A hericium erinaceus active glycopeptide with liver protection, blood sugar reduction and immune activity, and a preparation method and application thereof

Abstract

The present application provides a kind of active glycopeptide of Hericium erinaceus with liver protection, blood sugar reduction and immune activity and its preparation method and application, belong to edible fungus processing technical field.The present application provides an active glycopeptide of Hericium erinaceus, the amino acid sequence of the active glycopeptide of Hericium erinaceus is as shown in SEQ ID NO:1;The Thr site of the active glycopeptide of Hericium erinaceus is connected by O-connection mode with the oligosaccharide chain consisting of 7 mannose residues.The active glycopeptide of Hericium erinaceus of the present application can promote cell consumption glucose, stimulate macrophage cell production NO, and can relieve hydrogen peroxide induced liver injury.Animal model experiment of the present application shows that the active glycopeptide of Hericium erinaceus can effectively reduce fasting blood sugar level, regulate the tolerance of organism to glucose, and can relieve liver inflammatory response, improve fat metabolism in liver.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi processing technology, specifically relating to a Hericium erinaceus active glycopeptide with hepatoprotective, hypoglycemic and immune-enhancing activities, its preparation method and application. Background Technology

[0002] Glycopeptides are important biomolecules formed by covalently linking polypeptide chains and glycosyl groups. They are widely distributed in nature and participate in various physiological and pathological processes in the body. Currently, studies have isolated various active glycoproteins or glycopeptides from plants and macrofungi (such as enoki mushrooms and trachomatis), which possess multiple biological activities including immunomodulation, antiviral, antitumor, and antibacterial effects. However, systematic research reports on glycopeptides that simultaneously possess multiple functions such as high-efficiency liver protection, hypoglycemic activity, and immunomodulatory activity are still rare.

[0003] Lion's mane mushroom ( Hericium erinaceus Hericium erinaceus, a type of large fungus with high edible and medicinal value, is rich in various bioactive substances such as polysaccharides, proteins, glycoproteins, terpenes, and phenols. Current technologies have not yet revealed the functions of Hericium erinaceus-derived glycopeptides in liver protection, blood glucose regulation, and immune enhancement. Therefore, developing a multifunctional bioactive glycopeptide derived from Hericium erinaceus that possesses liver-protective, blood glucose-lowering, and immunomodulatory activities is of great significance in filling this gap in the field. Summary of the Invention

[0004] In view of this, the present invention provides a Hericium erinaceus active glycopeptide, which has the combined activities of protecting the liver, lowering blood sugar and regulating immunity.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a Hericium erinaceus active glycopeptide, the amino acid sequence of which is shown in SEQ ID NO: 1; The Thr site of the Hericium erinaceus active glycopeptide is connected to an oligosaccharide chain consisting of 7 mannose residues via an O-linking mechanism.

[0006] This invention provides a method for preparing Hericium erinaceus glycopeptides, comprising the following steps: Hericium erinaceus was enzymatically hydrolyzed using β-glucosidase, and the hydrolysate was extracted with sodium chloride to obtain Hericium erinaceus glycopeptides. The hericium erinaceus glycopeptide contains the hericium erinaceus active glycopeptide.

[0007] Preferably, the amount of β-glucosidase added is 1% to 5% of the mass of Hericium erinaceus.

[0008] Preferably, the enzymatic hydrolysis temperature is 40–60°C and the enzymatic hydrolysis time is 0.5–2 h.

[0009] Preferably, the final concentration of sodium chloride is 0.3 to 1.2 mol / L.

[0010] Preferably, the extraction temperature is 30–70°C and the extraction time is 1–6 hours.

[0011] This invention provides the application of the described Hericium erinaceus active glycopeptide and / or the Hericium erinaceus glycopeptide prepared by the described preparation method in the preparation of products that enhance immunity and / or have anti-inflammatory effects.

[0012] This invention provides the application of the described Hericium erinaceus active glycopeptide and / or the Hericium erinaceus glycopeptide prepared by the described preparation method in the preparation of products for lowering blood sugar and / or maintaining healthy blood sugar levels.

[0013] This invention provides the application of the described Hericium erinaceus active glycopeptide and / or the Hericium erinaceus glycopeptide prepared by the described preparation method in the preparation of products for protecting and / or assisting in the protection of liver damage.

[0014] Compared with the prior art, the present invention has the following advantages: This invention provides a Hericium erinaceus active glycopeptide, the amino acid sequence of which is shown in SEQ ID NO: 1; the Thr site of the active glycopeptide is linked to an oligosaccharide chain consisting of 7 mannose residues via an O-linking mechanism. The active glycopeptide of this invention can promote cellular glucose consumption, significantly stimulate macrophages to produce NO, and alleviate hydrogen peroxide-induced liver damage. Animal model experiments of this invention show that the active glycopeptide of this invention can effectively reduce fasting blood glucose levels, regulate the body's glucose tolerance, alleviate liver inflammation, and improve lipid metabolism in the liver.

