CN114106092B

CN114106092B - Active polypeptide with ACE inhibition effect and application thereof

Info

Publication number: CN114106092B
Application number: CN202111149814.8A
Authority: CN
Inventors: 和七一; 余晓东; 陈峻波
Original assignee: Chongqing Normal University
Current assignee: Chongqing Normal University
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2023-08-22
Anticipated expiration: 2041-09-29
Also published as: CN114106092A

Abstract

The invention discloses an active polypeptide with ACE inhibition effect, and the amino acid sequence of the active polypeptide is WGAP. The active polypeptide with ACE inhibition effect has continuous and stable inhibition effect on ACE activity and good blood pressure reduction effect, so that the active polypeptide can be used as an active ingredient in foods, health care products and blood pressure reduction medicines, and has good potential and application prospect.

Description

Active polypeptide with ACE inhibition effect and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an active polypeptide with an ACE inhibition effect and application thereof.

Background

Hypertension is one of the most common cardiovascular diseases and is also an important risk factor for diseases such as cerebral apoplexy, coronary heart disease, heart failure and the like. According to the statistics of the world health organization in 2020, about 11.3 hundred million people all over the world suffer from hypertension, and the health of the people is seriously threatened. Angiotensin converting enzyme @Angiotensin-I converting enzyme, ACE) Is a dipeptide carboxyl metalloprotease, which is used in renin-angiotensin systemRenin-Angiotensin System, RAS) And kallikrein-kallikrein system ]Kallikrein-Kinin System, KKS) Plays an important regulating and controlling role. ACE inhibitors can lower blood pressure by inhibiting ACE activity, preventing the conversion of angiotensin I to angiotensin II, and maintaining the activity of angiotensin bradykinin. Currently, ACE inhibitors commonly used for the treatment of hypertension are captopril, enalapril, fosinopril, lamipril, and the like. Although the effect is obvious, the side effects of the traditional Chinese medicine composition are large after long-term administration, including cough, gustatory deformation, rash, reduced renal function, increased lung cancer risk and the like. Thus, research has focused on finding natural ACE inhibitors that are beneficial to health and have no adverse effects.

The food-source active peptide has the advantages of easy solubility, no sensitization and good ADMETAbsorption, Distribution, Metabolism, Excretion and Toxicity) Characteristics and thus are increasingly subject to peopleAttention is paid. In recent years, food-derived biopeptides having various functions, such as ACE inhibitory peptides, anti-inflammatory peptides, dipeptidyl peptidase IV inhibitory peptides, and antioxidant peptides, etc., have been found. At present, scholars at home and abroad have found that a large number of animals and plants contain bioactive peptides for inhibiting ACE, especially in various frequently-eaten animals such as chickens, ducks, fishes, pigs, cattle and the like.

New Zealand white rabbits are used as the most edible rabbits fed in China, and are functional meat food with high nutritive value. The rabbit meat is rich in proteins, minerals, vitamins, lecithin and polyunsaturated fatty acids, can provide bioactive substances for consumers, and can play a key role in controlling cardiovascular diseases and other chronic diseases when being eaten frequently. At present, research on rabbit meat at home and abroad mainly focuses on nutritional value, and research reports on rabbit meat source ACE inhibitory peptide and action mechanism thereof are not yet reported.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide the ACE inhibitory peptide which is derived from rabbit meat, has a continuous and stable inhibitory effect on the activity of ACE and has a good blood pressure reducing effect.

1. An active polypeptide with ACE inhibition effect, which is characterized in that: the amino acid sequence is Trp-Gly-Ala-Pro (WGAP).

2. An application of the active polypeptide with ACE inhibition effect in preparing ACE inhibition medicines.

3. An application of the active polypeptide with ACE inhibition effect in preparing antihypertensive drugs.

4. A medicine with ACE inhibition effect is characterized in that: the active ingredient contains the active polypeptide with ACE inhibition effect.

5. A medicine with the function of reducing blood pressure is characterized in that: the active ingredient contains the active polypeptide with ACE inhibition effect.

