Hericium erinaceus hypoglycemic peptide, and preparation method and application thereof

Abstract

The application discloses a hericium erinaceus hypoglycemic peptide as well as a preparation method and application thereof. The amino acid sequence of the hericium erinaceus hypoglycemic peptide is GRVVPAPIPR. 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, and the hypoglycemic activity detection shows that the alpha-amylase inhibition rate is 80.63%+ / -0.23%, and the alpha-glucosidase inhibition rate is 70.77%+ / -0.29%. The hericium erinaceus hypoglycemic peptide can be used as a natural hypoglycemic peptide and has a potential application prospect in the field of preparation of blood sugar reducing drugs.

Description

Technical Field

[0001] This invention belongs to the field of biological extract technology, and relates to a hypoglycemic peptide from Hericium erinaceus, its preparation method, and its application. Background Technology

[0002] Diabetes has become the third leading chronic non-communicable disease after cardiovascular and cerebrovascular diseases and malignant tumors. Its most prominent characteristic is hyperglycemia, and controlling postprandial hyperglycemia is an effective and attractive target for diabetes treatment. Carbohydrates in our daily diet mainly exist in the form of polysaccharides, which cannot be directly absorbed and utilized by the body. They need to be broken down into monosaccharides by α-glucosidase and α-amylase in the gastrointestinal tract before they can be absorbed. Therefore, inhibiting the activity of these two enzymes can delay glucose absorption, thereby effectively reducing postprandial blood glucose levels.

[0003] Numerous studies have shown that edible fungi are rich in various active substances with hypoglycemic effects, such as proteins, peptides, polysaccharides, and terpenes. Furthermore, compared to plants and animals, edible fungi have a short growth cycle and are easy to cultivate, making them a high-quality material for hypoglycemic drug development. Li Jinglei et al. studied the in vitro hypoglycemic and hypolipidemic activities of morel extracellular polysaccharide (MEP). The study found that MEP inhibited α-glucosidase and α-amylase by 53.13% and 54.76% respectively, showing certain hypoglycemic activity, but it did not have a significant effect on the inhibition rate of α-amylase and α-glucosidase (Li Jinglei, Liu Yuting, Zong Shuai, et al. Study on the in vitro hypoglycemic and hypolipidemic activities of morel extracellular polysaccharide [J]. Food Research and Development, 2020, 41(16):39-45.). Hericium erinaceus contains various active ingredients, especially abundant proteins, but so far there are no reports on the preparation of hypoglycemic peptides from Hericium erinaceus fruiting bodies. Summary of the Invention

[0004] This invention provides a blood sugar-lowering peptide from Hericium erinaceus, its preparation method, and its application.

[0005] The Hericium erinaceus hypoglycemic peptide of the present invention has the amino acid sequence GRVVPAPIPR, namely Gly-Arg-Val-Val-Pro-Ala-Pro-Ile-Pro-Arg, as shown in SEQ ID NO.1.

[0006] The Hericium erinaceus hypoglycemic peptide described in this invention can be prepared by solid-phase synthesis.

[0007] This invention provides the application of the above-mentioned Hericium erinaceus hypoglycemic peptide in the preparation of drugs that inhibit α-amylase and α-glucosidase activity.

[0008] Furthermore, the present invention provides the application of the above-mentioned Hericium erinaceus hypoglycemic peptide in the preparation of hypoglycemic drugs.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] 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, GRVVPAPIPR, was determined by LC-MS / MS. The hypoglycemic activity assay showed an inhibition rate of 80.63%±0.23% against α-amylase and 70.77%±0.29% against α-glucosidase. It can be used as a natural hypoglycemic peptide and has potential application prospects in the preparation of hypoglycemic drugs. Attached Figure Description

[0011] Figure 1 The inhibition rates of three polypeptides with different molecular weights (C1, C2, and C3) on α-glucosidase and α-amylase are shown.

[0012] Figure 2 The separated products Q1, Q2, and Q3 are obtained by passing through an anion exchange column.

[0013] Figure 3 The separated products q1, q2, and q3 are obtained by passing through an anion exchange column.

[0014] Figure 4 The α-glucosidase inhibition rate of the separation products Q1, Q2, and Q3 obtained by passing through an anion exchange column.