[0015] This invention provides a method for preparing Hericium erinaceus glycopeptides, comprising the following steps: enzymatic hydrolysis of Hericium erinaceus using β-glucosidase; extraction of the hydrolysate with sodium chloride to obtain Hericium erinaceus glycopeptides; wherein the Hericium erinaceus glycopeptides contain the active Hericium erinaceus glycopeptides. This invention utilizes a β-glucosidase-assisted salt extraction method, which is simple, green, and safe, yielding a high-purity product. The obtained Hericium erinaceus glycopeptides exhibit good effects in lowering blood sugar, protecting the liver, and enhancing immunity.

[0016] The preparation method described in this invention yields Hericium erinaceus glycopeptide (HEGP), which promotes cellular glucose uptake and consumption, stimulates macrophages to release nitric oxide (NO) to activate the immune response, and enhances stem cell survival. Animal experiments show that this Hericium erinaceus glycopeptide can effectively improve the symptoms of type 2 diabetes induced by streptozotocin combined with a high-fat diet, significantly reduce hepatic steatosis and lipid droplet accumulation, alleviate hepatic inflammation, and lower the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the liver. Attached Figure Description

[0017] Figure 1 Image showing the identification results of active glycopeptides from Hericium erinaceus; Figure 2 This is a graph showing the results of fasting blood glucose testing in mice. Figure 3 The graph shows the results of oral glucose tolerance and insulin-related indicators in mice. This indicates a significant difference compared to the model group, P < 0.001. ### indicates a significant difference compared to the model group, P<0.01; ### indicates a difference compared to the control group, P<0.001. Figure 4 The image shows the results of HEGP's hepatoprotective effect on type 2 diabetic mice. A represents hematoxylin and eosin (H&E) staining of liver tissue sections (200×). B and C show the serum levels of liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), respectively. D-F show the activities of liver oxidative stress markers superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA), respectively. G-I show the mRNA levels of pro-inflammatory liver cytokines TNF-α, IL-1β, and IL-10, respectively. J and K show the results of Oil Red O staining and quantitative analysis of liver lipid droplet accumulation (200×), respectively. L-N show the liver lipid profiles of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), respectively. , and This indicates a significant difference compared to the T2DM group, among which This indicates that p < 0.05. This indicates that p < 0.01. The symbol indicates p < 0.001. #, ##, and ### indicate significant differences compared to the CTL group, where # indicates p < 0.05, ## indicates p < 0.01, and ### indicates p < 0.001. Figure 5 The figure shows the results of deglycosylation assay and corresponding glucose consumption data in insulin-resistant HepG2 cells. Detailed Implementation

[0018] This invention provides a Hericium erinaceus active glycopeptide, the amino acid sequence of which is shown in SEQ ID NO: 1 (LAPETRFPGPLHDAVSAYFR); the Thr site of the Hericium erinaceus active glycopeptide is linked by an O-linking oligosaccharide chain consisting of 7 mannose residues.

[0019] In this invention, the active glycopeptides from Hericium erinaceus can promote cellular glucose consumption, significantly stimulate cellular NO production, and alleviate hydrogen peroxide-induced liver damage. Animal model experiments of this invention show that the active glycopeptides from Hericium erinaceus can effectively reduce fasting blood glucose levels, regulate the body's glucose tolerance, alleviate liver inflammation, and improve lipid metabolism in the liver.

[0020] This invention provides a method for preparing Hericium erinaceus glycopeptides, comprising the following steps: Hericium erinaceus was enzymatically hydrolyzed using β-glucosidase, and the hydrolysate was extracted with sodium chloride to obtain Hericium erinaceus glycopeptides. The hericium erinaceus glycopeptide contains the hericium erinaceus active glycopeptide.

[0021] In this invention, the Hericium erinaceus preferably comprises Hericium erinaceus powder prepared by drying and pulverizing Hericium erinaceus. The moisture content of the Hericium erinaceus powder can be any value within the range of 1% to 5%, for example, 3%, 4%, or 5%. The drying method of this invention can include drying; the drying temperature can be any value within the range of 40 to 60°C, for example, 40, 50, or 60°C. This invention does not have a special limitation on the pulverization method; conventional pulverization methods are sufficient. The purpose of pulverization is to disrupt the cell structure and improve extraction efficiency.

[0022] In this invention, before the enzymatic hydrolysis, it is preferable to mix the Hericium erinaceus powder with water to prepare an aqueous solution of Hericium erinaceus. The ratio of Hericium erinaceus powder to water in this invention can be any value within the range of 1g:10 to 40mL, for example, 1g:10mL, 1g:20mL, 1g:30mL or 1g:40mL.

[0023] In this invention, the amount of β-glucosidase added as a percentage of the mass of *Hericium erinaceus* can be any value within the range of 1% to 5%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. Excessive enzyme addition will degrade the target protein, reducing the total amount, and over-hydrolysis may also affect the integrity of the protein structure. The hydrolysis temperature in this invention can be any value within the range of 40 to 60°C, for example, 40, 45, 50, 55, or 60°C; the hydrolysis time can be any value within the range of 0.5 to 2 hours, for example, 0.5, 1, 1.5, or 2 hours. Hydrolysis accelerates the decomposition of the cell wall, allowing intracellular proteins to be released more fully into the hydrolysate, indirectly improving the solubility and stability of the protein. After hydrolysis, this invention may also include enzyme inactivation. The enzyme inactivation temperature described in this invention can be any value within the range of 95 to 105°C, for example, 95, 100 or 105°C, and the enzyme inactivation time can be any value within the range of 5 to 15 min, for example, 5, 10 or 15 min.

