A multifunctional bioactive peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities.

Abstract

This invention discloses a multifunctional bioactive peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities. The multifunctional bioactive peptide NGVPL of this invention, exhibiting strong antioxidant, ACE inhibitory, and DPP-IV inhibitory activities, was demonstrated through molecular docking simulation and in vitro activity assays. It is suitable for preparing functional products that act as antioxidants, antihypertensive drugs, hypoglycemic drugs, or aid in the regulation of blood pressure and blood sugar. It can also be used in combination with other functional ingredients or food additives.

Description

Technical Field

[0001] This invention relates to the field of protein engineering, and in particular to a multifunctional active peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities. Background Technology

[0002] Enzyme inhibitors are a key strategy in modern drug development. Their core principle is to specifically block the activity of certain enzymes in the body, thereby regulating related physiological or pathological processes and achieving therapeutic effects. These drugs are typically designed based on a deep understanding of enzyme structure and function, enabling high targeting and low non-specific side effects. DPP-IV inhibitors and ACE inhibitors are two successful examples, targeting completely different metabolic pathways and revolutionizing their respective therapeutic areas. They are widely used due to their good efficacy and safety.

[0003] DPP-IV, or dipeptidyl peptidase-IV, is a protease found on the cell membrane surface. One of its physiological functions is to rapidly degrade a class of hormones in the body called incretins. These hormones are secreted by the intestines after meals, and their core function is to stimulate insulin secretion while inhibiting glucagon secretion, thereby intelligently lowering blood sugar. Based on this principle, DPP-IV inhibitors have been developed. These drugs, by efficiently and selectively inhibiting the activity of the DPP-IV enzyme, can prolong the duration of action of endogenous incretins, thereby promoting insulin release and lowering glucagon levels in a glucose concentration-dependent manner. This mechanism of action gives it a unique advantage in the treatment of type 2 diabetes, as it only works when blood sugar is elevated, significantly reducing the risk of hypoglycemia and making it a safe and effective new type of oral hypoglycemic agent.

[0004] ACE, or angiotensin-converting enzyme, is a core enzyme in the renin-angiotensin-aldosterone system. Its main function is to convert angiotensin I into angiotensin II, which has a strong vasoconstrictive effect, while simultaneously degrading bradykinin, which has a vasodilatory effect. Therefore, ACE activity is directly related to blood pressure regulation and the cardiovascular system's workload. ACE inhibitors competitively bind to the active site of this enzyme, blocking the conversion process, thereby reducing the production of angiotensin II and increasing the accumulation of bradykinin. This dual action leads to vasodilation and a decrease in peripheral resistance, producing a significant antihypertensive effect. More importantly, long-term use can reverse cardiac and vascular remodeling and improve the prognosis of heart failure, establishing its cornerstone position in the treatment of cardiovascular diseases such as hypertension and heart failure.

[0005] Furthermore, oxidative stress, as an independent damage mechanism at the cellular level, is characterized by the excessive accumulation of reactive oxygen species (ROS). Excessive ROS can attack biomembrane systems, inducing a chain reaction of lipid peroxidation. Oxidative stress is widely involved in the pathological processes of various metabolic-related diseases, such as atherosclerosis, obesity, non-alcoholic fatty liver disease, and chronic kidney disease, and is a key common link connecting metabolic disorders and target organ damage. In response to these issues, food-derived peptides with high bioavailability demonstrate strong potential for comprehensive intervention. Food-derived peptides can not only act as inhibitors to reduce ACE and DPP-IV activity, but also exert antioxidant effects by enhancing the activity of endogenous antioxidant defense enzymes, simultaneously improving the complex pathology of hypertension, hyperglycemia, and oxidative damage. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a multifunctional active peptide with antioxidant activity and ACE and DPP-IV inhibition capabilities.

[0007] Another object of the present invention is to provide applications of the above-mentioned multifunctional active peptides having antioxidant activity and ACE and DPP-IV inhibitory capabilities.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A multifunctional bioactive peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities, comprising at least one of the following polypeptides:

[0010] NGVPL, with the amino acid sequence Asn-Gly-Val-Pro-Leu;

[0011] NGAL, with the amino acid sequence Asn-Gly-Ala-Leu.

