Hericium erinaceus hypoglycemic peptide as well as preparation method and application thereof

Abstract

The invention discloses 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. 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 the peptide for alpha-amylase is 80.63% + / -0.23% through blood sugar reduction activity detection, the inhibition rate of the peptide for alpha-glucosidase is 70.77% + / -0.29%, the peptide can be used as a natural peptide for reducing blood sugar, and the peptide for reducing blood sugar 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 present application belongs to the technical field of biological extracts, and relates to a Hericium erinaceum hypoglycemic peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Diabetes has become the third chronic non-communicable disease after cardiovascular and cerebrovascular diseases and malignant tumors, and its most important feature is hyperglycemia. Controlling postprandial hyperglycemia is an effective and attractive target for treating diabetes. Carbohydrates in daily diet mainly exist in the form of polysaccharides, which cannot be directly absorbed and utilized by the human body, and need to be decomposed into monosaccharides by α-glucosidase and α-amylase in the gastrointestinal tract before being absorbed. Therefore, inhibiting the activity of the two enzymes can delay glucose absorption, thereby effectively reducing the postprandial blood glucose level.

[0003] A large number of studies have shown that edible fungi are rich in various active substances with hypoglycemic effect, such as proteins, peptides, polysaccharides, terpenoids, etc. At the same time, compared with animals and plants, edible fungi have a short growth cycle and are easy to cultivate, and are a high-quality hypoglycemic drug research and development material. Li Jinglei et al. studied the in vitro hypoglycemic and hypolipidemic activity of Morchella esculenta extracellular polysaccharide (MEP). It was found that the inhibition rates of MEP on α-glucosidase and α-amylase were 53.13% and 54.76% respectively, showing certain hypoglycemic activity, but the inhibition rate of MEP on α-amylase and α-glucosidase had no significant effect (Li Jinglei, Liu Yuting, Zong Shuai, et al. In vitro hypoglycemic and hypolipidemic activity of Morchella esculenta extracellular polysaccharide [J]. Food Research and Development, 2020, 41(16): 39-45.). Hericium erinaceum contains various active ingredients, especially rich proteins, but so far there has been no related report on the preparation of hypoglycemic peptides from Hericium erinaceum fruiting bodies. SUMMARY

[0004] The present application provides a Hericium erinaceum hypoglycemic peptide as well as a preparation method and application thereof.

[0005] The amino acid sequence of the Hericium erinaceum hypoglycemic peptide is GRVVPAPIPR, i.e. Gly-Arg-Val-Val-Pro-Ala-Pro-Ile-Pro-Arg, as shown in SEQ ID NO. 1.

[0006] The Hericium erinaceum hypoglycemic peptide can be prepared by a solid-phase synthesis method.

[0007] The present application provides the application of the above-mentioned Hericium erinaceum hypoglycemic peptide in the preparation of an α-amylase and α-glucosidase activity inhibiting drug.

[0008] Further, the present application provides the application of the above-mentioned Hericium erinaceum hypoglycemic peptide in the preparation of a hypoglycemic drug.

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

[0010] The present application first extracts and purifies polypeptides from Hericium erinaceus fruiting body proteins, finds multiple hypoglycemic peptides with good blood glucose lowering capacity, and determines the amino acid sequence of one of the hypoglycemic peptides as GRVVPAPIPR through LC-MS / MS, and through hypoglycemic activity detection, the α-amylase inhibition rate is 80.63%±0.23%, and the α-glucosidase inhibition rate is 70.77%±0.29%, which can be used as a natural hypoglycemic peptide and has potential application prospects in the field of preparing blood glucose lowering drugs. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 C1, C2, C3 are three polypeptides with different molecular weights, and the α-glucosidase and α-amylase inhibition rates thereof.

[0012] Figure 2 Q1, Q2, Q3 are products separated by an anion column.

[0013] Figure 3 q1, q2, q3 are products separated by an anion column.

[0014] Figure 4 The α-glucosidase inhibition rates of Q1, Q2, Q3 separated by an anion column.

[0015] Figure 5 The α-glucosidase inhibition rates of q1, q2, q3 separated by an anion column.

[0016] Figure 6 G1, G2, G3, G4 are products separated by a gel column.

[0017] Figure 7 The α-glucosidase inhibition rates of G1, G2, G3, G4.

