Stropharia rugoso-annulata double-target glycopeptide weight-reducing agent and application thereof

Abstract

The invention provides a stropharia rugoso-annulata double-target glycopeptide weight-reducing agent and application thereof, and belongs to the technical field of glycopeptides. The glycopeptide is composed of a polypeptide and a sugar chain, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1, the sugar chain is N-acetyl glucosamine, and the N-acetyl glucosamine is connected to a first serine hydroxyl group of the polypeptide through an alpha-glucosidic bond in an O-connection glycosylation mode. The glycopeptide disclosed by the invention can be combined with the leptin, stabilize the four-spiral-beam conformation of the leptin, delay the degradation of the leptin, enhance the binding effect of the leptin and a leptin receptor and activate a melanocortin-4 receptor, and has potential multi-path weight-losing effects of inhibiting appetite and promoting energy consumption.

Description

Technical Field

[0001] The present invention belongs to the technical field of glycopeptides, and in particular relates to a Stropharia rugosa dual-target glycopeptide weight-loss agent and applications thereof. Background Art

[0002] According to the World Health Organization (WHO), the number of obese adults worldwide has doubled since 1990, and the number of obese adolescents has quadrupled. By 2022, over one billion people worldwide will be obese. Obesity increases the risk of numerous chronic diseases, including cardiovascular disease, diabetes, and metabolic-related steatohepatitis, and has become a major global health issue.

[0003] In 2021, a glucagon-like peptide-1 (GLP-1) receptor agonist drug called semaglutide was approved by the U.S. FDA for long-term weight control in obese and overweight adult patients. This type of drug helps lose weight by suppressing appetite and delaying gastric emptying, but its side effects have also attracted much attention. About 60% of users will continue to experience symptoms of nausea, vomiting, and vomiting during medication, which are especially intense when facing food. For women who use semaglutide to lose weight, the risk of hair loss may double. The metabolic benefits of semaglutide are undoubted, but the development of safer and side-effect-free weight-loss products is also imperative.

[0004] Leptin, a hormone secreted by adipocytes, has a four-helix bundle fold and is involved in regulating food intake and energy balance. Leptin binds to the leptin receptor (LepR) in the hypothalamus, reducing neuropeptide secretion and increasing melanocyte-stimulating hormone secretion, leading to decreased appetite and increased energy expenditure. Leptin acts on the central nervous system to increase sympathetic nerve activity and activate adrenergic receptors on the adipocyte membrane, converting stored energy into heat. Leptin can directly inhibit lipogenesis and promote its degradation. Due to its important role in regulating body weight and fat metabolism, leptin has become a key target for drug development in the treatment of obesity. Natural leptin has a short half-life and is easily degraded by proteases, and leptin resistance is common in obese patients. Therefore, stabilizing leptin's four-helix bundle conformation, delaying its degradation, and enhancing leptin receptor binding are key to achieving leptin-based hypothalamic appetite regulation. The melanocortin 4 receptor (MC4R) is a core receptor in the central nervous system that regulates energy balance. It is primarily located in the hypothalamus. Activation of the MC4R can suppress appetite, enhance satiety signals, and reduce the drive to eat. Activating the melanocortin 4 receptor suppresses appetite through the MC4R while promoting lipolysis. This can achieve a weight loss effect through central-peripheral synergy, thereby achieving a multi-pathway weight loss effect. While existing MC4R agonists can suppress appetite, long-term use may lead to risks such as increased heart rate. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a glycopeptide that can delay the degradation of leptin, enhance the binding effect of leptin and leptin receptor, activate melanocortin 4 receptor, and has therapeutic potential for weight regulation.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A glycopeptide, comprising a polypeptide and a sugar chain, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1, the sugar chain is N-acetylglucosamine, and the N-acetylglucosamine is linked to the first serine hydroxyl group of the polypeptide via an α-glycosidic bond in an O-linked glycosylation manner.

[0008] Preferably, the molecular weight of the glycopeptide is 1006 Da.

