ACE inhibitory peptides derived from turtle meat and screening method thereof
The IEWEF and WK peptides were screened out through Shotgun technology and virtual screening methods, which solved the problem of low efficiency in screening ACE inhibitory peptides in turtle meat, achieved efficient screening and verification of the activity of ACE inhibitory peptides, and avoided adverse reactions.
Patent Information
- Application Number
- CN202310069577.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-07
- Publication Date
- 2025-09-19
- Estimated Expiration
- 2043-02-07
AI Technical Summary
The existing technology has problems such as long experimental cycle and low separation efficiency in the process of screening ACE inhibitory peptides from turtle meat, and long-term use of ACE inhibitors will cause adverse reactions.
Shotgun technology was used in combination with protein virtual enzymatic cleavage and activity virtual screening methods to screen out IEWEF and WK peptides. ACE inhibitory peptides were synthesized using FMOC solid-phase chemical synthesis, and their activity was verified by HPLC technology.
The efficient screening of ACE inhibitory peptides with strong inhibitory activity was achieved, which can tightly bind to ACE, solving the problems of long cycle and low efficiency in the existing technology, while avoiding adverse reactions.
Smart Images
Figure CN115873072B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of bioactive peptides and more specifically relates to a method for screening ACE inhibitory peptides derived from turtle meat. Background Art
[0002] Hepatic fibrosis (HF) is a liver tissue repair and reconstruction process characterized by parenchymal cell regeneration, interstitial cell activation and proliferation, and extracellular matrix (ECM) deposition following chronic liver damage from various causes. It is a common pathological basis for various chronic liver diseases and a necessary stage in the progression to cirrhosis. According to the 2020 Global Cancer Statistics Report, liver cancer ranks third among the causes of cancer deaths worldwide. Developing drug treatments to prevent further progression of fibrosis before it develops into cirrhosis or even liver cancer has become a key focus of liver cancer prevention and treatment research.
[0003] Experiments have demonstrated that the renin-angiotensin system (RAS) plays a crucial role in the development and progression of liver fibrosis. During liver fibrosis, the ACE-Ang II-AT1R pathway, comprised of angiotensin II (Ang II), angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor (AT1R), promotes fibrosis. Angiotensin-converting enzyme 2 (ACE2) degrades Ang II to produce Ang(l-7), which then acts on the Mas receptor to exert anti-fibrotic effects. This suggests that the ACE2-Ang(l-7)-Mas pathway negatively regulates the ACE-Ang II-AT1R pathway. In addition to their blood pressure-lowering function, clinically used ACE inhibitors such as captopril, lisinopril, and enalapril can also affect the main components of the local tissue RAS system, inhibiting organ fibrosis. However, long-term use of such drugs can produce obvious adverse reactions, such as dry cough, rash, tachycardia, and even lead to decreased renal function, increased risk of lung cancer, and systemic hypotension.
[0004] The soft-shell turtle (Pelodiscus sinensis Wiegmann), a member of the family Testudinidae in the order Testudinata, is a key freshwater aquaculture species, with Zhejiang, Jiangsu, Hubei, and Guangxi as the primary production areas. Since ancient times, people have recognized the high medicinal and edible value of soft-shell turtles. The Chinese Materia Medica records that soft-shell turtle meat nourishes yin and the kidneys, clears away asthenic heat, and is primarily used to treat consumptive emaciation, bone-steaming fever, chronic malaria, chronic dysentery, metrorrhagia, and leukorrhea. Researchers have discovered components with ACE inhibitory activity from soft-shell turtle meat hydrolysates. Chiu LH et al. used gastrointestinal digestive enzymes to hydrolyze soft-shell turtle meat proteins, resulting in enhanced ACE inhibitory activity in the hydrolyzed products. Xu Huaide et al. used papain to hydrolyze and purify soft-shell turtle meat proteins, identifying several peptide fractions with excellent ACE inhibitory activity. Liu LL et al. used neutral protease to hydrolyze and separate soft-shell turtle meat proteins, finding that ultrafiltration fractions with molecular weights less than 5 kDa exhibited significant ACE inhibitory activity.
