Use of a hypoglycemic peptide in the preparation of a medicine for inhibiting the activity of alpha-amylase and alpha-glucosidase

Abstract

The application discloses application of a hypoglycemic peptide in preparation of a medicine for inhibiting alpha-amylase and alpha-glucosidase activity. The hypoglycemic peptide is derived from Hericium erinaceus, and the amino acid sequence is AVFPSIVGRPR. The polypeptide is extracted and purified from Hericium erinaceus fruiting body protein, the amino acid sequence of one hypoglycemic peptide is determined by LC-MS / MS, the alpha-amylase inhibition rate is 71.86%±0.08% through hypoglycemic activity detection, and the alpha-glucosidase inhibition rate is 61.52%±0.15%, so the hypoglycemic peptide can be used as a natural hypoglycemic peptide and has a potential application prospect in the field of preparation of medicines for reducing blood sugar and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological extract technology and relates to the application of a hypoglycemic peptide in the preparation of drugs that inhibit the activity of α-amylase and α-glucosidase. Background Technology

[0002] Currently, diabetes has become the third leading chronic non-communicable disease after cardiovascular and cerebrovascular diseases and malignant tumors, and it is gradually becoming a prevalent disease. The number of people with diabetes worldwide has reached 420 million. High postprandial blood glucose is considered a prominent feature of prediabetic patients. Sustained hyperglycemia can lead to various complications, such as diabetic nephropathy, diabetic retinopathy, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, and diabetic neuropathy, among which severe cases can threaten the patient's life. Widely used hypoglycemic drugs in clinical practice include acarbose, miglitol, and voglibose; however, they are frequently reported to cause diarrhea, bloating, corresponding abdominal pain, and liver disease—these are the most common side effects—and are also expensive. Therefore, finding new hypoglycemic drugs with fewer side effects has become particularly important. Natural bioactive peptides, due to their high efficacy, safety, tolerability, high selectivity, and low accumulation rate in the body, have become an excellent source.

[0003] Studies have shown that edible fungi contain abundant hypoglycemic active ingredients, including proteins, polypeptides, polysaccharides, and terpenoids, among other bioactive substances. Compared with animal and plant sources, edible fungi have become an ideal source of raw materials for developing hypoglycemic drugs due to their short growth cycle and ease of cultivation. These characteristics not only reduce production costs but also make large-scale industrial production possible, demonstrating the important application value of edible fungi in the development of functional foods and drugs. Luo Liping et al. studied the in vitro antioxidant and hypoglycemic activities of extracts from Agrocybe aegerita, Lactarius deliciosus, and Dictyophora indica. The results showed that at a concentration of 5 mg / mL, the Agrocybe aegerita extract inhibited α-amylase and α-glucosidase by 62.15% and 57.08%, respectively, showing certain hypoglycemic activity, but the inhibitory effect was not significant (Luo Liping, Li Bingjing, Zhao Jingfang, et al. Study on in vitro antioxidant and hypoglycemic activities of three edible fungi extracts [J]. Food Industry Technology, 2020, 41(20):324-329.). Chinese patent application 202310490895.0 discloses a β-actin antimicrobial peptide PVQ9 from Litopenaeus vannamei and its application. Its amino acid sequence is AVFPSIVGRPR, and it has a significant inhibitory effect on Staphylococcus aureus, Bacillus cereus, Staphylococcus aureus, or Curella giardi.

[0004] Hericium erinaceus contains a variety of active ingredients, especially abundant protein. To date, there are no reports on hypoglycemic peptides derived from Hericium erinaceus fruiting bodies. Summary of the Invention

[0005] This invention provides the application of a hypoglycemic peptide in the preparation of drugs that inhibit α-amylase and α-glucosidase activity.

[0006] The hypoglycemic peptide described in this invention is derived from Hericium erinaceus and has the amino acid sequence AVFPSIVGRPR, namely Ala-Val-Phe-Pro-Ser-Ile-Val-Gly-Arg-Pro-Arg, as shown in SEQ ID NO.1.

[0007] The present invention also provides the application of the above-mentioned hypoglycemic peptide in the preparation of hypoglycemic drugs.

[0008] Compared with the prior art, the present invention has the following advantages:

[0009] This invention is the first to extract and purify polypeptides from Hericium erinaceus fruiting body protein, discovering several hypoglycemic peptides with good blood glucose-lowering ability. The amino acid sequence of one of these hypoglycemic peptides was determined by LC-MS / MS as AVFPSIVGRPR. The hypoglycemic activity test showed that the α-amylase inhibition rate was 71.86%±0.08% and the α-glucosidase inhibition rate was 61.52%±0.15%, which is significant. It can be used as a natural hypoglycemic peptide and has potential application prospects in the preparation of hypoglycemic drugs. Attached Figure Description

[0010] Figure 1 The inhibition rates of three polypeptides with different molecular weights (C1, C2, and C3) on α-glucosidase and α-amylase are shown.