[0015] Figure 5 The α-glucosidase inhibition rate of the separation products q1, q2, and q3 obtained by passing through an anion exchange column.

[0016] Figure 6 To separate products G1, G2, G3, and G4 using a gel column.

[0017] Figure 7 The α-glucosidase inhibition rate of the isolated products G1, G2, G3, and G4.

[0018] Figure 8 This is the TIC map of G2.

[0019] Figure 9 This is a secondary mass spectrum of the active polypeptide.

[0020] Figure 10 This is for the prediction of the structure of active polypeptides.

[0021] Figure 11 The figures show 2D (a) and 3D (b) diagrams of the docking of the active polypeptide with the α-glucosidase molecule.

[0022] Figure 12The images show 2D (a) and 3D (b) diagrams of the docking of the active polypeptide with the α-amylase molecule.

[0023] Figure 13 The effect of active peptides on glucose content in IR HepG2 cells. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0025] 1. Determination of α-amylase inhibition rate: The method was followed (Suna. Preparation and identification of hypoglycemic bioactive peptides in camel milk protein [D]. Hohhot: Inner Mongolia Agricultural University, 2020.).

[0026] 2. Determination of α-glucosidase inhibition rate: The method was followed (Zhao Hongxing. Preparation of hypoglycemic active peptides and purification and identification of α-glucosidase-inhibiting active peptides [D]. Harbin: Harbin Institute of Technology, 2018.).

[0027] Example:

[0028] (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.

[0029] (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.

[0030] (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%.

[0031] 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 in Figure 7, the four fractions obtained were named G1, G2, G3, and G4 according to the elution order. The fractions were separated and purified by gel column chromatography based on molecular weight. The inhibition rates of α-glucosidase by G1, G2, G3, and G4 were measured, and the results are shown in Figure 7. G2 showed significantly higher hypoglycemic activity than the other three fractions, with an inhibition rate of 83.24 ± 0.37% against α-glucosidase. Furthermore, its hypoglycemic activity was also improved compared to that of 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.

[0032] (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 the G2 region 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 GRVVPAPIPR, as shown in SEQ ID NO.1, and its mass spectrum is shown below. Figure 9 As shown, the molecular weight is 1061.66 Da.

[0033] (5) The performance of the GRVVPAPIPR peptide was 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 56.53, which is good; the predicted half-life in mammalian reticulocytes is 30 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.

[0034] 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.

[0035] (6) GRVVPAPIPR peptide was obtained by solid-phase synthesis at Sangon Biotech Co., Ltd., and its hypoglycemic activity was determined. The inhibition rate against α-amylase was 80.63% ± 0.23%, and the inhibition rate against α-glucosidase was 70.77% ± 0.29%. The structure of the obtained Hericium erinaceus hypoglycemic peptide was predicted by PepDraw, and its 2D structure was plotted by ChemDraw. The results are shown in […]. Figure 10 .

[0036] To explore the binding mechanism of GRVVPAPIPR with α-amylase and α-glucosidase, Chem3D was used to convert the drawn 2D structure into a 3D structure. Molecular docking was performed using MOE (2019), and the docking model was constructed to obtain 2D and 3D diagrams of the molecular docking of GRVVPAPIPR with α-glucosidase, as shown below. Figure 11 As shown in the figure. And the 2D and 3D diagrams of the docking of GRVVPAPIPR with α-amylase molecules, as shown in the figure. Figure 12 As shown. GRVVPAPIPR forms two hydrogen bonds with α-amylase, with binding sites Trp280 and Gly225, where the hydrogen bond energy at Trp280 is the largest; it forms five hydrogen bonds with α-glucosidase, with binding sites Asp451, Glu488, Lys35, and Arg10, where there are two hydrogen bonds at Lys35 and the bond energy at Asp451 is the largest.

[0037] (7) Extract HepG2 cells from the liquid nitrogen tank and quickly place them in a constant temperature water bath at 37°C to thaw them. After thawing, pipette and transfer them to centrifuge tubes. Then, add DMEM (DMEM complete culture medium is DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin, high glucose concentration 25 mmol / L) to 5 mL, pipette and centrifuge at 1100 r / min for 5 minutes, discard the supernatant, add 0.5 mL of DMEM complete culture medium and pipette, and finally add DMEM complete culture medium to 5 mL and transfer to a culture flask. Incubate at 37°C, 5% CO2 for 2 days. When the cell density reaches 80%-90% at the bottom of the culture flask, digest the cells with 1 mL of trypsin and passage them at a ratio of 1:3. During cell passage, closely monitor the cell growth status and keep the cells in the logarithmic growth phase.

