Lycium barbarum glycopeptide lbgp-i-b and use thereof

Abstract

This invention belongs to the technical field of natural product active ingredients, specifically relating to a glycopeptide LBGP-I-b derived from wolfberry and its uses. The glycopeptide LBGP-I-b has an average molecular weight of 700–1000 Da, a polypeptide content of 70%–80%, and a polysaccharide content of 10%–20%. The sugar chains are composed of glucose residues, and the sugar chains are linked to the peptide chains via O-glycopeptide bonds. The glycopeptide has functions such as regulating lipid metabolism, lowering blood lipid levels, and improving liver function. The glycopeptide of this invention has a clear source and stable properties, and has good application prospects in the prevention and treatment of metabolic diseases such as metabolic-related fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of natural product active ingredient technology, specifically relating to a glycopeptide LBGP-Ib derived from wolfberry and its uses. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic metabolic disease characterized by abnormal lipid deposition in hepatocytes. Its development is closely related to insulin resistance, lipid metabolism disorders, oxidative stress, and inflammatory responses. With the rising incidence of obesity and metabolic syndrome, the number of people suffering from MASLD is increasing annually, making it one of the most common chronic liver diseases worldwide. Current treatments mainly include lifestyle interventions and some pharmacological interventions, but these still have limitations in efficacy or adverse reactions. Therefore, developing safe and effective interventional substances is of great significance.

[0003] Goji berries (Lycium barbarum L.) are a traditional medicinal and edible plant with a long history of application. They are rich in various active ingredients such as polysaccharides, carotenoids, flavonoids, and amino acids, and have excellent biological functions in antioxidation, immune regulation, and lipid metabolism regulation. Among them, goji berry polysaccharides and their derivatives are considered to be one of the main material bases for their biological activity.

[0004] In recent years, glycopeptides, as natural biomolecules formed by covalently linked sugar and peptide chains, have gradually attracted attention. Glycopeptides possess the dual structural characteristics of both carbohydrates and peptides, exhibiting excellent biological activities in areas such as antioxidation, anti-inflammation, immunomodulation, and lipid metabolism regulation. Studies have shown that some glycopeptides derived from natural products have potential applications in lipid metabolism regulation and related diseases.

[0005] However, existing research on Lycium barbarum glycopeptides mainly focuses on components with polysaccharides as the main component, while research on glycopeptides with polypeptides as the main structural feature is relatively limited, and their structural characteristics and biological functions remain unclear. Furthermore, the roles of these glycopeptides in lipid metabolism regulation and other aspects still lack systematic research.

[0006] Therefore, providing a specific glycopeptide component derived from wolfberry and studying its structural characteristics and application potential is of great significance for promoting its development and utilization in related fields. Summary of the Invention

[0007] This invention provides a glycopeptide LBGP-Ib derived from wolfberry and its uses, in order to solve the problems of unclear structural characteristics and insufficient activity research of existing natural glycopeptides, and to provide a material basis and application evidence for their prevention and treatment of lipid metabolism disorder-related diseases (especially metabolic-related fatty liver disease).

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a glycopeptide LBGP-Ib, wherein the glycopeptide has the following characteristics:

[0010] (1) The average molecular weight is 700-1000 Da;

[0011] (2) The polypeptide content is 70%–80%, and the polysaccharide content is 10%–20%;

[0012] (3) The sugar chain is composed of monosaccharide residues, and the monosaccharide is glucose;

[0013] (4) The sugar chain and peptide chain are connected by O-glycopeptide bond.

[0014] Preferably, the average molecular weight of the glycopeptide LBGP-Ib is 850–900 Da. More preferably, the average molecular weight of the glycopeptide LBGP-Ib is 857.8 Da.

[0015] Preferably, the glycopeptide further comprises a uronic acid component, wherein the uronic acid content is 6.0% to 9.0%.

[0016] In this invention, the sugar chain ends with a terminal β-D-glucose residue. In some embodiments, the sugar residue of the sugar chain is T-β-Glcp-(1→), that is, it contains only one terminal glucose residue.

[0017] In this invention, the monosaccharide composition of the glycopeptide LBGP-Ib includes glucose. In some embodiments, the monosaccharide composition of the glycopeptide LBGP-Ib consists entirely of glucose and contains no other monosaccharides.

[0018] In this invention, the polypeptide comprises 1 to 10 amino acid residues. Preferably, the polypeptide comprises 2 to 9 amino acid residues.

[0019] In this invention, the amino acid composition of the polypeptide includes, but is not limited to, glutamic acid and / or aspartic acid. In some embodiments, the amino acid composition of the polypeptide includes aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and proline. Glutamic acid and aspartic acid are the main amino acids, with glutamic acid content of approximately 16.31% and aspartic acid content of approximately 13.13%.

[0020] In some embodiments, the polypeptide comprises the following amino acids:

[0021] .

[0023] In this invention, the peptide sequence of the polypeptide includes, but is not limited to, at least one of CGVNIPY, AGLPGKC, CSGPF, GVNIPYK, and TTSGVD. The method for determining the peptide sequence includes, but is not limited to, de novo sequencing.

[0024] In this invention, the glycopeptide LBGP-Ib is obtained by isolating and purifying wolfberry.

[0025] In this invention, the structural characteristics and average molecular weight of the glycopeptide LBGP-Ib can be determined using conventional methods in the art. The methods for determining the structural characteristics include, but are not limited to, nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), monosaccharide composition analysis, methylation analysis, and mass spectrometry; the methods for determining the average molecular weight include, but are not limited to, high-performance gel permeation chromatography (HPGPC), high-performance size exclusion chromatography (HPSEC), or multi-angle laser light scattering (MALLS).

[0026] In this invention, the glycopeptide LBGP-Ib can be prepared by methods including but not limited to the following:

[0027] (1) The wolfberry raw material was subjected to alcohol extraction to obtain an extract;

[0028] (2) The extract was subjected to alcohol precipitation, and the precipitate was collected to obtain wolfberry crude glycopeptide LBGP;

[0029] (3) The crude glycopeptide is dissolved and then separated by ion exchange chromatography. The target elution fraction is collected to obtain the LBGP-I fraction.

