Application of Hericium erinaceus hypoglycemic peptide in preparation of medicines for inhibiting activities of alpha-amylase and alpha-glucosidase

Abstract

The application discloses application of a Hericium erinaceum hypoglycemic peptide in preparation of a medicine for inhibiting alpha-amylase and alpha-glucosidase activity. The amino acid sequence of the hypoglycemic peptide is AGLQFPVGR. The polypeptide is extracted and purified from Hericium erinaceum fruiting body protein, the amino acid sequence of one hypoglycemic peptide is determined through LC-MS / MS, the alpha-amylase inhibition rate is 75.82%+ / -0.12% and the alpha-glucosidase inhibition rate is 63.33%+ / -0.28% through hypoglycemic activity detection, so the hypoglycemic peptide can be used as a natural hypoglycemic peptide and has a potential application prospect in the field of preparation of a medicine for reducing blood sugar and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological extracts and relates to application of a hericium erinaceus hypoglycemic peptide in preparation of a medicine for inhibiting activities of alpha-amylase and alpha-glucosidase. BACKGROUND

[0002] Diabetes has become the third chronic non-communicable disease after cardiovascular and cerebrovascular diseases and malignant tumors. Hyperglycemia is the most important feature of diabetes, and controlling postprandial hyperglycemia is an effective and attractive target for treating diabetes. In daily life, carbohydrates mainly exist in the form of polysaccharides, which makes them unable to be directly absorbed and utilized by the human body. In the gastrointestinal tract, alpha-glucosidase and alpha-amylase are responsible for the decomposition of oligosaccharides and disaccharides into monosaccharides for absorption by the human body. Therefore, inhibiting alpha-glucosidase and alpha-amylase can delay glucose absorption in the body, thereby reducing postprandial blood glucose levels. According to research, hypoglycemic peptides extracted from natural products have the activity of inhibiting alpha-amylase and alpha-glucosidase, and compared with chemically synthesized drugs, they have less toxicity and side effects on the human body. However, oral polypeptide drugs have the disadvantage of absorption barrier, and it is difficult for them to pass through the lipid barrier of the intestinal wall, resulting in poor absorption effect.

[0003] A large number of studies have shown that edible fungi are rich in various active substances with hypoglycemic effects, such as proteins, peptides, polysaccharides, and terpenoids. At the same time, compared with animals and plants, edible fungi have a short growth cycle and are easy to cultivate, making them a high-quality material for developing hypoglycemic drugs. Liu Bing et al. studied the isolation and purification of extracellular polysaccharides from mycelium of Morchella esculenta and their hypoglycemic and antioxidant activities. The study found that the inhibition rates of Morchella extracellular polysaccharides MEP-H and MEP-N on alpha-glucosidase were 74.93% ± 2.72% and 69.48% ± 2.97%, respectively, and the highest inhibition rates on alpha-amylase were 8.06% ± 1.93% and 11.08% ± 1.05%, respectively, showing certain hypoglycemic activity, but the inhibition rate on alpha-amylase was not significantly effective (Liu Bing, Li Pei, Zeng Xinyu, et al. Isolation and purification of extracellular polysaccharides from mycelium of Morchella esculenta and their hypoglycemic and antioxidant activities [J]. Journal of Food Safety and Quality Inspection, 2025, 16(05): 86-93.). Liu Z. et al. found seven umami peptides derived from Takifugu rubripes, and the amino acid sequence of one umami peptide was AGLQFPVGR (Liu Z, Zhu Y, Wang W, et al. Seven novel umami peptides from Takifugu rubripes and their taste characteristics [J]. Food Chemistry, 2020, 330: 127204.).

[0004] Hericium erinaceus contains various active ingredients, especially rich in protein. So far, there is no related report on hypoglycemic peptides derived from hericium erinaceus fruiting body. SUMMARY

[0005] The application provides application of the hericium erinaceus hypoglycemic peptide in preparation of a medicine for inhibiting activities of alpha-amylase and alpha-glucosidase.

[0006] The hericium erinaceus hypoglycemic peptide has an amino acid sequence of AGLQFPVGR, that is, Ala-Gly-Leu-Glu-Phe-Pro-Val-Gly-Arg, as shown in SEQ ID NO. 1.

[0007] The application also provides application of the above hypoglycemic peptide in preparation of a hypoglycemic medicine.