[0005] Screening and isolating ACE inhibitory peptides from natural proteins generally involves enzymatic hydrolysis of the protein to release the active peptide fragments. These peptides are then isolated and purified using techniques such as ultrafiltration, gel chromatography, ion exchange chromatography, and high-performance liquid chromatography (HPLC), combined with activity screening experiments. While these methods are widely used in the preparation of ACE inhibitory peptides, they suffer from drawbacks such as long experimental cycles and low separation efficiency.
[0006] Protein virtual enzymatic cleavage and activity-based virtual screening technologies are continuously developing and maturing, and are increasingly being used in the screening of bioactive peptides from natural proteins. Virtual enzymatic cleavage uses computer-assisted techniques to simulate the enzymatic digestion of various proteins by proteases, effectively identifying protein cleavage sites and rapidly obtaining small peptide fragments. Tools such as BIOPEP and Peptide Cutter can predict potential protease cleavage sites. Activity-based virtual screening is a well-established research strategy that utilizes computers to virtually evaluate compound activity to efficiently identify bioactive small molecules. This includes both small molecule-based and protein-target-based screening methods.
[0007] Shotgun technology is an analytical and identification method for proteins in complex mixtures. It combines HPLC technology and mass spectrometry technology to achieve the effect of analyzing all protein information as completely as possible. Virtual enzyme cleavage research based on the protein information analyzed by Shotgun technology is conducive to more accurate positioning of active peptides.
[0008] In summary, the use of Shotgun technology combined with protein virtual enzymatic cleavage and activity virtual screening will enable a faster and more efficient search for new ACE inhibitory peptides derived from turtle meat, which is of great significance for the treatment of hypertension and liver fibrosis. Summary of the Invention
[0009] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.
[0010] One object of the present invention is to provide an ACE inhibitory peptide derived from turtle meat, which binds tightly to ACE and has a strong inhibitory effect on ACE.
[0011] In order to achieve these purposes and other advantages of the present invention, a turtle meat-derived ACE inhibitory peptide is provided, including an IEWEF peptide segment and a WK peptide segment, wherein the amino acid sequence of the IEWEF peptide segment is Ile-Glu-Trp-Glu-Phe, and the amino acid sequence of the WK peptide segment is Trp-Lys.
[0012] A method for screening ACE inhibitory peptides derived from turtle meat, comprising the following steps:
[0013] 1) Shotgun analysis of water-soluble proteins in turtle meat was performed, and then the proteins identified by the shotgun analysis were subjected to virtual enzymatic digestion to screen for the optimal enzyme for virtual enzymatic digestion;
[0014] 2) Use the screened optimal enzymes for virtual enzymatic hydrolysis to perform virtual enzymatic digestion on the identified proteins, predict the theoretical release amount of ACE inhibitory peptides obtained by each enzyme digestion, and use the theoretical release amount of ACE inhibitory peptides as the evaluation index to analyze the optimal enzyme for virtual enzymatic hydrolysis of each protein, and count the number of proteins corresponding to each optimal enzyme. Use the active virtual screening tool combined with ADMET property evaluation and database search to screen new ACE inhibitory peptides.
[0015] Preferably, the preparation of turtle meat water-soluble protein includes the following steps: slaughtering and bleeding a turtle, removing its internal organs and attached tendons and skin, crushing the turtle meat tissue to make minced meat, and freeze-drying it for later use; weighing the freeze-dried turtle meat minced meat according to a material ratio of 1:10-15 (g / mL), adding pure water, stirring and extracting at room temperature for 2-4 hours, and then centrifuging at 2000×g for 20-30 minutes, repeating the extraction 2-3 times under the same conditions, collecting the supernatant after centrifugation, and obtaining turtle meat water-soluble protein.
[0016] Preferably, the turtle meat water-soluble protein is subjected to pre-treatment operations of trypsin digestion, centrifugation, desalting, and freeze-drying, and then subjected to LC-MS / MS analysis to identify the protein.
[0017] Preferably, the LC-MS / MS analysis is performed by: separating using a sodium upgraded flow rate HPLC liquid phase system Easy-nLC system;
[0018] Buffers used: Solution A was 0.1% formic acid in water, and Solution B was 0.1% formic acid in acetonitrile. The chromatographic column was equilibrated with 95% Solution A, and the analytical column was Thermo Scientific EASY column. The flow rate was 300 nL / min.
