Sophora moorcroftiana protein source antihypertensive peptide and application thereof
By extracting and synthesizing a specific sequence of protein-derived antihypertensive peptides from the seeds of Sophora alopecuroides, the problems of easy degradation and low bioavailability of ACE inhibitory peptides in existing technologies have been solved, achieving efficient ACE inhibition and long-term blood pressure regulation effects.
Patent Information
- Application Number
- CN202511750366.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-01-06
AI Technical Summary
Existing natural ACE inhibitory peptides are easily degraded by gastrointestinal proteases after oral administration, resulting in low bioavailability and a short half-life in vivo, making it difficult to achieve long-term blood pressure control and limiting their sustained efficacy in the long-term treatment of hypertension.
Protein-derived antihypertensive peptides with specific sequences, such as MGL PWYR, WHRPLR, INPLGFR, LPNSLFLFR, and LFLWPIYR, were extracted and synthesized from the seeds of Sophora japonica. Through enzymatic digestion, activity screening, and molecular docking, polypeptides with high ACE inhibitory activity were prepared for use in the preparation of drugs and functional foods.
It achieves highly efficient ACE inhibition, with peptides MGL PWYR and WHRPLR achieving ACE inhibition rates of 88.99% and 90.07%, respectively. This solves the problem of peptide degradation in existing technologies and provides a long-lasting blood pressure regulation effect.
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Figure CN121270656A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biopeptide drugs, and specifically relates to a blood pressure-lowering peptide derived from Sophora alopecuroides and its application. Background Technology
[0002] Hypertension is a prevalent cardiovascular disease worldwide, seriously threatening human health. Developing safe and effective antihypertensive active ingredients is a research hotspot in the pharmaceutical and food fields. Hypertension treatment mainly employs combination therapy, with antihypertensive drugs primarily falling into five categories: diuretics, beta-blockers, calcium channel blockers (CCBs), angiotensin-converting enzyme inhibitors (ACEDs), and angiotensin I receptor antagonists (ARBs). Current research indicates that food-derived antihypertensive peptides derived from animal, plant, and microbial sources possess excellent ACE inhibitory activity and have no toxic side effects on humans, thus showing broad application prospects in the prevention and adjuvant management of hypertension.
[0003] Angiotensin-converting enzyme (ACE) inhibitory peptides are a class of bioactive peptides that regulate blood pressure and improve hemodynamic disturbances in hypertensive patients by inhibiting ACE activity, blocking the conversion of angiotensin I to the pressor substance angiotensin II, and reducing bradykinin degradation. They represent one of the core directions in current research on natural antihypertensive active ingredients. Currently, research mainly focuses on the screening and discovery of naturally sourced ACE inhibitory peptides and the optimization of their artificial sequences. Dairy products and legumes are the main sources for preparing food-derived antihypertensive peptides, which are primarily isolated from the hydrolysis products of dairy products and legumes. Their activity depends on the C-terminus, which often contains Pro, Tyr, or Arg residues to enhance hydrophobic interactions with the ACE active site, and the N-terminus, which often contains Ala or Gly residues to reduce steric hindrance. The optimal amino acid count for these peptides is typically concentrated in the 3-10 range.
[0004] Computer-aided virtual enzymatic digestion (based on protease-specific cleavage site simulation), molecular docking (targeting the active pocket of the ACE crystal structure), and structure-activity relationship analysis (verifying the activity contribution by mutating key amino acid residues) have become important technical means to directionally enhance the ACE inhibitory activity of peptides and improve target binding specificity. However, existing natural ACE inhibitory peptides generally suffer from problems such as easy degradation and inactivation by gastrointestinal proteases (e.g., pepsin, trypsin) after oral administration, low oral bioavailability (<5%), and short in vivo half-life (mostly 0.5-2 hours), making it difficult to achieve long-term blood pressure control. These problems result in insufficient sustained efficacy in the long-term treatment of hypertension, limiting their transformation from food functional ingredients to clinical antihypertensive drugs.
