Angiotensin I converting enzyme inhibitory peptide and its application

Abstract

The present invention belongs to the technical field of active peptide preparation, and specifically relates to an angiotensin I converting enzyme inhibitory peptide and its application. The present invention uses Yinghong No. 9 black tea residue as raw material, and utilizes enzymatic hydrolysis, ultrafiltration, high performance liquid chromatography separation and purification, mass spectrometry, etc. to screen out angiotensin I converting enzyme inhibitory peptides from 59 peptide segments. The molecular docking results show that the inhibitory peptide can bind to multiple amino acid residues in the S1, S2 and S1′ active pockets of ACE, and it is mainly hydrogen bonds, metal ion binding and hydrophobic interactions that play the most important role in stabilizing the docking complex and inhibiting ACE activity. In addition, the EA.hy926 cell model was used to find that the inhibitory peptide can play an active role by promoting the release of NO and inhibiting the secretion of ET‑1. The inhibitory peptide is expected to be used as a potential antihypertensive drug or functional food ingredient.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptide preparation, and in particular relates to an angiotensin I converting enzyme inhibitory peptide and application thereof. Background Art

[0002] The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) are the primary mechanisms by which the human body regulates blood pressure, vascular tone, and cardiovascular function. Angiotensin-converting enzyme (ACE) is a key enzyme in these two blood pressure-regulating systems. ACE regulates blood pressure by catalyzing the conversion of angiotensin I to angiotensin II, triggering a series of reactions and inactivating bradykinin. This suggests that inhibiting ACE activity may be an effective way to block elevated blood pressure in the body. Although most chemically synthesized ACE inhibitors (such as captopril, enalapril, lisinopril, and benazepril) have good clinical therapeutic effects, they can cause toxic side effects such as renal impairment, dry cough, and rash, which should not be underestimated. Therefore, the development of new ACE inhibitors with fewer side effects has become a research hotspot. Currently, a large number of bioactive peptides have been isolated from fungi, bacteria, plants, and animals. To date, more than 900 bioactive peptides have been reported as ACE inhibitors in the BIOPEP database, most of which are derived from marine organisms, dairy products, cereals, and legumes.

[0003] Tea is a popular non-alcoholic beverage consumed worldwide, and China is the world's largest tea producer. For a long time, research on tea has largely focused on its water-soluble active ingredients. With the rapid development of the tea beverage industry, global production of tea waste has increased dramatically. The resulting tea waste is used as feed, soil fertilizer, and heavy metal adsorbent, but most of it is discarded without any further use, resulting in a significant waste of resources. The protein in tea waste accounts for approximately 21% to 28% of the dry matter, making it a high-quality plant protein with potential for further purification and utilization. The protein in tea waste exhibits a variety of biological activities and functional properties, making it a high-quality plant protein and a good source of active peptides. Currently, there are reports on active peptides from tea proteins, such as lipid-lowering peptides, antioxidant peptides, and hypoglycemic peptides. Summary of the Invention

[0004] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide an angiotensin I converting enzyme inhibitory peptide, which is an inhibitory peptide (AAASPGGGM or GIHETTYNS) that binds to the key residues of the ACE active pocket and Zn 2+The active site is bound by hydrogen bonds and metal ion interactions, and also exhibits a similar ACE inhibitory activity mechanism to captopril through a large number of hydrophobic interactions. Furthermore, AAASPGGGM and GIHETTYNS can exert cellular antihypertensive activity in the EA.hy926 cell model by promoting NO production and inhibiting ET-1 secretion.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned angiotensin I converting enzyme inhibitory peptide.

[0006] Another object of the present invention is to use the above-mentioned angiotensin I converting enzyme inhibitory peptide.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An angiotensin I converting enzyme inhibitory peptide, whose amino acid sequence is: AAASPGGGM or GIHETTYNS;

[0009] The method for preparing the angiotensin I converting enzyme inhibitory peptide comprises the following steps:

[0010] The angiotensin I converting enzyme inhibitory peptide is directly synthesized in vitro solid phase or is obtained by using Yinghong No. 9 black tea residue as raw material, extracting by alkali dissolution and acid precipitation, hydrolyzing with trypsin, and purifying the angiotensin I converting enzyme inhibitory peptide;

[0011] The trypsin hydrolysis conditions are preferably: hydrolysis at 37° C. and pH 7.20 for 3.20 h;

[0012] The substrate mass concentration of the trypsin hydrolysis is preferably 3%, and the enzyme-substrate mass ratio is preferably 0.40%;

[0013] The purification comprises the steps of ultrafiltration separation and liquid chromatography separation;

[0014] The specific operation of the ultrafiltration separation is:

[0015] The Yinghong No. 9 tea protein hydrolysate was initially separated using an ultrafiltration tube, centrifuged, and fractions with a molecular weight <3 kDa were collected;

[0016] The centrifugal condition is preferably 4000 r / min for 30 min;

[0017] The specific operation of the liquid chromatography separation is as follows: using Prep 150 preparative liquid phase system, reverse phase column SunFirePrep C18OBD TM(19 mm × 250 mm, 5 μm, Waters), the molecular weight <3 kDa fraction obtained by ultrafiltration was separated and purified; the active fraction was collected, concentrated by rotary evaporation and freeze-dried to obtain angiotensin I converting enzyme inhibitory peptide;

