Red kidney bean hypoglycemic active peptide and application thereof

Abstract

The invention belongs to the technical field of plant active peptides, and particularly relates to a red kidney bean hypoglycemic active peptide and application thereof. The amino acid sequences of the hypoglycemic active peptide are FFGGPAF, SAKPFFSGL and PPGHPFVTF. The invention further discloses a preparation method of the hypoglycemic active peptide. FF, SL and PF for short. The polypeptide is prepared through enzymolysis of red kidney bean protein, and the polypeptide is identified by utilizing ultrafiltration separation and LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry); the hypoglycemic active peptides which are high in hypoglycemic effect, free of toxicity, carcinogenicity and sensitization and capable of being absorbed by human intestinal tracts are screened out through a bioinformatics method and molecular docking, then the screened hypoglycemic active peptides and alpha-glucosidase are subjected to molecular docking, and finally three hypoglycemic active peptides FF, SL and PF are screened out. The invention finds that the hypoglycemic active peptides FF, SL and PF have high inhibitory activity on alpha-glucosidase, can obviously promote glucose consumption of insulin resistance model cells, and can be used for preparing hypoglycemic drugs, food and health care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant active peptides, and particularly relates to a red kidney bean hypoglycemic active peptide and its application. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by hyperglycemia caused by abnormal insulin metabolism or insufficient secretion, which can lead to disorders in the metabolism of carbohydrates, proteins, fats, etc. Severe diabetic patients may develop chronic progressive lesions, functional decline or even failure of tissues and organs such as the eyes, kidneys, nerves, heart, blood vessels, etc., and may also cause acute severe metabolic disorders. Currently, the methods for treating diabetes include injecting insulin and taking oral hypoglycemic drugs, but these treatment methods are prone to cause serious adverse reactions. Therefore, searching for safe and effective natural hypoglycemic drugs is a current research hotspot.

[0003] The main ways to prevent and treat diabetes include inhibiting α-glucosidase, α-amylase, etc. Among them, α-glucosidase belongs to the digestive enzymes in the intestine and can cleave carbohydrates into free glucose. Therefore, blocking the activity of α-glucosidase can effectively reduce blood sugar levels. Food-derived hypoglycemic peptides can play a hypoglycemic role by inhibiting α-glucosidase. Food-derived hypoglycemic peptides have the characteristics of safety, naturalness, high efficiency, etc. and have become an important part of the non-drug treatment of diabetes.

[0004] Red kidney bean is a herbaceous plant of the genus Phaseolus in the legume family, containing rich proteins and having high nutritional and medicinal values. According to ancient medical records, the fruit shell, seeds, and roots of red kidney bean can all be used as medicine. It tastes sweet and flat, and is warm in nature, and can warm the middle-jiao and lower qi, benefit the intestines and stomach, replenish the vital energy of the kidney, etc. Red kidney bean has functions such as improving the body's immune ability and promoting the body's detoxification. The dietary fiber in red kidney bean can make food pass through more quickly, reduce sugar absorption, and improve blood sugar. In addition, red kidney bean polysaccharide can effectively reduce hyperglycemia and hyperlipidemia in diabetic rats. However, so far, there are few research reports on red kidney bean hypoglycemic peptides at home and abroad. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to overcome the deficiencies of the above-mentioned existing technologies and provide a red kidney bean hypoglycemic active peptide and its application.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] The first aspect of the present invention is a red kidney bean hypoglycemic active peptide, and its amino acid sequence is FFGGPAF(FF), SAKPFFSGL(SL) or PPGHPFVTF(PF).

[0008] Furthermore, the molecular weights of the amino acid sequences FFGGPAF, SAKPFFSGL, and PPGHPFVTF are 741.3476 Da, 952.5003 Da, and 997.5007 Da, respectively.

[0009] The second aspect of the present invention is the application of the red kidney bean hypoglycemic active peptide described in the first aspect in the preparation of hypoglycemic drugs, hypoglycemic foods, and hypoglycemic health products.

[0010] The third aspect of the present invention is a hypoglycemic drug, which includes the red kidney bean hypoglycemic active peptide described in the first aspect.

[0011] The fourth aspect of the present invention is a hypoglycemic food, which includes the red kidney bean hypoglycemic active peptide described in the first aspect.