[0012] The multifunctional active peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities was extracted and isolated from cricket protein.

[0013] The multifunctional active peptide with antioxidant activity and ACE and DPP-IV inhibition capabilities is prepared by chemical synthesis.

[0014] The above-mentioned multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities are used in the preparation of DPP-IV and / or ACE inhibitors.

[0015] The above-mentioned multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities are used in the preparation of DPP-IV / ACE dual inhibitors.

[0016] The above-mentioned multifunctional bioactive peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities are used in the preparation of hypoglycemic and / or hypertension treatment drugs.

[0017] The above-mentioned multifunctional bioactive peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities are used in the preparation of drugs that can simultaneously lower blood sugar and treat hypertension.

[0018] The above-mentioned multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities are used in the preparation of health products with auxiliary hypoglycemic and / or auxiliary hypotensive functions.

[0019] The above-mentioned multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities are used in the preparation of health products that simultaneously have the functions of assisting in lowering blood sugar and assisting in lowering blood pressure.

[0020] The above-mentioned multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities are used in the preparation of antioxidant products.

[0021] The above-mentioned multifunctional bioactive peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities are used in the preparation of drugs with antioxidant effects.

[0022] The present invention has the following advantages and effects compared with the prior art:

[0023] (1) The multifunctional active peptide of the present invention, which has antioxidant activity and ACE and DPP-IV inhibition ability, has a clear and distinct structure. It can be prepared by solid phase chemical synthesis or by separating and purifying cricket protein hydrolysate.

[0024] (2) The multifunctional active peptide NGVPL of the present invention, which has antioxidant activity and ACE and DPP-IV inhibition ability, has strong antioxidant, ACE inhibition and DPP-IV inhibition activity as predicted by molecular docking simulation and in vitro activity determination. It is suitable for preparing antioxidant, antihypertensive drugs, hypoglycemic drugs or functional products that help regulate blood pressure and blood sugar. It can also be used in combination with other functional ingredients or food additives.

[0025] (3) The multifunctional active peptides of the present invention, which have antioxidant activity and ACE and DPP-IV inhibition capabilities, are derived from edible insect protein. The raw materials are safe and natural, conform to the concept of green and sustainable development, and are easy to prepare on a large scale, with good industrial transformation potential. Attached Figure Description

[0026] Figure 1 These are the results of ABTS activity assays in the cricket enzymatic hydrolysates of the examples and comparative examples.

[0027] Figure 2 These are the DPPH activity assay results of cricket enzymatic hydrolysates from the examples and comparative examples.

[0028] Figure 3 These are the results of ACE inhibitory activity assays of cricket enzymatic hydrolysates from the examples and comparative examples.

[0029] Figure 4 These are the results of the DPP-IV inhibitory activity assay of the cricket enzymatic hydrolysates in the examples and comparative examples.

[0030] Figure 5 This is a molecular weight distribution diagram of the cricket enzymatic hydrolysates from the examples and comparative examples.

[0031] Figure 6 This is the result of molecular docking of the peptide NGVPL. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0033] Example 1: Preparation of Cricket Protein Hydrolysate A

[0034] The crickets were washed, blanched, and then air-dried in a natural environment. The crickets were then crushed and sieved through a 40-mesh sieve. The cricket powder was defatted to obtain cricket protein powder. The cricket protein powder was dissolved in water (material ratio 1g:12mL), and the pH of the solution was adjusted to 7.0-8.0. Alcalase enzyme (10000 U / g) at 5% of the cricket protein substrate amount was added to the mixed solution, and the mixture was placed in a water bath shaker at 45-55℃ for 1-3 h for enzymatic hydrolysis. After the reaction was completed, the system was inactivated in boiling water for 10 min, and then centrifuged at 8000 rpm for 15 min at 4℃. The supernatant was collected and freeze-dried to obtain cricket protein hydrolysate powder A.

