Application of hypoglycemic peptide in preparation of medicine for inhibiting activity of alpha-amylase and alpha-glucosidase

Abstract

The invention discloses an application of a hypoglycemic peptide in preparation of a medicine for inhibiting activity of alpha-amylase and alpha-glucosidase. The hypoglycemic peptide is derived from hericium erinaceus, and the amino acid sequence of the hypoglycemic peptide is AVFPSIVGRPR. Polypeptides are extracted and purified from hericium erinaceus sporocarp protein, an amino acid sequence of one of the peptides for reducing blood sugar is determined through LC-MS / MS, through blood sugar reducing activity detection, the inhibition rate of alpha-amylase is 71.86% + / -0.08%, the inhibition rate of alpha-glucosidase is 61.52% + / -0.15%, the peptide can be used as a natural peptide for reducing blood sugar, and the peptide can be used as a natural peptide for reducing blood sugar. Potential application prospects are realized in the fields of preparation of medicines for reducing blood sugar and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extracts and relates to the application of a hypoglycemic peptide in the preparation of a drug for inhibiting the activities of alpha-amylase and alpha-glucosidase. Background Art

[0002] Diabetes has become the third most common chronic non-communicable disease, following cardiovascular and cerebrovascular diseases and malignant tumors, and has become a prevalent disease. Currently, the number of people with diabetes worldwide has reached 420 million. High postprandial blood sugar levels are considered a hallmark of prediabetes. Persistent hyperglycemia can lead to a variety of complications, including diabetic nephropathy, diabetic eye complications, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, and diabetic neuropathy. Severe cases can be life-threatening. Widely used clinically, hypoglycemic drugs include acarbose, miglitol, and voglibose. However, these drugs are frequently reported to cause diarrhea and bloating, along with abdominal pain and liver disease, as their most common side effects. Furthermore, they are expensive. Therefore, the search for new hypoglycemic drugs with fewer side effects is crucial. Natural biopeptides offer a promising source due to their high efficacy, safety, strong tolerability, high selectivity, and low body accumulation.

[0003] Studies have shown that edible fungi contain a wealth of hypoglycemic active ingredients, including proteins, peptides, polysaccharides, terpenoids and other bioactive substances. Compared with animal and plant sources, edible fungi have become an ideal source of raw materials for the development of hypoglycemic drugs due to their advantages such as short growth cycle and simple cultivation. These characteristics not only reduce production costs, but also provide the possibility for large-scale industrial production, showing the important application value of edible fungi in the field of functional food and drug development. Luo Liping et al. studied the in vitro antioxidant and hypoglycemic activities of extracts from Agrocybe tumefaciens, Lactarius pine and Dictyophora truncatula. The results showed that at a concentration of 5 mg / mL, the inhibition rates of Agrocybe tumefaciens extract on α-amylase and α-glucosidase reached 62.15% and 57.08%, respectively, showing a certain hypoglycemic activity, but the inhibitory effect was not significant (Luo Liping, Li Bingjing, Zhao Jingfang, et al. Study on the antioxidant and hypoglycemic activities of three edible fungi extracts in vitro [J]. Food Industry Science and Technology, 2020, 41(20): 324-329.). Chinese patent application 202310490895.0 discloses a white shrimp β-actin antimicrobial peptide PVQ9 and its application, whose amino acid sequence is AVFPSIVGRPR, and has a significant inhibitory effect on Staphylococcus aureus, Bacillus cereus, Staphylococcus squirrel or Kurtia gibbsii.

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

[0005] The present invention provides an application of a hypoglycemic peptide in the preparation of a drug for inhibiting the activities of alpha-amylase and alpha-glucosidase.

[0006] The hypoglycemic peptide of the present invention is derived from Hericium erinaceus, and has an amino acid sequence of AVFPSIVGRPR, i.e., 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 use of the hypoglycemic peptide in the preparation of hypoglycemic drugs.

[0008] Compared with the prior art, the present invention has the following advantages: The present invention is the first to extract and purify polypeptides from Hericium erinaceus fruiting body proteins, and discover multiple hypoglycemic peptides with good blood sugar lowering ability. The amino acid sequence of one of the hypoglycemic peptides was determined to be AVFPSIVGRPR by LC-MS / MS. After hypoglycemic activity detection, the α-amylase inhibition rate was 71.86%±0.08%, and the α-glucosidase inhibition rate was 61.52%±0.15%, with significant effects. It can be used as a natural hypoglycemic peptide and has potential application prospects in the preparation of blood sugar lowering drugs and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The inhibition rates of three peptides with different molecular weights, C1, C2 and C3, on α-glucosidase and α-amylase.