[0024] In this invention, sodium chloride is added to the enzyme hydrolysate after enzyme inactivation for extraction. The final concentration of sodium chloride can be any value within the range of 0.3 to 1.2 mol / L, for example, 0.3, 0.6, 0.9, or 1.2 mol / L. After adding sodium chloride, Na... + and Cl - Ions neutralize the surface charge of charged molecules such as polysaccharides and proteins, weakening their electrostatic adsorption and allowing glycopeptides to be released into the extract. The extraction temperature described in this invention can be any value within the range of 30–70°C, for example, 30, 40, 50, 60, or 70°C. Increased temperature accelerates protein diffusion, partially disrupts cell walls or membrane structures, increases solubility, and reduces protein precipitation tendency. However, excessively high temperatures can cause protein denaturation, aggregation, or precipitation, affecting extraction efficiency and content. The extraction time described in this invention can be any value within the range of 1–8 hours, for example, 1, 2, 3, 4, 5, or 6 hours. Soluble proteins (such as cytoplasmic proteins and some extracellular enzymes) are released in large quantities in the early stages of extraction, and extending the extraction time does not significantly improve extraction efficiency. However, the longer the extraction time, the more protease activity accumulates, gradually hydrolyzing the extracted proteins, leading to a decrease in the total protein detection value.

[0025] In this invention, the extract is centrifuged, the supernatant is collected, dialyzed, and dried to obtain Hericium erinaceus glycopeptides. The centrifugation speed can be any value within the range of 7000–9000 rpm, for example, 7000, 8000, or 9000 rpm; the centrifugation time can be any value within the range of 10–20 min, for example, 10, 15, or 20 min. Concentration may be included before dialysis in this invention; there are no special limitations on the concentration method, and conventional concentration methods are acceptable. In a specific embodiment of the present invention, concentration is achieved through rotary evaporation, concentrating to any value within the range of 1 / 10 to 1 / 12 of the original volume, for example, 1 / 10, 1 / 11, or 1 / 12; the pressure of the rotary evaporation can be any value within the range of 60 to 100 mbar, for example, 60, 70, 80, 90, or 100 mbar; the time of the rotary evaporation is any value within the range of 3 to 5 hours, for example, 3, 3.5, 4, 4.5, or 5 hours; the temperature of the rotary evaporation is any value within the range of 40 to 60°C, for example, 40, 45, 50, 55, or 60°C. The dialysis bag used in the dialysis of the present invention can have a specification of any value within the range of 3500 to 3500 Da, for example, 2500, 3000, or 3500 Da, and the dialysis time can be 60 to 84 hours, for example, 60, 66, 72, 78, or 84 hours. The drying method of the present invention includes freeze-drying, for example, low-temperature vacuum freeze-drying. The temperature of the low-temperature vacuum freeze-drying described in this invention can be any value within the range of -70 to -50°C, for example -70, -60, or -50°C; the vacuum degree of the low-temperature vacuum freeze-drying can be any value within the range of 0.1 to 0.3 mbar, for example 0.1, 0.2, or 0.3 mbar; and the time of the low-temperature vacuum freeze-drying is 3 to 5 days, for example 3, 4, or 5 days.

[0026] This invention provides Hericium erinaceus glycopeptides prepared by the aforementioned preparation method. The structure of the separated glycopeptides was identified by liquid chromatography-mass spectrometry. The results showed that the glycopeptide molecules with high mass spectrometry abundance in the Hericium erinaceus glycopeptides were the active Hericium erinaceus glycopeptides.

[0027] This invention provides the application of the described Hericium erinaceus active glycopeptide and / or the Hericium erinaceus glycopeptide prepared by the described method in the preparation of products that enhance immunity and / or have anti-inflammatory effects. Cellular experiments of this invention demonstrate that Hericium erinaceus glycopeptides can stimulate macrophages to produce NO, activating the immune response, and animal experiments demonstrate that Hericium erinaceus glycopeptides can improve glucose tolerance and alleviate liver inflammation.

[0028] This invention provides the application of the described Hericium erinaceus active glycopeptide and / or the Hericium erinaceus glycopeptide prepared by the described method in the preparation of products for lowering blood sugar and / or maintaining healthy blood sugar levels. The blood sugar-lowering products preferably include drugs for treating type 2 diabetes. The type 2 diabetes preferably includes streptozotocin-induced and high-fat diet-induced type 2 diabetes. The high-fat diet preferably includes a diet with 60% fat as its energy source. Results from embodiments of this invention show that Hericium erinaceus glycopeptides can promote cellular glucose consumption, and animal model experiments show that 200 mg / kg to 500 mg / kg of Hericium erinaceus glycopeptides can effectively alleviate streptozotocin-induced and high-fat diet-induced type 2 diabetes.