[0035] Comparative Example 1: Preparation of Cricket Protein Hydrolysate B

[0036] The preparation method of Example 1 was followed, except that Alcalase enzyme was replaced with flavor protease (500 MG), and cricket protease hydrolysate powder B was finally prepared.

[0037] Comparative Example 2: Preparation of Cricket Protein Hydrolysate C

[0038] The preparation method of Example 1 was followed, except that Alcalase enzyme was replaced with neutral protease (50,000 U / g), and cricket protease hydrolysate powder C was finally prepared.

[0039] Example 2 Characterization and determination of cricket protein hydrolysate

[0040] 2.1 Determination of DPPH free radical scavenging activity

[0041] The cricket protein hydrolysate solution was mixed with 0.1 mmol / L DPPH solution at a volume ratio of 1:1, and reacted at room temperature in the dark for 30 min. The absorbance value was then measured at a wavelength of 517 nm.

[0042] 2.2 Determination of ABTS free radical scavenging activity

[0043] 38.4 mg ABTS and 33.1 mg K₂S₂O₈ were dissolved in deionized water and the volume was adjusted to 100 mL to prepare an ABTS stock solution. This stock solution was reacted at 4°C in the dark for 16 h, and then diluted with 0.2 mol / L phosphate buffer to obtain an absorbance of 0.70 ± 0.02 at 734 nm, thus yielding the ABTS working solution. The cricket protein hydrolysate solution was mixed with the ABTS working solution at a volume ratio of 1:2 and reacted at room temperature in the dark for 6 min. The absorbance was then measured at 734 nm.

[0044] Vitamin C was the positive control for both DPPH and ABTS free radical scavenging activities.

[0045] Scavenging rate calculation: The free radical scavenging rate is calculated using the following formula:

[0046] Clearance rate (%) = (A0 - A1) / A0 × 100

[0047] In the formula, A0 is the absorbance value of the blank control group (using solvent instead of sample), and A1 is the absorbance value after adding sample.

[0048] IC 50 Value calculation: By measuring the clearance rate of samples at different concentrations, a dose-response curve is plotted, and the half-maximal inhibitory concentration (IC50) is calculated accordingly. 50 ).

[0049] 2.3 Assay for ACE inhibitory activity

[0050] The ACE inhibitory activity of cricket protein hydrolysates was determined using the ACE Kit-WST enzymatic assay kit, with a sample protein concentration of 0.2 mg / mL. The kit was operated strictly according to the instructions, and absorbance was measured at 450 nm using a microplate reader. The ACE inhibition rate was calculated. Captopril was used as a positive control for the ACE inhibitory activity assay.

[0051] 2.4 Assay for DPP-IV inhibitory activity

[0052] The DPP-IV inhibitory activity of cricket protein hydrolysates was determined using a DPP-IV inhibitor screening kit at a protein concentration of 1.0 mg / mL. The kit was operated strictly according to the instructions, and fluorescence intensity was measured using a multi-mode microplate reader at excitation wavelengths of 350–360 nm and emission wavelengths of 450–465 nm. The DPP-IV inhibition rate was calculated. Sitagliptin was used as a positive control for the DPP-IV inhibitory activity assay.

[0053] 2.5 Experimental Results

[0054] Experimental results are as follows Figures 1-5 As shown in Table 1, it can be seen that the cricket protein hydrolysate A prepared in the examples has a smaller molecular weight and greater bioactivity potential compared to the comparative examples B and C. Furthermore, Table 1 shows that the cricket protein hydrolysate A in the examples exhibits stronger antioxidant, antihypertensive, and hypoglycemic activities compared to the comparative examples B and C. Under the same experimental conditions, the cricket protein hydrolysates B and C prepared in Comparative Examples 1 and 2 using flavor proteases and neutral proteases to hydrolyze the protein showed lower antioxidant activity, ACE inhibitory activity, and DPP-IV inhibitory activity compared to the cricket protein hydrolysate A in the examples.

[0055] Table 1. Bioactivity assays of examples and comparative examples.