[0010] Figure 2 These are the separated products Q1, Q2, and Q3 after passing through the anion column.

[0011] Figure 3 These are the separated products q1, q2, and q3 after passing through the anion column.

[0012] Figure 4 It is the α-glucosidase inhibition rate of the separation products Q1, Q2, and Q3 passing through the anion column.

[0013] Figure 5 is the α-glucosidase inhibition rate of the separated products q1, q2, and q3 passing through the anion column.

[0014] Figure 6 The products G1, G2, G3 and G4 were separated by gel column.

[0015] Figure 7 is the α-glucosidase inhibition rate of the separated products G1, G2, G3, and G4.

[0016] Figure 8 This is the TIC map of G2.

[0017] Figure 9 This is the secondary mass spectrum of the active peptide.

[0018] Figure 10 For the prediction of active peptide structures.

[0019] Figure 11 2D (a) and 3D (b) images of the docking of the active peptide and α-glucosidase molecule.

[0020] Figure 12 2D (a) and 3D (b) images of the docking of active peptide and α-amylase molecule. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0022] In the following examples, the α-amylase inhibition rate was determined by referring to the method of (Su Na. Preparation and identification of hypoglycemic bioactive peptides from camel milk protein [D]. Hohhot: Inner Mongolia Agricultural University, 2020.), and the α-glucosidase inhibition rate was determined by referring to the method of (Zhao Hongxing. Preparation of hypoglycemic active peptides and purification and identification of α-glucosidase inhibitory active peptides [D]. Harbin: Harbin Institute of Technology, 2018.).

[0023] Example

[0024] (1) Wash and cut fresh Hericium erinaceus fruiting bodies into small pieces and freeze them in liquid nitrogen. After freeze-drying in a freeze dryer, crush the freeze-dried fruiting bodies into powder using a grinder and pass through a 60-mesh sieve. Mix the powder with ultrapure water in a mass ratio of 1:10, dissolve it by ultrasonication, and let it stand for 4 hours. Centrifuge the solution at 4°C and 8000 r / min for 20 minutes, remove the upper liquid, add ammonium sulfate to make the final concentration reach 80% saturation (add 561 g of solid ammonium sulfate per liter of solution), dissolve it by ultrasonication, and let it stand for 12 hours. Centrifuge the solution at 4°C and 8000 r / min for 20 minutes to obtain the precipitate. Dissolve the precipitate in ultrapure water, then place it in a 10 kDa dialysis bag and dialyze it in an aqueous solution environment at 4°C for 48 hours. After the dialyzation, freeze-dry it into powder to obtain Hericium erinaceus protein.

[0025] (2) Hericium erinaceus protein was dissolved in ultrapure water, and the environment was adjusted to the suitable conditions for alkaline protease (pH 9, 55 ℃) using 1 mol / L sodium hydroxide and hydrochloric acid. After the environment reached a stable state, alkaline protease with a substrate mass of 4% was added. After enzymatic hydrolysis for 4 hours, the reaction was terminated by water bath at 90 ℃ for 15 minutes. Finally, the mixture was centrifuged at 4 ℃ and 8 000 r / min for 15 minutes, and the supernatant was freeze-dried to obtain the enzymatic hydrolysis product. The Hericium erinaceus active peptide obtained after enzymatic hydrolysis was dissolved and filtered through a 0.22 μm filter membrane. It was centrifuged at 4000 r / min for 60 minutes using 10 kDa and 3 kDa ultrafiltration tubes to obtain three polypeptide components with different molecular weights, namely less than 3 kDa, between 3 kDa and 10 kDa, and greater than 10 kDa. They were named C1, C2, and C3. The inhibition rates of the three polypeptides with different molecular weights on α-glucosidase and α-amylase were tested respectively. The results are shown in Figure 2. Figure 1 As shown in the figure, it can be seen that the polypeptide component with the best inhibition rate of α-amylase and α-glucosidase activity is C1.