[0019] The liquid phase gradient was as follows: 0% to 55% solution B from 0 to 110 min; 55% to 100% solution B from 110 to 115 min; and 100% solution B from 115 to 120 min. The peptides were separated by chromatography and analyzed using a Q-Exactive mass spectrometer. Analysis time: 120 min. Detection: positive ion. Parent ion scan range: 300 to 1800 m / z.
[0020] The protein database is uniprot_Pelodiscus_sinensis_20923_20210307, and the parameters are set as follows: the enzyme is trypsin; the missing cleavage site is set to 2; the fixed modification is carbamidomethyl (C); the dynamic modification is set to oxidation (M) and acetyl (protein N-term); the proteins identified by database retrieval pass the set filtering parameter FDR ≤ 0.01.
[0021] Preferably, the method for virtual enzymatic cleavage of proteins is as follows: according to the results of LC-MS / MS analysis, the sequence information of the corresponding turtle meat water-soluble protein is obtained from the UniProt database, and then virtual enzymatic cleavage is performed through the enzymeaction program in the BIOPEP-UWM online tool, and trypsin, pepsin, papain, bromelain and alkaline protease are selected respectively to predict the theoretical release amount of ACE inhibitory peptides obtained by enzymatic cleavage of each enzyme, and the theoretical release amount of ACE inhibitory peptides is used as an evaluation index to analyze the optimal enzyme for virtual enzymatic hydrolysis of each protein, and the number of proteins corresponding to each optimal enzyme is counted, and the enzyme corresponding to the largest number of proteins is used as the optimal enzyme for enzymatic hydrolysis of turtle meat water-soluble protein.
[0022] Preferably, the specific method for screening new ACE inhibitory peptides using an activity virtual screening tool in combination with ADMET property evaluation and database search comprises the following steps:
[0023] a) Use the Peptide Ranker database to evaluate the activity of the peptides obtained by virtual enzyme digestion to determine their possibility of biological activity, and select peptides with a score ≥ 0.5 for the next step of analysis;
[0024] b) The Biopep-UWM database was used to search for peptides with a score ≥ 0.5 to screen for unreported ACE inhibitory peptides;
[0025] c) Use the Proteomics tools database to predict the water solubility of peptides, and select active peptides with good water solubility predictions for further research;
[0026] d) Use the Toxin Pred website to predict the toxicity of peptides and select non-toxic peptides for further analysis;
[0027] e) Use the ADMET evaluation module in the ADMETlab database to predict the HIA and BBB properties of peptides;
[0028] f) After screening the peptides obtained by virtual enzyme digestion with the two proteases, IEWEF peptide and WK peptide were obtained;
[0029] g) The IEWEF and WK peptides were used to predict potential ACE inhibitory activity using the Profiles of potential biological activity program in BIOPEP-UWM, and the active peptides were analyzed.
[0030] Preferably, the method further comprises molecular docking of the ACE inhibitory peptide with ACE to predict the binding mode and affinity of the ACE inhibitory peptide with ACE.
[0031] Preferably, the method further comprises synthesizing ACE inhibitory peptides by FMOC solid phase chemical synthesis method, and then evaluating ACE inhibitory activity in vitro by HPLC technology.
[0032] The present invention has at least the following beneficial effects:
[0033] First, the ACE inhibitory peptide of the present invention binds tightly to ACE, C-ACE, and N-ACE, and has strong inhibitory activity against ACE, C-ACE, and N-ACE.
[0034] Second, the present invention uses Shotgun technology for analysis, which can achieve the effect of analyzing all protein information as completely as possible, and based on the obtained protein information, virtual enzyme cleavage research is carried out, which is conducive to more accurate positioning of active peptide segments.
[0035] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1This is the SDS-PAGE electrophoresis result of turtle meat water-soluble protein;
[0037] Figure 2 3D binding map and interaction force diagram of IEWEF and ACE;
[0038] Figure 3 3D binding diagram and interaction force diagram of WK and ACE. DETAILED DESCRIPTION
[0039] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.
[0040] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.
[0041] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.
