An active peptide from *Flammulina velutipes* and its glycopeptides and their applications

Abstract

This invention belongs to the field of bioactive peptide technology, specifically relating to an active peptide from *Flammulina velutipes*, its glycopeptides, and their applications. The active peptide of this invention includes a first active peptide and / or a second active peptide. Both the first and second active peptides have targeting effects on ACE, GLP-1 receptors, and GIP receptors, achieving inhibitory effects on ACE and agonistic effects on GLP-1 and GIP receptors. The glycopeptides of the active peptide molecule exhibit stronger binding activity to ACE, GLP-1 receptors, and GIP receptors, enabling better targeting of binding sites and a more diversified mechanism of action. This invention experimentally discovered that the active peptide and its fucosylated glycopeptides are the key structural basis for enhancing hypotensive and weight-loss activities. This invention provides new insights for research related to hypoglycemia, weight loss, and hypotension.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an active peptide from *Deer Antler Mushroom*, its glycopeptide, and its applications. Background Technology

[0002] Deer antler mushroom, a rare edible and medicinal fungus, is rich in nutrients, high in protein, and low in fat. It also possesses anti-tumor, blood pressure-lowering, immune-regulating, and liver-protecting effects. Its main functional active substances are polysaccharides, sterols, and nucleosides.

[0003] In recent years, research on isolating and identifying functional proteins and bioactive peptides from *Agaricus esculentus* has gradually increased. For example, prior art CN115786312A discloses a novel fibrinolytic enzyme from *Agaricus esculentus*, which can sequentially degrade the α, β, and γ chains of human fibrinogen, exhibiting good thrombolytic effects; another prior art CN119119195A discloses a flavor-enhancing peptide from *Agaricus esculentus*, which has excellent effects on enhancing rich and savory flavors. However, existing technologies have not sufficiently investigated the functional bioactive peptide components in *Agaricus esculentus*, and exploring these functional bioactive peptides remains a pressing issue in this field.

[0004] EF1-alpha is a highly conserved translation elongation factor in eukaryotes, and its known biological functions mainly focus on promoting GTP-dependent binding of aminoacyl-tRNA to the ribosomal A site. Current research on EF1-alpha primarily focuses on its use as an internal reference gene, promoter element, gene engineering expression, and vaccine development. Functional studies on functional proteins and bioactive peptides in *Agaricus esculentus* are still very limited. Summary of the Invention

[0005] To address the lack of bioactive peptides and glycopeptides from *Deer Antler Mushroom* that possess antihypertensive, hypoglycemic, and / or weight-loss functions in existing technologies, this invention provides a *Deer Antler Mushroom* bioactive peptide and its glycopeptides, along with their applications, specifically including the following technical solutions: This invention provides an active peptide, which includes a first active peptide and / or a second active peptide; The sequence of the first active peptide is shown in SEQ ID NO:2; The sequence of the second active peptide is shown in SEQ ID NO:3.

[0006] Preferably, the glycopeptide comprises a first glycopeptide and / or a second glycopeptide; The first glycopeptide comprises an active peptide with an amino acid sequence as shown in SEQ ID NO:2 and two first glycan chains; the two first glycan chains are respectively linked to threonine residues and serine residues in the sequence shown in SEQ ID NO:2 in the form of O-glycosidic bonds; The second glycopeptide comprises an active peptide with an amino acid sequence as shown in SEQ ID NO:3 and a second sugar chain, the second sugar chain being linked to a threonine residue of the sequence shown in SEQ ID NO:3 via an O-glycosidic bond.

[0007] Preferably, both the first and second sugar chains are fucose residues.

[0008] The present invention also provides a deer antler mushroom glycoprotein, wherein the deer antler mushroom glycoprotein contains the active peptides and / or glycopeptides as described above.

[0009] The present invention also provides a method for preparing the antler mushroom glycoprotein as described above, comprising: Protein was extracted from *Flammulina velutipes* to obtain a crude extract of *Flammulina velutipes*. The crude extract of *Deer Antler Mushroom* was subjected to fractionation and precipitation, and the precipitate was collected to obtain crude protein from *Deer Antler Mushroom*. The crude protein from *Flammulina velutipes* was subjected to ion exchange chromatography to obtain negatively charged crude protein from *Flammulina velutipes*. The negatively charged crude protein from *Pleurotus ostreatus* was subjected to filtration chromatography, and the elution peak with ultraviolet absorption at 280 nm was collected to obtain *Pleurotus ostreatus* glycoprotein.

[0010] The present invention also provides the application of the active peptides, glycopeptides, velvet antler glycoproteins or velvet antler glycoproteins prepared by the above-mentioned methods in the preparation of products for treating hypertension.

[0011] The present invention also provides the application of the active peptides, glycopeptides, velvet antler glycoproteins or velvet antler glycoproteins prepared by the above-described methods in the preparation of angiotensin-converting enzyme inhibitors.

[0012] The present invention also provides the application of the active peptides, glycopeptides, velvet antler glycoproteins or velvet antler glycoproteins prepared by the above-described methods in the preparation of products for treating obesity.

[0013] The present invention also provides the use of the active peptides, glycopeptides, velvet hyaluronic acid glycoproteins or velvet hyaluronic acid glycoproteins prepared by the above-described methods in the preparation of GLP-1 receptor agonists and / or GIP receptor agonists.

[0014] The present invention also provides a product for treating hypertension and / or obesity, the product comprising active ingredients and excipients; The active ingredients include the active peptides, glycopeptides, velvet antler glycoproteins, or velvet antler glycoproteins prepared by the methods described above.

[0015] The beneficial effects of this invention are as follows: This invention provides an active peptide, comprising a first active peptide and / or a second active peptide; the sequence of the first active peptide is shown in SEQ ID NO:2; the sequence of the second active peptide is shown in SEQ ID NO:3. The active peptide of this invention is derived from the functional protein elongation factor 1α in *Agrocybe aegerita*. Through functional annotation of elongation factor 1α, this invention has discovered that this functional protein contains 7 active peptides and 14 glycopeptides, wherein the first and second active peptides all have targeting effects on ACE, GLP-1 receptor, and GIP receptor. In in vitro cell experiments, the active peptide exhibited good inhibitory activity against ACE, a secretory-promoting effect on GLP-1, and agonistic activity against GIP.