[0029] This invention provides the application of the described Hericium erinaceus active glycopeptide and / or the Hericium erinaceus glycopeptide prepared by the described method in the preparation of products for protecting and / or assisting in the protection of liver injury. In this invention, the liver injury preferably includes hydrogen peroxide-induced liver injury and / or hepatic steatosis. Examples of this invention demonstrate through cell experiments that Hericium erinaceus glycopeptides can alleviate hydrogen peroxide-induced liver injury. Animal model experiments show that Hericium erinaceus glycopeptides can also alleviate the accumulation of large amounts of lipid droplets in steatotic livers, significantly reduce lipid droplet formation, alleviate liver inflammatory responses, and dose-dependently reduce liver TC, TG, and LDL-C levels.

[0030] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Effects of different enzyme treatments on Hericium erinaceus glycopeptides Dried Hericium erinaceus was used as raw material, pulverized and sieved to obtain Hericium erinaceus powder with a mesh size <100. 10.00g of Hericium erinaceus powder was accurately weighed, and distilled water was added to a material-to-liquid ratio of 1g:20mL to prepare an aqueous solution of Hericium erinaceus. Five experimental groups were set up, and each group was supplemented with an enzyme at a mass of 3% of the Hericium erinaceus powder. The difference between groups was the type of enzyme added. The enzymes used were β-glucanase (product enzyme activity 20000 U / g, purchased from Beijing Solarbio Technology Co., Ltd., brand Solarbio), β-glucosidase (product enzyme activity 300 U / g, purchased from Beijing Solarbio Technology Co., Ltd., brand Solarbio), pectinase (product enzyme activity 100000 U / g, purchased from Beijing Solarbio Technology Co., Ltd., brand Solarbio), hemicellulase (product enzyme activity 200000 U / g, purchased from Beijing Solarbio Technology Co., Ltd., brand Solarbio), or lysozyme (product enzyme activity 200000 U / g, purchased from Shanghai Yuanye Biotechnology Co., Ltd.). After adding the enzymes, the mixture was enzymatically hydrolyzed at 50℃ for 1 hour to obtain the enzymatic hydrolysate.

[0032] The enzyme hydrolysate was subjected to enzyme inactivation treatment at 100℃ for 10 min. Sodium chloride solid was added to the enzyme hydrolysate after enzyme inactivation to achieve a final sodium chloride concentration of 0.5 mol / L. Extraction was continued at 50℃ for 2 h to obtain the extract.

[0033] The extract was centrifuged at 8000 rpm for 15 min, the supernatant was collected, and the mixture was rotary evaporated at 90 mbar and 50 °C for 4 h to concentrate it to 1 / 10 of the original volume. The solution was then placed in a 3000 Da dialysis bag and dialyzed in ultrapure water for 72 h. After that, it was frozen at -60 °C under vacuum for 4 days at a vacuum degree of 0.2 mbar. The resulting lyophilized extract yielded Hericium erinaceus glycopeptide.

[0034] At the same time, a group without enzymes was set up, which was the same as the experimental group except that no enzymes were added.

[0035] The polysaccharide and soluble protein contents of Hericium erinaceus glycopeptides extracted by different methods were determined, and the results are shown in Table 1. The polysaccharide content (w / w) was determined by the phenol-sulfuric acid method at 490 nm, and the soluble protein content (w / w) was determined by the BCA method at 562 nm.

[0036] Table 1. Polysaccharide and soluble protein content (w / w) of glycopeptides

[0037] Note: Under the same indicator, different letters represent statistical significance.

[0038] Table 1 shows that, compared to the control group, the total content of soluble protein and polysaccharides significantly increased after treatment with lysozyme, β-glucanidase, and β-glucanidase and pectinase. Among these, the total content of soluble protein and polysaccharides significantly increased after treatment with β-glucosidase compared to the control group and other treatments. Therefore, β-glucosidase was selected for subsequent extraction.

[0039] Example 2: Effects of different enzyme treatments on the hepatoprotective activity of Hericium erinaceus glycopeptides Normal hepatocytes (L-O2, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml-CC1440) were grown in RPMI1640 medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics at 37°C and 5% CO2. After cell adhesion, the cells were divided into a control group, a model group, a positive control group, and a group treated with different enzymes (or without enzymes) and Hericium erinaceus glycopeptides.

[0040] The control group was replaced with complete culture medium (purchased from Merck Gibco, catalog number 11875176) and cultured for another 24 hours. The model group was replaced with complete culture medium and cultured for another 18 hours. Then, H2O2 (final concentration 0.75 mmol / L) was added to induce oxidative stress hepatocyte damage, and the cells were cultured for another 6 hours. The difference between the positive control group and the model group was that the complete culture medium was replaced with a complete culture medium supplemented with vitamin C (final concentration of 400 μg / mL); The difference between the intervention group and the model group is that the complete culture medium was replaced with a complete culture medium containing 20 μg / mL Hericium erinaceus glycopeptides prepared in Example 1. The intervention group was divided into four groups according to the Hericium erinaceus glycopeptides extracted by different enzymes (or without enzymes): no enzyme group, β-glucanase group, β-glucosidase group, pectinase group, hemicellulase group, and lysozyme group.

[0041] A blank control group was set up, in which no cells were plated and all other operations were the same as those in the control group.

[0042] Afterwards, the culture medium in each well was aspirated, and the cells were washed three times with phosphate buffer solution at pH 7.5. The medium was then replaced with serum-free medium containing 0.05% thiazolyl bromide blue tetrazolium (MTT) solution. After incubation in the dark for 4 hours, the OD value was measured at a wavelength of 490 nm, and the cell viability was calculated according to the following formula.