[0030] (4) The LBGP-I fraction was separated by gel filtration chromatography and eluted with salt solution. The eluted fraction corresponding to the second target peak of the elution curve was collected to obtain the wolfberry glycopeptide LBGP-Ib.

[0031] In this invention, the "alcohol extraction treatment" refers to the extraction of wolfberry raw materials using a system containing alcohol solvents, including but not limited to ethanol, methanol, isopropanol, or a mixed solvent system formed by them and water.

[0032] In this invention, the alcohol extraction in step (1) is performed using ethanol or an ethanol-water mixture solvent. The volume fraction of the ethanol can be 30% to 95%, preferably 40% to 60%, and more preferably 50%. Within the above range, the volume fraction of the ethanol can be any value such as 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0033] In this invention, the ratio of the raw material of wolfberry to the extraction solvent in step (1) can be 1:5 to 1:50 (g / mL), preferably 1:10 to 1:30 (g / mL), and more preferably 1:15 (g / mL). Within the above range, the ratio can be any value such as 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, or 1:50.

[0034] In this invention, the material-liquid ratio can be adjusted according to the properties of the raw materials, the extraction method, and the equipment conditions. For example, the amount of solvent can be appropriately reduced under heating extraction conditions, while the amount of solvent can be appropriately increased under room temperature extraction conditions to improve the extraction efficiency of the target component.

[0035] Furthermore, the extraction process can be carried out under stirring, oscillation, or ultrasonic assistance to promote full contact between the solvent and the raw material, thereby further improving the extraction efficiency.

[0036] In this invention, the extraction process can be carried out under heating conditions, and the heating methods include, but are not limited to, water bath heating, constant temperature heating, or reflux heating. The extraction temperature can be 30–100°C, preferably 40–90°C, more preferably 60–80°C, for example, 40°C, 50°C, 60°C, 70°C, or 80°C.

[0037] In this invention, the extraction time can be 0.5 to 8 hours, preferably 1 to 6 hours, and more preferably 3 to 5 hours, for example, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours. Furthermore, the extraction can be performed once or multiple times, for example, once, twice, or three times.

[0038] In this invention, "alcohol precipitation" refers to the process of precipitating the target component by adding an alcohol solvent to the extract. Preferably, in step (2), the glycopeptide is precipitated by adjusting the volume fraction of the alcohol solvent in the system, and the alcohol solvent includes, but is not limited to, ethanol.

[0039] In some embodiments, ethanol is used for the alcohol precipitation treatment, such that the volume fraction of ethanol in the system is 60% to 100%, preferably 95%. The alcohol precipitation process can be carried out at low temperature conditions, including but not limited to 0 to 10°C, preferably 4°C, and the precipitation time can be 6 to 24 hours.

[0040] In this invention, step (3) refers to the process of separation using the charge interaction between the target component and the ion exchange packing material. Preferably, the ion exchange chromatography is a DEAE-type anion exchange column chromatography, including but not limited to a DEAE-52 ion exchange column. The elution process can be carried out using a salt solution, including but not limited to NaCl solution, and can be performed using isocratic elution or gradient elution.

[0041] In some embodiments, the ion exchange chromatography employs a gradient elution with NaCl solution. The gradient elution can be a salt concentration gradient of 0–1.0 mol / L, preferably 0–0.5 mol / L.

[0042] In this invention, the eluent obtained by ion exchange chromatography is concentrated and then desalted by semi-permeable membrane dialysis to remove inorganic salts, and then dried to obtain the LBGP-I fraction.

[0043] In this invention, in step (4), the isocratic elution is performed using a 0.05–0.50 mol / L sodium chloride solution.

[0044] In some embodiments, the isocratic elution is performed using a 0.10–0.20 mol / L sodium chloride solution.

[0045] In some embodiments, the isocratic elution is performed using a 0.15 mol / L sodium chloride solution.

[0046] In this invention, the elution volume of the elution component corresponding to the second target peak is 270–350 mL.

[0047] In some embodiments, the gel filtration chromatography is Sepharose CL-6B gel column chromatography.

[0048] In a second aspect, the present invention provides the use of the glycopeptide LBGP-Ib in the preparation of medicaments for the prevention and / or treatment of lipid metabolism disorder-related diseases.

[0049] In this invention, the lipid metabolism disorder-related diseases are selected from at least one of diabetes, metabolic-related fatty liver disease, insulin resistance, hyperglycemia, hyperlipidemia, obesity, atherosclerosis, and cardiovascular diseases.

[0050] In some implementations, the lipid metabolism disorder-related disease is metabolic-associated fatty liver disease.

[0051] In some embodiments, the drug has at least one of the following effects:

[0052] (1) Reduce lipid deposition;

[0053] (2) Lowering blood lipid levels; the lowering of blood lipid levels includes lowering at least one of TG, TC, and LDL-C;

[0054] (3) Improve liver function; the improvement of liver function includes reducing ALT and / or AST levels;

[0055] (4) Reduce oxidative stress level; the reduction of oxidative stress level includes reducing MDA content and / or increasing SOD activity.

[0056] Thirdly, the present invention also provides a pharmaceutical composition comprising the above-mentioned glycopeptide LBGP-Ib.

[0057] In this invention, the pharmaceutical composition may further include pharmaceutically acceptable excipients or carriers. The dosage forms of the pharmaceutical composition include, but are not limited to, tablets, capsules, granules, oral liquids, injections, or lyophilized powder for injection.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) This invention provides a novel wolfberry glycopeptide LBGP-Ib with a well-defined structure and stable properties. Its molecular weight, polypeptide content, polysaccharide content and uronic acid content have been clearly characterized, providing a reliable material basis for related research and application.

[0060] (2) The present invention confirms that the glycopeptide LBGP-Ib can effectively regulate lipid metabolism, reduce blood lipid levels, improve liver function, and has significant preventive and therapeutic effects on metabolic-related fatty liver disease. Attached Figure Description

[0061] Figure 1 : The process of extracting wolfberry glycopeptides;

[0062] Figure 2 Elution results using 0~1.0 mol / L NaCl solution;

[0063] Figure 3 Results of purification of LBGP-I fraction using Sepharose CL-6B gel column chromatography;

[0064] Figure 4Purity and average molecular weight of LBGP-Ia and LBGP-Ib ((A) LBGP-Ia; (B) LBGP-Ib);

[0065] Figure 5 Standard curves for peptides, polysaccharides, and uronic acids ((A) peptides; (B) polysaccharides; (C) uronic acids);

[0066] Figure 6 : Ion chromatograms of monosaccharide composition of LBGP-Ia and LBGP-Ib;

[0067] Figure 7 : Ion chromatograms of amino acid composition of LBGP-Ia and LBGP-Ib;

[0068] Figure 8 Total ion current chromatograms of LBGP-Ia and LBGP-Ib peptide sequencing ((A) LBGP-Ia; (B) LBGP-Ib).