[0016] This invention also discloses glycopeptides of the aforementioned bioactive peptides, wherein the glycopeptides include a first glycopeptide and / or a second glycopeptide; the first glycopeptide is a glycopeptide of the first bioactive peptide as described above; and the second glycopeptide is a glycopeptide of the second bioactive peptide as described above. The first and second glycopeptides of this invention have targeting effects on ACE, GLP-1 receptors, and GIP receptors, exhibiting more diverse mechanisms of action. Compared to the first and second bioactive peptides, the glycopeptides of the first and second bioactive peptides showed stronger binding activity in molecular binding experiments, enabling them to better target binding sites. This invention experimentally discovered that fucosylation modification is a key structural basis for enhancing hypotensive and weight-loss activities, and that glycopeptides are significantly superior to their non-glycosylated counterparts in both hypotensive and weight-loss / anti-obesity effects. This invention provides new insights for research related to hypoglycemia, weight loss, and hypotension. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The amino acid sequence of the functional protein LDP from *Flammulina velutipes* and the distribution of its contained active peptides and glycopeptide fragments are shown in the diagram. Figure 2 The results of the Ramachandran plot (plot) of the functional protein LDP from *Fragaria deer antler* are used to verify the structural rationality. Figure 3 This is the secondary mass spectrum of the peptide ETSGFIK; Figure 4 This is the secondary mass spectrum of the peptide VETGIIK. Figure 5 The mass spectrum of the glycopeptide ET(Fuc)S(Fuc)GFIK is shown. Figure 6 This is the secondary mass spectrum of the glycopeptide molecule VET(Fuc)GIIK; Figure 73D diagram of molecular docking between functional protein LDP and ACE; Figure 8 This is a diagram showing the intramolecular residue binding energy distribution of ACE and the functional protein LDP; Figure 9 2D diagram of the docking of peptide ETSGFIK and ACE molecules; Figure 10 3D diagram of the docking of the glycopeptide molecule ET(Fuc)S(Fuc)GFIK with the ACE molecule; Figure 11 2D diagram of the docking of the glycopeptide molecule ET(Fuc)S(Fuc)GFIK with the ACE molecule; Figure 12 2D diagram of the docking of the peptide VETGIIK with the ACE molecule; Figure 13 3D diagram of the docking of the glycopeptide molecule VET(Fuc)GIIK with the ACE molecule; Figure 14 2D diagram of the docking of the glycopeptide molecule VET(Fuc)GIIK with the ACE molecule; Figure 15 3D diagram of molecular docking between functional protein LDP and GLP-1; Figure 16 The diagram shows the intramolecular residue binding energy distribution of GLP-1 and the functional protein LDP; Figure 17 2D diagram of the docking of peptide ETSGFIK with GLP-1 molecule; Figure 18 2D diagram of the docking of peptide VETGIIK and GLP-1 molecule; Figure 19 2D diagram of the docking of the glycopeptide molecule ET(Fuc)S(Fuc)GFIK with the GLP-1 molecule; Figure 20 2D diagram of the docking of the glycopeptide molecule VET(Fuc)GIIK with the GLP-1 molecule; Figure 21 3D diagram of molecular docking between functional protein LDP and GIP; Figure 22 The diagram shows the intramolecular residue binding energy distribution of GIP and the functional protein LDP. Figure 23 2D diagram of the docking of peptide ETSGFIK and GIP molecules; Figure 24 2D diagram of the docking of peptide VETGIIK and GIP molecule; Figure 25 A 2D diagram of the molecular docking between the glycopeptide molecule ET(Fuc)S(Fuc)GFIK and GIP; Figure 26 2D diagram of the docking of the glycopeptide molecule VET(Fuc)GIIK with the GIP molecule; Figure 27 The results show the molecular interaction between deer antler mushroom protein and ACE; Figure 28 The results show the molecular interaction between the active peptide ETSGFIK and ACE; Figure 29 This is the result of the molecular interaction between the active peptide VETGIIK and ACE; Figure 30 The results are for the molecular interaction between the glycopeptide ET(Fuc)S(Fuc)GFIK and ACE; Figure 31 The results are from the molecular interaction between the glycopeptide VET(Fuc)GIIK and ACE; Figure 32 This is the result of the molecular interaction between captopril and ACE. Detailed Implementation

[0019] The present invention provides an active peptide, wherein the active peptide comprises a first active peptide and / or a second active peptide.

[0020] In one embodiment, the sequence of the first active peptide is shown in SEQ ID NO:2. In another embodiment, the molecular weight of the amino acid sequence shown in SEQ ID NO:2 is 780 Da.

[0021] SEQ ID NO:2: ETSGFIK.

[0022] In one embodiment, the sequence of the second active peptide is shown in SEQ ID NO:3. In another embodiment, the molecular weight of the amino acid sequence shown in SEQ ID NO:3 is 758 Da.

[0023] SEQ ID NO:3: VETGIIK.

[0024] It should be noted that the terms "first" and "second" used in this invention are merely for distinguishing between the two active peptides and do not have any limiting meaning. The active peptides described in this invention are derived from the functional protein extension factor 1α in *Flammulina velutipes*. Through extraction and processing using extension factor 1α, *Flammulina velutipes* functional protein is obtained. This functional protein contains a first active peptide and a second active peptide. Both the first and second active peptides have targeting effects on ACE, GLP-1 receptors, and GIP receptors, and can achieve therapeutic effects by acting on these receptors.

[0025] The present invention also provides glycopeptides of the active peptides described above, wherein the glycopeptides include a first glycopeptide and / or a second glycopeptide.

[0026] It should be noted that the terms "first" and "second" used in this invention are merely for distinguishing between the two glycopeptides and do not have any limiting meaning. In one embodiment, the first glycopeptide is a glycopeptide of the first active peptide as described above; the second glycopeptide is a glycopeptide of the second active peptide as described above. In one embodiment, the first glycopeptide comprises an active peptide with the amino acid sequence shown in SEQ ID NO:2 and two first glycan chains; the two glycan chains are respectively linked to threonine and serine residues in the sequence shown in SEQ ID NO:2 via O-glycosidic bonds. In one embodiment, both first glycan chains are fucose residues. In one embodiment, the sequence of the first glycopeptide corresponding to the active peptide molecule shown in SEQ ID NO:2 is: ET(Fuc)S(Fuc)GFIK. In one embodiment, the molecular weight of the first glycopeptide corresponding to the active peptide molecule shown in SEQ ID NO:2 is 1073 Da.

[0027] In one embodiment, the second glycopeptide comprises an active peptide with the amino acid sequence shown in SEQ ID NO:3 and a second glycan chain, the glycan chain being linked to a threonine residue of the sequence shown in SEQ ID NO:3 via an O-glycosidic bond. In one embodiment, the second glycan chain is a fucose residue. In one embodiment, the sequence of the second glycopeptide corresponding to the active peptide molecule shown in SEQ ID NO:3 is: VET(Fuc)GIIK. In one embodiment, the molecular weight of the second glycopeptide corresponding to the active peptide molecule shown in SEQ ID NO:3 is 905 Da.

[0028] The first and second glycopeptides of this invention are derived from elongation factor 1α. As one embodiment, the elongation factor 1α has the NCBI accession number Q2HXZ6, a molecular weight of 26873 Da, and a full length of 245 amino acids. This invention, through GO annotation, discovered that elongation factor 1α possesses GTP-binding activity, GTPase activity, and translation elongation factor activity. This protein can produce 21 bioactive peptide molecules, including 7 bioactive peptides and 14 glycopeptide molecules. Therefore, this invention further studies the bioactive peptide molecules produced by elongation factor 1α. In the prior art, elongation factor 1α, as a highly conserved translation elongation factor in eukaryotes, is known to have biological functions limited to GTP-dependent binding during protein translation, and has been applied in fields such as internal reference gene screening, promoter elements, gene engineering expression, and vaccine development. However, no other functions of this protein have been revealed. This invention reveals for the first time that EF1-alpha (elongation factor 1α) derived from *Amanita muscaria* has dual effects of ACE inhibition and GLP-1 / GIP receptor activation, laying the foundation for expanding the sources of natural bioactive proteins with multiple health benefits.

