Four angiotensin converting enzyme inhibitors derived from millet protein and uses thereof

Abstract

The application discloses four angiotensin transferase inhibitors derived from millet protein and application thereof. Active ingredients of the four angiotensin transferase inhibitors derived from millet protein are polypeptides with amino acid sequences of QDPLFPL, FPGVSPF, QPAGLLPF or SPAQLLPF. The four functional polypeptides discovered from the millet protein are pure natural, non-toxic and harmless plant source substances, and have significant ACE inhibiting efficacy. The four polypeptides can achieve the effect of reducing blood pressure by inhibiting the activity of ACE, and this is also confirmed by in-vitro enzyme activity inhibition experiments. Therefore, as a blood pressure reducing component, the four polypeptides have good ACE inhibiting effect, are non-toxic, and have clear action mechanism and clear target, meet the requirements of drug preparation development, are easy to be added into various health foods as functional factors, and have good market prospects in the medical and food industries.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to four angiotensin converting enzyme inhibitors derived from millet protein and application thereof. BACKGROUND

[0002] At present, hypertension has become one of the inducements of coronary heart disease, cerebrovascular disease, arrhythmia, stroke, atherosclerosis and other high-risk diseases. Angiotensin converting enzyme (ACE) is also known as peptidyl dipeptidase, which is a Zn 2+ dependent dipeptidase, and the catalytic site is mainly composed of C-terminal and N-terminal domains, wherein the C-terminal domain participates in the regulation of blood pressure in human body. ACE plays a role in promoting the increase of blood pressure in the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) for controlling blood pressure in human body. In the RAS system, angiotensin I can be hydrolyzed to angiotensin II under the action of ACE, and angiotensin II can cause vasoconstriction, resulting in an increase in systolic blood pressure; in the KKS system, bradykinin with vasodilatation activity can be generated from kininogen under the action of kallikrein, and the bradykinin is hydrolyzed into inactive polypeptide fragments under the action of ACE, thereby causing the failure of vasodilatation activity and the increase of blood pressure. Therefore, the inhibition of ACE is considered to be the key to reducing hypertension.

[0003] At present, the drugs used in the clinical treatment of hypertension mainly include diuretics, beta receptor blockers, calcium antagonists, ACE inhibitors and angiotensin receptor blockers, among which the use of ACE inhibitors is the most common. Traditional ACE inhibitors are artificially synthesized drugs, such as enalapril and captopril, which have significant effects but are often accompanied by side effects such as dizziness, cough, nausea, rapid heart rate and the like. Therefore, efficient, stable and safe ACE inhibitors have become a new research trend in the food and drug fields. From the perspective of edibility and safety, food-based bioactive peptides have become increasingly important, and a large number of studies have shown that food protein hydrolysates have a regulating effect on the blood pressure level of animals. SUMMARY

[0004] The purpose of the present application is to provide four angiotensin converting enzyme inhibitors derived from millet protein and application thereof. The four functional polypeptides discovered from millet protein are pure natural, non-toxic and harmless plant source substances, and have significant ACE inhibitory efficacy, and can be used for preparing blood pressure lowering products.

[0005] In a first aspect, the present application provides a polypeptide, which is as follows (a1), (a2), (a3) or (a4):

[0006] (a1) a polypeptide having an amino acid sequence of QDPLFPL;

[0007] (a2) a polypeptide having an amino acid sequence of FPGVSPF;

[0008] (a3) a polypeptide having an amino acid sequence of QPAGLLPF;

[0009] (a4) a polypeptide having an amino acid sequence of SPAQLLPF.