[0011] Figure 2 The separated products Q1, Q2, and Q3 are obtained by passing through an anion exchange column.

[0012] Figure 3 The separated products q1, q2, and q3 are obtained by passing through an anion exchange column.

[0013] Figure 4 The α-glucosidase inhibition rate of the separation products Q1, Q2, and Q3 obtained by passing through an anion exchange column.

[0014] Figure 5 The α-glucosidase inhibition rate of the separation products q1, q2, and q3 obtained by passing through an anion exchange column.

[0015] Figure 6 To separate products G1, G2, G3, and G4 using a gel column.

[0016] Figure 7 The α-glucosidase inhibition rate of the isolated products G1, G2, G3, and G4.

[0017] Figure 8 This is the TIC map of G2.

[0018] Figure 9 This is a secondary mass spectrum of the active polypeptide.

[0019] Figure 10 This is for the prediction of the structure of active polypeptides.

[0020] Figure 11 The figures show 2D (a) and 3D (b) diagrams of the docking of the active polypeptide with the α-glucosidase molecule.

[0021] Figure 12 The images show 2D (a) and 3D (b) diagrams of the docking of the active polypeptide with the α-amylase molecule. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0023] In the following examples, the α-amylase inhibition rate was determined according to the method described in (Su Na. Preparation and identification of hypoglycemic bioactive peptides in camel milk protein [D]. Hohhot: Inner Mongolia Agricultural University, 2020.), and the α-glucosidase inhibition rate was determined according to the method described in (Zhao Hongxing. Preparation of hypoglycemic bioactive peptides and purification and identification of α-glucosidase-inhibiting bioactive peptides [D]. Harbin: Harbin Institute of Technology, 2018.).

[0024] Example:

[0025] (1) Fresh Hericium erinaceus fruiting bodies were washed, cut into small pieces, and quick-frozen in liquid nitrogen. After freeze-drying using a freeze dryer, the freeze-dried fruiting bodies were pulverized into powder and passed through a 60-mesh sieve. The powder and ultrapure water were mixed at a mass ratio of 1:10, dissolved by sonication, and allowed to stand for 4 hours. The solution was centrifuged at 4 ℃ and 8000 r / min for 20 minutes, and the supernatant was removed. Ammonium sulfate was added to achieve a final concentration of 80% saturation (561 g of solid ammonium sulfate per liter of solution). After sonication to dissolve the solution, it was allowed to stand for 12 hours. The solution was then centrifuged at 4 ℃ and 8000 r / min for 20 minutes to collect the precipitate. The precipitate was dissolved in ultrapure water and placed in a 10 kDa dialysis bag. Dialysis was performed in an aqueous solution environment at 4 ℃ for 48 hours. After the dialysis, the product was freeze-dried into powder to obtain Hericium erinaceus protein.

[0026] (2) Hericium erinaceus protein was dissolved in ultrapure water, and the environment was adjusted to suitable conditions for alkaline protease (pH 9, 55 ℃) using 1 mol / L sodium hydroxide and hydrochloric acid. After the environment reached a stable state, 4% alkaline protease was added, and after 4 hours of enzymatic hydrolysis, the reaction was terminated by a water bath at 90 ℃ for 15 minutes. Finally, the mixture was centrifuged at 4 ℃ and 8000 r / min for 15 min, and the supernatant was lyophilized to obtain the enzymatic hydrolysis product. The Hericium erinaceus active peptides obtained after enzymatic hydrolysis were dissolved and filtered through a 0.22 μm filter membrane. The mixture was centrifuged at 4000 r / min for 60 min using 10 kDa and 3 kDa ultrafiltration tubes to obtain three polypeptide fractions with different molecular weights: less than 3 kDa, between 3 kDa and 10 kDa, and greater than 10 kDa, named C1, C2, and C3. The inhibition rates of the three polypeptides with different molecular weights on α-glucosidase and α-amylase were tested. The results are as follows: Figure 1 As shown in the figure, the polypeptide component with the optimal inhibition rate of α-amylase and α-glucosidase activity is C1.

[0027] (3) C1 was purified by a first stepwise gradient using a Q Sepharose FF anion exchange column (the eluent was NaCl (2 M pH 7.5) B solution). As shown in Figure 2, three elution peaks were generated, namely Q1, Q2, and Q3. The hypoglycemic activity of these three elution peaks was evaluated by the inhibition rate of α-glucosidase. The results are shown in Figure 4. Among the three components, Q2 showed better activity, with an α-glucosidase inhibition rate of 0.4974 ± 0.0166. Based on the fact that the gradient of Q2 appeared at 25% B (the proportion of Buffer B was 25%), the elution conditions were optimized again. C1 was linearly eluted through a Q Sepharose FF anion exchange column from 0 to 25% B for 20 column volumes, yielding the following results: Figure 3 As shown, three elution peaks were generated, namely q1, q2, and q3. The hypoglycemic activity of these three elution peaks was evaluated by the inhibition rate of α-glucosidase, and the results are as follows. Figure 5 As shown, q2 exhibited better activity, with an α-glucosidase inhibition rate of 62.95 ± 0.13%.