[0042] The process of the present invention is as follows: using Chinese soft-shelled turtle meat water-soluble protein as protein raw material, adopting Shotgun technology to analyze the protein contained therein, searching protein sequence information in the UniProt database, selecting several commonly used commercial proteases, applying the virtual enzyme cleavage tool of the BIOPEP platform in combination with the peptide activity prediction tool, screening out two proteases that can produce more ACE-inhibiting active peptides, performing ADMET property evaluation on the peptides obtained by the two proteases, comparing the final results, then adopting molecular docking technology to evaluate the affinity of the active peptides with ACE, and finally solid-phase synthesizing the peptides for activity verification.
[0043] The present invention can efficiently screen out new ACE inhibitory peptides from protein raw materials, providing a new research path for the rapid discovery of new active peptides in natural proteins.
[0044] A method for screening ACE inhibitory peptides derived from turtle meat, comprising the following steps:
[0045] 1) Shotgun analysis of water-soluble proteins in turtle meat was performed, and then the proteins identified by the shotgun analysis were subjected to virtual enzymatic digestion to screen for the optimal enzyme for virtual enzymatic digestion;
[0046] 2) Use the screened optimal enzymes for virtual enzymatic hydrolysis to perform virtual enzymatic digestion on the identified proteins, predict the theoretical release amount of ACE inhibitory peptides obtained by each enzyme digestion, and use the theoretical release amount of ACE inhibitory peptides as the evaluation index to analyze the optimal enzyme for virtual enzymatic hydrolysis of each protein, and count the number of proteins corresponding to each optimal enzyme. Use the active virtual screening tool combined with ADMET property evaluation and database search to screen new ACE inhibitory peptides.
[0047] In another technical solution, the preparation of turtle meat water-soluble protein includes the following steps: slaughtering and bleeding the turtle, removing its internal organs and attached tendons and skin, crushing the turtle meat tissue to make minced meat, and freeze-drying it for later use; weighing the freeze-dried turtle meat minced meat according to a material ratio of 1:10-15 (g / mL), adding pure water, stirring and extracting at room temperature for 2-4 hours, and then centrifuging at 2000×g for 20-30 minutes, repeating the extraction 2-3 times under the same conditions, collecting the supernatant after centrifugation, and obtaining turtle meat water-soluble protein.
[0048] In another technical solution, the turtle meat water-soluble protein is subjected to pre-treatment operations of trypsin digestion, centrifugation, desalting, and freeze-drying, and then LC-MS / MS analysis is performed to identify the protein.
[0049] In another technical solution, the method for performing LC-MS / MS analysis is: using a sodium upgraded flow rate HPLC liquid phase system Easy-nLC system for separation;
[0050] Buffers used: Solution A was 0.1% formic acid in water, and Solution B was 0.1% formic acid in acetonitrile. The chromatographic column was equilibrated with 95% Solution A, and the analytical column was Thermo Scientific EASY column. The flow rate was 300 nL / min.
[0051] The liquid phase gradient was as follows: 0% to 55% solution B from 0 to 110 min; 55% to 100% solution B from 110 to 115 min; and 100% solution B from 115 to 120 min. The peptides were separated by chromatography and analyzed using a Q-Exactive mass spectrometer. Analysis time: 120 min. Detection: positive ion. Parent ion scan range: 300 to 1800 m / z.
[0052] The protein database is uniprot_Pelodiscus_sinensis_20923_20210307, and the parameters are set as follows: the enzyme is trypsin; the missing cleavage site is set to 2; the fixed modification is carbamidomethyl (C); the dynamic modification is set to oxidation (M) and acetyl (protein N-term); the proteins identified by database retrieval pass the set filtering parameter FDR ≤ 0.01.
[0053] In another technical solution, the method for virtual enzymatic cleavage of proteins is as follows: the sequence information of the corresponding turtle meat water-soluble protein is obtained from the UniProt database according to the results of LC-MS / MS analysis, and then virtual enzymatic cleavage is performed through the enzyme action program in the BIOPEP-UWM online tool, and trypsin, pepsin, papain, bromelain and alkaline protease are selected respectively to predict the theoretical release amount of ACE inhibitory peptides obtained by enzymatic cleavage of each enzyme, and the theoretical release amount of ACE inhibitory peptides is used as an evaluation index to analyze the optimal enzyme for virtual enzymatic hydrolysis of each protein, and the number of proteins corresponding to each optimal enzyme is counted, and the enzyme corresponding to the largest number of proteins is selected as the optimal enzyme for enzymatic hydrolysis of turtle meat water-soluble protein.