The beneficial effects are that:

the active polypeptide with ACE inhibition effect has continuous and stable inhibition effect on ACE activity and good blood pressure reduction effect, so that the active polypeptide can be used as an active ingredient in foods, health care products and blood pressure reduction medicines, and has good potential and application prospect.

Drawings

FIG. 1 is a reversed phase high performance liquid chromatogram of fraction F-4;

FIG. 2 shows ACE inhibitory activity of various concentrations of active polypeptide before and after in vitro gastrointestinal digestion;

FIG. 3 is a line graph of the effect of single oral administration of physiological saline, captopril (20 mg/kg) and WGAP (25, 50 and 100 mg/kg) on systolic blood pressure in hypertensive rats (data mean.+ -. Standard deviation (n=6); x represents)P<0.05 captopril group and normal saline group, x representsP<0.01 captopril group and normal saline group, respectivelyP<0.05 25 mg/kg WGAP group and physiological saline group, respectivelyP<0.01 25 mg/kg WGAP group and physiological saline group, # representsP<0.05 50 mg/kg WGAP group and physiological saline group, # representsP<0.01 50 mg/kg WGAP group and physiological saline group, and DeltarepresentsP<0.05 100 mg/kg WGAP group and physiological saline group, and DeltaA representsP<0.01 100 mg/kg WGAP group and physiological saline group).

Concrete embodiments

The invention is further described below with reference to examples and figures.

Examples

Rabbit meat was chopped, brought to a concentration of 50mg/mL with distilled water, and homogenized for 2 min using a tissue homogenizer. The homogenate was inactivated in a boiling water bath for 10 min. After cooling, five different enzymes (alkaline protease: 50 ℃, ph7.0; pepsin: 37 ℃, ph2.0; trypsin: 37 ℃, ph8.0; complex protease: 50 ℃, ph7.0; bromelain: 55 ℃, ph 7.0) were used to hydrolyze for 2, 3, 4, 5 or 6 hours under optimal conditions with a mass ratio of enzyme to rabbit meat of 1:10. During the hydrolysis, the pH of the reaction mixture was maintained by the addition of 0.5N NaOH or 0.5N HCl. The mixture was then boiled for 10 minutes to terminate the reaction, cooled rapidly to room temperature, and adjusted to pH7.0. Then centrifuged at 10,000 r/min for 20 min at 4℃using a TGL-20 centrifuge. The supernatant was filtered with filter paper and lyophilized in a lyophilizer to give Rabbit Meat Protein Hydrolysate (RMPH). The ACE inhibitory activity of each RMPH was measured and the most active RMPH was stored at-20℃for further investigation. Then, the functional components in the zymolyte are screened by adopting an ultrafiltration method, sephadex G-15 Gel Filtration Chromatography (GFC) and high performance liquid chromatography (RP-HPLC), and finally F4-4 has the strongest ACE inhibitory activity (the final result is shown in figure 1); and then, identifying the functional peptide component by adopting liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine that the amino acid sequence of the polypeptide is Trp-Gly-Ala-Pro, namely WGAP.

2. ACE inhibition activity assay the inhibition of ACE activity by WGAP was determined. 20 μl of WGAP solution was pre-incubated with ACE solution (20 μl,25 mU/mL) in 50mM borate sodium buffer (containing 0.3M NaCl, pH 8.3) for 10 min at 37deg.C. Then Ma Niaoxian group aminoacyl leucine (HHL) solution (20 μl,5 mM) was added to the reaction mixture and incubated for 30min at 37 ℃. The reaction was terminated by adding 100 μl of 1M HCl. Ethyl acetate (1 mL) was added thereto, and after shaking, the mixture was centrifuged at 10,000 r/min at 4℃for 10 min. The upper layer was taken and evaporated to dryness in vacuo at 60 ℃. The residue was dissolved in 100 μl of primary water and absorbance was measured at 228 nm. The inhibitory activity of ACE was calculated according to the formula:

ACE inhibitory Activity (%) = (A) _Control – A _{Sample of} )/(A _Control – A _{Blank space} )

Wherein A is _Control Absorbance in the absence of inhibitor, A _{Sample of} Is the absorbance of the reaction mixture, A _{Blank space} Is the absorbance of the buffer. IC (integrated circuit) ₅₀ The value is defined as the concentration of inhibitor that causes 50% inhibition of ACE activity.

