Bifunctional edible fungus glycopeptide based on triple-frequency ultrasonic synergistic extraction as well as preparation method and application of difunctional edible fungus glycopeptide

Abstract

The invention relates to difunctional edible fungus glycopeptide based on triple-frequency ultrasonic synergistic extraction as well as a preparation method and application thereof, and belongs to the technical field of biology. The molecular sequence of the edible mushroom glycopeptide provided by the invention is AVS (GalNAc) VPLQ, the molecular sugar chain of the edible mushroom glycopeptide is composed of N-acetylgalactosamine, and the N-acetylgalactosamine is connected to a serine hydroxyl group of a peptide chain through an alpha-glucosidic bond in an O-connection glycosylation mode. The edible mushroom glycopeptide disclosed by the invention is derived from edible mushrooms and is safe to use; the activity of angiotensin converting enzyme can be effectively inhibited, the generation of angiotensin II (Ang II) is reduced, and the blood pressure is reduced; the activity of pancreatic lipase is inhibited, the absorption of fat is reduced, and the effects of losing weight and lowering lipid are achieved. The edible fungus glycopeptide can also inhibit Ang II induced NO reduction, promote NO rise, and play roles in relaxing blood vessels and reducing blood pressure; lipid droplet aggregation, induced by oleic acid, of human normal liver cells is inhibited, and the effect of reducing blood fat is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a bifunctional edible mushroom glycopeptide based on triple-frequency ultrasound-assisted extraction, a preparation method thereof, and an application thereof. Background Art

[0002] Angiotensin-converting enzyme (ACE), also known as kininase II or peptidyl-carboxypeptidase, is a membrane-bound enzyme on the vascular endothelial cell membrane. ACE has two main functions: one is to catalyze the conversion of angiotensin I to angiotensin II (Ang II); the other is to inactivate bradykinin. Angiotensin-converting enzyme has become an ideal target for the treatment of hypertension due to these two functions. Angiotensin-converting enzyme inhibitors (ACEIs) have antihypertensive effects, can delay and reverse ventricular remodeling, prevent the further development of myocardial hypertrophy, improve vascular endothelial function and cardiac function, reduce the occurrence of arrhythmias, and can also improve survival rate and prognosis. Commonly used ACEIs clinically include captopril, enalapril, benazepril, fosinopril, ramipril, etc.

[0003] Pancreatic lipase (PL) is the most important enzyme for hydrolyzing dietary fat, enabling the full digestion and absorption of dietary fat. Pancreatic lipase inhibitors can bind irreversibly to lipase in the stomach and small intestine, making the enzyme lose its activity, thereby reducing the hydrolysis and absorption of fat in the intestine and playing a role in reducing lipid and losing weight.

[0004] As important pharmaceutical ingredients for the treatment of cardiovascular diseases (such as hypertension and hyperlipidemia) and obesity, angiotensin-converting enzyme inhibitors and pancreatic lipase inhibitors have a huge market demand. Existing ACE / PL inhibitors, such as the chemical synthetic drugs captopril and orlistat, have side effects such as organ damage and gastrointestinal discomfort. Although natural inhibitors are safe, they often have insufficient activity, and even large-dose use cannot achieve ideal therapeutic effects. Therefore, there is an urgent need in this field for natural ACE / PL inhibitors that can effectively lower blood pressure and reduce lipids. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an edible mushroom glycopeptide that can effectively inhibit angiotensin-converting enzyme and pancreatic lipase, and has the effects of lowering blood pressure, reducing lipids, and losing weight.

[0006] The present invention provides an edible mushroom glycopeptide, and the molecular sequence of the edible mushroom glycopeptide is AVS(GalNAc)VPLQ; the sugar chain composition of the edible mushroom glycopeptide molecule is N-acetylgalactosamine, which is connected to the serine hydroxyl group of the peptide chain through an α-glycosidic bond in an O-linked glycosylation manner.

[0007] The present invention also provides the application of the edible mushroom glycopeptide in the preparation of an angiotensin-converting enzyme inhibitor.