[0010] In (a1), the amino acid sequence is a polypeptide as shown in SEQ ID NO: 1 (QDPLFPL) of the sequence listing, i.e., a polypeptide having an amino acid sequence of glutamine-aspartic acid-proline-leucine-phenylalanine-proline-leucine (Gln-Asp-Pro-Leu-Phe-Pro-Leu);

[0011] In (a2), the amino acid sequence is a polypeptide as shown in SEQ ID NO: 2 (FPGVSPF) of the sequence listing, i.e., a polypeptide having an amino acid sequence of phenylalanine-proline-glycine-valine-serine-proline-phenylalanine (Phe-Pro-Gly-Val-Ser-Pro-Phe);

[0012] In (a3), the amino acid sequence is a polypeptide as shown in SEQ ID NO: 3 (QPAGLLPF) of the sequence listing, i.e., a polypeptide having an amino acid sequence of glutamine-proline-alanine-glycine-leucine-leucine-proline-phenylalanine (Gln-Pro-Ala-Gly-Leu-Leu-Pro-Phe);

[0013] In (a4), the amino acid sequence is a polypeptide as shown in SEQ ID NO: 4 (SPAQLLPF) of the sequence listing, i.e., a polypeptide having an amino acid sequence of serine-proline-alanine-glutamine-leucine-leucine-proline-phenylalanine (Ser-Pro-Ala-Gln-Leu-Leu-Pro-Phe).

[0014] In a second aspect, the present application provides the use of the above-mentioned polypeptide in (b1), (b2) or (b3) as follows:

[0015] (b1) for preparing an angiotensin transferase inhibitor;

[0016] (b2) for preparing a blood pressure lowering product;

[0017] (b3) for preparing a product for preventing and treating hypertension.

[0018] In a third aspect, the present application provides an angiotensin transferase inhibitor, wherein the active ingredient is the above-mentioned polypeptide.

[0019] In a fourth aspect, the present application provides a blood pressure reducing drug, wherein the active ingredient is the polypeptide.

[0020] In a fifth aspect, the present application provides a preparation method of angiotensin transferase inhibiting peptide, comprising the following steps:

[0021] S1, performing first enzymolysis on defatted millet powder by using α-amylase, saccharifying enzyme and complex cellulase to obtain millet protein;

[0022] S2, performing second enzymolysis on the millet protein by using alkaline protease to obtain millet protein enzymolysis product;

[0023] S3, performing ultrafiltration on the millet protein enzymolysis product to collect a first component with MW < 3 kDa;

[0024] S4, performing separation on the first component by using reverse phase high performance liquid chromatography to collect a component with the highest angiotensin transferase inhibiting activity to obtain the angiotensin transferase inhibiting peptide.

[0025] In the above preparation method, preferably, the mass ratio of the α-amylase, the saccharifying enzyme and the complex cellulase is 2:2:1;

[0026] The enzymes in the complex cellulase are β-glucanase and xylanase;

[0027] The total amount of enzymes in the first enzymolysis is 2.2% of the mass of the defatted millet powder;

[0028] The pH value of the first enzymolysis is 4.7, the temperature is 48℃, the time is 10 h, and the solid-liquid ratio is 1 g:20 mL;

[0029] Step S1 further comprises: centrifuging the system after enzyme inactivation after the first enzymolysis to collect supernatant, washing the supernatant with water, adjusting the pH to neutral, freeze-drying to obtain the millet protein;

[0030] In step S2, the ratio of the alkaline protease to the millet protein is 4000 U:1 g;

[0031] The pH value of the second enzymolysis is 8.0, the temperature is 60℃, the time is 4 h, and the solid-liquid ratio is 1 g:20 mL;

[0032] Step S2 further comprises: centrifuging the system after enzyme inactivation after the second enzymolysis to collect supernatant, freeze-drying the supernatant to obtain the millet protein enzymolysis product;

[0033] In step S4, the conditions of the reverse phase high performance liquid chromatography are as follows:

[0034] The chromatographic column is Waters XBridge Prep C18 with a specification of 250mmx19mm, 5um;

[0035] The column temperature is 30℃;

[0036] The mixed gradient elution is adopted, A is a water solution containing 0.1% trifluoroacetic acid by volume percentage, B is a solution of acetonitrile containing 0.1% trifluoroacetic acid by volume percentage, and the gradient elution program is as follows: 0-5min, 10% B; 5-15min, 10-35% B; 15-40min, 35-70% B; 40-45min, 10% B;

[0037] The sample amount is 2mL;

[0038] The sample concentration is 2mg / mL;

[0039] The determination wavelength is 218nm;

[0040] The component with the highest angiotensin transferase inhibitory activity is the component with a retention time of 21-22.5min.