[0018] The liquid chromatography conditions are preferably as follows: mobile phase A: ultrapure water, 0.1% TFA; mobile phase B: methanol, 0.1% TFA; elution conditions: 0-30 min, 5%-15% B; 30-40 min, 15%-25% B; 40-60 min, 25%-35% B; 60-70 min, 35%-45% B; 70-80 min, 45%-95% B; the above percentages are all volume percentages; the injection volume is 4 mL, the elution flow rate is 10 mL / min, and the detection wavelengths are 214 nm and 280 nm;

[0019] Use of the angiotensin I converting enzyme inhibitory peptide in the preparation of angiotensin converting enzyme (ACE) inhibiting products;

[0020] Use of the angiotensin I converting enzyme inhibitory peptide in the preparation of a blood pressure lowering product;

[0021] An angiotensin-converting enzyme inhibitor, comprising the angiotensin-I converting enzyme inhibitory peptide as an active ingredient;

[0022] The angiotensin-converting enzyme inhibitor further comprises a pharmaceutically acceptable carrier or excipient;

[0023] The pharmaceutically acceptable carrier or excipient includes at least one of a cosolvent, a moisturizer, a surfactant, a matrix, an emulsifier, a preservative and a solvent;

[0024] A blood pressure lowering drug comprising the angiotensin I converting enzyme inhibitory peptide as an active ingredient;

[0025] The blood pressure-lowering drug further comprises a pharmaceutically acceptable carrier or excipient;

[0026] The pharmaceutically acceptable carrier or excipient includes at least one of a cosolvent, a moisturizer, a surfactant, a matrix, an emulsifier, a preservative and a solvent;

[0027] The present invention has the following advantages and effects compared to the prior art:

[0028] (1) The present invention uses Yinghong No. 9 black tea residue as raw material, and obtains ACE inhibitory peptides from Yinghong No. 9 tea protein by enzymatic hydrolysis, ultrafiltration, and high performance liquid chromatography separation and purification; then the ACE inhibitory peptides are structurally identified by mass spectrometry, and two new high-activity ACE inhibitory peptides are screened out from 59 peptide segments, whose amino acid sequences are: AAASPGGGM (903.3717Da, IC 50 =0.77mg / mL), GIHETTYNS (1053.3994Da, IC 50 =0.2 mg / mL). The present invention then used molecular docking technology to study their structure-activity relationship and ACE inhibition mechanism. The results showed that both AAASPGGGM and GIHETTYNS were able to bind to multiple amino acid residues in the S1, S2, and S1′ active pockets of ACE, and that hydrogen bonding, metal ion binding, and hydrophobic interactions played the most important roles in stabilizing the docked complex and inhibiting ACE activity.

[0029] (2) The present invention further found through the EA.hy926 cell model that AAASPGGGM and GIHETTYNS can exert their active effects by promoting the release of NO and inhibiting the secretion of ET-1.

[0030] In summary, tea protein is a good source of food-borne ACE inhibitory peptides. The angiotensin I-converting enzyme inhibitory peptide provided by the present invention is expected to be used as a potential antihypertensive drug or functional food ingredient, providing new ideas for the development and high-value utilization of tea protein in tea residues and the development of new healthy foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figures are the results of further separation and purification of the ultrafiltration fraction <3 kDa using RP-HPLC; wherein, A: chromatogram of the ultrafiltration fraction <3 kDa; B: ACE inhibitory activity of fractions F1 to F11 at a concentration of 1.0 mg / mL, different letters indicate significant differences (P<0.05).

[0032] Figure 2 This is the LC-MS / MS total ion current graph of purified fraction F7.

[0033] Figure 3 These are the secondary mass spectrometry and molecular docking results of AAASPGGGM and GIHETTYNS; A: secondary mass spectrometry of AAASPGGGM; B: secondary mass spectrometry of GIHETTYNS; C: 2D and 3D images of the docking of AAASPGGGM with the receptor protein 1O8A; D: 2D and 3D images of the docking of GIHETTYNS with the receptor protein 1O8A.

[0034] Figure 4ACE inhibitory IC of peptides AAASPGGGM and GIHETTYNS 50 Value result graph.

[0035] Figure 5 This is the result graph of the effect of Yinghong No. 9 tea protein ACE inhibitory peptide on EA.hy926 cell viability.

[0036] Figure 6 This is a graph showing the effect of Yinghong No. 9 tea protein ACE inhibitory peptide on NO production in EA.hy 926 cells; different letters indicate significant differences, P < 0.05.

[0037] Figure 7 This is a graph showing the effect of Yinghong No. 9 tea protein ACE inhibitory peptide on the ET-1 content in EA.hy 926 cells; different letters indicate significant differences, P < 0.05. DETAILED DESCRIPTION

[0038] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0039] In the embodiment, Yinghong No. 9 black tea was provided by Tea Research Institute of Guangdong Academy of Agricultural Sciences; trypsin (2.5×10 5 U / g), Nanning Pangbo Bioengineering Co., Ltd.; angiotensin-converting enzyme (from rabbit lung), hydroxyethylpiperazineethanesulfonic acid (HEPES), and N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) were purchased from Sigma-Aldrich, USA; MTT was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; DMEM high-glucose medium, fetal bovine serum, and double-streptomycin antibodies were purchased from Gibico, USA; nitric oxide (NO) detection kit was purchased from Beyotime Biotechnology Co., Ltd.; human endothelin-1 (ET-1) enzyme-linked immunosorbent assay kit was purchased from Wuhan Huamei Bioengineering Co., Ltd.; all other reagents were of analytical grade; EA.hy926 cells were obtained from the Cell Bank of Shanghai Institutes for Biological Studies, Chinese Academy of Sciences.