[0008] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a pancreatic lipase inhibitor.

[0009] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a product for treating hypertension.

[0010] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a product for treating hyperlipidemia.

[0011] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a product that helps maintain a healthy blood pressure level.

[0012] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a product that helps maintain a healthy blood lipid level.

[0013] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a product for promoting an increase in intracellular NO.

[0014] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a product for inhibiting the aggregation of lipid droplets in hepatocytes.

[0015] The present invention also provides the use of the edible mushroom glycopeptide in the preparation of a weight loss product.

[0016] Advantages of the present invention:

[0017] The edible mushroom glycopeptide provided by the present invention is derived from edible mushrooms and is safe to use; it can effectively inhibit the activity of angiotensin-converting enzyme, reduce the production of angiotensin II, and lower blood pressure; it can effectively inhibit the activity of pancreatic lipase, reduce the absorption of fat, and achieve the effect of weight loss and lipid reduction; cell experiments also show that the edible mushroom glycopeptide of the present invention can inhibit the decrease in NO induced by Ang II, promote the increase in NO, and play a role in dilating blood vessels and lowering blood pressure; it can effectively inhibit the aggregation of lipid droplets in normal human hepatocytes induced by oleic acid and play a role in lowering blood lipids. Description of the Drawings

[0018] Figure 1 It is the molecular mass spectrum of the edible mushroom glycopeptide.

[0019] Figure 2 It is the 3D structure diagram of the edible mushroom glycopeptide molecule; wherein, yellow is the peptide molecule, orange is N-acetylgalactosamine, red is the oxygen atom, blue is the carbon atom, and gray is the hydrogen atom.

[0020] Figure 3 It is the inhibitory activity of different concentrations of the edible mushroom glycopeptide solution on ACE.

[0021] Figure 4 It is the inhibitory activity of different concentrations of the edible mushroom glycopeptide solution and the positive drug on pancreatic lipase.

[0022] Figure 5Effect diagram of edible mushroom glycopeptide on the release of nitric oxide (NO) by human umbilical vein endothelial cells; among them, **** indicates P < 0.0001, and ns indicates no significant difference.

[0023] Figure 6 Effect diagram of edible mushroom glycopeptide inhibiting lipid droplet aggregation in LO-2 hepatocytes; among them, **** indicates P < 0.0001, and ns indicates no significant difference.

[0024] Figure 7 Docking diagram of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ with the ACE molecule. Among them, in the edible mushroom glycopeptide molecule, the red part is the peptide molecule, and the green part is the N-acetylgalactosamine molecule.

[0025] Figure 8 Docking diagram of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ with the PL molecule. Among them, in the edible mushroom glycopeptide molecule, the green part is the peptide molecule, and the red part is the N-acetylgalactosamine molecule. Detailed implementation mode

[0026] The present invention provides an edible mushroom glycopeptide, the molecular sequence of the edible mushroom glycopeptide is AVS(GalNAc)VPLQ; the sugar chain composition of the edible mushroom glycopeptide molecule is N-acetylgalactosamine, which is connected to the serine hydroxyl group of the peptide chain by an O-linked glycosylation method through an α-glycosidic bond.

[0027] In the present invention, in the molecular sequence AVS(GalNAc)VPLQ of the edible mushroom glycopeptide, (GalNAc) is located after serine (S), indicating that N-acetylgalactosamine O-linked glycosylation modification occurs on serine (S), resulting in an increase in molecular weight by 203.08 Da. The molecular weight of the edible mushroom glycopeptide is preferably 915 Da. The peptide sequence in the edible mushroom glycopeptide molecule is preferably AVSVPLQ (SEQ ID NO.1).