[0029] Existing technologies do not reveal specific heptapeptides modified with O-fucosylation, nor are there any reports on the ACE inhibition of specific heptapeptides. The first and second glycopeptides disclosed in this invention possess unique O-glycosylation modification patterns. Molecular docking experiments on these glycopeptides showed that, compared to their corresponding active peptides, the glycopeptides had lower docking scores, more binding bonds, and lower bond energies. The fucose residues themselves contributed multiple hydrogen bonds and binding energies, indicating that glycosylation modification significantly enhances ACE inhibitory activity.

[0030] This invention also discloses the multi-target characteristics of the aforementioned bioactive peptides and their glycopeptides. These bioactive peptides and their glycopeptides exhibit targeting effects on ACE, GLP-1 receptors, and GIP receptors, demonstrating more diverse mechanisms of action. Furthermore, compared to bioactive peptide molecules, the glycopeptides of bioactive peptide molecules showed stronger binding activity in molecular binding experiments, enabling them to better target binding sites. This invention experimentally discovered that fucosylation modification is the key structural basis for enhancing antihypertensive and weight-loss activities, and that glycopeptides are significantly superior to their non-glycosylated counterparts in both antihypertensive and weight-loss / anti-obesity effects.

[0031] In existing research on bioactive peptides, ACE-inhibiting peptide patents mainly focus on dipeptide, tripeptide, or pentapeptide structures. Heptapeptide sequences have been reported, but their existing heptapeptide sequences differ from the two bioactive peptide heptapeptide sequences of this invention. The ETSGFIK and VETGIIK disclosed in this invention are two novel heptapeptide sequences, and these sequences exhibit excellent ACE-inhibiting activity.

[0032] In one embodiment, those skilled in the art can obtain the above-mentioned active peptides through chemical synthesis. In another embodiment, those skilled in the art can obtain the above-mentioned glycopeptides through chemical enzymatic synthesis.

[0033] The present invention also provides a deer antler mushroom glycoprotein, wherein the deer antler mushroom glycoprotein contains the active peptides and / or glycopeptides as described above.

[0034] The present invention also provides a method for preparing the above-mentioned antler mushroom glycoprotein, the preparation method comprising the following steps: Protein was extracted from *Flammulina velutipes* to obtain a crude extract; the extraction included phosphate buffer extraction. The crude extract of *Deer Antler Mushroom* was subjected to fractionation and precipitation, and the precipitate was collected to obtain crude protein from *Deer Antler Mushroom*. The crude protein from *Flammulina velutipes* was subjected to ion exchange chromatography to obtain negatively charged crude protein from *Flammulina velutipes*. The negatively charged crude protein from *Pleurotus ostreatus* was subjected to filtration chromatography, and the elution peak with ultraviolet absorption at 280 nm was collected to obtain *Pleurotus ostreatus* glycoprotein.

[0035] In one embodiment, the extraction includes extraction using phosphate buffer, ammonium sulfate fractionation, DADE ion exchange chromatography, and gel filtration. In one embodiment, after phosphate buffer extraction, a crude extract of *Pleurotus ostreatus* is obtained. In another embodiment, the crude extract is subjected to sulfuric acid fractionation, and the fraction with 30%–80% saturation is collected to obtain crude *Pleurotus ostreatus* protein. In another embodiment, the crude *Pleurotus ostreatus* protein is subjected to DADE ion exchange chromatography, and the negatively charged eluent fraction is collected to obtain negatively charged crude *Pleurotus ostreatus* protein. In yet another embodiment, the negatively charged crude *Pleurotus ostreatus* protein is subjected to gel filtration, and the 280 nm absorption peak is collected to obtain *Pleurotus ostreatus* glycoprotein.

[0036] In one embodiment, the separation includes separation using polypropylene gel electrophoresis. In another embodiment, the separation involves performing polypropylene gel electrophoresis on the *Pleurotus ostreatus* glycoprotein, collecting the target protein band, and obtaining electrophoretically purified *Pleurotus ostreatus* glycoprotein.

[0037] In one embodiment, the purification method includes C18 reversed solid-phase extraction desalting. In another embodiment, the separation involves: performing C18 reversed solid-phase extraction desalting on electrophoretically purified *Pleurotus ostreatus* glycoprotein, and collecting the desalted eluent to obtain *Pleurotus ostreatus* glycoprotein. The *Pleurotus ostreatus* glycoprotein contains the active peptides and / or glycopeptides described above.

[0038] The present invention also provides the use of the above-mentioned active peptides, glycopeptides and / or deer antler glycoprotein in the preparation of products for treating hypertension.

[0039] In one implementation, the treatment of hypertension includes lowering blood pressure. In another implementation, this lowering of blood pressure is achieved by targeting ACE and inhibiting ACE activity. The active peptides and / or active glycopeptides of *Deer Antler Mushroom* described in this invention can specifically bind to key amino acid residues (such as Ala354, Glu384, etc.) in the active pocket of angiotensin-converting enzyme (ACE), competitively blocking the hydrolysis of the substrate angiotensin I by the ACE catalytic domain, thereby reducing the generation of the potent vasoconstrictor angiotensin II, ultimately achieving an auxiliary antihypertensive effect. Molecular docking results further confirm that the *Deer Antler Mushroom* glycopeptide, due to the partial participation of the glycan chain in forming an additional hydrogen bond network, has a lower binding energy and stronger affinity to ACE, indicating superior ACE inhibitory activity.

[0040] The present invention also provides the use of the active peptides, glycopeptides and / or velvet antler glycoproteins as described above in the preparation of angiotensin-converting enzyme inhibitors.

[0041] In one implementation, the active peptides and / or glycopeptides can target ACE, inhibiting ACE activity and preventing angiotensin I from being effectively converted into the potent vasoconstrictor angiotensin II, thereby causing vasodilation, decreased peripheral resistance, and lower blood pressure. Furthermore, the reduction in angiotensin II can also induce aldosterone secretion, leading to decreased renal tubular reabsorption of sodium and water, reduced blood volume, and further achieving a hypotensive effect.

[0042] The present invention also provides the use of the above-mentioned active peptides, glycopeptides and / or deer antler glycoproteins in the preparation of products for treating obesity.

[0043] In one implementation, the treatment of obesity includes lowering blood sugar and / or weight loss. In another implementation, the weight loss and / or lowering of blood sugar is achieved by activating GLP-1 or GIP receptors.