[0040] Data analysis in the examples: Experimental results are expressed as mean ± standard deviation, and SPSS 26.0 statistical software was used to perform one-way analysis of variance (ANOVA) on the data. Duncan test was used for significance analysis. P < 0.05 was considered a significant difference, and significant differences are marked with different letters.

[0041] Example 1 Purification, enrichment and identification of ACE inhibitory peptides

[0042] 1. Experimental Methods

[0043] 1. Preparation of Yinghong No. 9 Tea Enzyme Hydrolysate

[0044] Yinghong No. 9 tea protein crude extract and Yinghong No. 9 tea protein hydrolysate were prepared with Yinghong No. 9 tea residue as raw material, referring to the reference (Ma Feng, Ye Haoduo, Xia Zhen, et al. Preparation of ACE inhibitory peptides from Yinghong No. 9 tea protein, amino acid composition and activity evaluation of different ultrafiltration fractions [J]. Modern Food Science and Technology, 2023, 39(07): 237-245.). The enzymatic hydrolysis process was as follows: the tea protein crude extract was enzymatically hydrolyzed by trypsin at 37°C, 3.20h, pH 7.20, substrate mass concentration of 3%, and enzyme-substrate mass ratio of 0.40%. The Yinghong No. 9 tea protein hydrolysate was collected and stored at -20°C for later use.

[0045] 2. Purification and Enrichment of ACE Inhibitory Peptides from Yinghong No. 9 Tea Protein

[0046] (1) Ultrafiltration separation

[0047] The Yinghong No. 9 tea protein hydrolysate obtained in step 1 was initially separated using an ultrafiltration tube (3 kDa, Millipore USA) and centrifuged at 4000 r / min for 30 min. Then, the components with molecular weights <3 kDa and >3 kDa were collected, freeze-dried, and stored at -20°C for subsequent ACE inhibitory activity analysis and further separation and purification.

[0048] (2) High performance liquid chromatography separation

[0049] Prep 150 preparative liquid phase system and reversed phase column SunFire Prep C18 OBD were used. TM (19mm×250mm, 5μm, Waters), the ultrafiltration fraction with better activity obtained in step (2) was separated and purified. Wherein, mobile phase A: ultrapure water, 0.1% TFA; mobile phase B: methanol, 0.1% TFA; elution conditions: 0-30min, 5%-15% B; 30-40min, 15%-25% B; 40-60min, 25%-35% B; 60-70min, 35%-45% B; 70-80min, 45%-95% B (the above percentages are all volume percentages). The sample volume during injection was set to 4mL, the elution flow rate was 10mL / min, and the detection wavelengths were 214nm and 280nm. The active fractions were collected, concentrated by rotary evaporation and freeze-dried, and their ACE inhibitory activity was detected. The fraction with the strongest ACE inhibition rate after separation by reversed-phase high-performance liquid chromatography (RP-HPLC) was screened for mass spectrometry identification.

[0050] (3) ACE inhibitory activity and IC 50 Determination of

[0051] The ACE inhibitory activity was determined with slight modifications according to the method of Guo et al. (GUO Junbin, LU Aichun, SUN Yunan, et al. Purification and identification of antioxidant and angiotensin converting enzyme-inhibitory peptides from Guangdong glutinous rice wine [J]. LWT-Food Science & Technology, 2022, 169: 113953.). The principle of this method is that FAPGG is hydrolyzed into amino acid FAP and dipeptide GG when it is used as an ACE substrate. The production of the two substances will reduce the absorption wavelength of the solution at 340nm, so the ACE inhibition rate is determined by the rate of reduction of the absorption wavelength of the solution. The matrix buffer HEPES is 80mmol / L (pH=8.30, Cl -1 The concentration was 300mmol / L), and 1.0mmol / L FAPGG was prepared with HEPES and stored in the dark.

[0052] In a 96-well plate, 40 μL of sample (the enzymatic hydrolysate of Yinghong No. 9 tea prepared in step 1, the molecular weight <3 kDa, molecular weight >3 kDa fraction obtained by ultrafiltration separation in step 2 (1), or the fraction obtained by liquid chromatography separation in step 2 (2)) and HEPES were first added to the sample well and the blank well, and then 50 μL of FAPGG and 10 μL of 0.1 U / mL ACE were added in sequence. After shaking for 10 seconds after the addition of the sample, the absorbance at 340 nm was immediately measured. After incubation at 37°C in the dark for 30 minutes, the absorbance of the solution at 340 nm was measured again. The ACE inhibition rate was calculated as follows:

[0053]

[0054] Where: Y is the inhibition rate, %; A is the decrease in absorbance at 340 nm after incubation of blank well solution for 30 min; B is the decrease in absorbance at 340 nm after incubation of sample well solution for 30 min.

[0055] The samples were prepared into a series of concentration gradient solutions, and their ACE inhibition rates were determined. The data were fitted using SPSS 26.0 software, and the half-inhibitory concentration (IC50) of the ACE inhibitory peptide was calculated using the regression equation. 50 ).