[0054] In another technical solution, a specific method for screening new ACE inhibitory peptides using an activity virtual screening tool combined with ADMET property evaluation and database search includes the following steps:
[0055] a) Use the Peptide Ranker database to evaluate the activity of the peptides obtained by virtual enzyme digestion to determine their possibility of biological activity, and select peptides with a score ≥ 0.5 for the next step of analysis;
[0056] b) The Biopep-UWM database was used to search for peptides with a score ≥ 0.5 to screen for unreported ACE inhibitory peptides;
[0057] c) Use the Proteomics tools database to predict the water solubility of peptides, and select active peptides with good water solubility predictions for further research;
[0058] d) Use the Toxin Pred website to predict the toxicity of peptides and select non-toxic peptides for further analysis;
[0059] e) Use the ADMET evaluation module in the ADMETlab database to predict the HIA and BBB properties of peptides;
[0060] f) After screening the peptides obtained by virtual enzyme digestion with the two proteases, IEWEF peptide and WK peptide were obtained;
[0061] g) The IEWEF and WK peptides were used to predict potential ACE inhibitory activity using the Profiles of potential biological activity program in BIOPEP-UWM, and the active peptides were analyzed.
[0062] In another technical solution, the ACE inhibitory peptide is also included in molecular docking with ACE to predict the binding mode and affinity of the ACE inhibitory peptide with ACE.
[0063] In another technical solution, the ACE inhibitory peptide is synthesized by FMOC solid-phase chemical synthesis method, and then the ACE inhibitory activity in vitro is evaluated by HPLC technology.
[0064] <Example>
[0065] 1. Extraction of water-soluble protein from turtle meat
[0066] After slaughtering and bleeding the turtles, their internal organs and accompanying tendons and skin were removed. The turtle meat was minced and freeze-dried for later use. The freeze-dried turtle meat was weighed and added to pure water at a material ratio of 1:10 (g / mL). Extraction was performed at room temperature with stirring for 4 hours, followed by centrifugation at 2000 × g for 20 minutes. The extraction was repeated twice under the same conditions. The supernatant was collected, freeze-dried, and stored at –20°C until further use.
[0067] 2. Shotgun analysis of water-soluble protein in turtle meat
[0068] The results of protein quantitative analysis and SDS-PAGE identification of water-soluble proteins in turtle meat are shown in Figure 1 The sample bands were clearly separated and the quality met the experimental requirements. The water-soluble protein sample of turtle meat was subjected to trypsin digestion, centrifugation, desalting, freeze-drying and other pre-treatments, and then LC-MS / MS analysis was performed to identify the protein. The specific method was as follows:
[0069] The peptides were separated using the sodium-upgraded flow rate HPLC liquid phase system Easy-nLC system;
[0070] Buffer: Solution A is 0.1% formic acid in water, and Solution B is 0.1% formic acid in acetonitrile (84% acetonitrile concentration). The chromatographic column is balanced with 95% solution A. The chromatographic column is a Thermo scientific EASY column (2 cm × 100 μm, 5 μm-C 18 ), and then analyzed by Thermo scientific EASY column (75μm×100mm, 3μm-C 18 ) separation with a flow rate of 300 nL / min.
[0071] The HPLC gradient was as follows: 0% to 55% Solution B from 0 to 110 min; 55% to 100% Solution B from 110 to 115 min; and 100% Solution B from 115 to 120 min. Peptides were separated by chromatography and analyzed using a Q-Exactive mass spectrometer (ThermoScientific). Analysis time: 120 min; detection: positive ion; precursor ion scan range: 300 to 1800 m / z.
[0072] The protein database used was uniprot_Pelodiscus_sinensis_20923_20210307. The search parameters were as follows: trypsin as the enzyme; 2 missed cleavage sites; carbamidomethyl (C) as the fixed modification; and oxidation (M) and acetyl (Protein N-term) as the dynamic modifications. Proteins identified by database search had to pass the set filtering parameter, FDR ≤ 0.01.