[0005] Currently, scholars both domestically and internationally have prepared dietary ACE-inhibiting peptides from various animal and plant proteins, which have safe and effective antihypertensive effects. However, research on antihypertensive peptides derived from Sophora alopecuroides protein remains lacking and urgently needs further research and development for application. Summary of the Invention
[0006] Purpose of the invention: The first purpose of this invention is to provide a Sophora alopecuroides protein-derived antihypertensive peptide that inhibits ACE; the second purpose of this invention is to provide the application of the Sophora alopecuroides protein-derived antihypertensive peptide.
[0007] Technical solution: The antihypertensive peptide derived from Sophora alopecuroides in this invention includes at least one of the active peptides with the sequence structures MGLPBWYR (SEQ ID NO.1), WHRPLR (SEQ ID NO.2), INPLGFR (SEQ ID NO.3), LPNSLFLFR (SEQ ID NO.4), and LFLWPIYR (SEQ ID NO.5).
[0008] Preferably, the antihypertensive peptide is MGL PWYR and / or WHRPLR.
[0009] MGLPWYR has the amino acid sequence Met-Gly-Leu-Pro-Trp-Tyr-Arg and a molecular weight of 921 Da.
[0010] WHRPLR has the amino acid sequence Trp-His-Arg-Pro-Leu-Arg and a molecular weight of 863 Da.
[0011] INPLGFR has the amino acid sequence Ile-Asn-Pro-Leu-Gly-Phe-Arg and a molecular weight of 815 Da.
[0012] LPNSLFLFR has the amino acid sequence Leu-Pro-Asn-Ser-Leu-Phe-Leu-Phe-Arg and a molecular weight of 1105 Da.
[0013] LFLWPIYR has the amino acid sequence Leu-Phe-Leu-Trp-Pro-Ile-Tyr-Arg and a molecular weight of 1106 Da.
[0014] The preparation method of the antihypertensive peptide derived from Sophora alopecuroides protein of the present invention includes the following steps:
[0015] (1) Pretreatment of seeds of Sophora alopecuroides and extraction and sequence identification of proteins;
[0016] (2) The sequence obtained by identifying Sophora alopecuroides protein was digested with enzymes;
[0017] (3) Screening the activity of the peptides obtained by enzyme digestion;
[0018] (4) The highly active peptides obtained by screening were subjected to in vitro activity verification.
[0019] Preferably, in step (1), the pretreatment specifically involves: taking dried Sophora japonica seeds, and obtaining Sophora japonica seed powder by freeze grinding; and degreasing the obtained powder to remove flavonoids, polyphenols and alkaloids.
[0020] Preferably, in step (1), the extraction and sequence identification specifically involves: adding deionized water to the pretreated powder in step (1) and adjusting the pH, and extracting the protein from the seeds of Sophora japonica using an ultrasonic-assisted alkaline method; and using protein mass spectrometry to identify the sequence of the crude protein to obtain the protein amino acid sequence.
[0021] Preferably, step (2) specifically involves: using the protein sequence identified in step (1) as a basis, employing the Expasy-Peptide Cutter to simulate the enzymatic digestion process of the protease to obtain a polypeptide sequence library.
[0022] Preferably, step (3) specifically involves: scoring the peptide sequence library for activity using the PeptideRanker bioactivity prediction website, and screening out potential active peptides with a score ≥0.8; using angiotensin-converting enzyme as the antihypertensive target, performing molecular docking using MOE software.
[0023] Preferably, step (4) specifically involves: using solid-phase synthesis, the active peptides selected in step (4) are synthesized step by step to obtain a single active peptide; using the ACE inhibition activity assay to verify in vitro activity, and screening out highly active Sophora japonica antihypertensive peptides with an ACE inhibition rate ≥80%.
[0024] The application of the antihypertensive peptide derived from Sophora alopecuroides protein in the preparation of ACE inhibitors.
[0025] The application of the antihypertensive peptide derived from Sophora alopecuroides protein in the preparation of drugs for treating hypertension, as described in this invention.