[0056] 3. Identification and Characterization of ACE Inhibitory Peptides

[0057] An appropriate amount of the purified high-activity fraction (F7) obtained by liquid chromatography separation in step 2 (2) was desalted on a C18 desalting column, centrifuged and dried, and then redissolved in deionized water containing 0.1% (volume fraction) formic acid. The sample was then analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. A total of 3 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated by a 60-min gradient with a column flow rate of 300 nL / min, a column temperature of 40°C, an electrospray voltage of 2 kV, and a gradient starting from 4% phase B (80% acetonitrile, 0.1% formic acid). The gradient was equilibrated for 6 min, then increased to 28% in 40 min using a nonlinear gradient, and then increased to 90% in 5 min and maintained for 15 min. The above percentages are all volume percentages. The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200–1500; resolution: 120,000; AGC target: 2e5; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15,000; AGC target: 5e4; maximum injection time: 45 ms; collision energy: 30; dynamic exclusion time: 15 s.

[0058] 2. Results Analysis

[0059] 1. Purification and enrichment of ACE inhibitory peptides from Yinghong No. 9 tea protein

[0060] The inventors previously determined the preparation process parameters of the ACE inhibitory peptide of Yinghong No. 9 tea protein through enzymatic process optimization research (Ma Feng, Ye Haoduo, Xia Zhen, et al. Preparation, amino acid composition and activity evaluation of different ultrafiltration fractions of Yinghong No. 9 tea protein ACE inhibitory peptide [J]. Modern Food Science and Technology, 2023, 39(07): 237-245.). In order to further explore the active peptide composition and material basis of the ACE inhibitory peptide of Yinghong No. 9 tea protein, this example further separated and purified it and studied the activity evaluation of the highly active fraction. In order to obtain the highly active fraction, the enzymatic hydrolyzate of Yinghong No. 9 tea was first ultrafiltered and then further purified by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0061] As shown in Table 1, the ultrafiltration fraction with a molecular weight <3 kDa exhibited good ACE inhibitory activity, with an IC 50The value (0.85 mg / mL) was significantly lower than that of the enzymatic hydrolysate (1.37 mg / mL) and the ultrafiltration fraction with a molecular weight greater than 3 kDa (2.81 mg / mL). Chen et al. (CHEN Mengting, WANG Lan, ZHENG Changliang, et al. Novel ACE inhibitory peptides derived from bighead carp (Aristichthys nobilis) hydrolysates: Screening, inhibition mechanisms and the bioconjugation effect with graphene oxide [J]. Food Bioscience, 2023, 52: 102399.) used a 5 kDa ultrafiltration membrane to separate the bighead carp hydrolysate into three fractions. Among them, the ACE inhibition rate of the MW < 5 kDa fraction was significantly stronger than that of the enzymatic hydrolysate and the > 5 kDa fraction, which is similar to our results. The reason for this may be due to the steric hindrance effect, which makes it difficult for large molecular weight peptides to enter the spatial structure of ACE and cannot react with the active site.

[0062] Table 1 ACE inhibitory activity of tea protein hydrolysate and different ultrafiltration fractions

[0063]

[0064] The ultrafiltration fractions with molecular weight <3 kDa were collected and further separated by high performance liquid chromatography. Figure 1 As shown. HPLC separation yielded 11 components ( Figure 1 A), were named F1-F11, and their ACE inhibition rates were determined ( Figure 1 B). The results showed that F7 had the best in vitro ACE inhibitory activity and had significant differences in ACE inhibition rate with the other 10 fractions. At the same time, compared with our previous study (Ma Feng, Ye Haoduo, Xia Zhen, et al. Preparation, amino acid composition and activity evaluation of different ultrafiltration fractions of Yinghong No. 9 tea protein ACE inhibitory peptide [J]. Modern Food Science and Technology, 2023, 39(07): 237-245.), it was found that at a concentration of 1 mg / mL, the ACE inhibition rate of F7 (80.6%) was significantly higher than that of the <3kDa ultrafiltration fraction (54.61%), enzymatic hydrolysate (46.43%) and >3kDa ultrafiltration fraction (40.86%), indicating that highly active substances can be separated and obtained by high-performance liquid chromatography technology. Based on this result, the peptide composition and amino acid sequence of F7 were identified.

[0065] 2. Identification and Characterization of ACE Inhibitory Peptides from Yinghong No. 9 Tea Protein

[0066] The structure of F7 was identified by LC-MS / MS, and the total ion current is shown in Figure 2. A total of 59 peptide sequences with peptide chain length ≤10, -10lgP>20.0 and no modification groups were identified (Table 2). At the same time, the toxicity (http: / / crdd.osdd.net / raghava / toxinpred / ), hydrophobicity (http: / / www.innovagen.com / ) and isoelectric point (https: / / web.expasy.org / compute_pi / ) of the peptides were analyzed using online computer tools. It was found that all peptides were non-toxic, and 31 peptides such as AAASPGGGM and GIHETTYNS had strong hydrophobicity. Studies have shown that hydrophobicity plays a positive role in the biological activity of ACE inhibitory peptides. Since the C-terminal domain of ACE is a hydrophobic environment, peptides with strong hydrophobicity are more likely to bind to the ACE enzyme, such as the reported casein ACE inhibitory peptide. Xu et al. (XU Zhenqiu, Wu Changping, SUN Dongxiao, et al. Identification of post-digestion angiotensin-Iconverting enzyme (ACE) inhibitory peptides from soybean protein Isolate: Their production conditions and in silico molecular docking with ACE - Science Direct [J]. Food Chemistry, 2021, 345: 128855.) also found that among the 845 ACE inhibitory peptides reported in BIOPEP, hydrophobic amino acids accounted for 60% of the top 10 amino acids (Pro, Leu, Val, Gly, Tyr, Ala, His, Phe, Ile and Gln). In addition, the isoelectric point of the peptide is also one of the factors affecting the activity of ACE inhibitory peptides. Mohamad Ariff Hanafi found that acidic PI components tend to have higher ACE inhibitory activity than basic components (Hanafi, Mohamad, Ariff, et al. High angiotensin-I converting enzyme (ACE) inhibitory activity of Alcalase-digested green soybean (Glycine max) hydrolysates [J]. Food Research International, 2018, 106: 589-597.). Most of the peptides identified in this study have lower isoelectric points, which may also be the reason why the F7 component has higher ACE inhibitory activity.