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

[0009] The application extracts and purifies polypeptides from hericium erinaceus fruiting body protein for the first time, finds a plurality of hypoglycemic peptides with good blood sugar lowering capacity, and determines that the amino acid sequence of one of the hypoglycemic peptides is AGLQFPVGR through LC-MS / MS; the alpha-amylase inhibition rate is 75.82%±0.12%, and the alpha-glucosidase inhibition rate is 63.33%±0.28%, which is remarkable, and the hypoglycemic peptide can be used as a natural hypoglycemic peptide and has potential application prospect in the field of preparation of medicines for lowering blood sugar. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 C1, C2 and C3 are three polypeptides with different molecular weights, and the alpha-glucosidase and alpha-amylase inhibition rates thereof are shown in the table.

[0011] Figure 2 Q1, Q2 and Q3 are products separated through an anion column.

[0012] Figure 3 Q1, Q2 and Q3 are products separated through an anion column.

[0013] Figure 4 Q1, Q2 and Q3 are products separated through an anion column.

[0014] Figure 5 Q1, Q2 and Q3 are products separated through an anion column.

[0015] Figure 6 G1, G2, G3 and G4 are products separated through a gel column.

[0016] Figure 7To isolate the product G1, G2, G3, G4, the α-glucosidase inhibition rate.

[0017] Figure 8 TIC map of G2.

[0018] Figure 9 Secondary mass spectrum of the active polypeptide.

[0019] Figure 10 Prediction of the structure of the active polypeptide.

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

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

[0022] The application will be further described in detail below in combination with specific embodiments and the accompanying drawings.

[0023] In the following examples, the determination of α-amylase inhibition rate refers to the method in (Sun, A. Preparation and identification of hypoglycemic bioactive peptides in camel milk protein [D]. Hohhot: Inner Mongolia Agricultural University, 2020.) and the determination of α-glucosidase inhibition rate refers to the method in (Zhao, H. Preparation of hypoglycemic active peptides and purification and identification of α-glucosidase activity peptides [D]. Harbin: Harbin Institute of Technology, 2018.).

[0024] Example 1

[0025] (1) Fresh Hericium erinaceus fruiting bodies were washed and cut into small pieces for liquid nitrogen quick freezing, then freeze-dried using a freeze dryer, and then ground into powder using a grinder and passed through a 60-mesh sieve. The powder and ultrapure water were mixed at a mass ratio of 1:10, dissolved by ultrasonic, and then allowed to stand for 4 h. The solution was centrifuged at 4 ℃ and 8,000 r / min for 20 min, and the supernatant was taken out. Ammonium sulfate was added to make the final concentration reach 80% saturation (561 g of solid ammonium sulfate was added per liter of solution), and then dissolved by ultrasonic and allowed to stand for 12 h. The solution was then centrifuged at 4 ℃ and 8,000 r / min for 20 min to obtain the precipitate. The precipitate was dissolved in ultrapure water and then placed in a 10 kDa dialysis bag for dialysis in a 4 ℃ water solution for 48 h. After dialysis, the powder was freeze-dried to obtain Hericium erinaceus protein.

[0026] (2) The Hericium erinaceus protein was dissolved in ultrapure water, and 1 mol / L sodium hydroxide and hydrochloric acid were used to adjust the environment to the suitable conditions (pH 9, 55°C) for alkaline protease. After the environment reached a stable state, 4% of the substrate mass of alkaline protease was added, and the enzymolysis was performed for 4 hours. Then, the reaction was terminated by water bath at 90°C for 15 minutes. Finally, the supernatant was obtained by centrifugation at 4°C and 8,000 r / min for 15 min, and the enzymolysis product was obtained by freeze-drying. The active peptide obtained after enzymolysis was dissolved and filtered through a 0.22 μm filter membrane. Three polypeptide components with different molecular weights were obtained by centrifugation at 4,000 r / min for 60 min using 10 kDa and 3 kDa ultrafiltration tubes, and were named C1, C2, and C3, respectively. The inhibitory rates of the three polypeptide components with different molecular weights on α-glucosidase and α-amylase were tested. The results are shown in Figure 1 As can be seen from the figure, the polypeptide component with the optimal inhibitory rates on α-amylase and α-glucosidase is C1.

[0027] (3) C1 was purified by Q Sepharose FF anion column through first step gradient purification (eluent is NaCl (2 M pH 7.5) B solution). As shown in FIG. 2, three elution peaks were produced, which were Q1, Q2, and Q3, respectively. The hypoglycemic activity of the three elution peaks was evaluated by the inhibitory rate on α-glucosidase, and the results are shown in FIG. 4. Among the three components, Q2 showed better activity, and the inhibitory rate on α-glucosidase was 0.4974±0.0166. According to the gradient at which Q2 appeared, which was 25% B (the proportion of Buffer B was 25%), the elution conditions were optimized again, and C1 was eluted by Q Sepharose FF anion column at 0-25% B linearly, and 20 column volumes were eluted, as shown in Figure 3 Three elution peaks were produced, which were q1, q2, and q3, respectively. The hypoglycemic activity of the three elution peaks was evaluated by the inhibitory rate on α-glucosidase, and the results are shown in Figure 5 As can be seen from the figure, q2 showed better activity, and the inhibitory rate on α-glucosidase was 62.95±0.13%.