[0069] Figure 9 Distribution maps of LBGP-Ia and LBGP-Ib peptides ((A) LBGP-Ia; (B) LBGP-Ib).

[0070] Figure 10 : Glycopeptide bond types of LBGP-Ia and LBGP-Ib ((A) LBGP-Ia; (B) LBGP-Ib);

[0071] Figure 11 Infrared spectral analysis of LBGP-Ia and LBGP-Ib ((A) LBGP-Ia; (B) LBGP-Ib);

[0072] Figure 12 LBGP-Ia 1 H NMR (A) and 13 C NMR (B) spectrum;

[0073] Figure 13 LBGP-Ia 1 H- 1 HSCOSY (A) and HSQC (B) spectra;

[0074] Figure 14 : HMBC (A) and NOESY (B) spectra of LBGP-Ia;

[0075] Figure 15 LBGP-Ib 1 H NMR (A) and 13 C NMR (B) spectrum;

[0076] Figure 16 LBGP-Ib 1 H- 1 H COSY(A) and 1 H- 13 C HSQC(B) spectrum;

[0077] Figure 17 HMBC (A) and NOESY (B) spectra of LBGP-Ib

[0078] Figure 18 Scanning electron microscope images of LBGP-Ia and LBGP-Ib;

[0079] Figure 19 Atomic force microscopy images of LBGP-Ia and LBGP-Ib;

[0080] Figure 20 Changes in zebrafish growth performance indicators;

[0081] Figure 21 Biochemical indicators of liver lipids and liver function in zebrafish;

[0082] Figure 22 Effects of LBGP-Ib on growth indices in MASLD zebrafish;

[0083] Figure 23 Effects of LBGP-Ib on biochemical indicators of lipid accumulation in the liver of MASLD zebrafish;

[0084] Figure 24 Effects of LBGP-Ib on oxidative stress biochemical parameters in the liver of MASLD zebrafish. Detailed Implementation

[0085] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All features disclosed in this specification, or steps in all disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0087] Example 1

[0088] 1 Experimental Methods

[0089] 1.1 Extraction of crude glycopeptides from wolfberry

[0090] 15 kg of dried wolfberry fruit was extracted with 50% ethanol solution at a material-to-liquid ratio of 1:15 (kg:L), at an extraction temperature of 70°C, for 4 hours, and twice. After extraction, all extracts were combined and concentrated under reduced pressure. After cooling to room temperature, 95% ethanol was slowly added and stirred until the ethanol concentration reached 80%. The mixture was allowed to precipitate overnight, and the precipitate was collected by centrifugation. The precipitate was dried in a forced-air drying oven at 55°C to obtain wolfberry crude glycopeptide powder, named LBGP.

[0091] 1.2 Preparation process of Lycium barbarum glycopeptides

[0092] (1) Determination of the elution concentration of Lycium barbarum glycopeptides

[0093] A certain amount of DEAE-52 ion exchange resin was swollen with deionized water for 24 h to remove impurities. It was then treated with 0.5 mol / L NaOH solution for 1 h, washed with deionized water until neutral, and then treated with 0.5 mol / L HCl solution for 1 h, washed with deionized water until neutral, and then treated with 0.5 mol / L NaOH solution for 1 h, washed with deionized water until neutral. After wet packing, the column was equilibrated with deionized water for 24 h before sample loading. 0.301 g of crude wolfberry glycopeptide was accurately weighed and dissolved in 20 mL of deionized water. The solution was heated in a 50°C water bath until fully dissolved, and centrifuged at 5000 rpm for 15 min to obtain the supernatant. The supernatant was loaded onto a pre-equilibrated DEAE-52 ion exchange chromatography column (2.6 cm × 30 cm) and eluted at a flow rate of 0.45 mL / min. The eluent was collected using an automatic fraction collector (8 mL / tube). First, isocratic elution was performed with 240 mL of deionized water. Then, gradient elution was performed using a gradient mixing system with 1032 mL of 0–0.5 mol / L NaCl solution. Finally, isocratic elution was performed with 240 mL of 0.5 mol / L NaCl solution and 240 mL of 1.0 mol / L NaCl solution, respectively. The changes in polysaccharide and peptide content in each eluent tube were detected using the phenol-sulfuric acid method (490 nm) and the ultraviolet absorption difference method (238 nm), respectively. Elution curves were plotted based on the measurement results to determine the optimal elution concentration of Lycium barbarum glycopeptides.

[0094] (2) Preparation of Lycium barbarum glycopeptides

[0095] Weigh 50.02 g of crude wolfberry glycopeptides and dissolve them in 3400 mL of deionized water. Heat (50℃ water bath) until fully dissolved, centrifuge (5000 rpm, 15 min), concentrate the supernatant under reduced pressure to 500 mL, and then add it to a pre-equilibrated preparative DEAE-52 ion exchange chromatography column (Cl...). - The sample (7.5 cm × 60 cm) was used for elution at a flow rate of 10 mL / min. Neutral polysaccharides were first eluted with deionized water. Changes in polysaccharide and peptide content were monitored using the phenol-sulfuric acid method (490 nm) and the UV-Vis absorption difference method (238 nm). After complete elution of the neutral sugar fraction, isocratic elution was performed according to a determined NaCl solution concentration, and the target Lycium barbarum glycopeptide fraction was collected. The Lycium barbarum glycopeptide eluent was concentrated under reduced pressure. The concentrate was added to a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed against running water and pure water for 12 h to remove salts. After confirming the absence of NaCl using silver nitrate solution, the concentrate was concentrated under reduced pressure and freeze-dried to obtain the LBGP-I fraction.