[0067] Table 2 Identification of ACE inhibitory peptides (-101gP>20.0) in F7 by LC-MS / MS analysis

[0068]

[0069]

[0070]

[0071] Example 2 Screening of highly active antihypertensive peptides based on molecular docking technology and prediction of their mechanism

[0072] 1. Experimental Methods

[0073] Referring to the team's previous research method (ZHANG Liuyu, MIAO Jianyin, GUO Jinbin, et al. Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Rice (Oryzasativa L.) Bran Protein [J]. Journal of Agricultural and Food Chemistry, 2023, 71 (9), 4153-4162.), the crystal structure of the receptor protein 1O8A was downloaded from the RCSB Protein Data Bank (www.rcsb.org). The receptor protein was subjected to water molecule and original ligand removal using PyMol software and saved in PDB format. The processed PDB receptor protein was then opened with Autodock Tools 1.5.6 software, hydrogenated, and the Zn of ACE was quenched in its pdbqt format file. 2+ A positive charge of 0.95 e was added. The two-dimensional structure of the ligand-active peptide small molecule was drawn using Marvin Sketch software and saved in PDB format as the lowest-energy conformation. Hydrogenation, Gasteiger calculations, and bond rotations were then performed using Autodock Tools software, and saved as a pdbqt file. Molecular docking was performed using Autodock Vina 1.1.2, with the grid box center positions set to 43.821, 38.24, and 46.712 (X, Y, Z) and the box size set to 100.125 × 100.125 × 100.125. All other parameters remained at default values. Docking results were expressed as binding energy values, and the conformation with the lowest binding energy was selected as the optimal binding site. Finally, the molecular docking results were visualized and analyzed using Discovery Studio 2017 software.

[0074] 2. Results Analysis

[0075] Molecular docking, a virtual screening technique, is not only an important tool for studying receptor-ligand complex interactions but can also be used to analyze structure-activity relationships to elucidate the biological mechanisms of peptide activity. Currently, the ACE enzyme is considered a key target for the treatment of hypertension. ACE has three major active site regions (S1, S2, and S1′). The S1 active pocket contains amino acid residues ALA354, GLU384, and TYR523; the S2 active pocket contains amino acid residues GLN281, HIS353, LYS511, HIS513, and TYR520; and the S1′ contains amino acid residue GLU162.

[0076] In order to further explore the active action mode of ACE inhibitory peptides, this study selected ACE (1O8A) as the receptor protein, and carried out molecular docking simulation studies on 59 identified peptides and receptor proteins. By analyzing the binding energy between different peptides and receptor proteins, peptides with potential ACE inhibitory activity were screened out. As shown in Table 2, the peptides AAASPGGGM and GIHETTYNS have lower binding energies when docked with the receptor 1O8A, indicating that these two peptides can form stable complexes with the ACE receptor protein, and their molecular mechanisms are related to their interaction sites and forces. Studies have shown that inhibitory peptides are connected to ACE through various forces such as hydrogen bonds, hydrophobic interactions, van der Waals forces and electrostatics, among which the binding ratio of hydrogen bonds is relatively large. The secondary mass spectra of the peptides AAASPGGGM and GIHETTYNS and the molecular docking results with the receptor 1O8A are shown in Table 2. Figure 3and Table 3. AAASPGGGM formed 13 conventional hydrogen bonds and 1 non-conventional hydrogen bond with 1O8A at ARG522, HIS353, ALA354, LYS454, THR282, ASN277, LYS511, GLN281, TYR523, ALA356, and HIS513, indicating that AAASPGGGM can effectively bind to the S1 active pocket (ALA354 and TYR523) and the S2 active pocket (GLN281, LYS511, HIS353, and HIS513) through hydrogen bonds. GIHETTYNS formed 14 hydrogen bonds with 1O8A, acting on TYR360, ARG522, ALA356, TYR523, ALA354, LYS511, GLU162, THR282, GLN281, ASN277, and GLU376, respectively, and bound to amino acid residues in the S1, S2, and S1′ active pockets, which is consistent with the results of reported studies (LIN Kai, ZHANG Lanwei, HAN Xue, et al. Quantitative Structure-Activity Relationship Modeling Coupled with Molecular Docking Analysis in Screening of Angiotensin I-Converting Enzyme Inhibitory Peptidesfrom Qula Casein Hydrolysates Obtained by Two-Enzyme Combination Hydrolysis[J]. Journal of Agricultural&Food Chemistry, 2018, 66(12): 3221-3228.). The formation of hydrogen bonds enhances the interaction between peptides and ACE, which may be one of the reasons why AAASPGGGM and GIHETTYNS exert ACE inhibitory activity. Our results are similar to those of previous studies.The highly active ACE inhibitory peptide Tyr-Ser-Lys from rice bran protein also established hydrogen bonds with ALA354, GLN281, and HIS353 (WANG Xiuming, CHEN Haixia, FU Xuegang, et al. A novel antioxidant and ACE inhibitory peptide from rice bran protein: Biochemical characterization and molecular docking study [J]. LWT-Food Science & Technology, 2017, 75: 93-99.). AAASPGGGM and GIHETTYNS formed a total of 10 hydrophobic interactions with ACE, of which AAASPGGGM interacted with PHE457, HIS383, VAL379, MET450, HIS387, and TYR523, and GIHETTYNS interacted with PRO407, HIS383, PHE527, and HIS353. Previous reports have shown that the common ACE inhibitors lisinopril and enalaprilat interact with ALA354, HIS383, GLU384, HIS387, GLU411, and TYR523 at the ACE active site. This finding is similar to our results, indicating that AAASPGGGM and GIHETTYNS can effectively interact with the ACE active site and inhibit ACE activity.