[0044] The present invention also provides the use of the active peptides, glycopeptides and / or velvet spore glycoproteins as described above in the preparation of GLP-1 receptor agonists and / or GIP receptor agonists.

[0045] In one embodiment, the active peptides and / or glycopeptides can target GLP-1 receptors and / or GIP receptors. Activation of GLP-1 receptors or GIP receptors can suppress appetite, enhance satiety, or delay gastric emptying, thereby achieving a weight loss effect. The peptide molecules or glycopeptides can activate GLP-1 receptors or GIP receptors, which can promote pancreatic secretion in a glucose-dependent manner. Furthermore, GLP-1 can inhibit the release of glucagon, reducing hepatic glucose output and further lowering blood glucose levels. Therefore, the active peptides or glycopeptides of the present invention can achieve hypoglycemic and weight loss effects by activating the activity of GLP-1 or GIP.

[0046] The present invention also provides a product for treating hypertension and / or obesity, the product comprising active ingredients and excipients; the active ingredients comprising glycopeptides, active peptides and / or deer antler glycoprotein as described above.

[0047] In one embodiment, the product includes any one of pharmaceuticals, health supplements, and functional foods. In another embodiment, the health supplement includes any one or more of the following: health supplements that help control body fat, health supplements that help maintain healthy blood sugar levels, and health supplements that help maintain healthy blood pressure levels.

[0048] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a deer antler mushroom active peptide and its glycopeptide and applications provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1: Extraction and Identification of LDP, a Functional Protein from *Fungiformis deer* Powdered fruiting bodies of *Flammulina velutipes* were extracted using 0.05 mol / L phosphate buffer to obtain a crude extract. The crude extract was then subjected to fractional precipitation with 60% ammonium sulfate to obtain crude protein. The crude protein was eluted using DEAE ion-exchange chromatography (elution with 1.0 mol / L NaCl), and the negatively charged eluent fraction was collected to obtain negatively charged crude protein. This negatively charged crude protein was then subjected to Sephadex G25 gel filtration chromatography, and the protein elution peak was collected at 280 nm UV absorption to obtain *Flammulina velutipes* glycoprotein. The specific procedures for each method described above are as described in existing techniques.

[0050] The glycoprotein from *Deer Antler Fungus* was subjected to native-polyacrylamide gel electrophoresis (Native-PAGE) and its purity was identified as >95%. LC-MS / MS mass spectrometry identified the protein as extension factor 1α (EF1-alpha), with NCBI database accession number Q2HXZ6, a molecular weight of 26873 Da, and a full length of 245 amino acids.

[0051] The core functions of elongation factor 1α were verified using gene ontology (GO) annotation. It was found that elongation factor 1α possesses GTP binding (GO:0005525), GTPase activity (GO:0003924), and translation elongation factor activity (GO:0003746), making it a key factor mediating the delivery of aminoacyl-tRNA to the ribosomal A site during protein translation. It also participates in important physiological processes such as cytoskeleton organization, protein transport, and cell proliferation. This protein can produce 21 bioactive peptide molecules, including 7 bioactive peptides and 14 glycopeptides. Post-translational modification analysis showed that the protein exhibits N-terminal acetylation and has abundant glycosylation sites, totaling 12, specifically distributed at amino acid positions 5, 6, 12, 37, 47, 53, 137, 138, 181, 224, 232, and 242 on the protein chain. The protein sequence is shown in SEQ ID NO:1, and its structural and functional characteristics are as follows: Figure 1 As shown in Table 1, the protein has a well-defined structure and function, possessing a unique molecular structural basis as a functional protein.

[0052] SEQ ID NO:1:VPSRSSRRKLLSLLARAPSSTVPWVLDKLKAERERGITIDIALWKFETPKFMVTVIDAPGHRDFIKNMITGTSQADCAILIIAGGTGEFEAGISKDGQTREHALLAFTLGVRQLIVAVNKM DTTKWSEDRFNEIVKETSGFIKEVGYNPKTVAFVPISGWHGDNMLEESANMPWYKGWTRETKGGVVKGKTLLDAIDAIEPPVRPSEKPLRLPLQDVYKIGGIGTVPVGRVETGIIKAGMVVSFAP.

[0053] Table 1. Information on Functional Proteins from *Deer Antler Mushroom*

[0054] The EF1-alpha protein was structurally modeled using Alphafold (https: / / alphafold.ebi.ac.uk / ), resulting in a theoretical model of its three-dimensional structure. The rationality of the protein structure was then verified using SAVES v6.1 (https: / / saves.mbi.ucla.edu / ), and the Laplace plot evaluation results output by this platform are shown below. Figure 2 As shown, 100% of the amino acid residues of the EF1-alpha protein are located within the allowed regions of the Laplace diagram, with 96.1% located within the fully allowed regions of the Φ angle (Phiangle) and Ψ angle (Psi angle), and 3.9% located within the acceptable regions. This indicates that the dihedral angles of the amino acid residues in the protein structure are reasonable and the protein structure is reliable.

[0055] The 21 bioactive peptide molecules produced by the glycoprotein of *Agaricus esculentus* include 7 bioactive peptides and 14 glycopeptide molecules. Among them, 2 bioactive peptides can produce corresponding glycopeptide molecules. Therefore, the 2 bioactive peptides and their corresponding glycopeptide molecules were selected as the research objects.

[0056] Example 2: Identification of active peptides and glycopeptides Take the target protein band extracted in Example 1 into an EP tube, vortex wash twice with ultrapure water for 5 min each time, and remove the liquid; mix 50 mM NH4HCO3 and ACN (acetonitrile) at a volume ratio of 1:1 to obtain a mixed solution, sonicate for 15 min to decolorize, and remove the liquid; after removing the liquid, repeat the steps of decolorizing the gel block with a 1:1 mixture of sodium bicarbonate and acetonitrile and removing the liquid until the solution and gel block are colorless.

[0057] Mix 50 mM NH4HCO3 and ACN at a volume ratio of 1:1 to obtain a mixture. Wash the colorless gel block once with this mixture using a vortex oscillation. Then add 100% acetonitrile, oscillate, and dehydrate until the gel particles turn white. Remove the liquid and vacuum dry for 5 min. Add 150 μL of DTT solution to a final concentration of 10 mM until the gel block is submerged. Shake the DTT solution and gel block to mix thoroughly until the gel block swells and becomes transparent. Place the mixture of DTT and gel block in an oven at 56°C for 1 h.

[0058] After drying, remove the gel block, cool it to room temperature, blot off any residual reagent, and then quickly add 150 μL of 50 mM iodoacetamide (IAM) solution to the container to submerge the gel block. Place the gel block in the dark for 30 minutes. After this period, wash the gel block with different solutions, including: a mixture of 25 mM NH4HCO3, 50 mM NH4HCO3 and ACN in a 1:1 volume ratio, and 100% acetonitrile. After washing, dehydrate the gel block with acetonitrile until it turns white, and then vacuum dry the gel block for 5 minutes.