[0009] Another object of the present invention is to provide a method for preparing a Stropharia officinalis extract rich in the glycopeptide, comprising the following steps: crushing the dried Stropharia officinalis and mixing it with water, and extracting it using a slit quad-frequency ultrasonic method, wherein the ultrasonic frequency combination is 22-24 kHz + 24-26 kHz + 27-29 kHz + 39-41 kHz.

[0010] Preferably, the Stropharia rugosa is crushed and mixed with water at a material-liquid ratio of 1 g:18-25 mL.

[0011] Preferably, the ultrasonic conditions are: ultrasonic power density is 80-120 W / L, ultrasonic time is 20-40 min, ultrasonic intermittent ratio is ultrasonic working 4-8 s: stop 1-3 s, and each frequency in the four-frequency ultrasound works alternately for 1-2 s.

[0012] Preferably, after the ultrasound is completed, the supernatant is collected by centrifugation, the centrifugal speed is 7000-9000 rpm, and the centrifugal time is 10-20 min.

[0013] Another object of the present invention is to provide the Stropharia officinalis extract prepared by the preparation method.

[0014] Another object of the present invention is to provide the use of the glycopeptide or the Stropharia officinalis extract in any one or more of the following (1) to (3);

[0015] (1) preparing drugs for treating and / or preventing obesity;

[0016] (2) preparing foods for controlling body fat;

[0017] (3) Preparation of leptin enhancer.

[0018] Preferably, the glycopeptide delays the degradation of leptin, enhances the binding effect of leptin and leptin receptor, and activates melanocortin 4 receptor.

[0019] Another object of the present invention is to provide a product comprising the glycopeptide or the Stropharia officinalis extract.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a glycopeptide that can bind to leptin, stabilize its four-helix bundle conformation, delay its degradation, enhance the binding effect of leptin to leptin receptors, activate melanocortin 4 receptors, and has the potential to suppress appetite and promote energy consumption through multiple pathways of weight loss. It can be used in the development of health foods for the auxiliary treatment of obesity and long-acting leptin enhancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the mass spectrum of glycopeptide molecules;

[0023] Figure 2 The molecular structure of glycopeptide, where the left picture is the 2D structure of glycopeptide molecule, and the right picture is the 3D structure of glycopeptide molecule; in the 2D and 3D structures, the green one is the N-acetylglucosamine molecule;

[0024] Figure 3 Flowchart for receptor construction, molecular docking and activity evaluation;

[0025] Figure 4 The left figure is a 2D image of the docking of glycopeptide and leptin. The numbers on the bonds in the 2D image represent the bond energy. The right figure is a 3D image of the docking of glycopeptide and leptin. The peptide molecules are represented in ball-and-stick mode, and leptin is represented in surface mode. In the glycopeptide molecules, red represents the peptide molecules, and green represents the N-acetylglucosamine molecules.

[0026] Figure 5 The upper figure is a 3D docking diagram of the leptin receptor and leptin molecules. The leptin receptor and leptin are represented in surface mode, with orange representing the leptin receptor and white representing leptin. The lower figure is a binding energy diagram of the leptin receptor and leptin molecules.

[0027] Figure 6 The figure above shows the molecular docking diagram of the leptin receptor and the leptin-glycopeptide complex. The upper figure shows the 3D docking diagram of the leptin receptor and the leptin-glycopeptide complex. The leptin receptor and leptin are represented in surface mode, with the leptin receptor in orange and leptin in white. The glycopeptide molecules bound to leptin are represented in ball-and-stick mode, with the peptide molecules in red and the N-acetylglucosamine molecules in green. The lower figure shows the binding energy diagram of the leptin receptor and the leptin-glycopeptide complex.

