Alpha-amylase inhibitory active peptide and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN BUSINESS UNIV
- Filing Date
- 2025-12-23
- Publication Date
- 2026-05-01
AI Technical Summary
[0012] This invention isolates an active peptide with good α-amylase inhibitory activity from the free peptides of mandarin fish. It can reduce the hydrolysis of starch by inhibiting the activity of α-amylase, thereby reducing the conversion of starch to glucose. It can be used to prepare α-amylase inhibitors, hypoglycemic drugs, or drugs for the prevention and treatment of hyperglycemia-related diseases.
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Abstract
Description
α-Amylase inhibitory active peptides and their applications Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to an α-amylase inhibitory active peptide and its applications. Background Technology
[0002] Hyperglycemia, medically speaking, refers to a pathological state in which the concentration of glucose in the blood (i.e., blood glucose level) consistently exceeds the normal physiological range. It is typically diagnosed when fasting blood glucose is ≥7.0 mmol / L or 2-hour postprandial blood glucose is ≥11.1 mmol / L. Its core pathological mechanism is directly related to an imbalance in glucose metabolism regulation. Besides inherent patient deficiencies, most cases of hyperglycemia are caused by unhealthy dietary habits and lifestyle factors, such as a long-term high-carbohydrate diet, a sedentary lifestyle, or abdominal obesity.
[0003] Alpha-amylase is a hydrolytic enzyme that catalyzes the hydrolysis of α-1,4-glycosidic bonds in polysaccharide molecules such as starch and glycogen. In the human body, it is mainly derived from the salivary glands and pancreas. Salivary amylase has a molecular weight between 55 kDa and 62 kDa, while pancreatic amylase has a molecular weight between 54 kDa and 57 kDa. The actual molecular weight may vary slightly due to differences in amino acid sequence and degree of glycosylation. Its active site contains a hydrophobic region. Both amylases are synthesized and secreted by the acinar cells of their respective glands. In the human body, carbohydrates are initially hydrolyzed by salivary amylase in the oral cavity, completely hydrolyzed by pancreatic amylase in the small intestine, and then absorbed by the small intestinal epithelium, converting carbohydrates into absorbable small-molecule sugars. These sugars are then absorbed into the bloodstream, leading to a postprandial increase in blood glucose. Therefore, inhibiting α-amylase activity can reduce the hydrolysis of starch, thereby reducing the conversion to glucose and thus reducing the postprandial increase in blood glucose. Current research has found that food-derived α-amylase inhibitory peptides may serve as potential alternatives to chemically synthesized drugs for treating hyperglycemia. Therefore, it is necessary to develop a food-derived bioactive peptide that inhibits α-amylase. Summary of the Invention
[0004] The purpose of this invention is to address the aforementioned problems in the prior art by providing an α-amylase inhibitory peptide isolated from free peptides of mandarin fish, which has good α-amylase inhibitory activity and its applications.
[0005] To achieve the above objectives, the technical solution of the present invention is as follows:
[0006] In a first aspect, the present invention provides an α-amylase inhibitory active peptide, said active peptide being isolated from free peptides of mandarin fish, and its amino acid sequence being any one of the following:
[0007] Leu-Pro-Lys-Leu-Arg-Val-Lys-Val;
[0008] Val-Glu-Lys-Ser-Lys-Val-Tyr;
[0009] Glu-Val-Ile-Glu-Leu-Asp-Trp-Arg.
[0010] Secondly, the present invention provides the use of the aforementioned α-amylase inhibitory active peptide in the preparation of α-amylase inhibitors, hypoglycemic drugs, or drugs for the prevention and treatment of hyperglycemia-related diseases.
[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0012] This invention isolates an active peptide with good α-amylase inhibitory activity from the free peptides of mandarin fish. It can reduce the hydrolysis of starch by inhibiting the activity of α-amylase, thereby reducing the conversion of starch to glucose. It can be used to prepare α-amylase inhibitors, hypoglycemic drugs, or drugs for the prevention and treatment of hyperglycemia-related diseases. Attached Figure Description
[0013] Figure 1 shows the molecular docking results of active peptide 1 and α-amylase, where figures a and b are schematic diagrams of the docking complex formed by active peptide 1 and α-amylase and a schematic diagram of the two-dimensional interaction, respectively.