[0043] Cell viability (%) = [(OD1-OD2) / OD2-OD3] × 100% (Formula I) In the formula, OD1: analyte treatment group; OD2: control group, no analyte added; OD3: blank control group.

[0044] The above method was used to detect the glycopeptides extracted from Hericium erinaceus using different enzymes. The results are shown in Table 2.

[0045] Table 2 Hepatocyte survival rate

[0046] Note: Different letters indicate statistical significance.

[0047] The results showed that Hericium erinaceus glycopeptides had a protective effect against chemically induced liver injury. Compared with the untreated group and other enzyme-treated groups, the cell survival rate of Hericium erinaceus glycopeptides prepared by β-glucosidase was the highest, comparable to that of the positive group, and the survival rate was increased by 108.8% compared with the model group.

[0048] Example 3: Protective effect of different enzyme treatments on insulin resistance in Hericium erinaceus glycopeptides in HepG2 cells induced by insulin. HepG2 cells (purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml098066) were grown in 1640 medium (purchased from Gibco-Thermo Fisher Scientific, catalog number 11875176) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics, at 37°C and 5% CO2. Cells were cultured at a growth rate of 2 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 μL / well to 96-well plates, with 100 μL of cell suspension per well. The cells were divided into control, model, positive, and sample groups. After 24 hours of culture, the original culture medium was discarded. 100 μL of complete culture medium was added to each well of the control group, while 100 μL of insulin (final concentration 1 × 10⁻⁶) was added to each well of the model, positive, and sample groups. -8 The complete culture medium (mol / L) was placed in an incubator and incubated for 36 hours to induce an insulin resistance model.

[0049] After incubation, the blank group and model group were replaced with normal 1640 complete medium, the positive group was added with complete medium containing metformin (final concentration 1 mM), and the sample group was added with complete medium containing 400 μg / mL of Hericium erinaceus glycopeptides extracted with different enzymes (or without enzymes) in Example 1. After incubating the cells in the incubator for another 12 h, the glucose content in the cell culture medium was measured using a glucose assay kit (Merck Sigma, catalog number GAGO20). The results are shown in Table 3.

[0050] Table 3. Glucose Consumption

[0051] Note: Different letters indicate statistical significance.

[0052] The results showed that in the insulin-resistant diabetes model, cells treated with Hericium erinaceus glycopeptides prepared by β-glucosidase had the highest glucose consumption compared with the untreated group and other enzyme-treated groups, and the glucose consumption was significantly increased compared with the model group.

[0053] Example 4: Effects of different enzyme treatments on Hericium erinaceus glycopeptides on NO release from RAW264.7 cells Mouse monocyte / macrophage RAW264.7 cells (purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml088508) were cultured in DMEM medium (purchased from Gibco-Thermo Fisher Scientific) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics at 37°C and 5% CO2. Cells were sporulated at a rate of 5 × 10⁶ cells / year. 4 Cells were seeded at a density of 100 μL per well into 96-well plates, with three groups: blank, positive, and sample. After 24 hours of culture, the original culture medium was discarded.

[0054] 100 μL of complete culture medium was added to each well of the blank group, complete culture medium containing lipopolysaccharide LPS (final mass concentration of 10 μg / mL) was added to the positive group, and complete culture medium containing 20 μg / mL of Hericium erinaceus glycopeptides extracted by different enzymes (or without enzymes) in Example 1 was added to the sample group.

[0055] After the cells were incubated in the incubator for another 24 hours, the absorbance of the sodium nitrite solution at 543 nm was measured using the Griess reaction. A NO standard curve was plotted, and the amount of NO generated in the culture medium was calculated based on the standard curve. The results are shown in Table 4.

[0056] Table 4 NO generation status

[0057] Note: Different letters indicate statistical significance.

[0058] The results showed that, except for the hemicellulose glycopeptide group, all other groups could significantly stimulate the production of NO in cells. Among them, the β-glucosidase group showed the highest NO production compared with other groups, indicating that the glycopeptide has strong immunostimulatory activity.

[0059] Example 5: A method for preparing Hericium erinaceus glycopeptides The difference between the preparation method of Hericium erinaceus glycopeptide with β-glucosidase group in Example 1 and the method is that the amount of β-glucosidase added is 1% (m / m), and the low temperature vacuum freezing temperature is -70℃.

[0060] Example 6: A method for preparing Hericium erinaceus glycopeptides The difference between the preparation method of Hericium erinaceus glycopeptide with β-glucosidase group in Example 1 and the method is that the amount of β-glucosidase added is 6% (m / m), and the low temperature vacuum freezing temperature is -70℃.

[0061] Example 7: Structural Identification of Hericium erinaceus Glycopeptides The β-glucosidase group, the group without enzyme, and the Hericium erinaceus glycopeptides obtained in Examples 5 and 6 of the above-mentioned Example 1 were identified using liquid chromatography-mass spectrometry (Vanquish Neo / Orbitrap Fusion Lumos, Thermo Fisher Scientific). The specific methods are as follows.