[0026] (3) C1 was purified by the first step gradient on a Q Sepharose FF anion 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, and C1 was linearly eluted from 0 to 25% B on a Q Sepharose FF anion column for 20 column volumes to obtain the following. Figure 3 As shown in Figure 2, three elution peaks are generated, namely q1, q2, and q3. The hypoglycemic activity of these three elution peaks is evaluated by the inhibition rate of α-glucosidase. The results are shown in Figure 2. Figure 5 As shown, Q2 exhibited better activity, with an inhibition rate of 62.95 ± 0.13% for α-glucosidase.

[0027] The q2 separated from the anion column was further purified by Superdex 30 Increase10 / 300 GL gel column and detected by absorbance at 220 nm. Four different components were successfully separated. Figure 6As shown. According to the order of peaks, the four components obtained were named G1, G2, G3, and G4. The gel column was used to separate and purify the components according to their molecular weight, and the inhibition rates of G1, G2, G3, and G4 on α-glucosidase were measured respectively. The results are shown in Figure 7 The hypoglycemic activity of G2 was significantly higher than that of the other three components, with an inhibition rate of 83.24±0.37% on α-glucosidase. Compared with the q2 purified by ion column, the hypoglycemic activity of G2 was also improved to a certain extent, indicating that the hypoglycemic peptide of Hericium erinaceus was further purified, which improved its hypoglycemic activity, and that the Hericium erinaceus hypoglycemic peptide was mainly concentrated in G2.

[0028] (4) The amino acid sequence of the hypoglycemic peptide at Hericium erinaceus G2 was determined using LC-MS / MS. Figure 8 This is the TIC spectrum of Hericium erinaceus G2. 33 sequences were obtained and uploaded to PeptideRanker for activity prediction. PeptideRanker gave scores for 34 peptides in G2, among which one hypoglycemic active peptide scored higher than 0.5, indicating that this peptide showed a high possibility of 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 in Figure 9 As shown, the molecular weight is 1198.71 kDa.

[0029] (5) The performance of the AVFPSIVGRPR peptide was analyzed through bioinformatics. The physicochemical properties of the peptide were predicted using the ProParam online software: the theoretical isoelectric point is 12; it contains two positively charged amino acid residues; the stability is 39.33, indicating good stability; 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.

[0030] The safety of the Hericium erinaceus hypoglycemic peptide obtained was evaluated and tested using the Toixinpred software, and its toxicity and allergenicity were predicted. The results showed that it is non-toxic, non-allergenic, and has a high safety profile.

[0031] (6) The AVFPSIVGRPR peptide was synthesized 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 Hericium erinaceus hypoglycemic peptide, and ChemDraw drew its 2D structure. The results are shown in the figure. Figure 10 .

[0032] To explore the binding mechanism of AVFPSIVGRPR with α-amylase and α-glucosidase, the drawn 2D structure was converted into a 3D structure 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 the PDB https: / / www.rcsb.org / . The downloaded macromolecular protein was imported into MOE, water molecules and small molecule ligands were removed, redundant chains were deleted, and the macromolecular processing was completed by clicking QuickPerp. Since the docking pocket site was unknown, all-atom docking was performed between the small molecule library and the macromolecular protein. After docking, the structure with the lowest score and the most stable structure was selected from the docking results. Through docking, the 2D and 3D images of the docking of AVFPSIVGRPR and α-glucosidase molecules were obtained, as shown in Figure 11 AVFPSIVGRPR forms three hydrogen bonds with α-amylase, with the binding sites at Thr6, Ser3, and Lys227, and the hydrogen bond energy at Ser3 is the highest. It also forms five hydrogen bonds with α-glucosidase, with the binding sites at Asp351, Glu344, Lys200, and Lys347, with two hydrogen bonds at Glu344 and the highest bond energy at Lys347.

Claims

1. Use of a hypoglycemic peptide in the preparation of a drug for inhibiting the activity of α-amylase and α-glucosidase, characterized in that: The hypoglycemic peptide is derived from Hericium erinaceus, and has an amino acid sequence of AVFPSIVGRPR.

2. Use of a hypoglycemic peptide in the preparation of a hypoglycemic drug, characterized in that: The hypoglycemic peptide is derived from Hericium erinaceus, and has an amino acid sequence of AVFPSIVGRPR.

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