[0026] The present invention provides a pharmaceutical composition with antihypertensive effect, comprising the aforementioned Sophora alopecuroides protein-derived antihypertensive peptide or its derivatives, or pharmaceutically acceptable salts, solvates, or hydrates of both as active ingredients.
[0027] Preferably, the pharmaceutical composition further contains pharmaceutically acceptable excipients.
[0028] Preferably, the pharmaceutically acceptable excipients include one or more of the following: excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.
[0029] Preferably, the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository, or patch.
[0030] The application of the antihypertensive peptide derived from Sophora alopecuroides protein in the preparation of functional foods for blood pressure regulation, as described in this invention.
[0031] Preferably, the functional food also contains food-acceptable excipients.
[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) the antihypertensive peptide of Sophora alopecuroides protein source of the present invention is a polypeptide with ACE inhibitory activity; (2) the ACE inhibition rate of polypeptide MGL PWYR is 88.99% (0.9764 mg / mL); (3) the ACE inhibition rate of polypeptide WHRPLR is 90.07% (0.9970 mg / mL). Attached Figure Description
[0033] Figure 1 SDS-PAGE analysis bands of crude protein in Sophora japonica seeds;
[0034] Figure 2 The image shows the molecular docking results of MGLPWYR-ACE.
[0035] Figure 3 The image shows the molecular docking results of WHRPLR-ACE.
[0036] Figure 4 The image shows the molecular docking results of INPLGFR-ACE.
[0037] Figure 5 The image shows the molecular docking results of LPNSLFLFR-ACE.
[0038] Figure 6 The image shows the molecular docking results of LFLWPIYR-ACE.
[0039] Figure 7 The graph shows the ACE inhibition rate of the five peptides.
[0040] Figure 8 The figure shows the results of the ACE inhibitory activity test of the synthetic peptide MGLPWYR;
[0041] Figure 9 The figure shows the ACE inhibitory activity test results of the synthetic peptide WHRPLR. Detailed Implementation
[0042] The technical solution of the present invention will be further described below with reference to the embodiments.
[0043] Example 1
[0044] Pretreatment and impurity removal of *Sophora alopecuroides* seeds include the following steps:
[0045] (1) Cryogenic grinding: Weigh 100 g of Sophora japonica seeds, soak them in 200 mL of liquid nitrogen for 8 min, quickly transfer them to a high-speed cryogenic grinder, grind them 3 times, pass them through a 60-mesh sieve, and collect the powder that passes through the sieve for later use.
[0046] (2) Degreasing treatment: Transfer the above powder to a 2000 mL Erlenmeyer flask, add n-hexane at a material-to-liquid ratio of 1:10 (mass-to-volume ratio, g / mL), place in a 28℃ constant temperature water bath shaker, and extract by shaking at 150 rpm for 2 h. Then centrifuge at 5000 rpm for 5 min, discard the supernatant, and retain the precipitate; repeat this degreasing operation twice.
[0047] (3) Impurity removal: Take the precipitate obtained in step (2), add anhydrous ethanol at a material-to-liquid ratio of 1:10 (mass-to-volume ratio, g / mL), and shake in a shaker at 150 rpm for 2 h at 28℃. After centrifugation at 5000 rpm for 5 min, discard the supernatant and collect the precipitate; repeat this impurity removal operation twice. Spread the final precipitate evenly in a clean petri dish and air dry at room temperature to obtain the pretreated powder.
[0048] Example 2
[0049] Crude protein extraction and sequence identification from seeds of Sophora alopecuroides include the following steps:
[0050] (1) Ultrasonic alkaline dissolution: Accurately weigh 20 g of the pretreated powder prepared in Example 1, add 400 mL of deionized water (pH adjusted to 9.0), place in an ice-water bath, and use an ultrasonic disruptor (40% amplitude, 20 min) to obtain cell-breaking fluid.
