activity, indicating that they have the potential to be further developed into natural antihypertensive active ingredients;

[0020] (3) The present invention has conducted in vitro and in vivo joint verification of candidate peptides and screened out active peptides with practical application value, providing a new technical basis for the development of ACE inhibitory peptides and auxiliary antihypertensive products. Attached Figure Description

[0021] Figure 1 This is the mass spectrum of LVSKP.

[0022] Figure 2 This is the mass spectrum of LVAP.

[0023] Figure 3 This is the mass spectrum of VVAGP.

[0024] Figure 4This refers to the ACE inhibitory activity of LVSKP.

[0025] Figure 5 This indicates the ACE-inhibiting activity of LVAP.

[0026] Figure 6 This represents the ACE-inhibiting activity of VVAGP.

[0027] Figure 7 The graph shows the results of in vivo antihypertensive activity assays for LVSKP, LVAP, and VVAGP. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0029] Example 1: Solid-phase synthesis and characterization of polypeptides

[0030] In previous experiments, our research group identified several potential bioactive peptides in soybean protein hydrolysates. To further study their properties, we synthesized polypeptides using conventional solid-phase synthesis methods and used the synthesized peptides in subsequent experiments.

[0031] 1.1 Solid-phase synthesis of peptides

[0032] The dichlororesin was swollen and washed to remove the Fmoc protecting group. Amino acids were then added for a condensation reaction. This deprotection-condensation process was repeated until all amino acids were linked. The resin was then cleaved to obtain crude LVSKP peptide, which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain a pure product (purity >95%).

[0033] In addition, following the steps above, by adjusting the order of the added amino acids, a crude polypeptide with the amino acid sequence LVAP, VVAGP, AP, GP, AGP, LVS was obtained. The product was then purified by reversed-phase high-performance liquid chromatography to obtain a pure product (purity >95%) for subsequent experiments.

[0034] 1.2 Characterization of peptides

[0035] The peptides obtained by solid-phase synthesis were characterized using LC-MS. Figure 1 , Figure 2 and Figure 3 Mass spectrometry spectra of LVSKP, LVAP, and VVAGP are shown. Characterization results confirm the successful synthesis of peptides LVSKP, LVAP, and VVAGP.

[0036] Example 2: Assay of ACE inhibitory activity of peptides

[0037] 2.1 Method for Assaying ACE Inhibitory Activity

[0038] Based on the reaction principle of ACE cleavage of hippuryl-histyl-leucine (HHL) to generate hippuric acid (HA) in vitro, high-performance liquid chromatography (HPLC) was used to detect the peak area of ​​HA at a wavelength of 228 nm to evaluate the ACE inhibitory activity of the peptide. The specific operation is as follows:

[0039] 30 μL of 7.5 mM HHL substrate was mixed with 20 μL of pure peptide solutions of different concentrations. An equal volume of sodium borate buffer was used as a blank control instead of the peptide sample. The reaction system was incubated at 37 °C for 10 min, and then 30 μL of 0.05 U / mL ACE solution was added to start the reaction. After reacting at 37 °C for 30 min, 20 μL of 0.1 M HCl was added to terminate the reaction. Finally, the ACE inhibitory activity of the peptide was calculated according to the corresponding formula.

[0040] ACE inhibitory activity (%) = (A 对照 -A 样品 ) / A 对照 ×100%

[0041] Where A 对照 and A 样品 These represent the peak areas of HA formation in the blank control group and the peptide sample group, respectively. The half-maximal inhibitory concentration (IC50) is also shown. 50 ) is a key indicator for measuring ACE inhibitory activity, specifically referring to the peptide sample concentration that can reduce ACE activity to 50% of its original level.

[0042] 2.2 Measurement Results

[0043] The ACE inhibitory activities of peptides LVSKP, LVAP, and VVAGP under different concentration conditions are as follows: Figure 4 , Figure 5 and Figure 6 As shown. The assay results show that the IC50 values ​​of peptides LVSKP, LVAP, and VVAGP are... 50 The values ​​were 22.3 μM, 26.3 μM, and 103.5 μM, respectively. Generally, IC... 50 The lower the value, the stronger its inhibitory ability on ACE. Therefore, the above results indicate that the peptides LVSKP, LVAP, and VVAGP all have strong ACE inhibitory activity, among which LVSKP and LVAP show stronger in vitro ACE inhibitory ability, while VVAGP also has good inhibitory activity.

[0044] The IC of peptides AP, GP, AGP, and LVS 50The values ​​were 230.0 µM, 252.6 µM, 562.3 µM, and 600.0 μM, respectively. Compared with the peptide LVSKP, LVS showed significantly enhanced ACE inhibitory activity after the addition of two C-terminal amino acid residues, with IC50 values ​​of 230.0 µM, 252.6 µM, 562.3 µM, and 600.0 µM. 50 The value decreased from 600.0 μM to 22.3 μM.