[0028] The q2 separated from the anion exchange column was further purified using a Superdex 30 Increase10 / 300 GL gel column and analyzed by absorbance at 220 nm. Four distinct components were successfully separated, as shown in the results. Figure 6As shown. Based on the elution order, the four obtained components were named G1, G2, G3, and G4. Gel column chromatography was used to separate and purify the components according to their molecular weight. The inhibition rates of α-glucosidase by G1, G2, G3, and G4 were measured, and the results are shown below. Figure 7 As shown in the figure, G2 exhibited significantly higher hypoglycemic activity than the other three components, with an inhibition rate of 83.24±0.37% against α-glucosidase. Furthermore, its hypoglycemic activity was also improved compared to q2 after ion column purification. This indicates that the hypoglycemic peptides of Hericium erinaceus were further purified, enhancing their hypoglycemic activity, and that the hypoglycemic peptides of Hericium erinaceus were mainly concentrated in G2.

[0029] (4) The amino acid sequence of the hypoglycemic peptide at G2 of Hericium erinaceus was determined by LC-MS / MS. Figure 8 The TIC (Transmission Indication Characteristic) spectrum of G2 in *Hericium erinaceus* is shown. Thirty-three sequences were obtained and uploaded to PeptideRanker for activity prediction. PeptideRanker provided scores for 34 peptides in G2, with one hypoglycemic active peptide scoring above 0.5, indicating a high potential for biological activity. The amino acid sequence of this hypoglycemic active peptide is AVFPSIVGRPR, as shown in SEQ ID NO.1, and its mass spectrum is shown below. Figure 9 As shown, the molecular weight is 1198.71 Da.

[0030] (5) The properties of the AVFPSIVGRPR peptide were analyzed by bioinformatics. The physicochemical properties of this peptide were predicted by the ProParam online software: the theoretical isoelectric point is 12; it has two positively charged amino acid residues; the stability is 39.33, which is good; the predicted half-life in mammalian reticulocytes is 4.4 h, the half-life in yeast is greater than 20 h, and the half-life in Escherichia coli is greater than 10 h; it is a hydrophilic peptide.

[0031] The safety of the obtained Hericium erinaceus hypoglycemic peptide was evaluated using Toixinpred software, and its toxicity and sensitization were predicted. The results showed that it was non-toxic and non-sensitizing, and had high safety.

[0032] (6) The AVFPSIVGRPR peptide was obtained by solid-phase synthesis at Sangon Biotech Co., Ltd., and its hypoglycemic activity was determined. The α-amylase inhibition rate was 71.86%±0.08%, and the α-glucosidase inhibition rate was 61.52%±0.15%. PepDraw predicted the structure of the obtained Hericium erinaceus hypoglycemic peptide, and ChemDraw plotted its 2D structure. The results are shown in […]. Figure 10 .

[0033] To explore the binding mechanism of AVFPSIVGRPR with α-amylase and α-glucosidase, the drawn 2D structures were converted into 3D structures using Chem3D. Molecular docking was performed using MOE (2019). According to Zhao Kangqi et al. (Zhao Kangqi, Jin Aijing, Wang Xiaoyue, et al. Hypoglycemic effect of Angelica dahurica in zebrafish and molecular docking study [J]. Information on Traditional Chinese Medicine, 2023, 40(06): 32-7.), the PDB code for α-amylase is 1SMD, and the PDB code for α-glucosidase is 5KZW. The 3D structure of the protein was downloaded by entering the PDB code in PDBhttps: / / www.rcsb.org / . The downloaded large protein was imported into MOE, water molecules and small ligands were removed, and redundant chains were deleted. QuickPerp was then clicked to complete the large molecule processing. Since the docking pocket sites were unknown, the small molecule library and the large protein were subjected to full atom docking. After docking, the structure with the lowest score and the most stable structure was selected from the docking results. Through docking, 2D and 3D diagrams of the docking between AVFPSIVGRPR and α-glucosidase molecules were obtained, as shown below. Figure 11 As shown, AVFPSIVGRPR forms three hydrogen bonds with α-amylase, with binding sites Thr6, Ser3, and Lys227, where the hydrogen bond energy is the largest at Ser3; and five hydrogen bonds with α-glucosidase, with binding sites Asp351, Glu344, Lys200, and Lys347, where there are two hydrogen bonds at Glu344 and the largest bond energy at Lys347.

Claims

1. The application of a hypoglycemic peptide in the preparation of hypoglycemic drugs, characterized in that, The hypoglycemic peptide is derived from Hericium erinaceus, and its amino acid sequence is AVFPSIVGRPR.

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