[0028] Figure 7This is the docking diagram of glycopeptide and melanocortin 4 receptor molecule. The numbers on the binding bonds in the 2D docking diagram of glycopeptide molecule and melanocortin 4 receptor are the binding bond energies, red represents the peptide molecule, and green represents the N-acetylglucosamine molecule. DETAILED DESCRIPTION

[0029] The present invention provides a glycopeptide comprising a polypeptide and a sugar chain. The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1 (ILALSSEA), the sugar chain is N-acetylglucosamine, and the N-acetylglucosamine is linked to the first serine hydroxyl group of the polypeptide via an α-glycosidic bond in an O-linked glycosylation manner. The glycopeptide sequence is ILALS(GlcNAc)SEA. The molecular weight of the glycopeptide of the present invention is 1006 Da.

[0030] The present invention also provides a method for preparing a stropharia mushroom extract rich in the glycopeptide, comprising the following steps: crushing the dried stropharia mushroom and mixing it with water, and extracting it using a slit four-frequency ultrasonic method, wherein the ultrasonic frequency combination is 22-24kHz+24-26kHz+27-29kHz+39-41kHz, preferably 23kHz+25kHz+28kHz+40kHz.

[0031] In the present invention, it is preferred that the pulverized Stropharia rugosa be mixed with water at a material-liquid ratio of 1g:18-25mL, more preferably at a material-liquid ratio of 1g:20mL. The ultrasonic conditions are as follows: the ultrasonic power density is preferably 80-120W / L, more preferably 100W / L; the ultrasonic time is preferably 20-40min, more preferably 30min; the ultrasonic intermittent ratio is preferably ultrasonic working (4-8)s: stopping (1-3)s, more preferably ultrasonic working 6s: stopping 2s; in the four-frequency ultrasound, each frequency is preferably alternately worked for 1-2s, more preferably each frequency is alternately worked for 1.5s. Taking the ultrasonic intermittent ratio of ultrasonic working 6s: stopping 2s, and the alternating working time of each frequency in the four frequencies as an example of 1.5s, the slit four-frequency ultrasonic extraction in the present invention refers to: working at 23kHz for 1.5s, working at 25kHz for 1.5s, working at 28Hz for 1.5s, working at 40kHz for 1.5s (a total of 6s), then stopping for 2s, and repeating the above intermittent ultrasonic work.

[0032] In the present invention, after the ultrasonic treatment, the supernatant is collected by centrifugation. The centrifugation speed is preferably 7000-9000 rpm, more preferably 8000 rpm, and the centrifugation time is preferably 10-20 minutes, more preferably 15 minutes. In the present invention, the supernatant is preferably collected and freeze-dried at -70°C for 48 hours to obtain the Stropharia rugosa freeze-dried powder.

[0033] The present invention also provides a stropharia extract prepared by the preparation method. After LC-MS / MS identification, a glycopeptide molecule with high mass spectrum abundance (peak area 2.30×10 5 ).

[0034] The present invention also provides the use of the glycopeptide or the Stropharia officinalis extract in any one or more of the following (1) to (3);

[0035] (1) preparing drugs for treating and / or preventing obesity;

[0036] (2) preparing foods for controlling body fat;

[0037] (3) Preparation of leptin enhancer.

[0038] In the present invention, the glycopeptide delays the degradation of leptin, enhances the binding effect of leptin and leptin receptor, and activates melanocortin 4 receptor.

[0039] The present invention also provides a product comprising the glycopeptide or the Stropharia officinalis extract.

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] A stropharia mushroom extract, the preparation method of which is as follows:

[0043] Dried Stropharia officinalis was used as the raw material, which was crushed and sieved to obtain powder with a mesh size of less than 100. The powder was mixed with water (1g:20mL) and subjected to slit quad-frequency ultrasonic extraction of glycopeptides using a frequency combination of 23, 25, 28, and 40 kHz, an ultrasonic power density of 100 W / L, a sonication time of 30 minutes, and an ultrasound intermittent ratio of 6:2 s / s (6 seconds on, 2 seconds off). Each of the four frequencies alternated for 1.5 seconds. After ultrasonic treatment, the sample was centrifuged at 8000 rpm for 15 minutes. The supernatant was collected and freeze-dried at -70°C for 48 hours to obtain freeze-dried Stropharia officinalis powder.