[0059] The vacuum-dried gel blocks were treated as follows: A 0.1 μg / μL trypsin stock solution was mixed with 25 mM NH4HCO3 at a volume ratio of 1:20 to obtain an enzyme solution mixture. The final concentration of trypsin in the enzyme solution mixture was 0.005 μg / μL. The gel blocks were submerged in the enzyme solution mixture, with the amount of enzyme solution sufficient to grind and swell the gel blocks. The gel blocks and enzyme solution mixture were briefly centrifuged at 2000–3000 rpm for 10–20 s to ensure full contact between the gel blocks and the enzyme solution mixture. The gel blocks and enzyme solution mixture were then incubated at 4°C for 30 min; after the solution was fully absorbed by the gel blocks, excess enzyme solution was removed. 25 mM NH4HCO3 was then added to the gel blocks, and digestion was carried out overnight at 37°C. The amount of NH4HCO3 added was 20 μL more than enough to submerge the gel blocks.

[0060] After digestion overnight, the enzyme solution was collected. 100 μL of a mixture of 30% acetonitrile and 0.1% TFA (volume percentage) was added to the original tube of the gel block, and the mixture was sonicated for 15 min. The solution was then aspirated to obtain the first solution. Next, 100 μL of a mixture of 60% acetonitrile and 0.1% TFA (volume percentage) was added to the original tube of the gel block, and the mixture was sonicated for 15 min to obtain the second solution. The first solution was then added to the second solution, and the mixture was lyophilized to obtain the lyophilized sample. The lyophilized sample was thoroughly dissolved in 0.1% TFA and used as a sample for later use.

[0061] The sample was rinsed and pipetted 10 times with a mixture of 50 μL of 60% acetonitrile and 0.1% TFA (v / v); washed and pipetted 10 times with 10 μL of 0.1% TFA; the sample was then aspirated and expelled, pipetting 20 times to remove the liquid; and washed and pipetted 5 times with 10 μL of 0.1% TFA. The peptides were eluted into new EP tubes with a mixture of 10 μL of 60% ACN and 0.1% TFA (v / v), and vacuum dried to obtain the functional protein from *Pleurotus ostreatus*. The collected peptides were analyzed by LC-MS / MS, and the results are as follows: Mass spectrometry analysis revealed two bioactive peptide molecules and their corresponding glycopeptides in the collected *Flammulina velutipes* functional protein (labeled LDP). The results are shown in Table 2. Figures 3-6 As shown: Table 2. Information on bioactive peptides / glycopeptides in Deer Antler Mushroom Protein Q2HXZ6

[0062] Note: The starting amino acid number on the protein chain refers to the starting amino acid position of the polypeptide molecule in the EF1-alpha protein sequence, and the ending amino acid number on the protein chain refers to the ending amino acid position of the polypeptide molecule in the EF1-alpha protein sequence.

[0063] As shown in Table 2, in this embodiment, two peptide molecules were obtained from the functional protein (LDP) of *Deer Antler Fungus* by mass spectrometry identification, and their sequences are shown as SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0064] SEQ ID NO:2: ETSGFIK; the secondary mass spectrum of this peptide molecule is shown below. Figure 3 As shown, it contains 7 amino acid residues and has a molecular weight of 780 Da. The glycopeptide molecule of the peptide molecule described in SEQ ID NO:2 is: SEQ ID NO:4: ET(Fuc)S(Fuc)GFIK, with a molecular weight of 1073 Da. In this glycopeptide molecule, two fucose residues are linked to threonine and serine residues respectively by O-glycosidic bonds. The secondary mass spectrum of this glycopeptide molecule is shown below. Figure 5 As shown.

[0065] SEQ ID NO:3: VETGIIK. The secondary mass spectrum of this peptide molecule is shown below. Figure 4 As shown, it contains 7 amino acid residues and has a molecular weight of 758 Da. The peptide molecule described in SEQ ID NO:3 is: SEQ ID NO:5: VET(Fuc)GIIK, with a molecular weight of 905 Da. In this glycopeptide molecule, a fucose residue is linked to a threonine residue via an O-glycosidic bond. The secondary mass spectrum of this glycopeptide molecule is shown below. Figure 6 As shown.

[0066] Example 3 Evaluation of angiotensin-converting enzyme (ACE) inhibitory activity The crystal structure of the ACE receptor protein was optimized using MOE molecular docking software. The accession number of the ACE receptor protein in the PDB database is 1O8A. Water molecules were removed, and hydrogen atoms were added. The 3D structures of the functional protein LDP, active peptide, and glycopeptide were constructed using MOE software, and molecular energy minimization was performed. Molecular docking binding analysis between the ACE receptor and the functional protein LDP, active peptide, and glycopeptide was conducted using both protein-protein and protein-peptide docking modes. Tightly bound complexes were selected based on the docking fraction, number of bonds, and bond energy. The binding sites and binding modes between molecules were analyzed using MOE software.

[0067] The ACE inhibitory activity of functional proteins LDP, bioactive peptides, and glycopeptides was evaluated using a molecular docking method. The molecular docking method was adapted from the prior art "Structural characterization and angiotensin-converting enzyme (ACE) inhibitory mechanism of Stropharia rugosoannulata mushroom peptides prepared by ultrasound. Ultrasonics Sonochemistry, 2022, 88106074." The evaluation results are as follows: Figures 7-13 As shown in Tables 3-7.

[0068] Table 3. Molecular docking results of ACE and functional protein LDP

[0069] Table 4. Docking results between peptide molecules ETSGFIK and ACE molecules.

[0070] Table 5. Docking results of glycopeptide molecules ET(Fuc)S(Fuc)GFIK with ACE molecules.

[0071] Table 6. Docking results between peptide VETGIIK and ACE molecules.

[0072] Table 7. Docking results between peptide molecules VET(Fuc)GIIK and ACE molecules.

[0073] Note: Total bond energy is the sum of the bond energies of all bonds in a peptide / glycopeptide molecule; (1) By Figures 7-8 As shown in Table 3, the docking score between the functional protein LDP and the ACE molecule was -19.25, and the total intermolecular binding bond energy was -27.44 kcal / mol. The amino acid residue Arg8 in the protein contributed approximately 50% of the binding energy, playing a crucial role in the protein's ACE inhibition. The binding strength of LDP to ACE (average binding bond energy -4.57 kcal / mol) was significantly higher than that of intramolecular residues in ACE (average binding bond energy -0.74 kcal / mol) and intramolecular residues in LDP (average binding bond energy -1.18 kcal / mol), indicating that LDP possesses excellent ACE inhibitory potential.

[0074] (2) By Figures 9-12 As shown in Tables 4-6, the docking score between the peptide ETSGFIK and the ACE molecule is -9.54, with 7 hydrogen bonds, 2 metal bonds, and 3 ionic bonds forming between them. The binding bond energy is -80.2 kcal / mol. The low bond energy and strong binding force indicate that the peptide molecule can inhibit ACE activity. The docking score between the peptide VETGIIK and the ACE molecule is -8.89, with 12 hydrogen bonds, 2 ionic bonds, and 2 hydrogen-π interactions forming between them. The binding bond energy is -67 kcal / mol. The low bond energy and strong binding force also indicate that the peptide molecule can inhibit ACE activity.