[0073] The results of sample protein identification are shown in Table 1 below. 52 proteins were identified from the turtle meat water-soluble protein, of which 3 were contaminating proteins (removed).
[0074] Table 1 Statistics of sample protein identification results
[0075]
[0076] 3. Virtual Enzymatic Digestion of Turtle Meat Water-Soluble Protein
[0077] According to the protein identification results of the samples, the sequence information of 49 water-soluble proteins of turtle meat was obtained from the UniProt database (https: / / www.uniprot.org / ), and then the sequence information of 49 water-soluble proteins of turtle meat was obtained by BIOPEP-UWM online tool (http: / / www.uwm.edu.pl /
[0078] The “enzyme action” program in biochemia / index.php / pl / biopep was used for virtual enzyme digestion. Trypsin (EC 3.4.21.4), pepsin (pH 1.3) (EC 3.4.23.1), papain (EC3.4.22.2), bromelain (EC 3.4.22.32) and alkaline protease (EC 3.4.21.62) were selected respectively to predict the theoretical release amount of ACE inhibitory peptides obtained by each enzyme digestion (A E ) and A EAs the evaluation metric, we analyzed the optimal enzyme for each protein's virtual enzymatic digestion and counted the number of proteins corresponding to each optimal enzyme, as shown in Table 2. Bromelain was the optimal enzyme for 30 proteins, and papain was the optimal enzyme for 16 proteins. Based on these results, bromelain and papain were selected for virtual enzymatic digestion of water-soluble turtle meat proteins, respectively.
[0079] Table 2 Statistical results of optimal enzymes for virtual enzymatic hydrolysis
[0080]
[0081] 4. Screening of ACE Inhibitory Peptides
[0082] Bromelain (EC 3.4.22.32) and papain (EC 3.4.22.2) were selected for virtual enzymatic digestion of turtle meat water-soluble protein. New ACE inhibitory peptides were screened using activity virtual screening tools combined with ADMET property evaluation and database search. The process is as follows:
[0083] a) The activity of the peptides obtained by virtual enzyme digestion was evaluated using the Peptide Ranker database (http: / / distilldeep.ucd.ie / PeptideRanker / ) to determine their potential for biological activity. Peptides with a score ≥ 0.5 were selected for further analysis.
[0084] b) The Biopep-UWM database was used to search for peptides with a score ≥ 0.5 to screen for unreported ACE inhibitory peptides.
[0085] c) The water solubility of peptides was predicted using the Proteomics tools (Proteomics tools (innovagen.com)) database, and active peptides with good water solubility predictions were selected for further study.
[0086] d) Toxin Pred (ToxinPred (osdd.net)) website was used to predict the toxicity of peptides, and “Non-toxic” peptides were selected for further analysis.
[0087] e) Use the “ADMET evaluation” (ADMET
[0088] Prediction-Webserver-ADMElab:ADMET Prediction|ADMET Predictor|QSAR|ADMET Database(scbdd.com)) module predicts the HIA (Human Intestinal Absorption) and BBB (Blood–Brain Barrier) properties of peptides.
[0089] f) The peptides obtained by virtual enzyme digestion with the two proteases were screened. The results are shown in Table 3. IEWEF peptide and WK peptide were obtained. The amino acid sequence of IEWEF peptide is Ile-Glu-Trp-Glu-Phe, IC 50 The value was 42.34±7.23μM; the amino acid sequence of the WK peptide was Trp-Lys, IC 50 The value was 1687.78±31.29μM.
[0090] Table 3 ACE inhibitory peptide screening results
[0091]
[0092] g) The active peptide IEWEF was further analyzed for potential ACE inhibitory activity using the "Profiles of potential biological activity" program in BIOPEP-UWM. The peptides that exerted the activity were analyzed. The IE, EW, IEW, and EF peptides within the IEWEF peptide have all been shown to exhibit ACE inhibitory activity, whereas the ACE inhibitory activity of IEWEF has not been reported.
[0093] 5. Molecular docking of active peptide IEWEF and ACE
[0094] The structures of peptides IEWEF and WK were drawn using ChemBioDraw Ultra and then converted into 3D structures using ChemBio3D Ultra. ACE (PDB ID: 1086) was downloaded from the RCSB Protein Data Bank database and its crystal structure was modified by deleting water molecules and heteroatoms and adding hydrogen atoms.