[0028] Preferably, the structural formula of the edible mushroom glycopeptide is shown in Formula I:

[0029]

[0030] The edible mushroom glycopeptide of the present invention can inhibit angiotensin-converting enzyme and pancreatic lipase, reduce the generation of angiotensin II, inhibit the digestion and absorption of fat, and play a role in lowering blood pressure, reducing blood lipid and losing weight. In vitro experiments also show that the edible mushroom glycopeptide of the present invention can inhibit the decrease of NO induced by Ang II, promote the increase of NO, play a role in dilating blood vessels and lowering blood pressure, and at the same time can inhibit the lipid droplet aggregation of normal human hepatocytes induced by oleic acid, and play a role in reducing blood lipid.

[0031] The present invention also provides the use of edible mushroom glycoprotein in the preparation of an angiotensin-converting enzyme inhibitor or a pancreatic lipase inhibitor. The active ingredient of the inhibitor preferably includes the edible mushroom glycoprotein, and the inhibitor preferably further includes carriers commonly used in the art, and the carriers include but are not limited to fillers, diluents, disintegrants, colorants, and / or flavoring agents.

[0032] The present invention also provides the use of edible mushroom glycoprotein in the preparation of a product for treating hypertension, a product for treating hyperlipidemia, a product for helping to maintain a healthy blood pressure level, a product for helping to maintain a healthy blood lipid level, a product for promoting the increase of intracellular NO, a product for inhibiting the aggregation of lipid droplets in hepatocytes, or a weight loss product.

[0033] In the present invention, the product preferably includes a drug. The active ingredient of the product preferably includes the edible mushroom glycoprotein, and the product preferably further includes carriers commonly used in the art, and the carriers include but are not limited to fillers, diluents, disintegrants, colorants, and / or flavoring agents.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] In the following embodiments, unless otherwise specified, all are conventional methods.

[0036] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0037] Example 1

[0038] Structural identification of edible mushroom glycoprotein

[0039] 1. Preparation of freeze-dried powder containing edible mushroom glycoprotein

[0040] Select dry Stropharia rugoso-annulata as the raw material, and after crushing and sieving, obtain Stropharia rugoso-annulata powder with a particle size less than 100 mesh. Mix the Stropharia rugoso-annulata powder with water (material-liquid ratio 1:20, g / mL), and use a slit-type ultrasonic device to extract edible mushroom glycoprotein by using triple-frequency ultrasound. The ultrasonic frequency is the combined frequency of 23 + 25 + 28 (kHz), the ultrasonic power density is 100 W / L, the ultrasonic time is 30 min, and the ultrasonic intermittent ratio is 6:2 s / s, that is, the ultrasound works for 6 s (the 23 kHz, 25 kHz, and 28 kHz in the combined frequency work in sequence for 2 s each) and stops for 2 s. After the ultrasonic treatment, the ultrasonic sample is centrifuged at 8000 rpm for 15 min, and the supernatant is freeze-dried at -70 °C for 48 h to obtain the freeze-dried powder of Stropharia rugoso-annulata.

[0041] 2. Structural identification of edible mushroom glycoprotein

[0042] The peptide molecules in the freeze-dried powder of Stropharia rugoso-annulata obtained in Step 1 of this example were identified by mass spectrometry. A ZipTip C18 microchromatography column (Merck-Millipore, Shanghai Anpu Experimental Technology Co., Ltd.) was used for desalting pretreatment of the freeze-dried powder, and the desalting method was as follows: Accurately weigh 1.0 mg of the freeze-dried powder of Stropharia rugoso-annulata, add 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA) to dissolve it to obtain a dissolved solution of the freeze-dried powder of Stropharia rugoso-annulata; rinse the chromatography column 10 times with 50 μL of a solution prepared with 60% (v / v) acetonitrile (ACN) and 0.1% TFA pure water; rinse the chromatography column 10 times with 10 μL of 0.1% TFA; aspirate and discharge the dissolved solution of the freeze-dried powder of Stropharia rugoso-annulata through the chromatography column 20 times; rinse the chromatography column 5 times with 10 μL of 0.1% TFA; elute the chromatography column with 10 μL of a solution prepared with 60% ACN and 0.1% TFA pure water, collect the eluate, transfer the eluate to a polypropylene centrifuge tube, and vacuum dry it to obtain a dry powder. The dry powder was dissolved with 20 μL of a dissolved solution (0.1% (v / v) formic acid aqueous solution), vortexed, centrifuged at 17000 rpm and 4 °C for 20 min, the supernatant was collected and transferred to a sample injection tube, and the injection volume was 3 μL for LC-MS / MS sequence analysis. Among them, the mobile phase A of the liquid chromatography was 0.1% formic acid aqueous solution, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid; the LC-MS / MS setting parameters were shown in Table 1. The PEAKS software was used for identification sequence database retrieval.