[0096] (3) Isolation and purification of Lycium barbarum glycopeptides

[0097] 0.5021 g of LBGP-I fraction was weighed and dissolved in 15 mL of deionized water. After centrifugation at 5000 rpm for 15 min, the supernatant was obtained and loaded onto a pre-equilibrated Sepharose CL-6B gel chromatography column (2.6 cm × 100 cm). Isocratic elution was performed with 0.15 mol / L NaCl solution at a flow rate of 0.45 mL / min. The eluent (8 mL / tube) was collected using an automatic collector. The changes in polysaccharide and peptide content in each tube of eluent were detected by the phenol-sulfuric acid method (490 nm) and the UV absorption difference method (238 nm), respectively. The target eluent fraction was collected based on the characteristic peak of the elution curve. After desalting by dialyzing (molecular weight cutoff 500 Da), the sample was concentrated under reduced pressure and then freeze-dried after confirming the absence of NaCl using silver nitrate solution.

[0098] 1.3 Determination of the homogeneity and average molecular weight of Lycium barbarum glycopeptides

[0099] The homogeneity and average molecular weight of the obtained Lycium barbarum glycopeptide fractions LBGP-Ia and LBGP-Ib were determined using the HPGPC method. 7.05 mg of Lycium barbarum glycopeptide sample was weighed into a 1.5 mL EP tube, 0.5 mL of 0.15 mol / L NaCl solution was added, and the sample was filtered through a 0.22 μm aqueous filter membrane for later use. The sample was then processed using a TSK-gel G3000PW filter. XL Or TSK-gelG4000PW XLAnalysis was performed using a Shimadzu HPLC system (LC-10ADvp) with a 7.8 mm × 300 mm column. The mobile phase was 0.15 mol / L NaCl solution, the flow rate was 0.6 mL / min, and the injection volume was 20 μL. Under the same conditions, T-dextran series dextran standards with known molecular weights were detected, and a standard curve was generated. The results were based on K... av =(V e -V0) / (V t -V0) Calculate the effective allocation coefficient K av Where V0 is the volume of water outside the chromatographic column, V t V is the total volume. e This represents the actual elution volume of the sample to be tested. (Using K...) v A standard curve is plotted with IgMw on the x-axis and IgMw on the y-axis. The molecular weight (M) of the sample is estimated based on the standard curve. w )size.

[0100] 1.4 Determination of the chemical composition of Lycium barbarum glycopeptides

[0101] (1) Determination of polypeptide content

[0102] The content of polypeptides in wolfberry glycopeptides was determined by ultraviolet absorption difference method.

[0103] Table 1 Preparation of GSH Standard Solution

[0104]

[0105] A 0.1 mg / mL reduced glutathione (GSH) solution was prepared as the standard peptide solution. To prepare the standard curve, the stock solution and distilled water were added sequentially according to the order in Table 1, mixed thoroughly, and allowed to equilibrate at room temperature for 30 min. The absorbance of the reaction system was then measured at wavelengths of 215 nm and 225 nm to plot the standard curve. Absorption difference L = A 215 -A 225 A standard curve was plotted with the absorption difference L as the ordinate and the peptide concentration as the abscissa, and the peptide content in the sample was calculated.

[0106] (2) Determination of polysaccharide content

[0107] The polysaccharide content of Lycium barbarum glycopeptides was determined using the phenol-sulfuric acid method. To prepare a standard curve, 0, 0.4, 0.8, 1.2, 1.6, and 2 mL of a 0.1 mg / mL standard solution were transferred to glass test tubes (1.8 cm × 18 cm). Distilled water was added to adjust the total volume to 2.0 mL. Three replicates were prepared for each concentration. Subsequently, 1 mL of 6% phenol reagent and 5 mL of concentrated sulfuric acid were added to each test tube, and the mixture was gently shaken to ensure homogeneity. The absorbance (A) was measured at 490 nm after reacting for 30 min. 490 , with absorbance A 490 Plot a standard curve with the ordinate as the vertical axis and polysaccharide concentration as the horizontal axis. Calculate the polysaccharide content of the sample based on the standard curve.

[0108] (3) Determination of uronic acid content

[0109] The uronic acid content in Lycium barbarum glycopeptides was determined using the m-hydroxybiphenyl method. A 0.1 mg / mL D-galacturonic acid standard solution was prepared. 0, 0.05, 0.1, 0.2, 0.3, and 0.4 mL of the D-galacturonic acid standard solution were added to glass test tubes, respectively, and distilled water was added to adjust the total volume to 0.4 mL. 40 μL of sulfamic acid (pH 2.5) was added to each test tube, and the mixture was shaken well. Then, 2.5 mL of concentrated sulfuric acid was added, and the mixture was shaken to mix. The tubes were heated in a boiling water bath for 20 min, then cooled to room temperature with cold water. 40 μL of an aqueous solution containing 0.3% m-hydroxybiphenyl and 0.5% NaOH was added to each tube, and the mixture was shaken well. The tubes were incubated at room temperature for 30 min. The absorbance (A) at 525 nm was measured. 525 With absorbance value A 525 Plot a standard curve with the concentration of D-galacturonic acid standard solution on the x-axis and the vertical axis on the y-axis.

[0110] 1.5 Monosaccharide Composition Analysis

[0111] Weigh 6.01 mg of Lycium barbarum glycopeptide sample, add 1 mL of 2 mol / L trifluoroacetic acid (TFA) solution, and place in an oven for hydrolysis at 120℃ for 2 h. After cooling, transfer the solution to an evaporating dish, and add methanol in small amounts multiple times to thoroughly remove residual TFA. Add 1 mL of PMP and NaOH solution (0.5 mol / L PMP : 0.3 mol / L NaOH = 1 : 1) to the evaporating dish, mix well, and then take 0.3 mL of the sample solution into a 1.5 mL centrifuge tube. React in a water bath at 70℃ for 30 min. After cooling, add 150 μL of pure water and 150 μL of 0.3 mol / L HCl solution to the tube, mix thoroughly, add 1 mL of CHCl3 for extraction, discard the CHCl3 layer, and repeat the extraction 3 times.