[0044] Glycopeptide molecules in freeze-dried powder of Stropharia rugosa were identified by mass spectrometry.

[0045] The lyophilized powder was pretreated for desalting using a ZipTip C18 microchromatography column (Merck-Millipore, Shanghai Anpu Laboratory Technology Co., Ltd.). The desalting method was as follows: 1.0 mg of Stropharia rugosa lyophilized powder was accurately weighed and dissolved in 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA); the column was rinsed 10 times with 50 μL of a solution prepared from 60% (v / v) acetonitrile (ACN) and 0.1% TFA in pure water; the column was rinsed 10 times with 10 μL of 0.1% TFA; the dissolved Stropharia rugosa lyophilized powder was aspirated and discharged through the column 20 times; the column was rinsed 5 times with 10 μL of 0.1% TFA; the column was eluted with 10 μL of a solution prepared from 60% ACN and 0.1% TFA in pure water, the eluate was collected, transferred to a polypropylene centrifuge tube, and dried under vacuum. Dissolve the sample in 20 μL of dissolving buffer (containing 0.1% (v / v) formic acid), vortex, and centrifuge at 17,000 rpm at 4°C for 20 min. The supernatant was collected and transferred to a 3 μL injection tube for LC-MS / MS sequence analysis. Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. LC-MS / MS parameters are shown in Table 1. PEAKS software was used for database searches of the identified sequences.

[0046] Table 1 LC-MS / MS parameter settings

[0047]

[0048] Identification by LC-MS / MS ( Figure 1 ), a glycopeptide molecule with high mass spectrometry abundance (peak area 2.30×10 5 ), the glycopeptide sequence is ILALS(GlcNAc)SEA, with a molecular weight of 1006Da; in the glycopeptide molecule, the sugar chain is composed of N-acetylglucosamine (GlcNAc), which is connected to the first serine (Ser) hydroxyl group of the peptide chain through an α-glycosidic bond in an O-linked glycosylation manner ( Figure 2 ).

[0049] Example 2

[0050] Molecular docking glycopeptide activity analysis.

[0051] Receptor construction, molecular docking and activity evaluation process such as Figure 3 shown.

[0052] 1. Molecular docking analysis of leptin and glycopeptide.

[0053] The crystal structure of leptin (PDB: 1AX8) was downloaded from the RCSB database (https: / / www.rcsb.org / ). The leptin protein crystal structure was optimized using the MOE2019 molecular docking software (Chemical Computing Group ULC, Montreal, Canada). Water molecules and bound small molecule ligands were deleted from the protein sequence, and hydrogen atoms were completed using the MOE software's "Quick Prep" function. The 3D structure of the glycopeptide molecule was constructed according to the glycopeptide sequence using the "ProteinBuilder" module under the MOE software's "Protein" tab. Molecular energy minimization was performed using the "Minimize" module. The amino acid residue active sites of the leptin protein were identified using the "Site Finder" module under the MOE software's "Protein" tab. The leptin molecule and the glycopeptide molecule were docked using the "Dock" module under the MOE software's "Compute" tab. In the "Dock" panel, select the active site identified by "Site Finder" in the "Site" section and the glycopeptide molecule in the "Ligand" section. In the molecular docking result list (LigandInteractionReport), the number of bonds and the binding energy between the glycopeptide and the leptin protein were used as screening indicators to select the tightly bound complex between the glycopeptide and the leptin protein. The MOE software was used to analyze the binding sites and interaction modes between the glycopeptide molecule and the leptin protein in the complex.