[0014] Figure 2 shows the molecular docking results of active peptide 2 and α-amylase, where figures c and d are schematic diagrams of the docking complex formed by active peptide 2 and α-amylase and a schematic diagram of two-dimensional interaction, respectively.
[0015] Figure 3 shows the molecular docking results of active peptide 3 and α-amylase, where figures e and f are schematic diagrams of the docking complex formed by active peptide 2 and α-amylase and a schematic diagram of two-dimensional interaction, respectively. Detailed Implementation
[0016] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.
[0017] Example 1: Screening of α-amylase-inhibiting peptides
[0018] Traditional methods for isolating target peptides from food proteins involve enzymatic hydrolysis, followed by purification of the released peptides using ultrafiltration, gel size exclusion chromatography, and reversed-phase high-performance liquid chromatography, and then screening using α-amylase. These methods are time-consuming, labor-intensive, and prone to errors. This invention employs a combination of virtual screening and in vitro analysis to screen and isolate the target peptides, resulting in high precision and efficiency.
[0019] All free peptides from *Siniperca chuatsi* were identified using peptidomimetics, and the activity of the identified peptide sequences was predicted using an activity prediction website (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep). The top 30 abundant peptides with potential α-amylase inhibitory activity were screened. The 3D structure of α-amylase (PDB ID: 1B2Y) was obtained from the Protein Data Bank (PDB) database and used as the receptor protein. Molecular docking was performed using DS software, and docking energy was used as the screening index. From the top 30 peptides with potential α-amylase inhibitory activity, three active peptides that bind most tightly to the α-amylase active site were selected. The amino acid sequences of these three active peptides are as follows:
[0020] Active peptide 1: Leu-Pro-Lys-Leu-Arg-Val-Lys-Val;
[0021] Active peptide 2: Val-Glu-Lys-Ser-Lys-Val-Tyr;
[0022] Active peptide 3: Glu-Val-Ile-Glu-Leu-Asp-Trp-Arg.
[0023] The molecular docking results of the three active peptides 1-3 with α-amylase are shown in Figures 1 to 3, respectively. The molecular docking results include schematic diagrams of the docking complex structure and two-dimensional interaction diagrams. The two-dimensional interaction diagrams illustrate the specific interaction between the ligand and receptor in a simplified planar form, and label the amino acid residues involved in the binding. The docking energy, interaction energy, binding energy, van der Waals energy, and electrostatic attraction energy are shown in Table 1.
[0024] Table 1. Energy of docking complexes formed by three bioactive peptides and α-amylase
[0025]
[0026] The predicted α-amylase activity inhibition rates of the above-mentioned active peptides 1-3 were 32.75±1.06%, 28.54±2.17%, and 24.01±1.62%, respectively. PepBank analysis confirmed that none of the three active peptide sequences had been previously reported. The isoelectric point, water solubility, toxicity, hydrophobicity, and lipid index of active peptides 1-3 are shown in Table 2. The isoelectric point of the peptide sequences was calculated using the Expasy tool; the water solubility was calculated using the water solubility calculator in the proteomics tool of the Innovagen online website; the toxicity was calculated using the ToxinPred online program; the hydrophobicity was predicted using the PepDraw online program; the instability was calculated using the ProtParam tool; and the lipid index was virtually predicted using ProtParam.
[0027] Table 2. Isoelectric point, water solubility, toxicity, hydrophobicity, and lipid index of three bioactive peptides.
[0028]
[0029] In the amino acid sequences of the above peptides, D represents aspartic acid (Asp); E represents glutamic acid (Glu); I represents isoleucine (Ile); K represents lysine (Lys); L represents leucine (Leu); P represents proline (Pro); R represents arginine (Arg); S represents serine (Ser); V represents valine (Val); W represents tryptophan (Trp); and Y represents tyrosine (Tyr).
[0030] Example 2: In vitro α-amylase peptide inhibitory activity assay
[0031] 1. Reagents and Instruments
[0032] (1) Main reagents:
[0033] α-Amylase: Derived from porcine pancreas, enzyme activity ≥2000U / mg, purchased from Sigma-Aldrich (catalog number: A3176).
[0034] Starch: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (item number: 10021418);
[0035] DNS reagent: self-made (formulation: 1% 3,5-dinitrosalicylic acid, 20% sodium hydroxide, 30% potassium sodium tartrate, diluted to 1000mL).