[0112] The extracted sample solution was filtered through a 0.22 μm microporous membrane and subjected to isocratic elution on a Thermo Fisher Ultimate 3000 two-dimensional liquid chromatography system. The chromatographic column was a Kinetex. ® C 18 100A (4.6 mm × 100 mm, 2.6 μm); Detector: DAD; Mobile phase: 20% acetonitrile (V / V), 80% phosphate buffer (50 mmol / L, pH 7.0); Elution flow rate: 0.5 mL / min; Column temperature: 35℃; Injection volume: 5 μL.

[0113] 1.6 Amino acid composition analysis

[0114] Accurately weigh 34.50 mg of Lycium barbarum glycopeptide sample and place it in a hydrolysis tube. Add 10 mL of 6 mol / L HCl solution to the hydrolysis tube, and then add 3 drops of phenol. Vacuum the hydrolysis tube, then purge it with nitrogen, repeating this process three times. Seal the tube under nitrogen purging. Place the hydrolysis tube in a 110℃ incubator for 22 h to hydrolyze, then remove and cool to room temperature. Filter the hydrolysate into a 50 mL volumetric flask and dilute to the mark. Accurately pipette 1.0 mL of the filtrate into a 15 mL test tube, dry at 60℃, dissolve the residue in 1.0 mL of water, and then dry again. Dissolve the dried sample in 1.0 mL of sample diluent, mix well, filter through a 0.22 μm filter membrane, and determine the amino acids according to GB5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food".

[0115] 1.7 Peptide Sequencing

[0116] (1) Reductive alkylation: Take 100 μg of peptide into a centrifuge tube, add pure water to make up to 100 μL, accurately transfer 1 μL of 1 mol / L dithiothreitol (DTT) solution into the sample to make the final DTT concentration 10 mmol / L, reduce in a 56℃ water bath for 1 h, accurately transfer 2 μL of 1 mol / L iodoacetamide (IAM) solution into the sample to make the final IAM concentration 20 mmol / L, react in the dark at room temperature for 40 min, accurately transfer 1 μL of 1 mol / L DTT solution into the sample to make the final DTT concentration 10 mmol / L, to neutralize the unreacted IAM.

[0117] (2) Desalting: Take the reduced alkylated sample solution and pass it through C 18 Stage-tip desalting was performed by eluting once with 300 μL of 50% acetonitrile and 50% 0.1% TFA, and the eluent was then vacuum dried at 45°C.

[0118] (3) Liquid chromatography conditions: The chromatographic column was Reprosil-Pur120 C 18 -AQ (150 μm × 170 mm × 1.9 μm), mobile phase A is 0.1% formic acid solution, mobile phase B is a mixed solution of 0.1% formic acid and 80% acetonitrile, flow rate is 600 nL / min, analysis time is 66 min, and specific chromatographic conditions are shown in Table 2.

[0119] Table 2 Liquid Chromatography Elution Procedure

[0120]

[0121] (4) Mass spectrometry conditions: The resolution of the first-stage mass spectrometer was set to 70,000, the full scan range was 100~1500 m / z, and the automatic gain control (AGC) was 3.0×10⁻⁶ m / z. 6 The maximum IT (ion implantation time) was set to 100 ms. The resolution of the secondary mass spectrometer was set to 17500, and the AGC was 1.0 × 10⁻⁶. 5 The raw mass spectrometry data were generated, and the peptide sequence was finally analyzed using the PEAKS De novo method.

[0122] 1.8 Glycopeptide bond type analysis

[0123] The presence of O-glycopeptide bonds in the two components of Lycium barbarum glycopeptides was determined using the β-elimination method. The specific procedure was as follows: 2.00 mg of sample was dissolved in 1 mL of 0.2 mol / L NaOH, and another 2.00 mg of sample was dissolved in 1 mL of distilled water as a control. The solutions were heated in a water bath at 45℃ for 1 h, and UV scans were performed in the range of 200–350 nm. The changes in absorbance at 240 nm were compared between the NaOH-treated and distilled water-treated groups to determine the presence of O-glycopeptide bonds.

[0124] 1.9 Infrared Spectroscopy Analysis

[0125] Two 2.00 mg samples of the wolfberry glycopeptide fraction were weighed separately, mixed with potassium bromide (KBr) and compressed into tablets. The tablets were then analyzed using a Fourier transform infrared spectroscopy (FTIR) instrument at 4000–400 cm⁻¹. -1 The sample is scanned within the wavenumber range, and the spectrum is recorded.

[0126] 1.10 Nuclear Magnetic Resonance Spectroscopy Analysis

[0127] Weigh 0.50 mg of each of the two wolfberry glycopeptide samples, dissolve them in 0.5 mL of D2O, and determine the sample composition using nuclear magnetic resonance spectroscopy. 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC, HMBC, and NOESY spectra.

[0128] 1.11 SEM Analysis

[0129] A suitable amount of dried Lycium barbarum glycopeptide sample was taken, and the sample was adhered to the sample stage using conductive adhesive. Gold plating was then performed, and the sample was observed under vacuum. The morphology of the Lycium barbarum glycopeptide sample was observed and photographed using a scanning electron microscope at 150, 1000, and 5000 magnification.

[0130] 1.12 AFM Analysis

[0131] The sample was dissolved in deionized water to prepare a 5 μg / mL solution. After centrifugation to remove insoluble matter, 5 mL of the clear solution was added to a clean mica sheet. After drying at room temperature, the sample was observed under an atomic force microscope in tapping mode with a scanning frequency of 1 Hz and a scanning range of 3000 nm.

[0132] 1.13 Statistical Analysis

[0133] The physicochemical properties of Lycium barbarum glycopeptides were analyzed, and the results are expressed as mean ± standard deviation.

[0134] 2. Experimental Results

[0135] 2.1 Extraction of crude glycopeptides from wolfberry

[0136] The process of extracting crude glycopeptides from wolfberries is as follows: Figure 1 As shown, 15 kg of dried wolfberry fruit was extracted with 50% ethanol to obtain 450 L of extract. After alcohol precipitation, impurities were washed with 95% ethanol solution, and the extract was dried at 55℃ to obtain 806 g of wolfberry crude glycopeptide (LBGP) powder, with an extraction yield of 5.37% ± 0.05%.

[0137] 2.2 DEAE-52 ion exchange column chromatography separation

[0138] Since the extracted crude glycopeptides from wolfberry are a complex mixture, DEAE-52 ion exchange column chromatography was used to separate and purify them in order to obtain a uniform wolfberry glycopeptide component.