The results show that polypeptide WGAP exhibits ACE inhibition IC ₅₀ 140.70.+ -. 4.51. Mu.M.

3. In vitro digestion stability WGAP was dissolved in 0.1M KCl and adjusted to pH2.0 with 1M HCl. Pepsin (E/S2%, w/w) was added and the polypeptide 2 h was digested in a 37℃water bath. The reaction was stopped by heating at 100℃for 10 min and the pH was quickly adjusted to 8.0 with 2M NaOH. Trypsin (E/S2%, w/w) was then added and incubated for 3 hours at 37 ℃. Again inactivated at 100℃for 10 minutes.After cooling to room temperature, the hydrolysate was adjusted to pH7.0 and centrifuged at 10,000 r/min for 10 minutes. Finally, the ACE inhibitory activity of the supernatant was examined and compared with the peptide before digestion. As shown in FIG. 2, there was no significant change in the ACE inhibitory activity of WGAP following pepsin and trypsin treatmentp>0.05). These results indicate that WGAP is resistant to gastrointestinal digestion and may exhibit hypotensive effects in vivo.

4. In vivo antihypertensive effect male SD rats (10 weeks, body weight 250±20 g) were fed by the university of Chongqing medical laboratory animal center in china, and were fed under standard conditions (12 hours light/dark cycle, 22±1 ℃) and were fed and drunk freely. After conditioning for one week, rats were intraperitoneally injected with 15mg/kg L-N-nitroarginine once a day for four weeks to establish a model of hypertension before the start of the experiment. Subsequently, rats were randomized into 5 treatment groups of 6: positive control, negative control, high, medium, low dose. The rats of the positive control group were orally administered captopril (20 mg/kg), and the rats of the negative control group were orally administered physiological saline. Rats in the high, medium and low dose groups were orally administered WGAP at doses of 100, 50 and 25 mg/kg body weight, respectively. Systolic Blood Pressure (SBP) was measured at 0, 2, 4, 6, 8, 10 and 12 hours post-administration using a noninvasive blood pressure measurement analysis system. As a result, as shown in FIG. 3, the negative control group (0.9% physiological saline) showed no decrease in SBP within 12 hours after administration, while the positive control group (captopril, 20 mg/kg) showed a significant decreaseP<0.01). 6 hours after captopril administration, SBP was decreased by 41.79 + -2.34 mmHg ±P< 0.01). However, the WGAP treated groups (25, 50 and 100 mg/kg) showed a decrease in SBP after only 4 hoursP< 0.01), 23.45.+ -. 1.63, 32.85.+ -. 1.94 and 42.66.+ -. 2.87 mmHg (FIG. 3), respectively, indicating that WGAP is better absorbed than captopril. In addition, the extent of SBP variation is WGAP dose dependent. 12 After h, the SBP of the rat is obviously reduced by 13.80+ -1.24 mmHg in the high-dose WGAP groupPLess than 0.01), and 9.28+/-0.76 mmHg is reduced in captopril groupP< 0.05), but there was no significant difference between the medium and low WGAP groups compared to the negative control groupP＞0.05）。

Thus, WGAP significantly reduces SBP after oral administration in hypertensive rats and is a potential natural ACE inhibiting peptide.

Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. An active polypeptide with ACE inhibiting effect, characterized in that: the amino acid sequence is WGAP.

2. Use of an active polypeptide having ACE inhibitory activity as claimed in claim 1 in the manufacture of an ACE inhibitory drug.

3. Use of an active polypeptide having ACE inhibiting activity as claimed in claim 1 in the manufacture of a medicament for lowering blood pressure.

4. A medicament with ACE inhibition, characterized in that: an active ingredient comprising the active polypeptide having ACE inhibitory activity as claimed in claim 1.

5. A medicament with blood pressure reducing effect, which is characterized in that: an active ingredient comprising the active polypeptide having ACE inhibitory activity as claimed in claim 1.