[0036] Phosphate-buffered saline (PBS): prepared in-house (sodium dihydrogen phosphate and disodium hydrogen phosphate at a ratio of 0.05 mol / L, pH=6.8).
[0037] (2) Main instruments:
[0038] Absorbency meter: Thermo Fisher Multiskan FC microplate reader;
[0039] Incubator: Shanghai Yiheng Scientific Instruments Co., Ltd., Model DHP-9052;
[0040] Ultrapure water system: Milli-Q model from Millipore.
[0041] 2. Synthesis of bioactive peptides
[0042] Synthesis Method: The Fmoc solid-phase synthesis method was employed, utilizing an APEX 396 automated peptide synthesizer (AAPPTEC, USA) to synthesize active peptide samples with a purity >95%. Specific steps included: using the corresponding single-letter protecting amino acids from the C-terminus to the N-terminus. Taking the active peptide 1 sequence LPKLRVKV as an example, it includes Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, and Fmoc-Leu-OH. 1g of Fmoc-Val-OH (0.3 mmol / g degree of substitution) was weighed and placed in the reactor, and dichloromethane was added for swelling for half an hour, after which the dichloromethane was removed. Add the second amino acid in the sequence (0.6 mmol / L equivalent), 1-hydroxybenzotriazole (0.6 mmol / L), N,N-diisopropylcarbodiimide (0.6 mmol / L), and an appropriate amount of dichloromethane solution (the appropriate amount of dichloromethane can fully agitate the resin). React under nitrogen for 1 hour, then wash with dimethylformamide, methanol, and dichloromethane (three times each, for a total of 9 times). Dry the solvent and use ninhydrin reagent to determine if the reaction is complete; no blue color indicates a complete reaction. Remove the Fmoc protecting group, add 8-10 mL of 20% piperidine in dimethylformamide solution, react for 20 minutes, and wash with dimethylformamide, methanol, and dichloromethane (three times each, for a total of 9 times). The ninhydrin test should show a blue color. The third amino acid in the sequence was added to the reactor, and this process was repeated until the last amino acid in the sequence (Fmoc-Leu-OH) was added, indicating the reaction was complete. After the second-to-last amino acid in the sequence, Fmoc-Pro-OH, was added, ninhydrin should show a reddish-brown color when the solvent was washed away. 10 mL of lysis buffer (v / v, trifluoroacetic acid: p-cresol: water: Tis: thiol = 82.5:5:5:5:2.5) was added, and lysis was carried out at room temperature for 2-3 hours. The lysis buffer was then poured into 25 mL of pre-cooled anhydrous diethyl ether, and the mixture was stirred, allowed to stand, and centrifuged. The mixture was then washed four times with anhydrous diethyl ether and dried under vacuum to obtain the crude peptide. The crude peptide was purified to a purity >95% using an LC3000 high-performance liquid chromatography system. Finally, purified peptides containing three bioactive peptides were obtained.
[0043] The molecular weights of the three bioactive peptides were determined. The results showed that the measured molecular weight of bioactive peptide 1 was 1131.7136 Da, while its theoretical molecular weight was 1131.71 Da; the measured molecular weight of bioactive peptide 2 was 982.484 Da, while its theoretical molecular weight was 982.48 Da; and the measured molecular weight of bioactive peptide 3 was 832.39479 Da, while its theoretical molecular weight was 832.39 Da. The relative errors between the measured and theoretical molecular weights of the three bioactive peptides were all less than 0.1%, proving that the bioactive peptides were successfully synthesized.
[0044] 3. α-Amylase inhibitory activity and IC50 50 Determination of value
[0045] The specific steps for determining α-amylase inhibitory activity are as follows:
[0046] (1) Standard curve: Prepare maltose solutions of different concentrations, add DNS reagent, boil in water for 5 min, cool and make up to 10 mL, measure absorbance at 540 nm wavelength and plot the standard curve.
[0047] (2) Control group: Take a test tube and add 2 mL of 1% (w / v) starch solution and 1 mL of 0.05 mol / L pH 6.8 phosphate buffer. After preheating at 37℃ for 5 min, add 0.5 mL of 1 mg / mL α-amylase. Incubate at 37℃ for 10 min, add 2 mL of DNS reagent, boil in water for 5 min, cool and measure absorbance. Obtain the amount of reducing sugar in the control group through the standard curve.