[0043] Table 1 LC-MS / MS Parameter Settings

[0044]

[0045] After LC-MS / MS identification, in the sample extracted by ultrasonic wave at 23 + 25 + 28 (kHz), high-abundance glycopeptide molecules (peak area 9.79×10 4 ) were obtained. The mass spectrometry diagram of the glycopeptide molecules is shown in Figure 1 . The glycopeptide molecule sequence is AVS(GalNAc)VPLQ, and the molecular weight is 915 Da; the sugar chain composition of the glycopeptide molecule is N-acetylgalactosamine (GalNAc), in an O-linked glycosylation manner, and is connected to the hydroxyl group of serine (Ser) of the peptide chain through an α-glycosidic bond, named edible mushroom glycopeptide molecule, and its structural formula is shown in Formula I, and the 3D structure diagram is shown in Figure 2 .

[0046]

[0047] Example 2

[0048] Efficacy Verification of Edible Mushroom Glycopeptide

[0049] The edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ used in this example was synthesized by Genscript Biotech (Shanghai) Corporation Limited according to the edible mushroom glycopeptide structure identified in Example 1, and the purity of the synthesized sample was greater than 98%.

[0050] 1. Analysis of angiotensin-converting enzyme (ACE) inhibitory activity

[0051] The method for analyzing the ACE inhibitory activity of edible mushroom glycopeptides was the kit method. The ACE Kit-WST kit method was based on ACE catalyzing the substrate 3-Hydroxybutyryl-Gly-Gly-Gly (3HB-GGG) to release the product 3-Hydroxybutyric acid (3HB), and the product was further reduced to a colored product. The depth of the color was proportional to the ACE activity. By measuring the absorbance of the colored product, the ACE activity could be quantitatively analyzed, and the ACE inhibitor activity value (inhibition rate) was calculated through the inhibited part.

[0052] Accurately weigh 0.1 g of edible mushroom glycopeptide, add 50 mL of pure water to dissolve it, and gradually dilute it to sample solutions with concentration gradients of 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, and 0.0032 mg / mL. Take 20 μL of the sample solution in the sample group, add 20 μL of the kit matrix buffer and 20 μL of the kit enzyme working solution (containing ACE and aminoacylase), incubate at 37 °C for 60 min, add 200 μL of the kit indicator working solution (containing 3-hydroxybutyric acid dehydrogenase (3HBDH), coenzyme, and indicator), incubate at 25 °C for 10 min, measure the absorbance value A at 450 nm, and calculate the inhibition rate according to the following formula. The inhibition rates of edible mushroom glycopeptides at different concentrations are shown in Figure 3 .

[0053] ACE inhibition rate (%) = (A Blank1 - A Sample ) ÷ (A Blank1 - A Blank2 ) × 100%

[0054] Among them, Blank1 was the fully colored group without inhibition, which was a reaction system composed of 20 μL of pure water, 20 μL of the kit matrix buffer, 20 μL of the kit enzyme working solution, and 200 μL of the kit indicator working solution; Blank2 was the blank control group of the reagent, which was a reaction system composed of 40 μL of pure water, 20 μL of the kit matrix buffer, and 200 μL of the kit indicator working solution. The reaction temperature, time, and detection wavelength of Blank1 and Blank2 were the same as those of the sample group.