[0051] (2) Acid precipitation purification: The cell-breaking solution obtained in step (1) was centrifuged at 6000 rpm for 5 min, the supernatant was collected, the pH was adjusted to 5.0 with 1 mol / L HCl, and the mixture was allowed to stand at 4℃ for 2.5 h. The mixture was then centrifuged at 8000 rpm for 20 min, and the precipitate was collected. The precipitate was washed three times with deionized water until the pH of the washing solution reached 7.0. After freeze-drying, the crude protein sample was obtained.
[0052] (3) SDS-PAGE analysis: The crude protein prepared in step (2) was diluted with deionized water to prepare a protein solution with a concentration of 2 mg / mL. 16 μL of this solution was mixed with 4 μL of 5× Loading buffer, heated in a boiling water bath for 5 min, and then centrifuged at 12000 rpm for 3 min. The supernatant was then subjected to SDS-PAGE electrophoresis analysis. The results are as follows: Figure 1 As shown;
[0053] (4) Protein sequence identification: Cut all target bands from the SDS-PAGE electrophoresis in step (3), place them in an EP tube, and wash twice with ultrapure water for 5 min each time. Add a 1:1 (v / v) mixture of 50 mM NH4HCO3 and acetonitrile (ACN), sonicate for 15 min, discard the supernatant, and repeat the decolorization operation until the gel and solution are colorless. Wash once with a 1:1 (v / v) mixture of 50 mM NH4HCO3 and acetonitrile; add 100% acetonitrile and shake to dehydrate until the gel particles are white, discard the acetonitrile, and vacuum dry for 5 min. Add 150 μL of 50 mM dithiothreitol (DTT) solution to submerge the gel block and shake to mix until the gel block swells and becomes transparent. Incubate at 56 °C for 1 h. Cool to room temperature, discard the supernatant, and quickly add 150 μL of 100 mM iodoacetamide (IAM) solution to submerge the gel block. Place in the dark for 45 min. Wash once each with 25 mM NH4HCO3 solution, a 1:1 (v / v) mixture of 50 mM NH4HCO3 and acetonitrile, and 100% acetonitrile. After dehydration with acetonitrile until the gel particles turn white, vacuum dry for 5 min. Take 0.1 μg / μL of enzyme stock solution, dilute it appropriately with 25 mM NH4HCO3 solution, and add the diluted enzyme solution until it just submerges the swollen gel block, ensuring full contact between the gel block and the enzyme solution. Incubate at 4℃ for 30 min. After the enzyme solution is completely absorbed by the gel block, aspirate the excess enzyme solution, add 25 mM NH4HCO3 until it submerges the gel block, and digest overnight at 37℃. Collect the enzyme digest. Add 100 μL of a mixture of 30% acetonitrile / 0.1% trifluoroacetic acid (TFA) to the original EP tube, sonicate for 15 min, and collect the supernatant. Add 100 μL of a mixture of 60% acetonitrile / 0.1% TFA, sonicate for 15 min, combine the two collected solutions, and freeze-dry. The lyophilized peptides were desalted using a ZipTip C18 column, then reconstituted with 20 μL of 0.1% formic acid solution. After thorough vortexing, the peptides were centrifuged at 14,000 rpm for 20 min at 4 °C. 3 μL of the supernatant was then used for mass spectrometry identification. Database search analysis was performed using PEAKS software, ultimately identifying 71 protein sequences.
[0054] Example 3
[0055] Virtual enzymatic digestion, activity screening, and targeted docking of peptide sequences:
[0056] (1) Virtual enzymatic digestion: The enzymatic digestion process of protease was simulated using Expasy-Peptide Cutter software, and a sequence library containing 1343 polypeptides was generated based on the identified protein sequences.
[0057] (2) Bioactivity screening: The bioactivity of the above 1343 peptides was scored and screened using the PeptideRanker database. Generally, peptides with a PeptideRanker score > 0.5 are considered to have potential bioactivity. To narrow the screening range and improve screening efficiency, 46 peptides with a score > 0.8 were selected for subsequent bioactivity analysis.