[0054] like Figure 4 As shown in Table 2, the glycopeptide molecule ILALS(GlcNAc)SEA forms 10 hydrogen bonds and 2 ionic bonds with the leptin molecule, with a binding energy of -47.9 kcal / mol. The binding energy between the peptide molecule and leptin is -43 kcal / mol, and the binding energy between the sugar molecule and leptin is -4.9 kcal / mol. The binding energy between the glycopeptide molecule ILALS(GlcNAc)SEA and the leptin molecule is low, the number of bonds is large, and the binding force is strong. The glycopeptide binds to leptin to form a glycopeptide-leptin binding complex (LEPGP), which stabilizes the four-helix bundle conformation of leptin and delays its degradation.

[0055] Table 2 Docking results of glycopeptide (ILALS(GlcNAc)SEA) and leptin

[0056]

[0057] 2 Docking analysis of leptin receptor and leptin molecule.

[0058] The crystal structure of the leptin receptor (LepR) (PDB: 3V6O) was downloaded from the RCSB database (https: / / www.rcsb.org / ). The MOE2019 molecular docking software was used to optimize the leptin receptor protein crystal structure. Water molecules in the receptor protein sequence were deleted, and hydrogen atoms were completed using the MOE software's "QuickPrep" function. Under the "Protein" tab of the MOE software, the "Protein-Protein Docking" module was selected. The receptor was "Lepin Receptor" and the ligand was "Lepin." All-atom rigid protein docking was performed, with the Pose to Retain parameters set to "Pre-Placement, 10000; Placement, 1000; Refinement, 100." The MOE software was used to analyze the binding score and binding energy between the leptin receptor and leptin in the complex.

[0059] Leptin receptor and leptin binding information Figure 5 As shown in Table 3, the results show that the leptin receptor and leptin generate 268 binding sites, with a binding score of -17.21 and a binding energy of -386.7 kcal / mol. The lower the binding score and binding energy, the better the intermolecular binding. These results suggest that leptin achieves its weight loss effect by binding to the leptin receptor with high affinity.

[0060] Table 3 Docking results of leptin receptor and leptin molecule

[0061]

[0062]

[0063]

[0064] Note: 3V6O.A and 3V6O.B represent the A and B protein chains of the leptin receptor, respectively.

[0065] 3 Molecular docking analysis of leptin receptor and leptin-glycopeptide complex.

[0066] The glycopeptide-leptin binding complex (LEPGP) obtained in "1. Molecular docking analysis of leptin and glycopeptide" was used to perform molecular docking of the complex with the leptin receptor (LepR). Click the "Protein-Protein Docking" module under the "Protein" tab of the MOE software, select "Lepin receptor" as the receptor, and "Glycopeptide-leptin complex" as the ligand, and perform all-atom rigid protein docking. Set the structure Pose to Retain parameters to "Pre-Placement, 10000; Placement, 1000; Refinement, 100". Other molecular docking parameter settings are the same as those in "2. Molecular docking analysis of leptin receptor and leptin". The MOE software analyzes the binding score and binding energy between the leptin receptor and the glycopeptide-leptin binding complex in the complex.

[0067] Leptin receptor and glycopeptide-leptin complex binding information Figure 6 As shown in Table 4, the results showed that the leptin receptor and the glycopeptide-leptin complex generated 395 binding sites, with a binding score of -17.48 and a binding energy of -461.38 kcal / mol. Comparison with the "2-Leptin Receptor and Leptin Molecular Docking Analysis" revealed that the glycopeptide induced an optimization of the leptin-leptin receptor binding interface, reducing the binding energy by -74.68 kcal / mol and significantly enhancing leptin's activation of the leptin receptor. Therefore, glycopeptide ingestion can significantly enhance the weight loss effect of leptin.

[0068] Table 4 Molecular docking results of leptin receptor and leptin-glycopeptide complex

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Note: 3V6O.A and 3V6O.B represent the A and B protein chains of the leptin receptor, respectively; LEPGP represents the leptin-glycopeptide binding complex.

[0075] 4. Docking analysis of melanocortin 4 receptor and glycopeptide molecules.