[0055] Taking the mass concentration of the sample solution and the inhibition rate as the horizontal and vertical coordinates, an inhibition curve was plotted, and the mass concentration of the sample at an inhibition rate of 50% (IC 50 ) was calculated from the inhibition curve.

[0056] The results of in vitro activity analysis showed that the IC 50 value of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ inhibiting ACE was 0.25 mg / mL.

[0057] 2. Analysis of the inhibitory activity against pancreatic lipase

[0058] In the sample group, 80 μL of 50 mmol / L Tris-HCl buffer (pH 8.0), 40 μL of the aqueous solution of edible mushroom glycopeptide (set at different concentrations according to the concentration gradient in the range of 0.1 - 5.0 mg / mL), and 120 μL of 10 mg / mL pancreatic lipase (PL) solution were mixed evenly and incubated at 37 °C for 10 min. Immediately, 160 μL of 0.8 mmol / L p-nitrophenyl palmitate (p-nitrophenyl phosphate, pNPP) was added as the substrate and reacted at 37 °C for 20 min. After the reaction, a water bath at 100 °C was used for 5 min to terminate the reaction. The precipitate was removed by centrifugation, and 200 μL of the supernatant was taken to measure the absorbance (A 样品 ) at a wavelength of 405 nm. In the blank group, an equal volume of buffer was used to replace the pancreatic lipase solution and the aqueous solution of edible mushroom glycopeptide, and the absorbance (A 空白 ) of the corresponding supernatant was measured. In the positive group, an equal volume of the positive drug orlistat (10 μg / mL) solution was used as the inhibitor to replace the aqueous solution of edible mushroom glycopeptide, and the absorbance (A 阳性 ) of the corresponding supernatant was measured. In the control group, an equal volume of buffer was used to replace the edible mushroom glycopeptide solution, and the absorbance (A 对照 ) of the corresponding supernatant was measured. Except for the different solution compositions in each group, the remaining reaction steps and measurement conditions were the same. The solution composition of each group is shown in Table 2.

[0059] Table 2 Solution composition for the analysis of pancreatic lipase inhibitory activity

[0060] experimental group Tris-HCl buffer (μL) glycopeptide (μL) pancreatic lipase (μL) orlistat (μL) sample group 80 40 120 0 blank group 240 0 0 0 positive group 80 0 120 40 control group 120 0 120 0

[0061] Each experiment was repeated 3 times, and the inhibition rate of the edible mushroom glycopeptide against pancreatic lipase was calculated according to the following formula:

[0062] Inhibition rate of pancreatic lipase activity (%) = [1 - (A 样品 - A 空白 ) / (A 对照 - A 空白 )] × 100%

[0063] Regarding A in the above formula 样品Replace with A 阳性 Calculate the inhibition rate of the positive drug orlistat on pancreatic lipase activity.

[0064] The inhibition rates of edible mushroom glycopeptides and positive drugs (positive controls) at different concentrations on pancreatic lipase are shown in Figure 4 . Using the mass concentration of the edible mushroom glycopeptide solution and the inhibition rate as the horizontal and vertical coordinates, fit the concentration-inhibition rate curve and calculate the half-inhibitory concentration (IC 50 ).

[0065] The results of in vitro activity analysis showed that the IC 50 value of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ inhibiting PL was 3.9 mg / mL.

[0066] 3. Effect on hypertension

[0067] Human umbilical vein endothelial cells (HUVECs) were induced by angiotensin II (Ang II) to establish a hypertension cell model. The effect of the edible mushroom glycopeptide AVS(GalNAc)VPLQ on the release of nitric oxide (NO) by HUVECs cells was detected.