[0077] In addition, ACE is a metalloprotein involved in regulating blood pressure homeostasis, and its active site has a Zn 2+ , which can coordinate with amino acid residues HIS383, HIS387, and GLU411 to form the active center site. The catalytic regulation ability of ACE inhibitors is not only related to hydrogen bonds and hydrophobic interactions, but also to Zn 2+It is related to the interaction between ACE inhibitors. Jimsheena and Gowda proposed that peanut ACE inhibitory peptides do not directly coordinate with Zn(II) atoms, but the interaction between the peptide and His383, His387 and Glu411 can promote the inhibitory activity of the peptide (Jimsheena VK, Gowda L R. Arachin derived peptides as selective angiotensin I-converting enzyme (ACE) inhibitors: Structure-activity relationship [J]. Peptides, 2010, 31 (6): 1165-1176.). However, the MEVFVP peptide derived from soleus muscle protein can bind to the enzyme active site through metal ion interaction (Zn701) and hydrogen bond interaction (GLU411). As shown in Table 3, the two tea protein ACE inhibitory peptides can not only produce metal ion interactions with Zn701, but also with Zn 2+ The coordinating residues of 1O8A form hydrophobic interactions. AAASPGGGM forms Pi-sigma and Pi-Alkyl interactions with HIS387 and HIS383, respectively, and GIHETTYNS forms Pi-Pi-T-shaped interactions with HIS383. Existing literature indicates that captopril can form Pi-alkyl and Pi-sulfur interactions with His387 and His353 in 1O8A, respectively, thereby interacting with Zn 2+ Closely linked, thus exerting ACE inhibitory activity. This suggests that the ACE inhibitory activity of AAASPGGGM and GIHETTYNS is similar to that of captopril. Therefore, hydrogen bonding, hydrophobic interactions, and metal interactions are the main interactions affecting the stability and inhibitory effect of the tea protein ACE inhibitory peptide-ACE complex.

[0078] Table 3 Interaction sites of AAASPGGGM, GIHETTYNS and ACE (PDB: 108A)

[0079]

[0080] Example 3 Synthesis of ACE inhibitory peptides and cell experiments

[0081] 1. Experimental Methods

[0082] 1. Synthesis of ACE inhibitory peptides

[0083] The ACE inhibitory peptides (AAASPGGGM, GIHETTYNS) screened by molecular docking were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. and analyzed by high performance liquid chromatography (HPLC) with a purity greater than 98%.

[0084] 2. ACE inhibitory activity and IC 50 Determination of

[0085] Referring to Example 1, tea protein ACE inhibitory peptides (AAASPGGGM, GIHETTYNS) were prepared into a series of concentration gradient solutions, and their ACE inhibition rates were measured. The data were fitted using SPSS 26.0 software, and the half-inhibitory concentration (IC50) of the ACE inhibitory peptides was calculated using the regression equation. 50 ).

[0086] 3. Cell Experiment

[0087] (1) Cell culture and cell viability assay

[0088] EA.hy926 cells were cultured according to the method of Zhang et al. (ZHANG Liuyu, MIAO Jianyin, GUO Jinbin, et al. Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Rice (Oryza sativa L.) Bran Protein [J]. Journal of Agricultural and Food Chemistry, 2023, 71(9), 4153-4162.). EA.hy926 cells were cultured at 25 cm 2 Culture the cells in culture flasks using complete DMEM medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin in an incubator with 5% CO2 at 37°C. Change the medium every 2 days and passage the cells when the cell density reaches 80%.

[0089] The cell viability was determined using the MTT method described by Chen et al. (CHEN Bingbing, MIAO Jianyin, YE Haoduo, et al. Purification, Identification, and Mechanistic Investigation of Novel Selenium-Enriched Antioxidant Peptides from Moringa oleifera Seeds. [J]. Journal of Agricultural and Food Chemistry, 2023, 71(11): 4625-4637.). 100 μL of EA.hy926 cell suspension (1×10 5 Cells / mL) were inoculated into 96-well plates. After 24 hours of adherent culture, the culture medium was discarded. 100 μL of ACE inhibitory peptide solution and captopril (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 mg / mL) were added to the active peptide group and the positive control group. The blank control group was replaced with complete culture medium. After a further 24 hours of culture, the culture medium was discarded, and after washing once with PBS, 100 μL of MTT (0.5 mg / mL) solution was added to each well. The cells were incubated for 4 hours, the MTT solution was discarded, and 100 μL of DMSO was added. After shaking for 10 minutes to completely dissolve the blue-purple crystals, the OD value at 490 nm was measured with a microplate reader.