[0058] (3) Molecular structure preparation: The crystal structures of ACE enzymes were downloaded from the RCSB PDB database, and the water of crystallization and ligands were removed using AutoDock Tools software. After charge partitioning, the structures were ready for use. PDB format structural files of 46 peptides were obtained using AlphaFold 3 software and imported into the software for structural optimization, including hydrogenation and charge partitioning.
[0059] (4) Molecular docking and result analysis: Molecular docking was performed using MOE software to identify the active pocket region of the ACE enzyme. Forty-six active peptides were targeted and docked with the ACE enzyme to evaluate their binding affinity. The docking results showed that the binding energies of the 46 active peptides to the ACE enzyme ranged from -3.5961 to -11.1073 kcal / mol. Five high-affinity peptides with lower binding energies (-8.1149 to -11.2737 kcal / mol) were further screened out: MGLPWYR, INPLGFR, WHRPLR, LPNSLFLFR, and LFLWPIYR.
[0060] The polypeptide MGLPWYR has a binding energy of -11.2737 kcal / mol with ACE enzyme, indicating a tight binding (see results). Figure 2 This polypeptide can form hydrogen bonds with residues such as ASP, ARG, THR, and LYS on the receptor, enabling it to effectively bind to the antihypertensive target and exert its effects.
[0061] The peptide WHRPLR has a binding energy of -10.6020 kcal / mol and exhibits strong binding affinity (see Figure 3). It exhibits various non-covalent interactions with the receptor, including hydrogen bonds with residues such as MET 832, GLU 1012, and TYR 744, an ionic bond with GLU 1012, and a π-cation interaction between the ligand's 5-membered ring and LYS 727. These multiple interactions synergistically ensure binding stability, laying the structural foundation for its potential antihypertensive activity.
[0062] The peptide INPLGFR exhibits a binding energy of -9.4093 kcal / mol and good thermodynamic stability (see Figure 4). Strong interactions are achieved through hydrogen bonds formed between ND2 and ALA 368, NH1 and SER 95, and O 35 and ALA 368; ionic bonds formed between 2OCT 120 and ARG 415; and hydrophobic interactions and van der Waals forces from surrounding Ala, Ser, and Phe residues.
[0063] The peptide LPNSLFLFR has a binding energy of -8.1149 kcal / mol (see Figure 5). Stable binding is achieved through strong hydrogen bonds between N and ASP 176, ionic bonds between N and ASP 176, and the direct involvement of residues such as ASP, GLU, and ARG, as well as the auxiliary effects of surrounding residues.
[0064] The peptide LFLWPIYR has a binding energy of -9.1362 kcal / mol (see Figure 6). Stable interactions are achieved through strong hydrogen bonds formed between NH1 155 and GLU 357, strong ionic bonds formed between 2OCT 163 and LYS 355, π-cation interactions between the 6-membered ring and LYS 160, and the direct participation of residues such as GLU, LYS, and TRP, as well as the auxiliary effects of residues such as Pro and Ala.
[0065] Example 4
[0066] Determination of ACE inhibitory activity of peptides: To verify the ACE inhibitory activity of the peptides screened in the above implementation cases, this embodiment sets up an experiment to detect their inhibition rate against ACE enzyme. The specific implementation steps are as follows:
[0067] (1) MGLPWYR, INPLGFR, WHRPLR, LPNSLFLFR, and LFLWPIYR peptides were obtained by Nanjing Jietai Biotechnology Co., Ltd. using solid-phase synthesis and purification methods, with a purity >95%.
[0068] (2) The ACE inhibition rate was determined using the FAPGG method. FAPGG (FA-Phe-Gly-Gly) was used as a mimic substrate for angiotensin I, and the ACE inhibitory activity of the above five peptides was measured using an ELISA reader. When FAPGG is used as an ACE substrate, it is hydrolyzed into FAP and GG, resulting in a decrease in absorbance at 340 nm, which was used to determine the ACE inhibition rate.