[0062] 1 mg of Hericium erinaceus glycopeptide sample was dissolved in 20 L of 0.1% (v / v) formic acid aqueous solution, vortexed, and centrifuged at 17000 rpm and 4℃ for 20 min. The supernatant was collected and transferred to a sample tube. The injection volume was 3 μL and the flow rate was 250 nL / min. The mobile phase A of the liquid chromatography was 0.1% (v / v) formic acid aqueous solution, and the mobile phase B was acetonitrile solution containing 0.1% (v / v) formic acid. The specific liquid chromatography conditions were: 0 min, 55% B; 8 min, 10% B; 33 min, 15% B; 43 min, 28% B; 50 min, 40% B; 60 min, 95% B; 65 min, 95% B; 66 min, 5% B; 70 min, 5% B. The mass spectrometry conditions were as follows: Primary mass spectrometry parameters: resolution: 70000, maximum ion implantation time: 80 ms, mass scan range: 100–1400 m / z; Secondary mass spectrometry parameters: resolution: 17500, maximum ion implantation time: 80 ms, NCE mode collision energy: 27. Sequence database searches were performed on the LC-MS / MS data, and the results are as follows: Figure 1 .

[0063] LC-MS / MS identification revealed a high abundance of *Hericium erinaceus* active glycopeptide (peak area: 142333210796.9) in the *Hericium erinaceus* glycopeptide sample obtained from the β-glucosidase group in Example 1. The *Hericium erinaceus* active glycopeptide was less abundant in the samples prepared in Examples 5 and 6, with peak areas of 5892.3 and 10238.1, respectively. This *Hericium erinaceus* glycopeptide molecule was not identified in the *Hericium erinaceus* glycopeptide obtained from the β-unenzyme group in Example 1. The peptide sequence of the *Hericium erinaceus* active glycopeptide is shown in SEQ ID NO: 1, with 7 mannose ions modified at Thr, glycosylated in an O-linked manner.

[0064] Example 9: Protective effect of Hericium erinaceus glycopeptides on type 2 diabetic mice Five-week-old male ICR mice were subjected to a one-week acclimatization period before being divided into groups for experiments. The control group (CTL group) mice were fed a normal diet (4%–8% fat energy), while the remaining mice were fed a high-fat diet (60% fat energy). The control group (CTL group) mice were administered 10 μL / g physiological saline by gavage from week 7 to week 8; the remaining mice were fed a high-fat diet (60% fat energy) for 4 weeks (weeks 1–5) before initiating a type 2 diabetes model. Mice were separated by weight and fasted for 16 hours. Mice were then injected intraperitoneally with streptozotocin (STZ) at a dose of 40 mg / kg for 5 consecutive days, followed by blood glucose level measurement (week 6). Blood was collected from the tail tip of the mice, and a fasting blood glucose (FBG) level ≥11.1 mmol / L was considered a successful model. Male mice that successfully developed the model were randomly divided into a model group, a positive drug group, a low-dose group of Hericium erinaceus glycopeptide, and a high-dose group of Hericium erinaceus glycopeptide, with 10 mice in each group. The mice were administered the drug by gavage for 2 weeks (i.e., week 7 to week 8).

[0065] Model group (T2DM group): The drug was 10 μL / g of physiological saline; Positive drug group (MET group): The drug was 200 mg / kg Metformin; Low-dose Hericium erinaceus glycopeptide group (HEGP-L group): The drug was 200 mg / kg of Hericium erinaceus glycopeptide obtained from the β-unenzyme group in Example 1; High-dose Hericium erinaceus glycopeptide group (HEGP-H group): The drug was 500 mg / kg of Hericium erinaceus glycopeptide obtained from the β-unenzyme group in Example 1.

[0066] 1. Measurement of fasting blood glucose (FBG) levels in mice Fasting blood glucose levels in mice were measured during the experiment. The method was as follows: Mice were placed in clean cages and fasted for 16 hours, during which time they were allowed to drink water normally. The mice were then removed from their cages and gently placed on a wire mesh. Approximately 1-2 mm of the tail was cut off, and the tail was gently squeezed to collect a single drop of blood. This first drop was discarded, and fasting blood glucose was measured using a blood glucose meter.

[0067] Results of fasting blood glucose level measurement in mice are shown below Figure 2 The results showed that, over time, the blood glucose levels in the control group remained relatively constant, while the fasting blood glucose levels in the model group continuously increased. The fasting blood glucose levels in the positive control group and the Hericium erinaceus glycopeptide group showed a trend of first increasing and then decreasing. Specifically, blood glucose levels gradually increased from week 0 to 6, and after week 7, FBG began to decrease after intervention with the positive control group and Hericium erinaceus glycopeptide. Compared with the model group, the fasting blood glucose level of HEGP showed a significant decreasing trend, and the blood glucose level of the HEGP-L group showed a more significant decreasing trend, gradually approaching the blood glucose level of the positive control group metformin.

[0068] 2. Oral glucose tolerance test (OGTT) Preparation of glucose solution: Weigh an appropriate amount of glucose accurately, add pure water to dissolve and dilute to a glucose solution with a concentration of 0.1 g / mL, and store at 4℃ for later use.