[0018] Figure 8 The TIC spectrum of G2.

[0019] Figure 9 The secondary mass spectrum of the active polypeptide.

[0020] Figure 10 The prediction of the structure of the active polypeptide.

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

[0022] Figure 122D (a) and 3D (b) diagrams for docking of active polypeptide with a-amylase molecule.

[0023] Figure 13 Effect of active polypeptide on glucose content of IR HepG2 cells. DETAILED DESCRIPTION

[0024] The application will be further described below in conjunction with specific examples and drawings.

[0025] 1. Determination of a-amylase inhibition rate: the method of (Sunna. Preparation and identification of hypoglycemic bioactive peptides in camel milk protein [D]. Hohhot: Inner Mongolia Agricultural University, 2020.) was referred to for determination.

[0026] 2. Determination of a-glucosidase inhibition rate: the method of (Zhao Hongxing. Preparation of hypoglycemic active peptides, purification and identification of a-glucosidase activity peptides [D]. Harbin: Harbin Institute of Technology, 2018.) was referred to for determination.

[0027] Example 1

[0028] (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. The freeze-dried fruiting bodies were pulverized into powder using a pulverizer 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 allowed to stand for 4 h. The solution was centrifuged at 4 ℃ and 8000 r / min for 20 min, and the upper liquid 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 the solution was dissolved by ultrasonic and allowed to stand for 12 h. The solution was 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 environment for 48 h. After dialysis, the powder was freeze-dried to obtain Hericium erinaceus protein.

[0029] (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.

[0030] (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. 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 q2 showed better activity, and the inhibitory rate on α-glucosidase was 62.95±0.13%.

[0031] 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, and G4 in turn according to the elution order. The gel column separates and purifies the components according to the molecular weight. The α-glucosidase inhibition rates of G1, G2, G3, and G4 are determined, and the results are shown in FIG. 7. The hypoglycemic activity of G2 is significantly higher than that of the other three components, and the inhibition rate of 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 also been improved to a certain extent, indicating that the hypoglycemic peptide of Hericium erinaceus has been further purified, the hypoglycemic activity has been improved, and the hypoglycemic peptide of Hericium erinaceus is 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 map of G2 of Hericium erinaceus is shown. 33 sequences are obtained and 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 high possibility of 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 in FIG. 6, with a molecular weight of 1061.66 kDa. Figure 9

[0033] (5) The performance of the GRVVPAPIPR peptide was analyzed by bioinformatics. The ProParam online software predicts the physicochemical properties of the peptide: the theoretical isoelectric point is 12; it has two positively charged amino acid residues; the stability is 56.53, which has good stability; 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 E. coli is greater than 10 h; it is a hydrophilic polypeptide.

[0034] The Toixinpred software is used to test the safety of the obtained hypoglycemic peptide of Hericium erinaceus, and the toxicity and allergenicity are predicted. The results show that it is non-toxic and non-allergic, and has high safety.

[0035] (6) The GRVVPAPIPR peptide is obtained by solid-phase synthesis method in Shenguo Biological Engineering Co., Ltd., and the hypoglycemic activity of the GRVVPAPIPR peptide is determined. The inhibition rate of α-amylase is 80.63%±0.23%, and the inhibition rate of α-glucosidase is 70.77%±0.29%. The structure of the obtained hypoglycemic peptide of Hericium erinaceus is predicted by PepDraw, and its 2D structure is drawn by ChemDraw. The results are shown in FIG. 7. Figure 10 .

[0036] ​To explore the binding mechanism of GRVVPAPIPR with a-amylase and a-glucosidase, the drawn 2D structure was converted into 3D structure using Chem3D. Molecular docking was performed with MOE (2019), and the constructed docking model obtained the 2D and 3D graphs of GRVVPAPIPR molecular docking with a-glucosidase, as shown in Figure 11 . And the 2D and 3D graphs of GRVVPAPIPR molecular docking with a-amylase, as shown in Figure 12 . GRVVPAPIPR formed 2 hydrogen bonds with a-amylase, and the binding sites were Trp280 and Gly225, respectively, in which the hydrogen bond energy at Trp280 was the largest; and 5 hydrogen bonds with a-glucosidase, and the binding sites were Asp451, Glu488, Lys35, and Arg10, respectively, in which there were two hydrogen bonds at Lys35, and the bond energy at Asp451 was the largest.