[0068] HUVECs were cultured in complete medium containing 10% fetal bovine serum (including 1% non-essential amino acids, 100 μg / mL streptomycin and 100 U / mL penicillin) until the logarithmic growth phase (containing 1×10 6 cells). Set up a blank group (HUVECs cells cultured normally), an HUVEC cell group induced by Ang II (Ang II group), and an HUVECs cell group induced by Ang II and then added with the edible mushroom glycopeptide AVS(GalNAc)VPLQ (10 μM) (glycopeptide group). The Ang II group was cultured and induced in HUVECs cells with a culture solution containing Ang II (10 μM) at 37 °C for 12 h. The glycopeptide group was to discard the cell culture solution induced by Ang II for 12 h and add complete culture solution containing 0.5 mg / mL edible mushroom glycopeptide AVS(GalNAc)VPLQ and culture at 37 °C for 12 h. The cell supernatants of each group were collected, and the concentration of nitrite (NO metabolite) was detected using a Griess kit (Hangzhou Gaosheng Biotechnology Co., Ltd.). Compare the NO release amounts of each group to verify whether AVS(GalNAc)VPLQ promotes vasodilation.

[0069] The effect of the edible mushroom glycopeptide AVS(GalNAc)VPLQ on the NO content of HUVECs cells is shown in Figure 5, The results showed that the NO content in the Ang II group was the lowest (22.2 ± 3.1 μmol / mL), indicating that the hypertensive cell model was successfully established (significance analysis: P value < 0.0001). After treatment with edible mushroom glycoprotein, the NO release was significantly higher than that in the Ang II group, and the NO concentration in HUVECs cells increased by 3 times, with the NO content reaching 71.68 ± 4.5 μmol / mL, indicating that edible mushroom glycoprotein promoted the increase of intracellular NO content, and there was no significant difference compared with the blank group. Since the NO content is positively correlated with the content of endothelial relaxation factor, and endothelial relaxation factor helps with vasodilation, it shows that edible mushroom glycoprotein promotes the increase of NO level and the increase of endothelial relaxation factor content, which can effectively reduce blood pressure.

[0070] 4. Effect on hyperlipidemia

[0071] Human normal hepatocytes (LO-2) were induced by oleic acid to establish a hyperlipidemia cell model. The inhibitory effect of edible mushroom glycoprotein AVS(GalNAc)VPLQ on lipid droplet aggregation in LO-2 hepatocytes was evaluated by the proportion of lipid droplets in LO-2 cells. LO-2 cells were cultured in RPMI1640 cell medium containing 10% fetal bovine serum and 1% penicillin / streptomycin until the logarithmic growth phase (containing 1×10 6 cells). A blank group (normally cultured LO-2 cells), an LO-2 cell group induced by oleic acid (OA group), and an LO-2 cell group treated with edible mushroom glycoprotein (1.5 mg / mL) after induction (glycoprotein group) were set up. The OA group was cultured and induced at 37 °C for 24 h by adding serum-free culture medium containing 1 mmol / L oleic acid to LO-2 cells. In the glycoprotein group, the cell culture medium induced by oleic acid for 24 h was discarded, and the culture medium containing 10 μM edible mushroom glycoprotein AVS(GalNAc)VPLQ was added and cultured at 37 °C for 24 h. The oil red O staining method was used to measure the intracellular lipid droplet content.

[0072] The statistical results of the proportion of positive staining area of oil red O are shown in Figure 6 , The results showed that compared with the blank group, the lipid droplet content in LO-2 cells of the OA group increased significantly, indicating that the hyperlipidemia cell model was successfully established (significance analysis: P value < 0.0001). The lipid droplet content in LO-2 cells of the glycoprotein group was lower and close to the level of the blank group. This shows that edible mushroom glycoprotein AVS(GalNAc)VPLQ has a good inhibitory effect on lipid droplet aggregation in hepatocytes in vitro.

[0073] 5. Molecular docking of the inhibitory activity of edible mushroom glycoprotein

[0074] Download the crystal structures of angiotensin-converting enzyme (PDB: 1O86) and pancreatic lipase (PDB: 1LPB) from the RCSB database (https: / / www.rcsb.org / ). Optimize the crystal structures of the receptor proteins using the MOE 2019 molecular docking software, remove water molecules, and complete the hydrogen atoms. Construct the 3D structure of the edible mushroom glycopeptide using the MOE software and perform molecular energy minimization. Use the Site Finder module of the MOE to determine the amino acid residue active sites of the receptor proteins. Select the complex of the edible mushroom glycopeptide and the receptor protein with tight binding based on the docking score, number of bonds formed, and binding bond energy between the edible mushroom glycopeptide and the receptor protein. Analyze the binding sites and interaction modes between the glycopeptide molecule and the receptor protein in the complex using the MOE software.