[0069] Eight weeks later, the mice were transferred to clean cages and fasted for 16 hours before undergoing an oral glucose tolerance test. During the fasting period, the mice were allowed normal water intake. Approximately 1-2 mm of the mouse's tail was cut off with scissors, and the tail was gently squeezed to collect a single drop of blood. This first drop was discarded, and fasting blood glucose was measured using a glucometer, recording the value as the 0-minute blood glucose level. Subsequently, the mice were administered glucose solution by gavage at a dose of 0.5 g / kg.

[0070] Immediately after gavage, timing was started. Blood glucose levels were measured and recorded for each mouse at 15, 30, 60, 90, and 120 minutes post-gavage. A line graph was plotted with time on the X-axis and blood glucose levels on the Y-axis. The area under the curve (AUC) was calculated using GraphPad software. Fasting insulin levels were measured using an Elabscience ELISA kit according to the manufacturer's instructions, and the insulin resistance index and insulin sensitivity index were calculated using the following formulas.

[0071] Insulin resistance index = (fasting blood glucose × fasting insulin level) / 22.5 (Formula II) Insulin sensitivity index = Ln[1 / (fasting blood glucose × fasting insulin level)] (Formula III) The results are as follows Figure 3 Figures (a) and (b) show that the oral glucose tolerance test (AUC) was significantly higher in the model group compared to the normal group (P<0.05). After two weeks of continuous intervention with metformin-positive drugs, the AUC was significantly lower in the model group compared to the model group (P<0.05). After two weeks of low- and high-dose HEGP intervention, HEGP significantly (P<0.05) reduced the AUC in diabetic model mice in a dose-dependent manner. Based on the oral glucose tolerance test and the effect of HEGP on fasting blood glucose (FBG) in diabetic model mice, this study demonstrates that HEGP can treat diabetes by regulating blood glucose levels, i.e., glucose tolerance.

[0072] like Figure 3 As shown in (c), the insulin resistance index was significantly higher in the model group compared to the normal group (P<0.05). After two weeks of continuous intervention with the positive control drug, the insulin resistance index was significantly lower in the model group compared to the model group (P<0.05). After two weeks of low-dose and high-dose HEGP intervention, HEGP significantly (P<0.05) reduced the insulin resistance index in diabetic model mice in a dose-dependent manner. Figure 3In the middle (d) period, compared with the normal group, the insulin sensitivity index of the model group was significantly decreased (P<0.05). After metformin positive drug intervention, the insulin resistance index was significantly increased compared with the model group (P<0.05). After HEGP intervention, it was found that HEGP could significantly increase the insulin sensitivity index of diabetic model mice (P<0.05). In conclusion, Hericium erinaceus glycopeptide has a good therapeutic effect on type 2 diabetes.

[0073] 3. Regulatory effects on key genes or proteins in glucose metabolism Immunohistochemical analysis of key proteins was performed after 8 weeks to elucidate the antidiabetic effects of HEGP in vivo, including GLUT4, IRS1, and GSK-3β (antibody purchased from Cell Signaling Technology). Results showed that, compared to the control group (CTL), the model group (T2DM) exhibited significantly downregulated expression of GLUT4 and IRS1, while significantly upregulated expression of GSK-3β. Decreased GLUT4 and IRS1 expression impaired glucose uptake and insulin signaling, while increased GSK-3β further inhibited glycogen synthesis and promoted hepatic glucose output, exacerbating hyperglycemia and insulin resistance. Notably, HEGP treatment significantly reversed these diabetes-related protein expression changes. Both low- and high-dose HEGP interventions significantly upregulated GLUT4 and IRS1 expression and downregulated GSK-3β in the liver of model mice, with effects comparable to the positive control metformin. The PI3K / Akt pathway plays a crucial role in regulating glucose homeostasis through downstream effectors, including IRS1, GLUT4, and GSK3β. In cell models and liver tissue of type 2 diabetic mice, insulin-resistant cells showed significantly reduced phosphorylation levels of PI3K and AKT, while HEGP treatment significantly increased the ratios of pPI3K to total PI3K and pAKT to total AKT. These data suggest that the hypoglycemic and insulin-sensitizing effects of HEGP are closely related to the activation of the PI3K / AKT signaling pathway, providing a mechanistic basis for its therapeutic potential in the management of type 2 diabetes.

[0074] 4. Protective effect against liver injury in type 2 diabetic mice Eight weeks later, the degree of liver injury in mice was assessed by liver H&E staining, biomarker detection, and liver lipid profiling. The liver H&E staining method was as follows: after dewaxing the sections to water, hematoxylin was stained for 8–15 min, washed with water, and differentiated with 1% hydrochloric acid alcohol for a few seconds to 30 seconds, followed by rinsing with running water until the cell nuclei turned blue. Then, eosin was stained for 1–3 min, and excess stain was washed away with water. Finally, the sections were dehydrated sequentially with 80% ethanol, 95% ethanol, and anhydrous ethanol, cleared with xylene, and mounted with neutral resin. Biomarkers detected (biomarker detection kits purchased from Nanjing Jiancheng Biotechnology) included serum levels of liver injury biomarkers AST and ALT, liver oxidative stress biomarkers SOD activity, GSH-Px activity, and MDA content, and mRNA levels of pro-inflammatory liver cytokines TNF-α, IL-1β, and IL-10. Primer sequences are shown in Table 5. The levels of TC, TG, and LDL-C in liver tissue were measured by mass spectrometry.