[0037] (7) Extract HepG2 cells from the liquid nitrogen tank and quickly put them into a constant temperature water at 37°C for oscillation to melt them. After melting, blow them and transfer them into a centrifuge tube. Then, add DMEM (DMEM complete culture medium is a complete culture medium containing 10% fetal bovine serum and 1% of the concentration of 25 mmol / L) to 5 mL, centrifuge at 1100 r / min for 5 minutes after blowing, discard the supernatant, add 0.5 mL of DMEM complete culture medium for blowing, and finally add DMEM complete culture medium to 5 mL and transfer it into a culture bottle, and culture it in a 37°C, 5% CO2 incubator for 2 days. When the cell density reaches 80%-90% of the bottom of the culture bottle, use 1 mL of trypsin for digestion treatment, and perform cell passage according to the ratio of 1:3. During the cell passage process, the growth state of the cells should be closely observed to keep the cells in the logarithmic growth phase.

[0038] Take the 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 in the incubator for two minutes, then add 4 mL of DMEM complete culture medium, blow after centrifugation 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 5The cell suspension was inoculated in a 96-well plate, 100 μL of the cell suspension was added to each well, four replicates were set, and the plate was incubated at 37°C in a 5% CO2 incubator for 12 h. The culture solution in each well was aspirated, 100 μL of DMEM complete culture solution was added to the blank group, and 100 μL of Hericium erinaceus hypoglycemic peptide at different concentrations (0.01, 0.1, 0.25, 0.5, 1, 2, 4, 8 mg / mL) was added to the test group for continued incubation for 12 h. 10 μL of 5 mg / mL MTT was added, and the plate was incubated in the incubator for 4 h. 100 μL of Formazan dissolving solution was added. The 96-well plate was placed on a shaker for 10 min, and the plate was observed under a general optical microscope until the formazan was completely dissolved. The absorbance was measured at 570 nm on a microplate reader.

[0039] Referring to the studies of Fang Fei (Fang Fei. Study on the improvement of HepG2 insulin resistance by effective parts of mulberry leaves [D]. Guangzhou: South China University of Technology, 2012.) and Zhou Ming (Zhou Ming. Screening of collagen peptides from pheasant skin and study on their hypoglycemic mechanism [D]. Luoyang: Henan University of Science and Technology, 2022.), the optimal concentration of insulin for action was determined to be 10 μg / mL, and the action time was 36 h. Under the above conditions of insulin concentration and action time, HepG2 cells were cultured. The cells were prepared into a suspension, and the cell density was adjusted to 110 5 After 80% of the cells adhered, DMEM complete medium containing 10 μg / mL insulin was used, and the plate was incubated in the incubator for 36 h. The culture solution was aspirated, and serum-free and phenol red-free high-glucose culture solution was added, and the cells were incubated for 12 h to synchronize the cells. The IR HepG2 cell model was established.

[0040] According to the obtained IR HepG2 model, when the cells are in an insulin-resistant state, the uptake of glucose content is reduced, leading to an increase in blood glucose concentration. Therefore, by detecting the consumption of glucose, the uptake of glucose by the cells can be determined. IR HepG2 cells were treated with Hericium erinaceus hypoglycemic peptide at different concentrations (0.01, 0.1, 0.25, 0.5, 1, 2 mg / mL), and the results are shown in Table 1. Figure 13As shown in the figure, it can be observed that the glucose consumption of the model group is far lower than that of the normal group, which indicates that the modeling of IR HepG2 cells is successful. After treatment with Hericium erinaceus hypoglycemic peptide, the glucose consumption of IR HepG2 cells is increased at most concentrations, and the glucose consumption of Hericium erinaceus hypoglycemic peptide at 2 mg / mL is significantly lower than that of the model group, which may be due to the toxicity of the high concentration to the cells, resulting in the decrease of glucose consumption. Hericium erinaceus hypoglycemic peptide at 1, 0.5, 0.25, 0.1 mg / mL has a significant difference compared with the model group, which indicates that Hericium erinaceus hypoglycemic peptide can play a role in lowering blood sugar at these concentrations.

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 drugs that inhibit α-amylase and α-glucosidase activity.

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

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