[0076] The MC4R crystal structure (PDB: 6W25) was downloaded from the RCSB database (https: / / www.rcsb.org / ). The MC4R receptor protein crystal structure was optimized using the MOE2019 molecular docking software (Chemical Computing Group ULC, Montreal, Canada). Water molecules and bound small molecule ligands were deleted from the MC4R receptor protein sequence. Hydrogen atoms were completed using the "Quick Prep" function in the MOE software. The 3D structure of the glycopeptide was constructed based on the glycopeptide sequence using the "ProteinBuilder" module under the "Protein" tab of the MOE software. Molecular energy minimization was performed using the "Minimize" module. The amino acid residue active site of the MC4R receptor protein was determined using the "SiteFinder" module under the "Protein" tab of the MOE software. The MC4R receptor and glycopeptide were docked using the "Dock" module under the "Compute" tab of the MOE software. In the "Dock" panel, select the active site identified by "SiteFinder" in the "Site" section and the glycopeptide in the "Ligand" section. In the molecular docking result list (LigandInteractionReport), the number of bonds and the binding energy between the glycopeptide and the MC4R receptor protein were used as screening indicators to select the tightly bound complex between the glycopeptide and the MC4R receptor protein. The MOE software was used to analyze the binding sites and interaction modes between the glycopeptide molecule and the MC4R receptor in the complex.

[0077] according to Figure 7 As shown in Table 5, eight hydrogen bond interactions are formed between the glycopeptide molecule ILALS(GlcNAc)SEA and the MC4R receptor molecule, with a binding energy of -14.6 kcal / mol. The binding energy between the peptide molecule and the melanocortin 4 receptor is -10.1 kcal / mol, and the binding energy between the sugar molecule and the MC4R receptor is -4.5 kcal / mol. Although the binding strength between the glycopeptide molecule ILALS(GlcNAc)SEA and the MC4R receptor molecule is lower than the binding strength between the glycopeptide molecule and leptin, the binding energy contributed by the sugar molecule to the intermolecular binding is not much different, indicating that N-acetylglucosamine molecules play a similar role in activating weight loss-related receptors (LepR, MC4R), cross-talking with the leptin receptor pathway, and enhancing the overall regulatory ability of weight loss.

[0078] Table 5 Docking results of glycopeptide (ILALS(GlcNAc)SEA) and melanocortin 4 receptor molecule

[0079]

[0080]

[0081] 5 Analysis of leptin receptor-leptin-glycopeptide molecular interaction.

[0082] Glycopeptide molecules were synthesized by Jier Biochemical (Shanghai) Co., Ltd., with a purity of greater than 98%. Leptin and leptin receptor were purchased from Shanghai Xinyu Biotechnology Co., Ltd. under the brand name Bioss.

[0083] Isothermal titration calorimetry (ITC) was used to verify the enhancing effect of glycopeptide on leptin-leptin receptor binding. The concentration of the titrated solution prepared with PBS buffer in the ITC sample cell was 0.02 μmol / L, the concentration of the titration solution prepared with PBS buffer was 0.2 μmol / L, and the volume of a single titration solution was 2 μL. The sample cell temperature was 25°C, the stirrer speed was 1000 rpm, the interval from the start to the first titration was 60 s, the total number of titrations was 20 times, the time interval between two titrations was 150 s, and the single titration time was 2 s. The heat change of the reaction system during the molecular binding process was measured, and the ITCNano analysis software was used to calculate the binding affinity (K) between the molecules. D ), stoichiometric value (N), enthalpy change (ΔH) and entropy change (ΔS) thermodynamic parameters.

[0084] ① Analysis of the interaction between leptin receptor and leptin molecules. A leptin solution (titrated solution, 0.02 μmol / L) was placed in an ITC sample cell and titrated with a leptin receptor solution (0.2 μmol / L). The titration and analysis procedures were the same as above.

[0085] ② Analysis of the interaction between the leptin receptor and the leptin-glycopeptide molecule. A leptin solution (titrated solution, 0.02 μmol / L) was placed in an ITC sample cell and titrated with a glycopeptide ILALS(GlcNAc)SEA solution (0.2 μmol / L). After the leptin-glycopeptide complex formed, the leptin receptor solution was titrated (0.2 μmol / L). The titration and analysis procedures were the same as above.