[0056] Enzymatic hydrolysate number Does it contain the target peptide? <![CDATA[ABTS clearance rate IC 50 (mg / mL)]]> <![CDATA[DPPH scavenging rate IC 50 (mg / mL)]]> ACE inhibitory activity (%) DPP-IV inhibitory activity (%) A yes 0.182±0.01 1.114±0.07 70.87±0.42 31.25±0.27 B no 0.253±0.09 1.665±0.02 64.01±0.15 23.43±0.13 C no 0.303±0.12 2.025±0.13 63.19±0.28 15.35±0.22

[0057] Note: ACE inhibitory activity (%) is compared with the positive control captopril, and DPP-IV inhibitory activity (%) is compared with the positive control sitagliptin, the same below.

[0058] Example 3

[0059] The peptide sequences in the above-mentioned cricket protein hydrolysate were identified using LC-MS / MS liquid chromatography-mass spectrometry, and multifunctional small molecule bioactive peptides with potential for antioxidant, ACE inhibition, and DPP-IV inhibition activities were further screened, including the following steps:

[0060] (1) The enzymatic hydrolysate powder prepared in Example 1 was dissolved in ultrapure water to a final concentration of 1.0 mg / mL. After filtration through a 0.22 μm filter membrane, it was analyzed using an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap mass spectrometry system. Chromatographic separation was performed on a Hypersil GOLD C18 column at a column temperature of 30 °C. Mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was ultrapure water. Gradient elution was performed at a flow rate of 0.20 mL / min, with an injection volume of 2 μL. The gradient program was as follows: 0-1 min, 5% A; 1-2.5 min, 5%-10% A; 2.5-12.5 min, 10%-25% A; 12.5-20 min, 25%-52.5% A; 20-22 min, 52.5%-95% A; and then reequilibrated to the initial conditions within 22-30 min. Mass spectrometry acquisition was performed in positive ion mode, with a spray voltage of 3.2 kV, a capillary temperature of 350 °C, and a scan range of m / z 200-2000. 1 The resolution is 70,000, MS 2 The resolution is 35,000, using data-dependent MS. 2 Data collection method.

[0061] (2) The obtained mass spectrometry data were analyzed using the PepOS 3.2 proteomics analysis platform to identify, extract, and predict the bioactivity of peptides. The identification parameters were set as follows: precursor mass tolerance was 20 ppm, fragment mass tolerance was 0.02 Da, peptide length was limited to 2–30 amino acid residues, and the minimum identification score was 36. Subsequently, based on the platform prediction results, peptides with simultaneous antioxidant, ACE inhibitory, and DPP-IV inhibitory activities were screened. Finally, peptides NGVPL and NGAL, which may have potential multiple bioactivities, were screened and synthesized into pure peptides using solid-state synthesis for subsequent experiments.

[0062] Example 4 Characterization of small molecule peptides

[0063] 4.1 Molecular docking verification

[0064] (1) Preparation of receptor and ligand structures

[0065] First, the crystal structures of target proteins, including the Keap1 Kelch domain (PDB: 2FLU), human ACE (PDB: 1O8A), and human DPP-IV (PDB: 4PNZ), were downloaded from the RCSB Protein Database. The receptor structures were preprocessed using molecular simulation software to remove water molecules, primitive ligands, and irrelevant ions, followed by hydrogenation, charge distribution, and energy minimization. Simultaneously, the three-dimensional structures of the active peptides were generated using a molecular building block and optimized for conformation.

[0066] (2) Molecular docking simulation

[0067] Semi-flexible molecular docking was performed using Discovery Studio 2019 software. Before docking, corresponding protonation models were generated based on the active cavities of each receptor, with a threshold set to 0.5. The optimized active peptides were docked into the active pockets of Keap1, ACE, and DPP-IV. Docking calculations were run, and the binding conformation and the result with the highest score were retained by default.

[0068] (3) Interaction analysis and visualization

[0069] After docking, Discovery Studio 2019 software was used to analyze the key molecular interactions between the active peptide and each target site, including hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0070] 4.2 Molecular docking verification results

[0071] Molecular docking results showed that the cricket bioactive peptide NGVPL exhibited high binding affinity to three target proteins: oxidative stress, ACE, and DPP-IV, demonstrating its potential as a natural multi-target inhibitor with promising applications in synergistic regulation of hypertension, hyperglycemia, and oxidative stress. In contrast, NGAL failed to form an effective binding posture with its target Keap1 during conformational search and refinement, thus lacking antioxidant activity. Furthermore, NGAL showed low binding affinity to both ACE and DPP-IV, exhibiting lower antihypertensive and hypoglycemic activities.