[0139] Specifically, isocratic elution was first performed with deionized water, and the washed fraction, named LBP, was collected with a yield of 1.18%. Subsequently, linear gradient elution was performed using 0–0.5 mol / L NaCl solutions. After gradient elution, isocratic elution was performed again with 0.5 mol / L and 1.0 mol / L NaCl solutions, respectively. The changes in polysaccharide and polypeptide content in each eluent were detected using the phenol-sulfuric acid method and the UV absorption difference method, respectively. Elution curves were plotted based on the detection results. The elution results are shown below. Figure 2As shown.

[0140] The results showed that four main elution fractions could be separated during the linear gradient elution process of 0–0.5 mol / L NaCl solution, named LBGP-I, LBGP-II, LBGP-III, and LBGP-IV, with yields of 4.64% ± 0.02%, 1.22% ± 0.03%, 1.34% ± 0.06%, and 1.12% ± 0.03%, respectively.

[0141] Further analysis revealed that the LBGP-I fraction (corresponding to the elution range of approximately 0.13 mol / L NaCl) had the highest peptide content, reaching 58.21% ± 0.06%, and its yield was also higher than that of other fractions. Therefore, the LBGP-I fraction was selected as the target fraction for subsequent separation and purification.

[0142] 2.3 Purification using Sepharose CL-6B gel chromatography column

[0143] To obtain high-purity Lycium barbarum glycopeptide samples, the LBGP-I fraction was purified using Sepharose CL-6B gel column chromatography with 0.15 mol / L NaCl as the eluent. The changes in polysaccharide and peptide content in each eluent tube were analyzed. Results are as follows: Figure 3 As shown, two components, LBGP-Ia and LBGP-Ib, were obtained with yields of 36.93% ± 0.05% and 30.46% ± 0.03%, respectively.

[0144] 2.4 Results of homogeneity and average molecular weight determination of Lycium barbarum glycopeptides

[0145] The homogeneity and average molecular weight of the two components were determined using the HPGPC method, and the results are as follows: Figure 4 As shown, both LBGP-Ia and LBGP-Ib exhibited uniform and symmetrical chromatographic peaks, and the average molecular weights of LBGP-Ia and LBGP-Ib, calculated from the dextran molecular weight standard curve, were 48.9 kDa and 857.8 Da, respectively.

[0146] 2.5 Results of chemical composition determination of Lycium barbarum glycopeptides

[0147] The chemical composition results of wolfberry glycopeptides are as follows: Figure 5 As shown in Table 3. A standard curve for the peptide was plotted with the concentration of reduced glutathione (GSH) on the x-axis and the difference in absorbance at wavelengths of 215 nm and 225 nm on the y-axis. The regression equation for the standard curve of the peptide is Y = 4.4253X + 0.0133, R0 2= 0.9992, the peptide contents of LBGP-Ia and LBGP-Ib were 46.84% ± 0.76% and 76.00% ± 0.18%, respectively. A polysaccharide standard curve was plotted with glucose concentration on the x-axis and absorbance at 490 nm on the y-axis. The regression equation for the polysaccharide standard curve was Y = 9.2504X + 0.0051, R0 = 0.9992. 2 =0.9992. The polysaccharide contents of LBGP-Ia and LBGP-Ib were 44.56% ± 1.8% and 14.61% ± 0.79%, respectively. A standard curve of uronic acid was plotted with D-galacturonic acid concentration as the x-axis and absorbance at 525 nm as the y-axis. The regression equation for the standard curve of uronic acid was Y = 9.2504X - 0.0071, R0 = 0.9992. 2 = 0.9989, the uronic acid content of LBGP-Ia and LBGP-Ib is 3.13% ± 0.01% and 7.62% ± 0.01%, respectively. In summary, the sum of polysaccharides, uronic acids, and polypeptides is 94.53% ± 2.57% for LBGP-Ia and 98.23% ± 0.98% for LBGP-Ib.

[0148] Table 3 Chemical composition of LBGP-Ia and LBGP-Ib

[0149]

[0150] 2.6 Monosaccharide Composition Analysis

[0151] The monosaccharide composition analysis results of the two components of Lycium barbarum glycopeptides LBGP-Ia and LBGP-Ib are as follows: Figure 6 As shown in Table 4.

[0152] Table 4. Monosaccharide composition results of LBGP-Ia and LBGP-Ib

[0153]

[0154] The ion chromatograms of the monosaccharide standards, from left to right, show mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), rhamnose (Rha), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc). Comparison with the standards revealed that LBGP-Ia mainly consists of mannose, glucuronic acid, galacturonic acid, rhamnose, glucose, galactose, and arabinose, with proportions of 3.00%, 1.44%, 2.89%, 3.37%, 3.86%, 38.85%, and 46.58%, respectively. Only glucose was detected in LBGP-Ib.

[0155] 2.7 Amino acid composition analysis

[0156] Amino acids are not only the backbone connecting sugar chains, but also the foundation for the biological functions of glycopeptides. The amino acid composition results of the two components of the wolfberry glycopeptide LBGP-Ia and LBGP-Ib are as follows: Figure 7 As shown in Table 5.

[0157] Analysis revealed that both components had the same amino acid composition, containing all eight essential amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and histidine) and eight non-essential amino acids (aspartic acid, serine, glutamic acid, glycine, alanine, tyrosine, arginine, and proline). However, they differed significantly in amino acid content. LBGP-Ia was primarily composed of glutamic acid and serine, accounting for 29.66% of the total amino acids. In contrast, LBGP-Ib was mainly composed of glutamic acid and aspartic acid, accounting for 29.44% of the total amino acids.

[0158] In addition, the average molecular weight of LBGP-Ib is 857 Da, but its amino acid composition analysis revealed that it contains 16 amino acids. It is inferred that it may be a peptide formed by combining 1 to 10 amino acids and then linking them with sugar chains to form a glycopeptide.