[0041] In a sixth aspect, the present application provides the angiotensin transferase inhibitory peptide prepared by the preparation method.

[0042] In a seventh aspect, the present application provides the angiotensin transferase inhibitory peptide for use in (b1), (b2) or (b3):

[0043] (b1) preparing an angiotensin transferase inhibitor;

[0044] (b2) preparing a blood pressure lowering product;

[0045] (b3) preparing a product for preventing and treating hypertension.

[0046] In an eighth aspect, the present application provides an angiotensin transferase inhibitor, wherein the active ingredient is the angiotensin transferase inhibitory peptide.

[0047] In a ninth aspect, the present application provides a blood pressure lowering drug, wherein the active ingredient is the angiotensin transferase inhibitory peptide.

[0048] The present application has the following beneficial effects:

[0049] (1) The present application screens the ACE inhibitory peptide in millet protein hydrolysate by ultrafiltration, liquid chromatography, mass spectrometry sequencing and other technologies and performs function verification, thereby providing technical support for developing safe substitutes for blood pressure lowering drugs.

[0050] (2) The four functional polypeptides (QDPLFPL, FPGVSPF, QPAGLLPF, SPAQLLPF) discovered from millet protein are pure natural, non-toxic and harmless plant source substances, and have significant ACE inhibiting efficacy. ACE plays an important role in catalyzing the conversion of angiotensin I to angiotensin II and regulating bradykinin, and is often studied as a target for controlling blood pressure level. QDPLFPL, FPGVSPF, QPAGLLPF and SPAQLLPF can achieve the effect of lowering blood pressure by inhibiting the activity of ACE, and this is also confirmed by in vitro enzyme activity inhibition experiments. Therefore, the QDPLFPL, FPGVSPF, QPAGLLPF and SPAQLLPF as blood pressure lowering components have good ACE inhibiting effect, are non-toxic, and have clear mechanism of action and clear target, meeting the requirements of drug preparation development, and are also easy to be added to various health foods as functional factors, having good market prospects in the medical and food industries. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The ACE inhibitory rates of different ultrafiltration fractions in the embodiments of the present application, including protein hydrolysate, <3kDa component, 3-10kDa component and >10kDa component, are shown by different letters.

[0052] Figure 2 The liquid chromatography separation chart of active peptides in the embodiments of the present application, including component 1, component 2, component 3, component 4, component 5, component 6 and component 7.

[0053] Figure 3 The ACE inhibitory rates of different ultrafiltration fractions in the embodiments of the present application, including protein hydrolysate, <3kDa component, 3-10kDa component and >10kDa component, are shown by different letters.

[0054] Figure 4 The mass spectrum of four ACE inhibiting peptides, including QDPLFPL (1), FPGVSPF (2), QPAGLLPF (3), SPAQLLPF (4).

[0055] Figure 5 The ACE inhibitory rates of different ultrafiltration fractions in the embodiments of the present application, including protein hydrolysate, <3kDa component, 3-10kDa component and >10kDa component, are shown by different letters. 50 DETAILED DESCRIPTION

[0056] ​The application will be further described in conjunction with the specific embodiments. The examples given below are only for the purpose of illustrating the application and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0057] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0058] The α-amylase used in the following examples is a product produced by Shanghai Yuan Ye Co., Ltd., with the trade name α-amylase (Bacillus subtilis) and the product code S10003, and the enzyme activity is 50000 U / g;

[0059] The glucoamylase is a product produced by Shanghai Yuan Ye Co., Ltd., with the trade name glucoamylase and the product code S10017, and the enzyme activity is 100000 U / g;

[0060] The complex cellulase is a product produced by Novozymes Denmark Co., Ltd., with the trade name β-glucanase (complex temperature-resistant Max type), the model number is Ultraflo Max, the glucanase activity is 700 EGU / g, and the xylanase activity is 250 FXU / g;

[0061] The alkaline protease is a product produced by Shanghai Yuan Ye Co., Ltd., with the trade name alkaline protease and the product code S10154, and the enzyme activity is 200000 U / g.