[0072] Table 2 Molecular docking results

[0073] peptide sequence Antioxidant activity (kcal / mol) ACE inhibitory activity (kcal / mol) DPP-IV inhibitory activity (kcal / mol) NGAL - -80.24 -61.98 NGVPL -69.74 -132.45 -118.79

[0074] 4.3 Determination of antioxidant capacity, ACE inhibitory activity and DPP-IV inhibitory activity

[0075] Following the method in Example 2, the antioxidant capacity, ACE inhibitory activity, and DPP-IV inhibitory activity of the peptides NGVPL and NGAL screened in Example 3 were tested.

[0076] Activity assays showed that NGVPL exhibited multi-target activity, with an IC50 scavenging rate of ABTS free radicals. 50 The concentration of nitric oxide was 0.09 mg / mL, the DPPH clearance rate was 0.41 mg / mL, the ACE inhibition activity was 90.22%, and the DPP-IV inhibition activity was 76.88%, showing good synergistic potential for antioxidation, blood pressure reduction, and blood glucose reduction; while the antioxidant activity of NGAL was significantly weaker (ABTS clearance rate IC50 was 0.09 mg / mL). 507.95 mg / mL, DPPH clearance IC50 50 The concentration was 13.22 mg / mL, and both ACE and DPP-IV inhibitory activities were low (ACE inhibition activity 38.16%, DPP-IV inhibition activity 25.63%), with poor antihypertensive and hypoglycemic activities. These activity assay results are consistent with the trends observed in molecular docking analysis. NGVPL exhibits high binding affinity to all three target proteins, thus demonstrating multi-target activity. NGAL, however, failed to form an effective binding posture with Keap1 during molecular docking and showed weak affinity for the other two protein classes; however, weak free radical scavenging ability, ACE, and DPP-IV inhibitory activities were still detected in actual assays.

[0077] Table 3 Results of Pure Peptide Activity Assay

[0078] sequence <![CDATA[ABTS clearance rate IC 50 (mg / mL)]]> <![CDATA[DPPH scavenging rate IC 50 (mg / mL)]]> ACE inhibitory activity (%) DPP-IV inhibitory activity (%) NGAL 7.95±0.28 13.22±0.16 38.16±0.41 25.63±0.15 NGVPL 0.09±0.01 0.41±0.02 90.22±0.35 76.88±0.56

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A multifunctional bioactive peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities, characterized in that... It is at least one of the following polypeptides: NGVPL, with the amino acid sequence Asn-Gly-Val-Pro-Leu; NGAL, with the amino acid sequence Asn-Gly-Ala-Leu.

2. The multifunctional active peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities according to claim 1, characterized in that: The multifunctional active peptide with antioxidant activity and ACE and DPP-IV inhibitory capabilities was extracted and isolated from cricket protein.

3. The use of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities as described in claims 1 to 2 in the preparation of DPP-IV and / or ACE inhibitors.

4. The application of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities as described in claims 1-2 in the preparation of DPP-IV / ACE dual inhibitors.

5. The use of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities as described in claims 1-2 in the preparation of hypoglycemic and / or hypertension treatment drugs.

6. The use of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibitory capabilities as described in claims 1-2 in the preparation of drugs that can simultaneously lower blood sugar and treat hypertension.

7. The use of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities as described in claims 1 to 2 in the preparation of health products with auxiliary hypoglycemic function and / or auxiliary hypotension function.

8. The application of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities as described in claims 1-2 in the preparation of health products that simultaneously have the functions of assisting in lowering blood sugar and assisting in lowering blood pressure.

9. The application of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities as described in claims 1-2 in the preparation of antioxidant products.

10. The use of the multifunctional active peptides with antioxidant activity and ACE and DPP-IV inhibition capabilities as described in claims 1-2 in the preparation of drugs with antioxidant effects.

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