[0038] Collect cells in the logarithmic growth phase, aspirate the DMEM culture medium, wash once with 2-3 mL of PBS buffer, add 1 mL of trypsin, incubate for two minutes, then add 4 mL of complete DMEM culture medium, pipette and centrifuge at 1100 r / min for 5 minutes, discard the supernatant, add DMEM complete culture medium to prepare a cell suspension, and adjust the cell density to 1:10. 5Cells / mL. Inoculate 100 μL of cell suspension into each well of a 96-well plate, setting up four replicates. Incubate at 37℃ and 5% CO2 for 12 h. Aspirate the culture medium from each well. Add 100 μL of DMEM complete culture medium to the control group, and add 100 μL of different concentrations (0.01, 0.1, 0.25, 0.5, 1, 2, 4, 8 mg / mL) of Hericium erinaceus hypoglycemic peptide to the experimental groups and continue incubation for 12 h. Add 10 μL of 5 mg / mL MTT and incubate for another 4 h. Add 100 μL of Formazan dissolving solution. Gently shake the 96-well plate on a shaker for 10 min until the formazan is completely dissolved under a regular optical microscope. Measure the absorbance at 570 nm using a microplate reader.

[0039] Referring to the research by Fang Fei (Fang Fei. Study on the effect of effective parts of mulberry leaves on improving insulin resistance in HepG2 cells and its mechanism [D]. Guangzhou: South China University of Technology, 2012.) and Zhou Ming (Zhou Ming. Screening of salamander skin collagen hypoglycemic peptides and its hypoglycemic mechanism [D]. Luoyang: Henan University of Science and Technology, 2022.) on insulin concentration and duration of action in an IR HepG2 cell model, the optimal insulin concentration was determined to be 10 μg / mL and the action time to be 36 h. Under these conditions, HepG2 cells were cultured, and the cells were made into a suspension and the cell density was adjusted to 1:10. 5 Cells / mL. They were seeded into 96-well plates, with 100 μL of cell suspension added to each well, in quadruplicate. The plates were incubated at 37°C with 5% CO2 for 12 h. After 80% cell attachment, the medium was replaced with DMEM complete medium containing 10 μg / mL insulin, and incubated for 36 h. The culture medium was then aspirated, replaced with serum-free, phenol red-free, high-glucose medium, and incubated for 12 h to synchronize the cells. The IR HepG2 cell model was established.

[0040] Based on the obtained IR HepG2 model, when cells are in a state of insulin resistance, they reduce glucose uptake, leading to elevated blood glucose levels. Therefore, the amount of glucose consumed can indicate the cells' glucose uptake status. IR HepG2 cells were treated with different concentrations (0.01, 0.1, 0.25, 0.5, 1, 2 mg / mL) of Hericium erinaceus peptide to lower blood glucose, and the results are as follows. Figure 13As shown in the figure, it is evident that the glucose consumption in the model group was significantly lower than that in the normal group, indicating that the IR HepG2 cell model was successful. Treatment with Hericium erinaceus hypoglycemic peptide increased glucose consumption in IR HepG2 cells at most concentrations. However, the glucose consumption at 2 mg / mL was significantly lower than that in the model group, possibly due to the high concentration causing cytotoxicity and thus reducing glucose consumption. The hypoglycemic peptide at concentrations of 1, 0.5, 0.25, and 0.1 mg / mL showed significant differences compared to the model group, indicating that at these concentrations, the hypoglycemic peptide can lower blood sugar.

Claims

1. Hericium erinaceus hypoglycemic peptide, characterized in that, The amino acid sequence is GRVVPAPIPR.

2. The method for preparing the Hericium erinaceus hypoglycemic peptide according to claim 1, characterized in that, It was prepared using a solid-phase synthesis method.

3. The use of the Hericium erinaceus hypoglycemic peptide according to claim 1 in the preparation of hypoglycemic drugs.

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