[0075] The amino acid sites that can bind within the cavity pocket of the ACE receptor protein consist of 133 amino acid residues, including the three active pockets S1 (ALA354, GLU384, and TYR523), S2 (GLN281, HIS353, HIS513, LYS511, and TYR520), and S1' (GLU162) reported in the literature. The active center contains a divalent zinc ion, and the HEXXH structure composed of zinc ion, HIS383, HIS387, and GLU411 is also the active center of ACE.

[0076] Six hydrogen bonds, three metal bonds, and four ionic bond interaction forces are formed between the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ and the ACE molecule, with a binding bond energy of -56.8 kcal / mol (the lower the binding bond energy, the higher the intermolecular binding strength). Among them, the binding bond energy of the peptide molecule binding to ACE is -50.7 kcal / mol, and the binding bond energy of the sugar molecule binding to ACE is -6.1 kcal / mol. In the edible mushroom glycopeptide molecule, the peptide molecule forms a binding interaction force with the active zinc ion ZN of the ACE enzyme with a binding bond energy of -34.7 kcal / mol, breaking the binding bonds between the zinc ion and GLU411, HIS387, and HIS383; the peptide molecule forms a hydrogen bond with ALA354. The oxygen atom in the N-acetylgalactosamine molecule forms a hydrogen bond interaction force with GLU384 and also forms a binding bond with the active zinc ion ZN 2+ forming a binding bond that breaks the HEXXH active structure center. In summary, the binding of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ to ACE significantly inhibits the activity of ACE (Table 3, 2+ ) Figure 7 )

[0077] Table 3 Docking results of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ and the ACE molecule

[0078]

[0079]

[0080] Pancreatic lipase (PDB 1LPB) consists of a catalytic triad (Ser153-His264-Asp177), a dynamic lid structure (residues 238-262), and an active site composed of hydrophobic residues such as Phe78, Leu153, and Ile209, which has the activity of guiding lipid substrates to enter.

[0081] Twelve hydrogen bonds and three ionic bond interaction forces are formed between the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ and the PL molecule, and the binding energy is -48.3 kcal / mol. Among them, the binding energy of the peptide molecule binding to PL is -40.3 kcal / mol, and the binding energy of the sugar molecule binding to PL is -8 kcal / mol. The edible mushroom glycopeptide binds to the amino acid residues in the dynamic lid structure of pancreatic lipase, occupies the substrate binding pocket of pancreatic lipase, and blocks the contact of the substrate. In summary, the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ binding to PL significantly inhibits the activity of PL (Table 4, Figure 8 ).

[0082] Table 4 Docking results of the edible mushroom glycopeptide molecule AVS(GalNAc)VPLQ and the PL molecule

[0083]

[0084] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An edible mushroom glycoprotein, characterized in that, The molecular sequence of the edible mushroom glycopeptide is AVS(GalNAc)VPLQ; the sugar chain composition of the edible mushroom glycopeptide molecule is N-acetylgalactosamine, which is connected to the serine hydroxyl group of the peptide chain through an O-glycosylation mode by an α-glycosidic bond.

2. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of an angiotensin converting enzyme inhibitor.

3. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a pancreatic lipase inhibitor.

4. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a product for treating hypertension.

5. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a product for treating hyperlipidemia.

6. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a product that helps maintain a healthy blood pressure level.

7. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a product that helps maintain a healthy blood lipid level.

8. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a product that promotes an increase in intracellular NO.

9. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a product that inhibits the aggregation of lipid droplets in hepatocytes.

10. Use of the edible mushroom glycopeptide according to claim 1 in the preparation of a weight loss product.

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