[0062] Example 1

[0063] (1) Preparation of millet protein

[0064] The millet powder was passed through a 60-mesh sieve, and the millet powder and n-hexane were mixed at a ratio of 1:5 (w / v) and defatted in a 37℃ water bath shaker for 4h, the n-hexane was recovered, and the millet powder was air-dried in a fume hood for 24h for standby use. The defatted millet protein was extracted by the three-enzyme complex method of α-amylase, glucoamylase and complex cellulase, and the extraction process was pH 4.7, enzyme hydrolysis temperature 48℃, enzyme addition amount 2.2wt%, enzyme hydrolysis time 10h, solid-liquid ratio 1g:20mL, and the specific operation was as follows: according to the solid-liquid ratio of 1g:20mL, water was added to the defatted millet powder, then α-amylase, glucoamylase and complex cellulase (α-amylase: glucoamylase: complex cellulase = 2:2:1) were added to the solution at an enzyme addition amount of 2.2wt%, the pH of enzyme hydrolysis was adjusted to 4.7 with 0.1M HCl, the temperature was 48℃, and the enzyme hydrolysis time was 10h. Then the enzyme was inactivated at 90℃ for 10min and centrifuged at 7000rpm for 20min, the supernatant was taken, washed with water multiple times, and the pH was adjusted to neutral. Finally, freeze-drying was performed and stored at -20℃.

[0065] (2) Preparation of protease hydrolysate and freeze-drying

[0066] Millet protein and distilled water were mixed at a ratio of 1:20 (w / v), and then alkaline protease was added to the solution at a ratio of 4000 U / g, the pH of the enzymolysis was adjusted to 8.0 with 0.1 M NaOH, the enzymolysis temperature was 60°C, and the enzymolysis time was 4 h. After the enzymolysis was completed, the enzyme was inactivated by boiling water, the enzymolysis solution was centrifuged at 7000 rpm for 20 min, the supernatant was freeze-dried, and stored at -20°C for standby use.

[0067] (3) Preparation of ACE inhibitory crude peptide

[0068] In order to purify the millet ACE inhibitory peptide, the enzymolysis product was prepared into a concentration of 10 mg / mL as a primary solution (MPH). The MPH was sequentially passed through ultrafiltration membranes with molecular weights of 3 kDa and 10 kDa, and three components with molecular weights of MW < 3 kDa, 3-10 kDa and > 10 kDa were obtained, which were freeze-dried and stored at -20°C for ACE inhibitory activity evaluation. The effects of three components (> 10 kDa, 3-10 kDa and < 3 kDa) on ACE activity were determined at a concentration of 1 mg / mL, and the results showed that the < 3 kDa fraction had the best inhibitory effect on ACE activity (see Figure 1 ).

[0069] The specific process of ACE activity inhibition experiment is as follows:

[0070] Preparation of hippurylhistidylleucine (HHL) solution: accurately weigh 0.107 g of HHL and 0.877 g of NaCl, accurately measure 5 ml of sodium borate buffer (pH 8.3), and make up to 50 ml with ultrapure water in a volumetric flask for standby use.

[0071] Mix 150 μL of the sample with the appropriate concentration and 100 μL (100 mU / mL) of ACE thoroughly at 37°C for 10 min, then add 500 μL of HHL solution, react at 37°C for 60 min, then add 750 μL of HCl (1 mol / L), 1500 μL of pyridine, and 750 μL of benzenesulfonyl chloride in sequence. Vortex the solution for 1 min and immediately cool it in an ice bath, and measure the absorbance at a wavelength of 410 nm.

[0072] The ACE inhibition rate is calculated according to formula (1):

[0073]

[0074] In formula (1), A C is the absorbance of the control group; A S is the absorbance of the sample; and A B is the absorbance of the blank.