[0086] Molecular interaction results (Table 6) show that the leptin receptor binds to leptin with high affinity (nM level). When the leptin receptor binds to more than one leptin molecule, an exothermic binding process occurs between the molecules, dominated by hydrogen bonding and electrostatic interactions. The leptin receptor binds to the leptin-glycopeptide complex with even higher affinity (approaching the nM level), and the number of bound molecules increases further, further reducing the enthalpy change. Glycosylation introduces strong polar interactions, leading to significant exotherm. Molecular binding significantly increases the conformational entropy of the receptor, indicating that the structural rigidity of the receptor decreases after molecule binding. The binding free energy ΔG decreases by -52.88 kcal / mol, indicating a significant leptin receptor activation effect. The molecular interaction results are consistent with the molecular docking results, indicating that the glycopeptide enhances leptin-leptin receptor binding.

[0087] Table 6 Results of interaction analysis between leptin receptor, leptin and leptin-glycopeptide molecules

[0088] interacting molecules <![CDATA[K D (M)]]> N △H (kcal / mol) △S (cal / mol·K) Leptin receptor and leptin <![CDATA[8.51×10 -6 ]]> 1.22 -52.84 -75.57 Leptin receptor and leptin-glycopeptide complex <![CDATA[1.33×10 -8 ]]> 3.64 -95.15 -40.11

[0089] In summary, the glycopeptide ILALS (GlcNAc) SEA can bind to leptin, stabilize its four-helix bundle conformation, delay its degradation, enhance the binding effect of leptin and leptin receptor, activate melanocortin 4 receptor, and has the potential to suppress appetite and promote energy consumption through multiple pathways of weight loss.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A glycopeptide, characterized in that The glycopeptide consists of a polypeptide and a sugar chain. The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1. The sugar chain is N-acetylglucosamine. The N-acetylglucosamine is linked to the first serine hydroxyl group of the polypeptide through an α-glycosidic bond in an O-linked glycosylation manner.

2. The glycopeptide according to claim 1, characterized in that The molecular weight of the glycopeptide is 1006 Da.

3. A method for preparing a Stropharia officinalis extract rich in the glycopeptide according to claim 1 or 2, characterized in that: The steps include: The dried Stropharia rugosa was crushed and mixed with water, and extracted by slit quad-frequency ultrasound, wherein the frequency combination of the ultrasound was 22-24 kHz+24-26 kHz+27-29 kHz+39-41 kHz.

4. The preparation method according to claim 3, characterized in that After crushing the Stropharia officinalis, mix it with water at a material-liquid ratio of 1g:18-25mL.

5. The preparation method according to claim 3, characterized in that The ultrasonic conditions are as follows: ultrasonic power density is 80-120 W / L, ultrasonic time is 20-40 min, ultrasonic intermittent ratio is ultrasonic working 4-8 s: rest 1-3 s, and each frequency in the four-frequency ultrasound works alternately for 1-2 s.

6. The preparation method according to claim 3, characterized in that After the ultrasound is completed, the supernatant is collected by centrifugation. The centrifugal speed is 7000-9000 rpm and the centrifugal time is 10-20 minutes.

7. The Stropharia rugosa extract obtained by the preparation method according to any one of claims 3 to 6.

8. Use of the glycopeptide according to claim 1 or 2 or the Stropharia rugosa extract according to claim 7 in any one or more of the following (1) to (3); (1) preparing drugs for treating and / or preventing obesity; (2) preparing foods for controlling body fat; (3) Preparation of leptin enhancer.

9. The use according to claim 8, characterized in that The glycopeptide delays the degradation of leptin, enhances the binding effect of leptin and leptin receptor, and activates melanocortin 4 receptor.

10. A product comprising the glycopeptide according to claim 1 or 2 or the Stropharia rugosa extract according to claim 6.

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