[0012] The fifth aspect of the present invention is a hypoglycemic health product, which includes the red kidney bean hypoglycemic active peptide described in the first aspect.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The present invention uses red kidney beans as raw materials to enzymatically prepare active polypeptides, separates polypeptides by ultrafiltration, and identifies polypeptides by LC-MS / MS; screens hypoglycemic active peptides using bioinformatics methods; artificially synthesizes hypoglycemic active peptides and verifies the hypoglycemic activity of hypoglycemic active peptides through experiments. The obtained red kidney bean hypoglycemic active peptides can significantly inhibit the activity of α-glucosidase, promote glucose consumption in the hepatic insulin resistance cell model, and can be used in the preparation of hypoglycemic drugs, foods, or health products. Description of the Drawings

[0015] Figure 1 Structures of the red kidney bean hypoglycemic active peptides FF, SL, and PF;

[0016] Figure 2 Molecular docking results of the red kidney bean hypoglycemic active peptide FF with α-glucosidase;

[0017] Figure 3 Molecular docking results of the red kidney bean hypoglycemic active peptide SL with α-glucosidase;

[0018] Figure 4 Molecular docking results of the red kidney bean hypoglycemic active peptide PF with α-glucosidase;

[0019] Figure 5 Effects of the red kidney bean hypoglycemic active peptides FF, SL, and PF on the activity of α-glucosidase;

[0020] Figure 6 Effects of the red kidney bean hypoglycemic active peptides FF, SL, and PF on the viability of HepG2 cells.

[0021] Figure 7 Effect of red kidney bean hypoglycemic active peptides FF, SL, and PF on glucose consumption in IR-HepG2 cells induced by high glucose. Detailed implementation manners

[0022] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Unless otherwise defined, all the technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0024] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0025] Example 1

[0026] Screening of red kidney bean hypoglycemic active peptides

[0027] Using red kidney beans as raw materials, active polypeptides were prepared by pepsin hydrolysis, ultrafiltration separation was carried out according to molecular weight size, and the components with a molecular weight less than 3KD were identified by LC-MS / MS. PeptideRanker was used to perform potential function scoring on the polypeptides. A score greater than 0.5 indicates that the polypeptide has potential biological activity; the BIOPEP database was used to analyze the polypeptides screened by PeptideRanker with a score greater than 0.5, and a score prediction was made on whether they have hypoglycemic activity. The ADMET program was used to evaluate the toxicity, carcinogenicity, and intestinal absorption ability of the polypeptides, and polypeptides without acute oral toxicity, without carcinogenicity, and with good human intestinal absorption ability were screened out; the AllergenFP program was used to evaluate the allergenicity of the polypeptides, and polypeptides without allergenicity were screened out.

[0028] Table 1 shows the functional characteristics of red kidney bean hypoglycemic active peptides FF, SL, and PF. Figure 1 Structures of red kidney bean hypoglycemic active peptides FF, SL, and PF.

[0029] Table 1 Functional characteristics of red kidney bean hypoglycemic active peptides FF, SL, and PF

[0030]

[0031] Example 2

[0032] Molecular docking of red kidney bean hypoglycemic active peptides with α-glucosidase

[0033] The secondary structures of the red kidney bean hypoglycemic active peptides FF, SL, and PF were drawn using Chemdraw 3D software; the x-ray crystal structure of the receptor protein α-glucosidase (PDB ID: 5NN3) was downloaded from the RCSB protein database. In the Discovery studio 2.5 software, the hypoglycemic active peptides were respectively docked with the receptor protein α-glucosidase.

[0034] The docking results of the hypoglycemic active peptide FF with α-glucosidase are as Figure 2 shown. The hypoglycemic active polypeptide FF binds to Ile 780 and Gln 776 of the α-glucosidase protein residues through hydrogen bonds, and binds to Pro 397, Pro 829, Arg 696, Lys 697, Val 934, and Leu 701 of the residues through hydrophobic interactions; the "LibDockScore" between the hypoglycemic active peptide FF and α-glucosidase is 188.801, indicating its tight binding to α-glucosidase.

[0035] The docking results of the hypoglycemic active peptide SL with α-glucosidase are as Figure 3 shown. The hypoglycemic active peptide SL binds to Val 934, Thr 927, Asn 925, Ilf 780, Cso 938, and Gln 776 of the α-glucosidase protein residues through hydrogen bonds, and binds to Lys 697 and Leu 701 of the residues through hydrophobic interactions; the "LibDockScore" between the hypoglycemic active peptide SL and α-glucosidase is 177.186, indicating its tight binding to α-glucosidase.

[0036] The docking results of the hypoglycemic active peptide PF with α-glucosidase are as Figure 4 shown. The hypoglycemic active peptide PF binds to Gln 693 and Thr 600 of the α-glucosidase protein residues through hydrogen bonds, and binds to Pro 779, Val 934, Arg 696, Lys 697, Leu 831, and Pro 397 of the residues through hydrophobic interactions; the "LibDockScore" between the hypoglycemic active peptide PF and α-glucosidase is 167.976, indicating its tight binding to α-glucosidase.