[0075] (4) Screening of ACE inhibitory peptides

[0076] The alkaline protease hydrolysate with a molecular weight less than 3 kDa was separated by RP-HPLC using a Waters XBridge Prep C18 column (250 mm x 19 mm, 5 μm) at 30 °C with a mixed gradient elution, A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile. The gradient elution program was as follows: 0-5 min, 10% B; 5-15 min, 10-35% B; 15-40 min, 35-70% B; 40-45 min, 10% B. The sample loading was 2 mL at a concentration of 2 mg / mL, and the absorption peaks were determined at 218 nm, and the fractions were collected for storage. The liquid chromatography separation chart of each component (components 1-7) is shown in Figure 2 . Subsequently, the effect of each absorption peak component (components 1-7) on ACE activity was determined at a concentration of 1 mg / mL, and the results showed that component 4 had the best inhibitory effect on ACE activity (see Figure 3 ), which was lyophilized and stored at -20 °C for storage.

[0077] (5) Synthesis of ACE inhibitory peptides

[0078] According to the ACE inhibitory activity analysis results of step (4), the amino acid sequence of component 4 was analyzed by liquid chromatography-tandem mass spectrometry, and the amino acid composition was determined as QDPLFPL, FPGVSPF, QPAGLLPF, and SPAQLLPF (mass spectrometry of QDPLFPL (1), FPGVSPF (2), QPAGLLPF (3), and SPAQLLPF (4) is shown in Figure 4 ), and the four kinds of polypeptides were prepared by Fmoc solid-phase synthesis method, and the purity of each peptide was determined to be greater than 95% by HPLC chromatography and mass spectrometry analysis.

[0079] (6) Functional evaluation

[0080] It was found through ACE activity inhibition experiments that the IC 50 of QDPLFPL, FPGVSPF, QPAGLLPF, and SPAQLLPF were 0.6912, 0.1261, 0.4063, and 0.4858 mg / mL, respectively (see Figure 5 ).

[0081] Computer software was used to predict the function of QDPLFPL, FPGVSPF, QPAGLLPF, SPAQLLPF, where toxicity and steric hindrance were assessed by ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), grand average of hydropathicity by ExPasy (https: / / web.expasy.org / protparam / ), human intestinal absorption by admetSAR (http: / / lmmd.ecust.edu.cn / admetsar1 / home / ), isoelectric point by Pepdraw (http: / / www.tulane.edu / ~biochem / WW / PepDraw / ). The experimental results are shown in Table 1.

[0082] Table 1 Function prediction of ACE inhibitory peptides

[0083]

[0084] Note: + / - means good / poor human intestinal absorption of the polypeptide

[0085] As can be seen from Table 1, the four ACE inhibitory peptides are all non-toxic, and in addition to QDPLFPL, the other three polypeptides have good human intestinal absorption. The isoelectric points of the four polypeptides are all less than 7, indicating that they are acidic. The grand average of hydropathicity can be used to represent the hydrophilicity or hydrophobicity of a protein, where the greater the positive value indicates the stronger the hydrophobicity, and the greater the negative value indicates the stronger the hydrophilicity, so FPGVSPF, QPAGLLPF and SPAQLLPF have good hydrophilicity, while QDPLFPL has better hydrophobicity. The steric hindrance values of the four ACE inhibitory peptides are between 0.53 and 0.58, of which SPAQLLPF has the lowest steric hindrance value, which is conducive to enhancing the inhibition of ACE.

[0086] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. A polypeptide which is (al), (a2), (a3) or (a4) as follows: (al) a polypeptide whose amino acid sequence is represented by SEQ ID NO: 1 in the Sequence Listing; (a2) a polypeptide whose amino acid sequence is represented by SEQ ID NO: 2 in the Sequence Listing; (a3) a polypeptide whose amino acid sequence is represented by SEQ ID NO: 3 in the Sequence Listing; (a4) a polypeptide whose amino acid sequence is represented by SEQ ID NO: 4 in the Sequence Listing.

2. Use of the polypeptide according to claim 1 in (bl), (b2) or (b3) as follows: (bl) the production of an angiotensin converting enzyme inhibitor; (b2) the production of a blood pressure lowering product; (b3) the production of a product for the prevention and treatment of hypertension.

3. An angiotensin converting enzyme inhibitor whose active ingredient is the polypeptide according to claim 1.

4. A blood pressure lowering drug whose active ingredient is the polypeptide according to claim 1. ​ ​ ​ ​ ​ ​ ​

Images