[0090]

[0091] Where A t is the absorbance value of the experimental group, and A0 is the absorbance value of the blank control group.

[0092] (2) Determination of cellular NO release

[0093] According to Zhang's method (ZHANG Liuyu, MIAO Jianyin, GUO Jinbin, et al. Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Rice (Oryza sativa L.) Bran Protein [J]. Journal of Agricultural and Food Chemistry, 2023, 71 (9), 4153-4162.), 100 μL EA.hy926 cells (1 × 10 5cells / mL), after 24 hours of adherent culture, the culture medium was discarded, and 100 μL of different concentrations of ACE inhibitory peptide (0.1, 0.25, 0.5 mg / mL) was added to the sample group, the positive control group was captopril at the same concentration, and the blank control group was complete culture medium. After 24 hours of treatment, the cell supernatant of each well was collected. Referring to the instructions of the NO kit, the standard was diluted to a series of concentrations (1, 2, 5, 10, 20, 40 μmol / L) using DMEM supplemented with 20% FBS. The operation was then carried out according to the instructions, and the standard concentration was used as the horizontal axis and the absorbance value (Abs 540 ) as the ordinate, a standard curve was drawn, resulting in a fitted regression equation of y = 0.0069x + 0.048, with R² = 0.9992. NO content in the cell supernatant was calculated using this formula. Using the blank control group as the standard, the effect of the tea protein ACE inhibitory peptide on NO release from EA.hy926 cells was evaluated.

[0094] (3) Determination of cellular ET-1 release

[0095] According to Zhang's method (ZHANG Liuyu, MIAO Jianyin, GUO Jinbin, et al. Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Rice (Oryza sativa L.) Bran Protein [J]. Journal of Agricultural and Food Chemistry, 2023, 71 (9), 4153-4162.), EA.hy926 cells (1 × 10 5 cells / mL) were seeded in a 96-well plate so that the number of cells per well was 1×10 4 After 24 hours of adherent culture, the culture medium was removed and the cells were treated with different concentrations of ACE inhibitory peptide (0.1, 0.25, and 0.5 mg / mL) for 24 hours. Captopril was used as a positive control, and the blank control was treated with complete culture medium alone. After treatment, the cell supernatant from each well was collected. The ET-1 content in the cell supernatant was determined using an ET-1 ELISA kit according to the manufacturer's instructions to evaluate the effect of the tea protein ACE inhibitory peptide on the amount of ET-1 released by EA.hy926 cells.

[0096] 2. Results Analysis

[0097] 1. Analysis of in vitro ACE inhibitory activity of two novel ACE inhibitory peptides

[0098] In order to verify the exact ACE inhibitory activity of the peptides AAASPGGGM and GIHETTYNS obtained by molecular docking screening, we performed in vitro solid phase synthesis and determined the ACE inhibition rate. Figure 4 It can be seen that the IC of AAASPGGGM and GIHETTYNS 50 The values ​​were 0.77 mg / mL and 0.20 mg / mL, respectively. Compared with >3 kDa, <3 kDa and enzymatic hydrolysate, the ACE inhibitory activity of the peptide monomer was stronger. This shows that molecular docking can be used as a means of screening bioactive peptides, effectively improving screening efficiency and reducing research costs. After literature search, we found that the peptides AAASPGGGM and GIHETTYNS have not been reported in research. They are the first new peptide segments discovered and proven to have biological activity. At the same time, compared with other tea protein-derived ACE inhibitory peptides, their IC 50 The values ​​are more advantageous, for example: LQPSLGFP (13.39 mg / mL) and AETGEIKGHY (23.56 mg / mL) from selenium-enriched tea protein and ordinary tea protein respectively. GIHETTYNS is better than IPPGVPYWT (IC 50 value:0.4mg / mL) showed stronger ACE inhibitory activity.

[0099] 2. Study on the antihypertensive activity of tea protein ACE inhibitory peptide based on cell model

[0100] (1) Effect of tea protein ACE inhibitory peptide on EA.hy926 cell viability

[0101] The effects of AAASPGGGM and GIHETTYNS on EA.hy926 cell viability were detected by MTT assay using captopril as a positive control. Figure 5 As shown, when the concentration was less than 0.5 mg / mL, the cell viability of the AAASPGGGM, GIHETTYNS, and captopril treatment groups was above 100%, with no significant toxic effects on EA.hy926 cells. Furthermore, within the concentration range of 0.01-0.5 mg / mL, AAASPGGGM and GIHETTYNS also promoted cell proliferation. However, at a concentration of 1.0 mg / mL, the cell viability of the AAASPGGGM, GIHETTYNS, and captopril treatment groups was less than 90%, indicating a certain impact on cell viability. Therefore, AAASPGGGM and GIHETTYNS can be used as safe ACE inhibitors within the concentration range of 0.01-0.5 mg / mL.