[0075] (3) By Figures 10-11 Table 5 shows that the docking score between the glycopeptide ET(Fuc)S(Fuc)GFIK and the ACE molecule is -17.05, with 23 hydrogen bonds, 6 ionic bonds, and 2 H-pi interactions forming between the molecules, and a binding bond energy of -103.9 kcal / mol. The two fucose residues bind to the same residues in the ACE molecule, and their binding bond energies are comparable. The number of binding bonds formed between the glycopeptide ET(Fuc)S(Fuc)GFIK and the ACE molecule increased from 12 to 29, and the binding bond energy decreased from -80.2 kcal / mol to -103.9 kcal / mol. Compared to the peptide ETSGFIK, the glycopeptide molecule has a lower bond energy and a significantly increased number of binding bonds, resulting in stronger binding forces and a more significant inhibitory effect on ACE activity. The ACE inhibitory activity of the glycopeptide is significantly better than that of its corresponding peptide ETSGFIK.

[0076] (4) By Figures 13-14As shown in Table 7, the molecular docking score between the glycopeptide molecule VET(Fuc)GIIK and ACE is -13.48, with 16 hydrogen bonds and 5 ionic bonds forming intermolecular interactions, and a binding bond energy of -67.3 kcal / mol. Among these, the binding bond energy of the fucose residues to ACE is -12.7 kcal / mol, accounting for 19% of the total binding bond energy. The binding bond energy between the glycopeptide molecule VET(Fuc)GIIK and ACE is comparable to that between the peptide molecule VETGIIK and ACE.

[0077] Example 4: Evaluation of Glucagon-Like Peptide-1 (GLP-1) Receptor Activation Effect Following the experimental method in Example 3, molecular docking binding analysis was performed between the GLP-1 receptor and functional proteins LDP, peptides, and glycopeptides. Using docking fraction, number of bonds, and bond energy as screening indicators, tightly bound complexes were selected. MOE software was used to analyze the binding sites and binding modes between molecules. The results are as follows: Figures 15-20 And as shown in Tables 8-12.

[0078] Table 8 Molecular docking results of GLP-1 and functional protein LDP

[0079] Table 9. Docking results between peptide ETSGFIK and GLP-1 molecules.

[0080] Table 10. Docking results of peptide VETGIIK and GLP-1 molecules.

[0081] Table 11. Docking results of glycopeptide molecules ET(Fuc)S(Fuc)GFIK with GLP-1 molecules.

[0082] Table 12. Docking results of glycopeptide molecules VET(Fuc)GIIK with GLP-1 molecules.

[0083] (1) By Figures 15-16As shown in Table 8, the docking score between the functional protein LDP and the GLP-1 molecule was -20.47, with a total intermolecular binding bond energy of -55 kcal / mol. Amino acid residues Asp128 and Glu127 in the protein contributed approximately 65% ​​of the binding energy, playing a crucial role in the protein's activation of GLP-1. LDP bound to amino acid residues in the A, B, and G chains of GLP-1. The binding strength of LDP to GLP-1 (average binding bond energy -4.53 kcal / mol) was significantly higher than that of intramolecular GLP-1 residues (average binding bond energy -2.41 kcal / mol), indicating that LDP possesses excellent GLP-1 receptor activation potential.

[0084] (2) By Figure 17 As shown in Table 9, the docking score between the peptide ETSGFIK and the GLP-1 molecule is -9.88. 16 hydrogen bonds and 5 ionic bonds are formed between the molecules, with a binding bond energy of -36.9 kcal / mol. The large number of binding bonds formed between the molecules indicates a strong binding force, and the peptide ETSGFIK can activate the GLP-1 receptor.

[0085] Depend on Figure 18 As shown in Table 10, the docking score between the peptide VETGIIK and GLP-1 molecules is -10.90. 15 hydrogen bonds and 3 ionic bonds are formed between the molecules, with a binding bond energy of -27.1 kcal / mol. The large number of binding bonds formed between the molecules indicates a strong binding force. The peptide VETGIIK can activate the GLP-1 receptor to exert a weight loss and anti-obesity effect.

[0086] (3) By Figure 19 As shown in Table 11, the docking score between the glycopeptide ET(Fuc)S(Fuc)GFIK and the GLP-1 molecule was -18.55, with 26 hydrogen bonds and 2 ionic bonds forming between the molecules, and a binding bond energy of -47 kcal / mol. The fucose residues themselves contributed 7 hydrogen bonds and the corresponding binding energy. Compared with the binding of VETGIIK to GLP-1, the number of binding bonds formed between the glycopeptide ET(Fuc)S(Fuc)GFIK and GLP-1 was significantly increased, and the binding force was stronger. The glycopeptide molecule's activation of the GLP-1 receptor and its weight loss and anti-obesity effects were more significant than those of its peptide counterpart.

[0087] Depend on Figure 20 As shown in Table 12, the docking score between the glycopeptide VET(Fuc)GIIK and the GLP-1 molecule was -15.12, with 19 hydrogen bonds and 3 ionic bonds forming between the molecules. The binding bond energy was -31 kcal / mol. The number of binding bonds formed between the molecules was higher than that between the peptide molecule VETGIIK and GLP-1, indicating a stronger binding force. The glycopeptide molecule also had a better weight loss effect by activating the GLP-1 receptor than its peptide molecule.

[0088] Example 5: Evaluation of GIP receptor activation effect Following the experimental method in Example 3, molecular docking binding analysis was performed between the GIP receptor and functional proteins LDP, peptides, and glycopeptides. Using docking fraction, number of bonds, and bond energy as screening indicators, tightly bound complexes were selected. MOE software was used to analyze the binding sites and binding modes between molecules. The results are as follows: Figures 21-26 And as shown in Tables 13-17.

[0089] Table 13 Molecular docking results of GIP and functional protein LDP

[0090] Table 14. Docking results of peptide molecules ETSGFIK and GIP molecules

[0091] Table 15. Docking results of peptide VETGIIK and GIP molecules

[0092] Table 16. Docking results of glycopeptide molecules ET(Fuc)S(Fuc)GFIK with GIP molecules.

[0093] Table 17. Docking results of glycopeptide molecules VET(Fuc)GIIK with GIP molecules.

[0094] (1) By Figures 21-22 As shown in Table 13, the docking score of the functional protein LDP with the GIP molecule was -22.84, and the total intermolecular binding bond energy was -31.8 kcal / mol. The amino acid residue Gly220 in the protein contributed approximately 47% of the binding energy, playing a crucial role in activating GIP. LDP bound to amino acid residues in the A, B, and N chains of GIP. The binding strength of LDP to GIP (average binding bond energy -3.53 kcal / mol) was higher than that of intramolecular GIP residues (average binding bond energy -2.82 kcal / mol), indicating that LDP possesses excellent GIP receptor activation potential.

