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

Abstract

The invention discloses an application of hericium erinaceus hypoglycemic peptide in preparation of a medicine for inhibiting activity of alpha-amylase and alpha-glucosidase. The amino acid sequence of the hypoglycemic peptide is AGLQFPVGR. 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, the inhibition rate of alpha-amylase is 75.82% + / -0.12% through blood sugar reduction activity detection, the inhibition rate of alpha-glucosidase is 63.33% + / -0.28%, 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 Hericium erinaceus hypoglycemic peptide in the preparation of drugs for inhibiting the activities of α-amylase and α-glucosidase. Background Art

[0002] Diabetes has become the third most common chronic non-communicable disease, following cardiovascular and cerebrovascular diseases and malignant tumors. Hyperglycemia is the most prominent characteristic of diabetes, and controlling postprandial hyperglycemia is an effective and attractive target for diabetes treatment. In daily life, carbohydrates primarily exist as polysaccharides, which prevent them from being directly absorbed and utilized by the body. In the gastrointestinal tract, α-glucosidase and α-amylase are responsible for breaking down oligosaccharides and disaccharides into monosaccharides for absorption. Therefore, inhibiting α-glucosidase and α-amylase can delay glucose absorption in the body, thereby lowering postprandial blood glucose levels. Research has shown that hypoglycemic peptides extracted from natural products have the ability to inhibit the activity of α-amylase and α-glucosidase, and are less toxic and less prone to side effects than chemically synthesized drugs. However, oral peptide drugs suffer from absorption difficulties, making them difficult to cross the lipid barrier of the intestinal wall, resulting in poor absorption.

[0003] Numerous studies have shown that edible fungi are rich in a variety of active substances with hypoglycemic effects, such as proteins, peptides, polysaccharides, and terpenes. Furthermore, compared to plants and animals, edible fungi have a shorter growth cycle and are easier to cultivate, making them a valuable resource for the development of hypoglycemic drugs. Liu Bing et al. investigated the isolation and purification of extracellular polysaccharides from Morchella mycelium in three locations and their hypoglycemic and antioxidant activities. The study found that the inhibition rates of Morchella exopolysaccharides MEP-H and MEP-N on α-glucosidase were 74.93%±2.72% and 69.48%±2.97%, respectively, and the highest inhibition rates on α-amylase were 8.06%±1.93% and 11.08%±1.05%, respectively, showing a certain hypoglycemic activity, but it had no significant effect on the inhibition rate of α-amylase (Liu Bing, Li Pei, Zeng Xinyu, et al. Isolation and purification of extracellular polysaccharides from Morchella mycelium in three places and their hypoglycemic and antioxidant activities [J]. Journal of Food Safety and Quality, 2025, 16(05):86-93.). Liu Z. et al. discovered seven novelumami peptides from pufferfish, one of which has the amino acid sequence AGLQFPVGR (Liu Z, Zhu Y, Wang W, et al. Seven novelumami peptides from Takifugu rubripes and their taste characteristics[J].Food Chemistry, 2020, 330:127204.).

[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 Hericium erinaceus hypoglycemic peptide in the preparation of a drug for inhibiting the activities of α-amylase and α-glucosidase.

[0006] The Hericium erinaceus hypoglycemic peptide of the present invention has an amino acid sequence of AGLQFPVGR, namely Ala-Gly-Leu-Glu-Phe-Pro-Val-Gly-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:

[0009] 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 AGLQFPVGR by LC-MS / MS. After hypoglycemic activity testing, the inhibition rate of α-amylase was 75.82%±0.12%, and the inhibition rate of α-glucosidase was 63.33%±0.28%, 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

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

[0011] Figure 2 These are the separated products Q1, Q2, and Q3 after passing through the anion column.

[0012] Figure 3 These are the separated products q1, q2, and q3 after passing through the anion column.

[0013] Figure 4 It is the α-glucosidase inhibition rate of the separation products Q1, Q2, and Q3 passing through the anion column.

[0014] Figure 5 is the α-glucosidase inhibition rate of the separated products q1, q2, and q3 passing through the anion column.

[0015] Figure 6 The products G1, G2, G3 and G4 were separated by gel column.

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

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

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

[0019] Figure 10 For the prediction of active peptide structures.

[0020] Figure 11 2D (a) and 3D (b) images of the docking of the active peptide and α-glucosidase molecule.

[0021] Figure 12 2D (a) and 3D (b) images of the docking of active peptide and α-amylase molecule. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0023] 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.).

[0024] Example 1

[0025] (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.

[0026] (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.

[0027] (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.

[0028] 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.

[0029] (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 AGLQFPVGR, as shown in SEQ ID NO.1, and its mass spectrum is shown in Figure 9 As shown, the molecular weight is 944.53 kDa.

[0030] (5) The performance of the AGLQFPVGR peptide was analyzed through bioinformatics. The physicochemical properties of the peptide were predicted using the ProParam online software: the theoretical isoelectric point is 7.97; it contains one positively charged amino acid residue; the stability is 70.1, 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.

[0031] 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.

[0032] (6) AGLQFPVGR peptide was synthesized by solid-phase synthesis at Sangon Biotech Co., Ltd., and its hypoglycemic activity was determined. The inhibition rate against α-amylase was 75.82% ± 0.12%, and the inhibition rate against α-glucosidase was 63.33% ± 0.28%. PepDraw predicted the structure of the obtained Hericium erinaceus hypoglycemic peptide, and ChemDraw drew its 2D structure. The results are shown in the figure. Figure 10 .

[0033] To explore the binding mechanism of AGLQFPVGR 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 AGLQFPVGR and α-glucosidase molecules were obtained, as shown in Figure 11 As shown. The 2D and 3D images of AGLQFPVGR docking with α-amylase molecule are shown. Figure 12 AGLQFPVGR forms five hydrogen bonds with α-amylase, with the binding sites being Gly334, Asp402, Asn408, Arg398, and Gly403, with Gly334 having the highest hydrogen bond energy. It also forms four hydrogen bonds with α-glucosidase, with the binding sites being Asp397, Gly398, Arg416, and Phe401, with Phe401 having the highest bond energy.

Claims

1. The use of Hericium erinaceus hypoglycemic peptide in the preparation of a drug for inhibiting the activity of α-amylase and α-glucosidase, characterized in that: The amino acid sequence of the Hericium erinaceus hypoglycemic peptide is AGLQFPVGR.

2. The use of Hericium erinaceus hypoglycemic peptide in the preparation of hypoglycemic drugs, characterized in that: The amino acid sequence of the Hericium erinaceus hypoglycemic peptide is AGLQFPVGR.

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