[0102] (2) Effect of tea protein ACE inhibitory peptide on cell NO secretion

[0103] Endothelial cells EA.hy926 are a classic cell evaluation model commonly used in antihypertensive activity research. NO is one of the vasoconstrictor factors that mainly regulates vascular tone and platelet aggregation, has a dilating effect on vascular smooth muscle, and plays a very important role in balancing human blood pressure. At the same time, NO production disorders caused by immune inflammatory responses are endothelial dysfunction and are also related to hypertension. EA.hy926 cells were treated with different concentrations of tea protein ACE inhibitory peptides, and captopril was used as a positive control. The results are shown in Figure 6 . Compared with the blank control group, the NO concentrations in the three sample treatment groups were significantly increased, and showed a certain concentration dependence. When the sample concentration reached 0.5 mg / mL, the NO content in the AAASPGGGM, GIHETTYNS and captopril treatment groups increased by 22.22%, 41.67% and 114.82%, respectively. Although captopril induced cells to produce more NO, AAASPGGGM and GIHETTYNS also had a significant effect on the production of cellular NO. Therefore, both tea protein ACE inhibitory peptides can promote the production of NO in EA.hy926 cells, thereby exerting a potential hypotensive effect, and there is a dose effect.

[0104] (3) Effect of tea protein ACE inhibitory peptide on cell secretion of ET-1

[0105] In addition to the renin-angiotensin system, the role of the endothelin system in blood pressure regulation is increasingly recognized, among which ET-1 has powerful vasoconstrictor and pressor properties. ET-1 is a vasoconstrictor peptide that is highly expressed in vascular endothelial cells and smooth muscle cells. In this study, the EA protein ACE inhibitory peptide was used to treat EA.hy926 cells. By detecting changes in ET-1 levels, the potential antihypertensive activity of the EA protein ACE inhibitory peptide can be reflected. Figure 7 It can be seen that after 24 hours of treatment, compared with the blank control group (41.16±3.83pg / mL), the tea protein ACE inhibitory peptide group had a significant inhibitory effect on the production of cellular ET-1 (p<0.05), and there was a clear dose-dependency between the low, medium, and high dose groups. At a low concentration of 0.1mg / mL, AAASPGGGM and GIHETTYNS significantly reduced the cellular ET-1 content by 16.72% and 30.88%, respectively, compared with the blank control group. Among them, GIHETTYNS was more effective in reducing the cellular ET-1 content than captopril (24.52%). The results show that tea protein ACE inhibitory peptide has a good antihypertensive effect at the cellular level. The above results prove that ACE inhibitory peptides can exert a antihypertensive effect by inhibiting the expression of ET-1 in EA.hy926 cells.

[0106] In summary, AAASPGGGM and GIHETTYNS have no significant toxic effects on endothelial cells at certain concentrations, and can promote the production of NO and inhibit the secretion of ET-1. This suggests that tea protein ACE inhibitory peptides may exert their antihypertensive effects by increasing NO release and reducing ET-1 secretion.

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An angiotensin I converting enzyme inhibitory peptide, characterized in that Its amino acid sequence is: AAASPGGGM or GIHETTYNS.

2. The method for preparing the angiotensin I converting enzyme inhibitory peptide according to claim 1, characterized in that The following steps are included: The angiotensin I converting enzyme inhibitory peptide according to claim 1 is directly synthesized by in vitro solid phase synthesis or is obtained by using Yinghong No. 9 black tea residue as raw material, extracting by alkali dissolution and acid precipitation, hydrolyzing by trypsin, and purifying.

3. The method for preparing angiotensin I converting enzyme inhibitory peptide according to claim 2, characterized in that: The purification comprises ultrafiltration separation and liquid chromatography separation steps.

4. The method for preparing angiotensin I converting enzyme inhibitory peptide according to claim 3, characterized in that: The specific operation of the ultrafiltration separation is: The Yinghong No. 9 tea protein hydrolysate was initially separated using an ultrafiltration tube and centrifuged to collect components with a molecular weight of <3 kDa.

5. The method for preparing angiotensin I converting enzyme inhibitory peptide according to claim 3, characterized in that: The specific operation of the liquid chromatography separation is as follows: using a Prep 150 preparative liquid phase system and a reversed-phase column SunFirePrep C18 OBD™ to separate and purify the components with a molecular weight <3 kDa obtained by ultrafiltration separation; The active components were collected, concentrated by rotary evaporation and freeze-dried to obtain angiotensin I converting enzyme inhibitory peptide.

6. The method for preparing angiotensin I converting enzyme inhibitory peptide according to claim 5, characterized in that: The liquid chromatography conditions are as follows: mobile phase A: ultrapure water, 0.1% TFA; mobile phase B: methanol, 0.1% TFA; elution conditions: 0-30 min, 5%-15% B; 30-40 min, 15%-25% B; 40-60 min, 25%-35% B; 60-70 min, 35%-45% B; 70-80 min, 45%-95% B; the above percentages are all volume percentages; the injection volume is 4 mL, the elution flow rate is 10 mL / min, and the detection wavelengths are 214 nm and 280 nm.

7. Use of the angiotensin I converting enzyme inhibitory peptide according to claim 1 in the preparation of antihypertensive drugs.

8. An angiotensin-converting enzyme inhibitor, characterized in that The invention comprises the angiotensin I converting enzyme inhibitory peptide according to claim 1 as an active ingredient.

9. A blood pressure lowering drug, characterized in that The invention comprises the angiotensin I converting enzyme inhibitory peptide according to claim 1 as an active ingredient.

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