[0045] Compared to peptide AP, LVAP exhibits significantly enhanced ACE inhibitory activity after the addition of two N-terminal amino acid residues, with an IC50 value of [missing value]. 50 The value decreased from 230.0 μM to 26.3 μM.

[0046] Compared to peptide GP and peptide AGP, VVAGP exhibits stronger ACE inhibitory activity after the addition of 2–3 N-terminal amino acid residues, with an IC50 value of [missing value]. 50 The values ​​decreased from 252.6 μM and 562.3 μM to 103.5 μM, respectively.

[0047] Example 3: Determination of the in vivo antihypertensive activity of the peptide

[0048] 3.1 Feeding and Grouping of Laboratory Animals

[0049] Thirty 10-week-old male essential hypertensive rats (SHRs) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used in this experiment. The animal housing conditions were as follows: a 12-hour light / 12-hour dark circadian rhythm was used; the room temperature was controlled at 22 ± 3 ℃; and the relative humidity was maintained at 50%–70%. All animals underwent a one-week acclimatization period before the formal experiment. The entire experimental process strictly followed the relevant requirements of the "Ethical Guidelines for the Housing and Use of Laboratory Animals".

[0050] After the adaptation period, SHRs were randomly divided into 5 groups of 6 each: model group (saline), positive control group (captopril, 10 mg / kg·bw / d), peptide LVSKP group (50 mg / kg·bw / d), peptide LVAP group (50 mg / kg·bw / d) and peptide VVAGP group (50 mg / kg·bw / d).

[0051] 3.2 Measurement of blood pressure in experimental animals

[0052] Arterial blood pressure in shrews (SHRs) was measured using the tail-cuff method. The instrument used was the BP-2010A intelligent non-invasive blood pressure monitor from Beijing Ruanlong Biotechnology Co., Ltd. During measurement, rats were secured in a cuff and preheated at 37 ℃ for 5–10 minutes. Systolic blood pressure was measured after the instrument displayed a stable and normal pulse waveform. At least six sets of valid data were collected from each animal. All measurements were performed by the same experimenter in a quiet environment to minimize measurement interference.

[0053] 3.3 Experiment on the antihypertensive effect of a single gavage administration

[0054] SHRs were administered a single gavage according to the above grouping and dosage. Systolic blood pressure was measured in each group at 0 h, 2 h, 4 h, 6 h, and 8 h post-gavage. Results are as follows: Figure 7 As shown in the figure. Experimental results showed that after gavage administration of physiological saline to the model group, systolic blood pressure did not decrease significantly during the observation period; after gavage administration of captopril to the positive control group, blood pressure showed a highly significant decreasing trend, reaching its lowest value at 8 h, with a maximum decrease of 31.6 mmHg (p<0.01). In the LVSKP group, blood pressure continued to decrease within 8 h after gavage, reaching its maximum decrease at 6 h, significantly reducing by 19.7 mmHg (p<0.05). The VVAGP group also showed a continuous decreasing trend, reaching its maximum decrease at 6 h, significantly reducing by 26.5 mmHg (p<0.05). In contrast, although the LVAP group showed strong ACE inhibitory activity in vitro, no significant systolic blood pressure reduction effect was observed under the experimental conditions (p>0.05). This further illustrates that there is no simple one-to-one correspondence between in vitro ACE inhibitory activity and in vivo antihypertensive effect. The above results indicate that the peptides LVSKP and VVAGP provided by this invention have good in vivo acute antihypertensive activity, and their antihypertensive effect is close to that of the positive drug captopril, showing high application potential.

[0055] The above comparison results show that the peptides LVSKP, LVAP and VVAGP described in this invention not only have clear ACE inhibitory activity, but also that peptides LVSKP and VVAGP further exhibit better in vivo antihypertensive activity, showing higher application value.

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide having ACE inhibitory activity, characterized in that at least one of the following polypeptides: LVSKP, having an amino acid sequence of Leu-Val-Ser-Lys-Pro; LVAP, having an amino acid sequence of Leu-Val-Ala-Pro; VVAGP, having an amino acid sequence of Val-Val-Ala-Gly-Pro.

2. Use of the polypeptide having ACE inhibitory activity according to claim 1 in the preparation of an ACE inhibitor.

3. Use of the polypeptide having ACE inhibitory activity according to claim 1 in the preparation of a drug for treating or preventing cardiovascular diseases.

4. Use of the polypeptide having ACE inhibitory activity according to claim 1 in the preparation of a drug for treating or preventing hypertension.

5. Use of the polypeptide having ACE inhibitory activity according to claim 1 in the preparation of a health product with the function of assisting blood pressure reduction.

6. Use of the polypeptide having ACE inhibitory activity according to claim 1 in the preparation of a health food, a functional food or a special medical purpose formula food.

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