[0075] Table 5 shows the primer sequences.

[0076] Test results are shown Figure 4 The results showed that HEGP treatment significantly improved liver histology, restored normal tissue morphology, and dose-dependently reduced serum AST and ALT levels, indicating a significant reduction in diabetes-induced liver injury. Figure 4 (A~C). Oxidative stress is a key factor in diabetic liver complications. In the type 2 diabetes group, the activities of antioxidant enzymes SOD and GSH-Px were significantly reduced, while MDA levels were increased. HEGP significantly reversed these changes and enhanced the liver's antioxidant defense system (…). Figure 4 Chronic inflammation further exacerbates liver damage in type 2 diabetes mellitus (T2DM). Liver levels of pro-inflammatory cytokines (including TNF-α and IL-1β) were significantly elevated in T2DM mice, and HEGP treatment significantly downregulated these cytokines, especially at high doses. Figure 4 (G and H). HEGP treatment can also upregulate IL-10 levels ( Figure 4 (I). Oil Red O staining showed a large accumulation of lipid droplets in the liver of T2DM mice, along with elevated levels of TC, TG, and LDL-C. HEGP intervention significantly reduced lipid droplet formation and dose-dependently decreased liver TC, TG, and LDL-C levels, indicating improved hepatic steatosis and lipid metabolism. Figure 4 (J~N).

[0077] 5. The physical form in which HEGP exerts its hypoglycemic activity To clarify whether the hypoglycemic effect of HEGP is mediated by its intact glycopeptide structure, the procedure was performed according to the instructions of the protein deglycosylation kit (Cat.HY-KE8014, MedChemExpress LLC.). Briefly, 100 µg of HEGP was mixed with 5 µL of 10×Glyco buffer, 2 µL of PNGase F, 2 µL of O-glycosidase, 2 µL of α2-3,6,8,9 neuraminidase, 2 µL of β1-4 galactosidase, and 2 µL of β-N-acetylhexosaminease. Purified water was added to bring the total reaction volume to 50 µL, and the mixture was gently mixed. The reaction mixture was incubated at 37 °C for 24 h, followed by incubation at 50 °C for 15 min to obtain a HEGP sample containing protein and residual carbohydrates (HE). The experimental protocol for the insulin resistance cell model was consistent with Example 3, except for the cell batch. The groups were divided according to the different drugs added. The working concentration of Metformin in the MET group was 1 mM; the working concentration of Hericium erinaceus glycopeptide obtained in the β-unenzyme group of Example 1 in the HEGPL group was 100 μg / mL; the working concentration of Hericium erinaceus glycopeptide obtained in the β-unenzyme group of Example 1 in the HEGPH group was 400 μg / mL; the working concentration of HE in the HEL group was 100 μg / mL; and the working concentration of HE in the HEH group was 400 μg / mL.

[0078] The results showed that, when tested at equal doses in the insulin-resistant HepG2 cell model, deglycosylated HE significantly reduced insulin resistance compared to HEGP. Figure 5 These findings suggest that the hypoglycemic effect of HEGP requires the complete structural integrity of the glycopeptide, and that the protein backbone and covalently linked carbohydrate chains are essential for its biological activity.

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of active glycopeptide from Hericium erinaceus, characterized in that, The amino acid sequence of the Hericium erinaceus active glycopeptide is shown in SEQ ID NO: 1; The Thr site of the Hericium erinaceus active glycopeptide is connected to an oligosaccharide chain consisting of 7 mannose residues via an O-linking mechanism.

2. A method for preparing Hericium erinaceus glycopeptides, characterized in that, Includes the following steps: Hericium erinaceus was enzymatically hydrolyzed using β-glucosidase, and the hydrolysate was extracted with sodium chloride to obtain Hericium erinaceus glycopeptides. The hericium erinaceus glycopeptide contains the active hericium erinaceus glycopeptide of claim 1.

3. The preparation method according to claim 2, characterized in that, The amount of β-glucosidase added is 1% to 5% of the mass of Hericium erinaceus.

4. The preparation method according to claim 2, characterized in that, The enzymatic hydrolysis temperature is 40–60℃, and the enzymatic hydrolysis time is 0.5–2 hours.

5. The preparation method according to claim 2, characterized in that, The final concentration of sodium chloride is 0.3–1.2 mol / L.

6. The preparation method according to claims 2-5, characterized in that, The extraction temperature is 30–70℃, and the extraction time is 1–6 hours.

7. The use of the Hericium erinaceus active glycopeptide of claim 1 and / or the Hericium erinaceus glycopeptide prepared by any one of claims 2 to 6 in the preparation of products that enhance immunity and / or have anti-inflammatory effects.

8. The use of the Hericium erinaceus active glycopeptide of claim 1 and / or the Hericium erinaceus glycopeptide prepared by any one of claims 2 to 6 in the preparation of products for lowering blood sugar and / or maintaining healthy blood sugar levels.

9. The use of the Hericium erinaceus active glycopeptide of claim 1 and / or the Hericium erinaceus glycopeptide prepared by any one of claims 2 to 6 in the preparation of products for protecting and / or assisting in the protection of liver damage.

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