[0028] q2 separated from the anion column was further purified by Superdex 30 Increase 10 / 300 GL gel column and detected by 220 nm absorbance, and four different components were successfully separated, and the results are shown in Figure 6The four components obtained are named G1, G2, G3, G4 in turn according to the elution order. The gel column separates and purifies the components according to the molecular weight, and the inhibitory rates of G1, G2, G3, G4 on α-glucosidase are determined respectively, and the results are shown in Table 1. Figure 7 The hypoglycemic activity of G2 is significantly higher than that of the other three components, and the inhibitory rate on α-glucosidase is as high as 83.24±0.37%. At the same time, compared with the hypoglycemic activity of q2 after ion column purification, the hypoglycemic activity has been improved to a certain extent, indicating that the hypoglycemic peptide of Hericium erinaceus has been further purified, and its hypoglycemic activity has been improved, and the hypoglycemic peptide of Hericium erinaceus is mainly concentrated in G2.

[0029] (4) The amino acid sequence of the hypoglycemic peptide at G2 of Hericium erinaceus is determined by LC-MS / MS. Figure 8 The TIC map of G2 of Hericium erinaceus is obtained. It is uploaded to PeptideRanker for activity prediction. PeptideRanker gives scores of 34 peptides in G2, one of which has a score higher than 0.5, indicating that this peptide shows a higher 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 FIG. 3. Figure 9 The molecular weight is 944.53 Da.

[0030] (5) The performance of AGLQFPVGR peptide is analyzed by bioinformatics. The online software ProParam predicts the physicochemical properties of the peptide: the theoretical isoelectric point is 7.97; it has one positively charged amino acid residue; the stability is 70.1, which has good stability; the predicted half-life in mammalian reticulocytes is 4.4 h, the half-life in yeast is more than 20 h, and the half-life in E. coli is more than 10 h; it is a hydrophilic polypeptide.

[0031] The safety of the obtained hypoglycemic peptide of Hericium erinaceus is tested by Toixinpred software, and its toxicity and allergenicity are predicted. The results show that it is non-toxic and non-allergic, and has high safety.

[0032] (6) AGLQFPVGR peptide is obtained by solid-phase synthesis method in Shenguo Biological Engineering Co., Ltd., and the hypoglycemic activity of AGLQFPVGR peptide is determined. The inhibitory rate on α-amylase is 75.82%±0.12%, and the inhibitory rate on α-glucosidase is 63.33%±0.28%. The structure of the obtained hypoglycemic peptide of Hericium erinaceus is predicted by PepDraw, and its 2D structure is drawn by ChemDraw, and the results are shown in FIG. 4. Figure 10 .

[0033] To explore the binding mechanism of AGLQFPVGR with α-amylase and α-glucosidase, the drawn 2D structure was converted into 3D structure using Chem3D. Molecular docking was performed with 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]. Chinese Journal of Information on Traditional Chinese Medicine, 2023, 40(06): 32-7.), the PDB code of α-amylase was 1SMD, and the PDB code of α-glucosidase was 5KZW. The 3D structure of the protein was downloaded by inputting the PDB code in PDB https: / / www.rcsb.org / . The downloaded macromolecular protein was imported into MOE, and the water molecules and small molecule ligands were removed, and the redundant chains were deleted. Click QuickPerp to complete the macromolecular processing. Since the docking pocket site is unknown, the small molecule library and the macromolecular protein are subjected to all-atom docking. After docking, the structure with the smallest score and the most stable structure is selected from the docking results. Through docking, the 2D and 3D graphs of AGLQFPVGR and α-glucosidase molecular docking are obtained, as shown in Figure 11 The 2D and 3D graphs of AGLQFPVGR and α-amylase molecular docking are shown in Figure 12 AGLQFPVGR forms 5 hydrogen bonds with α-amylase, and the binding sites are Gly334, Asp402, Asn408, Arg398, and Gly403, respectively. The hydrogen bond energy at Gly334 is the largest; and forms 4 hydrogen bonds with α-glucosidase, and the binding sites are Asp397, Gly398, Arg416, and Phe401, respectively, and the bond energy at Phe401 is the largest.

Claims

1. The application of Hericium erinaceus hypoglycemic peptide in the preparation of hypoglycemic drugs, characterized in that, The amino acid sequence of the Hericium erinaceum blood sugar reducing peptide is AGLQFPVGR.

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