[0095] (2) By Figure 23 As shown in Table 14, the docking score between the peptide ETSGFIK and the GIP molecule is -10.62, with 10 hydrogen bonds and 5 ionic bonds forming between the molecules, and a binding bond energy of -47.2 kcal / mol. The high number of intermolecular bonds and strong binding forces indicate that the peptide molecule can activate the GIP receptor to exert a weight-loss effect.

[0096] Depend on Figure 24 As shown in Table 15, the docking score between the peptide VETGIIK and the GIP molecule is -10.84, with 15 hydrogen bonds and 2 ionic bonds forming between the molecules, and a binding bond energy of -33.9 kcal / mol. The large number of intermolecular bonds and the strong binding forces indicate that the peptide molecule activates the GIP receptor, thus exerting a weight-loss effect.

[0097] (3) By Figure 25 As shown in Table 16, the docking score of the glycopeptide ET(Fuc)S(Fuc)GFIK with the GIP molecule is -19.51. It forms 21 hydrogen bonds and 4 ionic bonds between molecules, with a binding bond energy of -60.3 kcal / mol. The number of binding bonds formed between molecules is higher than that between the peptide molecule ETSGFIK and GIP, indicating a stronger binding force. The glycopeptide molecule has a better weight loss effect by activating the GLP-1 receptor than its peptide molecule.

[0098] Depend on Figure 26 As shown in Table 17, the docking score between the glycopeptide VET(Fuc)GIIK and the GIP molecule is -15.04. 19 hydrogen bonds and 2 ionic bonds are formed between the molecules, with a binding bond energy of -38.9 kcal / mol. The number of intermolecular bonds is higher than that between the peptide molecule VETGIIK and GIP, indicating a stronger binding force. The glycopeptide molecule also has a better weight loss effect by activating the GIP receptor than its peptide counterpart.

[0099] Example 6: In vitro validation of angiotensin-converting enzyme (ACE) inhibitory activity (ICP-C) 50 (Measurement) Hippuryl-His-Leu (HHL) was used as a substrate, and glycopeptides ET(Fuc)S(Fuc)GFIK, ETSGFIK, VET(Fuc)GIIK, and VETGIIK were used as samples. High-performance liquid chromatography (HPLC) was used to determine the inhibitory activity of the samples against ACE. Sample concentration gradients of 0.1, 0.5, 1, 5, 10, 50, and 100 μM were set, and the half-maximal inhibitory concentration (IC50) was determined. 50 In this embodiment, captopril at the same concentration as the sample was used as a positive control. Each sample was tested in triplicate, and the experiment was repeated three times. The results are expressed as mean ± standard deviation, as shown in Table 18.

[0100] Table 18 Inhibitory activity of each sample against ACE (IC50) 50 )

[0101] As shown in Table 18, the inhibitory activity of the glycopeptide ET(Fuc)S(Fuc)GFIK on ACE was approximately 5.2 times higher than that of its corresponding peptide ETSGFIK, and the difference was statistically significant. p <0.01).

[0102] As shown in Table 18, the inhibitory activity of the glycopeptide VET(Fuc)GIIK on ACE is approximately 3.4 times higher than that of its corresponding peptide VETGIIK on ACE. p <0.05).

[0103] The above results indicate that fucosylation significantly enhances the ACE inhibitory activity of peptide molecules, and the molecular docking results are highly consistent with the in vitro activity data.

[0104] Example 7: Validation of GLP-1 secretion-promoting activity NCI-H716 cells (human enteric endocrine cell line that secretes GLP-1) were used as an in vitro model, and glycopeptides ET(Fuc)S(Fuc)GFIK, ETSGFIK, VET(Fuc)GIIK, and VETGIIK were used as samples for experiments.

[0105] NCI-H716 cells were seeded in 24-well plates and treated with different concentrations of sample (10, 50, and 100 μM) for 2 h. The supernatant was collected, and the secretion of GLP-1 (7-36) amide was detected by ELISA. The positive control was 10 μM sitagliptin (a DPP-IV inhibitor). Each sample was tested in triplicate, and the experiment was repeated three times. Results are expressed as mean ± standard deviation.

[0106] Table 19. The promoting effect of each sample on GLP-1 secretion (100 μM treatment)

[0107] As shown in Table 19, the effect of glycopeptide ET(Fuc)S(Fuc)GFIK in promoting GLP-1 secretion was 35.6±2.9 pM, which was significantly higher than that of its corresponding peptide ETSGFIK (18.5±1.8 pM) and exceeded that of the positive control sitagliptin (25.4±2.1 pM).

[0108] As shown in Table 19, the GLP-1 secretion level of the glycopeptide VET(Fuc)GIIK was 28.7±2.3 pM, which was significantly better than that of its corresponding peptide VETGIIK (15.3±1.4 pM). Glycosylation modification increased the GLP-1 secretion-promoting activity by about 1.9 times (p<0.01).

[0109] In summary, compared with the peptides corresponding to glycopeptides, the glycopeptides ET(Fuc)S(Fuc)GFIK and VET(Fuc)GIIK screened in this invention have better anti-obesity potential.

[0110] Example 8: Verification of GIP receptor agonist activity Using the HEK293 cell line expressing the human GIP receptor as a model, GIP receptor agonist activity was verified by using glycopeptides ET(Fuc)S(Fuc)GFIK, ETSGFIK, VET(Fuc)GIIK, and VETGIIK as samples.

[0111] HEK293 cells were seeded in 96-well plates and stimulated for 30 min with different concentrations of the sample (1, 10, 100 μM). After stimulation, the accumulation of intracellular cAMP was detected by homogeneous time-resolved fluorescence (HTRF). 100 nM GIP (1-42) was used as a positive control, where GIP (1-42) represents the complete GIP sequence. Each sample was tested in triplicate, and the experiment was repeated three times. Results are expressed as mean ± standard deviation. The experimental results are shown in Table 20.

[0112] Table 20. Agonistaltic activity of each sample against the GIP receptor (cAMP accumulation)

[0113] As shown in Table 20, after the glycopeptide ET(Fuc)S(Fuc)GFIK activated the GIP receptor, the cAMP accumulation was 18.4±1.7 nM, which was 15.3 times higher than that of the blank group. The corresponding peptide ETSGFIK was only increased by 7.2 times, and the glycopeptide activity was increased by about 2.1 times (p<0.01).

[0114] As shown in Table 20, the cAMP accumulation of the glycopeptide VET(Fuc)GIIK was 14.2±1.3 nM, which was 11.8 times higher than that of the blank group, while the corresponding peptide VETGIIK was only 5.8 times higher, and the activity was about 2.0 times higher (p<0.05).

[0115] The above results indicate that fucosylation significantly enhances the agonistic effect of peptide molecules on GIP receptors, and glycopeptides have superior weight loss and anti-obesity effects.