[0095] Autodock was used to investigate the interaction between human ACE and peptides. ACE and peptides were converted to PDBQT format using AutodockTools 1.5.6. To increase accuracy, the exhaustiveness parameter was set to 20, and all other parameters were left at their default values. Finally, the highest-scoring conformation was used for analysis using PyMOL.
[0096] The binding energies of IEWEF and WK docking with ACE are -8.7 and -7.8 kcal·mol, respectively. -1 , indicating that IEWEF and WK bind tightly to ACE, with IEWEF binding more tightly to ACE. Based on the molecular docking results, the interaction between the peptide and ACE was further analyzed. The ACE binding site consists of three active pockets (S1, S2, and S1') and a zinc-binding domain. The S1 pocket contains three key residues, namely Ala354, Glu384, and Tyr523; the S2 pocket includes five key residues, namely Gln281, His353, Lys511, His513, and Tyr520; the S1' pocket has one key residue, Glu162; and the zinc-binding domain contains three cooperating residues, namely His383, His387, and Glu411.
[0097] The interaction between peptides IEWEF and WK and ACE is as follows Figure 2 、 Figure 3 ,Depend on Figure 2 、 Figure 3 As can be seen, the peptide IEWEF forms five hydrogen bonds with ACE (Asn66, Arg522, Glu411, Tyr523), two of which are with Tyr523, a key residue in the S1 pocket of ACE. The peptide WK forms three hydrogen bonds with ACE (Glu376, Asp415, Asp453). The interaction force analysis indicates that the peptide IEWEF forms more hydrogen bonds with ACE than WK. Hydrogen bonding is considered one of the most important non-covalent interactions in the binding process between ACE inhibitors and ACE. Analysis of the interaction force also indicates that IEWEF has a strong affinity for ACE, which is consistent with the binding energy results.
[0098] VI. Experimental Verification of ACE Inhibitory Activity of IEWEF
[0099] IEWEF and WK peptides were synthesized by FMOC solid-phase chemical synthesis, and their in vitro ACE inhibitory activity was evaluated by HPLC. Peptide fragments (IEWEF peptide fragment and WK peptide fragment) solutions of different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mg / mL) were dissolved in 0.1 mol / L boric acid buffer (containing 0.3 mol / L NaCl). 60 μL of peptide fragment solutions of different concentrations or blank BBS buffer was added to 40 μL of 0.10 U / mL ACE solution (dissolved in BBS buffer), vortexed, and reacted at 37°C for 10 min. Then, 40 μL of 5.40 mmol / L HHL solution (dissolved in BBS buffer) was added to react, vortexed, and reacted at 37°C for 15 min. Finally, 60 μL of 1 mol / L HCl was added to terminate the reaction. The mixture was filtered through a 0.45 μm microporous membrane, and 20 μL of the reaction solution was sampled and analyzed for the amount of HA generated.
[0100] Chromatographic conditions: Thermo C18 column (150 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid solution (15:85); flow rate: 1.0 mL / min; detection wavelength: 228 nm.
[0101]
[0102] Wherein: A represents the peak area of HA in the solution after sample reaction, mAU; B represents the peak area of HA in the solution after blank BBS reaction, mAU.
[0103] Log(peptide concentration) and ACE inhibition rate were plotted, and the IC of the peptide segment to inhibit ACE was calculated. 50 The values were 42.34 ± 7.23 μM and 1687.78 ± 31.29 μM, respectively, and the IC 50 The lower the value, the better the inhibitory effect. The results of ACE inhibitory activity assay showed that both IEWEF and WK peptides obtained through virtual screening were confirmed to have ACE inhibitory activity, and the inhibitory activity of peptide IEWEF against ACE was significantly higher than that of WK.
[0104] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.
Claims
1. A composition of ACE inhibitory peptides derived from turtle meat, characterized in that: The composition comprises an IEWEF peptide segment and a WK peptide segment, wherein the amino acid sequence of the IEWEF peptide segment is Ile-Glu-Trp-Glu-Phe, and the amino acid sequence of the WK peptide segment is Trp-Lys.