[0037] Example 3

[0038] Synthesis of red kidney bean hypoglycemic active peptides

[0039] The peptide segments were commissioned to Shanghai Sangon Biotech Co., Ltd. for synthesis, with a purity ≥ 95%, meeting the requirements of related experiments for activity detection.

[0040] Example 4

[0041] Effect of Red Kidney Bean Hypoglycemic Active Peptide on α-Glucosidase Activity

[0042] Take 40 μL of FF, SL, and PF sample solutions with different concentrations respectively, mix them with 40 μL of 1 U / mL α-glucosidase solution, incubate at 37 °C for 15 min, add 20 μL of 2.5 mM pNPG solution, react at 37 °C for 15 min, and then add 150 μL of 0.2 M Na2CO3 solution to stop the reaction. Measure the absorbance at a wavelength of 405 nm. The sample group is A1, the blank group A0 is replaced by PBS instead of the sample, and the control group A2 is replaced by PBS instead of the α-glucosidase solution. Calculate the α-glucosidase inhibitory activity according to the following formula.

[0043]

[0044] The results are as Figure 5 shown. The red kidney bean hypoglycemic active peptides FF, SL, and PF have obvious inhibitory effects on α-glucosidase. The IC50 values of the active peptides FF, SL, and PF are 6.357 μg / mL, 8.685 μg / mL, and 11.830 μg / mL respectively.

[0045] Example 5

[0046] Effect of Red Kidney Bean Hypoglycemic Active Peptide on the Survival of HepG2 Cells

[0047] Take HepG2 cells in the logarithmic growth phase and seed them into a 96-well culture plate at a density of 1×10 4 cells / well. Culture overnight at 37 °C. Add the red kidney bean hypoglycemic active peptide of Example 1 respectively to make the final concentrations 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, and 10 μg / mL. Set six replicates for each concentration, and use the medium containing the same volume of Tris-Hcl buffer as the control. After incubation for 24 h, add 20 μL of MTT solution (5 mg / mL) to each well. After 4 h, discard the supernatant, add DMSO, and shake for 10 min. Measure the absorbance at a wavelength of 570 nm.

[0048] The results are as Figure 6 shown. The red kidney bean hypoglycemic active peptides FF, SL, and PF have no obvious effect on the growth of HepG2 cells in the concentration range of 0 - 10 μg / mL.

[0049] Example 6

[0050] Effect of Red Kidney Bean Hypoglycemic Active Peptide on Glucose Consumption in a Hepatic Insulin Resistance Cell Model

[0051] Take HepG2 cells in the logarithmic growth phase and seed them into a 96-well culture plate at a density of 1×10 4Add them / holes into a 96-well plate. After the cells adhered to the wall, culture them in starvation for 12 h, and then culture them with a medium containing 30 mM glucose for 24 h to induce the establishment of insulin-resistant HepG2 cells (IR-HepG2). Finally, add FF, SL, and PF active peptide solutions at 5 μg / mL and 10 μg / mL and culture for 24 h. Take the cell culture medium and measure the glucose consumption according to the instructions of the glucose assay kit.

[0052] The results are as Figure 7 shown. The hypoglycemic active peptides FF, SL, and PF from red kidney beans significantly promoted the glucose consumption of IR-HepG2 cells.

[0053] The above are only examples for better explaining the present invention, not limitations thereof. Any modification or equivalent replacement without departing from the spirit and scope of the present invention shall fall within the scope covered by the present invention.

Claims

1. A red kidney bean hypoglycemic active peptide, characterized in that: Its amino acid sequence is FFGGPAF, SAKPFFSGL or PPGHPFVTF.

2. The red kidney bean hypoglycemic active peptide according to claim 1, characterized in that The molecular weights of the amino acid sequence FFGGPAF, the amino acid sequence SAKPFFSGL and the amino acid sequence PPGHPFVTF are 741.3476Da, 952.5003Da and 997.5007Da respectively.

3. Use of the red kidney bean hypoglycemic active peptide according to claim 1 in the preparation of hypoglycemic drugs, hypoglycemic foods and hypoglycemic health products.

4. A hypoglycemic drug, characterized in that: The invention comprises the red kidney bean hypoglycemic active peptide according to claim 1.

5. A hypoglycemic food, characterized in that: The invention comprises the red kidney bean hypoglycemic active peptide according to claim 1.

6. A blood sugar lowering health product, characterized in that: The invention comprises the red kidney bean hypoglycemic active peptide according to claim 1.

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