[0116] Example 9: Molecular Interactions Experimental Methods: The thermodynamic binding reaction type of the interaction between ACE and *Vitis vinifera* protein, bioactive peptides, and bioactive glycopeptides was analyzed using isothermal titration calorimetry (ITC). The ITC method is briefly described below: A 0.02 μmol / L ACE solution prepared with PBS buffer was injected into the ITC sample cell. The sample cell temperature was 25°C, and the stirrer speed was 1000 rpm. Sixty seconds after injecting ACE into the ITC sample cell, the first titration was initiated. A total of 20 titrations were performed. Each titration used 2 μL of a 0.2 μmol / L solution of *Vitis velutipes* protein, active peptides, or active glycopeptides prepared with PBS buffer. The time interval between titrations was 150 s, and the titration time was 2 s.

[0117] The heat change of the reaction system during molecular binding was measured, and the binding affinity (K0.05) between the salty taste receptor protein and the salty taste bioactive peptide molecule was calculated using ITC Nano analysis software. D The thermodynamic parameters, including stoichiometry (N), enthalpy change (ΔH), and entropy change (ΔS), are shown in Table 21 and [other tables]. Figures 27-32 As shown.

[0118] Table 21 Thermodynamic parameters of ITC binding of deer antler mushroom protein, bioactive peptides and bioactive glycopeptides to ACE receptors

[0119] From Table 21 and Figures 27-32 visible: Combining affinity: K of ET(Fuc)S(Fuc)GFIK and VET(Fuc)GIIK D The values ​​were 1.8±0.2 μM and 2.2±0.2 μM, respectively, which were significantly lower than (i.e., had stronger affinity than) ETSGFIK (8.2±0.7 μM), VETGIIK (9.5±0.8 μM), and LDP of *Pleurotus ostreatus* protein (12.5±1.2 μM). This indicates that the binding affinity of the active glycopeptide of *Pleurotus ostreatus* to the ACE receptor is approximately 4–5 times that of the simple peptide.

[0120] Stoichiometry (N): The N values ​​of all samples were in the range of 1.0 ± 0.1, indicating that the test samples and ACE receptors formed a 1:1 stoichiometric binding ratio.

[0121] Thermodynamic driving force: ETSGFIK and VETGIIK exhibit typical enthalpy-driven binding (ΔH is significantly negative, and -TΔS is negative), indicating that hydrogen bonding and van der Waals forces are the main driving forces for binding.

[0122] ET(Fuc)S(Fuc)GFIK and VET(Fuc)GIIK exhibit more negative ΔH values ​​(-12.3±0.6 kcal / mol and -11.6±0.5 kcal / mol, respectively), but are accompanied by a slight entropy penalty (-TΔS is positive), indicating that the glycan portion of the glycopeptide provides additional hydrogen bond interactions, while the constraint of glycan flexibility has a certain compensating effect on conformational entropy.

[0123] The binding free energy (ΔG) of ET(Fuc)S(Fuc)GFIK and VET(Fuc)GIIK is -11.8±0.4 kcal / mol and -11.3±0.3 kcal / mol, respectively, which is comparable to or even slightly better than that of the positive control captopril (-10.7±0.4 kcal / mol).

[0124] In summary, this invention provides a *Pleurotus ostreatus* glycoprotein, an active peptide, and its glycopeptides. The *Pleurotus ostreatus* glycoprotein and active peptide exhibit targeting effects on ACE, GLP-1 receptors, and GIP receptors. The glycopeptides of the active peptide molecule demonstrate stronger binding activity to ACE, GLP-1 receptors, and GIP receptors, enabling better targeting of their binding sites. This invention experimentally discovered that the active peptide and its fucosylated glycopeptides are the key structural basis for enhancing hypotensive and weight-loss activities. This invention provides new insights for research related to hypoglycemia, weight loss, and hypotension.

[0125] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An active peptide, characterized in that, The active peptide includes a first active peptide and / or a second active peptide; The sequence of the first active peptide is shown in SEQ ID NO:2; The sequence of the second active peptide is shown in SEQ ID NO:

3.

2. The glycopeptide of the active peptide according to claim 1, characterized in that, The glycopeptide includes a first glycopeptide and / or a second glycopeptide; The first glycopeptide comprises an active peptide with an amino acid sequence as shown in SEQ ID NO:2 and two first glycan chains; the two first glycan chains are respectively linked to threonine residues and serine residues in the sequence shown in SEQ ID NO:2 in the form of O-glycosidic bonds; The second glycopeptide comprises an active peptide with an amino acid sequence as shown in SEQ ID NO:3 and a second sugar chain, the second sugar chain being linked to a threonine residue of the sequence shown in SEQ ID NO:3 via an O-glycosidic bond.

3. The glycopeptide as described in claim 2, characterized in that, Both the first and second sugar chains are fucose residues.

4. A deer antler mushroom glycoprotein, characterized in that, The deer antler mushroom glycoprotein contains the active peptide described in claim 1 and / or the glycopeptide described in claim 2 or 3.

5. The method for preparing the glycoprotein from *Deer Antler Fungus* according to claim 4, characterized in that, include: Protein was extracted from *Flammulina velutipes* to obtain a crude extract of *Flammulina velutipes*. The crude extract of *Deer Antler Mushroom* was subjected to fractionation and precipitation, and the precipitate was collected to obtain crude protein from *Deer Antler Mushroom*. The crude protein from *Flammulina velutipes* was subjected to ion exchange chromatography to obtain negatively charged crude protein from *Flammulina velutipes*. The negatively charged crude protein from *Pleurotus ostreatus* was subjected to filtration chromatography, and the elution peak with ultraviolet absorption at 280 nm was collected to obtain *Pleurotus ostreatus* glycoprotein.

6. The use of the active peptide of claim 1, the glycopeptide of claim 2 or 3, the antler velvet glycoprotein of claim 4, or the antler velvet glycoprotein prepared by the preparation method of claim 5 in the preparation of products for treating hypertension.

7. The use of the active peptide of claim 1, the glycopeptide of claim 2 or 3, the velvet spore glycoprotein of claim 4, or the velvet spore glycoprotein prepared by the method of claim 5 in the preparation of angiotensin-converting enzyme inhibitors.

8. The use of the active peptide of claim 1, the glycopeptide of claim 2 or 3, the velvet antler glycoprotein of claim 4, or the velvet antler glycoprotein prepared by the preparation method of claim 5 in the preparation of products for treating obesity.

9. The use of the active peptide of claim 1, the glycopeptide of claim 2 or 3, the velvet glycoprotein of claim 4, or the velvet glycoprotein prepared by the method of claim 5 in the preparation of GLP-1 receptor agonists and / or GIP receptor agonists.

10. A product for treating hypertension and / or obesity, characterized in that, The product includes active ingredients and excipients; The active ingredients include the active peptide of claim 1, the glycopeptide of claim 2 or 3, the velvet spore glycoprotein of claim 4, or the velvet spore glycoprotein prepared by the preparation method of claim 5.

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