[0159] Table 5. Amino acid composition (mass content) of LBGP-Ia and LBGP-Ib

[0160]

[0161] 2.8 Peptide Sequencing

[0162] This invention employs de novo sequencing to determine the peptide sequence. This method utilizes the mass information of fragment ions in a tandem mass spectrum, calculates the mass difference between adjacent ions, and matches it with the theoretical mass of amino acid residues to directly read the amino acid sequence of the peptide. This method is database-independent. The total ion current chromatograms and peptide distribution maps of the two components, LBGP-Ia and LBGP-Ib, of the Lycium barbarum glycopeptide are shown below. Figure 8 and 9 As shown.

[0163] LBGP-Ia peptide fragments are mainly short peptides of 2-6 units in length, within this range -log 10 (P) The top five peptides by score were GFFTMT, PLETKK, FGDDSV, AFF, and FTNSA. LBGP-Ib mainly consists of short peptides of 2-9 units in length, within this range -log 10(P) The top five peptides by score were CGVNIPY, AGLPGKC, CSGPF, GVNIPYK, and TTSGVD. This indicates that there are significant differences between the two peptide groups.

[0164] 2.9 Glycopeptide bond type analysis

[0165] The UV spectra of Lycium barbarum glycopeptides LBGP-Ia and LBGP-Ib before and after dilute alkali treatment are shown below. Figure 10 As shown, before dilute alkali treatment, the absorbance values ​​of LBGP-Ia and LBGP-Ib at 240 nm were 1.2 and 1.22, respectively. After dilute alkali treatment and the β-elimination reaction, the absorbance values ​​at 240 nm increased to 1.48 and 1.94, respectively, showing obvious characteristic absorption, indicating that both Lycium barbarum glycopeptides LBGP-Ia and LBGP-Ib contain O-glycopeptide bonds.

[0166] 2.10 Infrared Spectroscopy Analysis

[0167] The FT-IR scanning analysis results of Lycium barbarum glycopeptides LBGP-Ia and LBGP-Ib are as follows: Figure 11 As shown. 3356 / 3473 cm -1 The strong peak at 2918 / 2935 cm⁻¹ is the characteristic OH stretching vibration peak of polysaccharides. -1 The peak is attributed to the CH stretching vibration in CH2 or CH3, 1643 / 1647 cm⁻¹ -1 The nearby absorption peak represents uronic acid (COO) - The asymmetric vibrational absorption peaks indicate that both components contain uronic acid structures. (1438 / 1404 cm⁻¹) -1 Absorption peaks of the deformation vibrations of CH are visible nearby, at 1066 / 1073 cm⁻¹. -1 The absorption peaks on the left and right indicate the presence of a pyranose ring.

[0168] In summary, both components of the wolfberry glycopeptide exhibit typical polysaccharide characteristics and contain uronic acid and pyranose rings.

[0169] 2.11 Nuclear Magnetic Resonance Spectroscopy Analysis

[0170] Nuclear magnetic resonance (NMR) spectroscopy can effectively obtain information about monosaccharide composition, anomeric configuration, and the linkage sequence between some glycoside residues, thereby providing more structural information about Lycium barbarum glycopeptides. 1 In ¹H NMR, the chemical shift values ​​of anomeric hydrogens range from 4.4 to 5.5 ppm. Generally, the chemical shift values ​​of anomeric hydrogens in α-configured sugars are greater than 5.0 ppm, while those in β-configured sugars are less than 5.0 ppm.

[0171] LBGP-Ia 1 H NMR and 13 C NMR spectra as follows Figure 12 As shown, in δ H Multiple anomeric hydrogen signals appeared between 4.3 and 5.3 ppm, indicating that it simultaneously possesses α and β glycosidic bonds. A total of 9 signal peaks were identified, which, combined with HSQC ( Figure 13 B) The spectrum confirmed that the sample contained at least nine sugar residues, denoted as residues A through I, with corresponding H-1 chemical shift values ​​of 5.24, 5.24, 5.09, 5.09, 4.72, 4.51, 4.53, and 4.75 ppm, and corresponding C-1 chemical shift values ​​of 109.20, 109.28, 107.49, 101.49, 103.25, 103.25, 101.17, 100.61, and 103.25 ppm.

[0172] Combination 1 H- 1 H COSY ( Figure 13 A) Spectrum and HSQC ( Figure 13 B) Assign the above 9 sugar residues to the spectra. Based on H-1 and H-2... 1 H- 1 The chemical shift value of H-2 can be obtained from the H COSY correlation signal, and further, based on the relationships between H-2 and H-3, H-3 and H-4, H-4 and H-5, and H-5 and H-6... 1 H- 1 The chemical shifts of H-3, H-4, H-5, and H-6 were obtained from the H COSY correlation signal. Furthermore, the corresponding carbon signals were located in the HSQC spectrum based on the HSQC correlation signal of the proton already assigned. The specific chemical shifts corresponding to each sugar residue are shown in Table 6.

[0173] Table 6. Chemical shift assignments of sugar residues in LBGP-Ia

[0174]

[0175] Based on the relevant literature and the results of monosaccharide composition, the A~I sugar residues were assigned as →5)-α-L-Araf-(1→, →3)-α-L-Araf-(1→, α-L-Araf-(1→, β-L-Araf-(1→, →3)-β-D-

[0176] Galp-(1→,→6)-β-D-Galp-(1→,→4)-α-D-GlcpA-(1→,α-L-Rhap-(1→,→3,6)-β-D-

[0177] Galp-(1→).

[0178] Subsequently, the sequence between key residues was analyzed in this invention. (By HBMC (...) Figure 14 A) The spectrum shows that there is a cross-peak (δ) between C-5 of sugar residue A and H-1 of residues C and D. C 66.52 / δ H (5.09 ppm), there is a cross peak (δ) at C-4 of sugar residue G and H-1 of residue H. C 79.26 / δ H (4.44 ppm), there is a cross peak (δ) at C-1 of sugar residue E and H-3 of residue I. C 103.25 / δ H (3.75 ppm), there is a cross peak (δ) at C-1 of sugar residue C and H-5 of residue A. C 107.49 / δ H 3.82 ppm), the H-3 of sugar residue E has a cross peak with the C-1 of residues E and F (δ). H 3.68 / δ C 103.25 ppm). (Based on NOESY spectra). Figure 14 B) It can be seen that H-1 of sugar residue A and H-5 of sugar residue A are spatially coupled (δ). H 5.24 / δ H 3.82 ppm), there is spatial coupling between H-1 and H-3 of sugar residue B (δ). H 5.24 / δ H 3.91 ppm), the H-5 of sugar residue A is spatially coupled with the H-1 of residue C and the H-1 of sugar residue D (δ). H 3.82 / δ H (5.09 ppm), there is spatial coupling between H-1 of sugar residue E and H-3 of residue I (δ). H 4.72 / δ H 3.75 ppm), H-1 of residue G and H-4 of residue G are spatially coupled (δ H 4.53 / δ H 3.60ppm).