[0048] (2) Experimental group: Take a test tube and add 2 mL of 1% (m / v) starch solution, 1 mL of 0.05 mol / L pH 6.8 phosphate buffer and 0.1 mL of 1 mg / mL active peptide sample solution. After preheating at 37℃ for 5 min, add 0.5 mL of 1 mg / mL α-amylase. Incubate at 37℃ for 10 min, add 2 mL of DNS reagent, boil in water for 5 min, cool and measure absorbance. Obtain the amount of reducing sugar in the experimental group through the standard curve.
[0049] (4) Based on the reducing sugar content of the control group and the experimental group, the α-amylase inhibitory activity (%) was calculated using the following formula: (1 - reducing sugar content of the experimental group / reducing sugar content of the control group) × 100%.
[0050] IC 50 The specific steps for value determination are as follows:
[0051] (1) Prepare sample solutions of the active peptide to be tested with gradient concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL and 80 μg / mL, and store at 4℃ for later use;
[0052] (2) Referring to the above method for determining the inhibitory activity of α-amylase, the enzyme inhibitory activity (%) corresponding to each gradient concentration of active peptide was determined. Three parallel groups were set up for each concentration and the average value was taken.
[0053] (3) Using the concentration of active peptide (μg / mL) as the x-axis and the enzyme inhibitory activity (%) as the y-axis, a nonlinear regression fitting (logistic equation) was performed using Origin software to obtain the dose-response curve. The concentration of active peptide corresponding to 50% inhibitory activity was read, which is the IC50 of the active peptide. 50 Value (μg / mL).
[0054] IC 50 The determination of enzyme activity was carried out in accordance with the relevant provisions on enzyme inhibition kinetics in GB / T 23747-2009 General Rules for Determination of Enzyme Activity in Feed Additives, and optimized by combining the gradient concentration-activity fitting method for evaluating the bioactivity of peptides in the "Detection Methods for Functional Components in Food".
[0055] The measurement results are shown in Table 3:
[0056] Table 3 Enzyme inhibitory activity and IC50 50 Value measurement results
[0057]
[0058] As can be seen, all three bioactive peptides exhibited good α-amylase inhibitory activity at low microgram concentrations (final concentration 27.8 μg / mL), with the bioactive peptide of sequence LPKLRVKV showing the highest inhibitory activity (32.75%), IC50... 50 The value was 35.2 μg / mL. This is in contrast to egg yolk high-phosphorus peptide (IC50). 50 Value is 0.8 μg / mL), acarbose (IC50) 50 Compared to the value of 3.17 μg / mL, the IC50 values of the three bioactive peptides mentioned above were significantly higher. 50 A higher value indicates that its enzyme inhibitory activity is lower than that of highly effective inhibitors such as egg yolk high-phosphorus protein peptide and acarbose, while it is lower than that of sea cucumber peptide SCP1 (IC50). 50 Compared to (with a value of 0.92 mg / mL), the IC50 values of the above three bioactive peptides were significantly higher. 50 The value is much lower than that of sea cucumber peptide SCP1, indicating that its enzyme inhibitory activity is significantly lower than that of sea cucumber peptide SCP1. In summary, the enzyme inhibitory activity of the three active peptides obtained in this invention is at a medium to high level.
[0059] Example 3: Application of α-amylase inhibitory active peptides
[0060] An α-amylase inhibitory peptide having the amino acid sequence described in Example 1 is obtained by solid-phase synthesis or directed enzymatic hydrolysis and purification. This peptide is then combined with a pharmaceutically acceptable carrier to prepare it into powder, granules, tablets, or liquid form, thereby creating an α-amylase inhibitor, a hypoglycemic drug, or a drug for the prevention or treatment of hyperglycemia-related diseases.
Claims
1. An α-amylase-inhibiting peptide, characterized in that: The active peptide was isolated from the free peptides of mandarin fish, and its amino acid sequence is shown in any one of the following: Leu-Pro-Lys-Leu-Arg-Val-Lys-Val; Val-Glu-Lys-Ser-Lys-Val-Tyr; Glu-Val-Ile-Glu-Leu-Asp-Trp-Arg.
2. The use of the α-amylase inhibitory active peptide according to claim 1 in the preparation of α-amylase inhibitors, hypoglycemic drugs, or drugs for the prevention and treatment of hyperglycemia-related diseases.