[0069] Reagent preparation:
[0070] ① Matrix buffer: HEPES-NaOH (80 mmol / L, pH 8.3), containing 0.3 mol / L NaCl. The HEPES content is 1.910 g, and the NaCl content is 1.755 g. After dissolving in deionized water, adjust the pH to 8.3 with a suitable concentration of NaOH solution, then add water to a final volume of 100 mL. Store at 4℃ for later use.
[0071] ②FAPGG (1.0 mmol / L): Accurately weigh 3.994 mg FAPGG and dissolve it in matrix buffer, bring the volume up to 10 mL, mix well, and store at 4°C protected from light.
[0072] ③ACE (0.1 U / mL): Dissolve 2U of ACE in matrix buffer, bring the volume to 4 mL to make a concentration of 0.5 U / mL, aliquot into 1.5 mL centrifuge tubes, 200 μL per tube, and store at -20℃. Dilute the concentration to 0.1 U / mL before use.
[0073] Add 40 μL of the test sample, 50 μL of FAPGG, and 10 μL of ACE to a 96-well plate in sequence. After shaking for 10 s, immediately measure the absorbance at 340 nm using a microplate reader, and record it as A1. After incubating at 37℃ in the dark for 30 min, measure the absorbance again, and the value is A2. Calculate ΔA = A1 - A2. The change in absorbance value per unit time is expressed as the ACE activity. Use matrix buffer instead of the test sample as a blank control.
[0074] ACE inhibitor inhibition rate (%) = (ΔA) b -ΔA a )÷ΔA b ×100%
[0075] In the formula: ΔA b —The change in absorbance over 30 minutes after the addition of matrix buffer;
[0076] ΔA a —The change in absorbance within 30 minutes after the sample to be tested is added.
[0077] Test results are as follows Figures 7-9 As shown.
[0078] Depend on Figure 7The inhibition rates were as follows: 88.99% for heptapeptide MGLPWYR at 0.9764 mg / mL; 90.07% for hexapeptide WHRPLR at 0.9970 mg / mL; 3.28% for nonapeptide LPNSLFLFR at 0.9923 mg / mL; 76.89% for octapeptide LFLWPIWR at 0.9899 mg / mL; and 3.05% for heptapeptide INPLGFR at 0.9515 mg / mL.
[0079] The ACE inhibitory activity of heptapeptide MGLPWYR at different concentrations is as follows: Figure 8 As shown, the inhibition rate was 58.71% at a concentration of 0.9764 μg / mL.
[0080] The ACE inhibitory activity of hexapeptide WHRPLR at different concentrations is as follows: Figure 9 As shown, the inhibition rate was 52.75% when the concentration was 0.9970 μg / mL.
Claims
1. A hypotensive source of peptides derived from Sophora japonica, characterized in that, at least one of the active peptides having a sequence structure of MGLPWYR, WHRPLR, INPLGFR, LPNSLFLFR, LFLWPIYR.
2. The amorphophellus sandalwood protein-derived antihypertensive peptide according to claim 1, characterized by, The antihypertensive peptide is MGLPWYR and / or WHRPLR.
3. Use of the antihypertensive peptide derived from the protein of Haloxylon ammodendron in claim 1 or 2 in the preparation of an ACE inhibitor.
4. Use of the antihypertensive peptide derived from the protein of Haloxylon ammodendron in claim 1 or 2 in the preparation of a drug for treating hypertension.
5. A pharmaceutical composition having a blood pressure lowering effect, characterized by comprising the compound of claim 1. The pharmaceutical composition further contains any one or more of excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, or pH buffering substances.
6. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition is a capsule, a powder, a tablet, a granule, a pill, an injection, a syrup, an oral solution, an inhalant, an ointment, a suppository, or a patch.
7. The pharmaceutical composition of claim 6, wherein, 9. Use of the antihypertensive peptide derived from the protein of Haloxylon ammodendron in claim 1 or 2 in the preparation of a functional food for regulating blood pressure.
8. The pharmaceutical composition of claim 5, wherein, The functional food further contains food-acceptable excipients. 10. Use according to claim 9, characterized in that,