[0179] Based on the above two-dimensional NMR spectrum results and monosaccharide composition analysis, it is speculated that the linkage mode of each residue of LBGP-Ia may be as follows: α-L-Araf-(1→5)-α-L-Araf-(1→5)-α-L-Araf-(1→;β-L-Araf-(1→5)-α-L-Araf-(1→5)-α-L-Araf-(1→;α-L-Rhap-(1→4)-β-D-GlcpA-(1→4)-β-D-GlcpA-(1→;→3)-α-L-Araf-(1→3)-α-L-Araf-(1→;→6)-β-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Galp-(1→3,6)-β-D-Galp-(1→;。

[0180] LBGP-Ib 1 H NMR and 13 C NMR spectra as follows Figure 15 As shown. One signal peak was identified in the eccentric signal region, combined with... 1 H- 1 H COSY and HSQC ( Figure 17 The two-dimensional spectrum of the sample indicates that it contains one sugar residue, denoted as sugar residue J, with an H-1 chemical shift of 4.13 ppm and a corresponding C-1 chemical shift of 102.85 ppm.

[0181] According to H-1 and H-2 1 H- 1 H COSY ( Figure 16 A) Based on the relevant signals, the chemical shift value of H-2 of sugar residue J is 3.89 ppm. Further analysis is performed based on the relationships between H-2 and H-3, H-3 and H-4, H-4 and H-5, and H-5 and H-6. 1 H- 1 Based on the H COSY correlation signal, the chemical shift values ​​of H-3, H-4, H-5, and H-6 were found to be 3.64, 3.99, 3.72, and 4.07 ppm, respectively. From HSQC ( Figure 16 B) and HMBC Figure 17 A) The chemical shift values ​​of the C-2 to C-6 signals corresponding to sugar residue J can be found in the spectrum, which are 69.45, 75.49, 72.89, 80.86, and 60.68 ppm, respectively. The specific chemical shifts corresponding to sugar residues are shown in Table 7.

[0182] Table 7 Chemical shift assignments of sugar residues in LBGP-Ib

[0183]

[0184] By comparing relevant literature and combining it with monosaccharide composition analysis, it can be inferred that the J sugar residue is T-β-Glcp-(1→). After completing the assignment, based on HMBC and NOESY ( Figure 17 Based on the spectrum and monosaccharide composition analysis results, it is speculated that LBGP-Ib may contain only one terminal glucose.

[0185] 2.12 Morphological Feature Analysis

[0186] The morphological characteristics of Lycium barbarum glycopeptides LBGP-Ia and LBGP-Ib were observed using scanning electron microscopy. Scanning electron micrographs at different magnifications (150 ×, 1000 ×, and 5000 ×) are shown below. Figure 18 As shown.

[0187] LBGP-Ia exhibits an irregular, flake-like appearance with irregular debris in some areas. LBGP-Ib also presents an irregular, fragmented appearance, but the debris is larger and the surface is relatively rough. A 5000× magnified SEM image reveals that LBGP-Ia has a porous, interconnected mesh-like surface, while LBGP-Ib has a smoother surface.

[0188] AFM two-dimensional and three-dimensional morphology analysis clearly revealed the distinctly different surface structure features of the wolfberry glycopeptides LBGP-Ia and LBGP-Ib at the nanoscale, such as... Figure 19 As shown.

[0189] The surface morphology of LBGP-Ia exhibits significant inhomogeneity and complex topology at the nanoscale. Two-dimensional height maps reveal dramatic surface undulations and distinct height contrasts. Quantitative data analysis indicates a surface height range of -1.6 to 3.9 nm, with a height difference of 5.5 nm, a result visually confirmed in the three-dimensional morphology images. In contrast, the surface morphology of LBGP-Ib displays distinctly different characteristics. Both two-dimensional and three-dimensional AFM images show a flatter and more uniform surface. Its surface height ranges from -1.1 to 2.4 nm, with a height difference of only 3.5 nm. Three-dimensional structural analysis reveals that the surface undulations of LBGP-Ib are more gradual, resulting in smaller, more uniformly distributed aggregates, lacking the sharp peaks and valleys observed in LBGP-Ia.

[0190] Experimental Example 1

[0191] 1 Experimental Methods

[0192] 1.1 Laboratory Animals

[0193] Wild-type (AB strain) adult male zebrafish aged 4-5 months were purchased from Hubei Chuangxin Biotechnology Co., Ltd. The experimental animals were raised in a zebrafish-specific water circulation system under standard conditions: water temperature maintained at 26-28℃, pH value at 7.0-7.4, humidity at 40%-60%, and a light cycle of 14 hours of light / 10 hours of darkness.

[0194] 1.2 Preparation of the test substance

[0195] Weigh 18.00 mg of LBGP-Ib powder and dissolve it in 0.18 mL of phosphate-buffered saline (PBS) to prepare a stock solution with a concentration of 100 mg / mL. Before use, dilute the stock solution with 0.9% NaCl solution to the required concentration. After preparation, store the solution at 4°C.

[0196] 1.3 Preparation of high-fat feed

[0197] Mix regular feed, egg yolk powder, and cholesterol in a mass ratio of 4:5:1. Use ether as a solvent to cover the feed and stir thoroughly. After the ether has completely evaporated in a fume hood, transfer the mixture to a sealed container, dispense it into 50 mL centrifuge tubes, and store at 4°C for later use.

[0198] 1.4 Establishment of the zebrafish MASLD model and treatment with test substance

[0199] Male adult zebrafish aged 4-5 months were randomly assigned to groups of 16 each. The high-fat diet group (HFD) was fed high-fat diet four times a day. The normal diet group (ND) zebrafish were raised in normal water system and fed normal diet twice a day. The entire modeling period was 28 days.

[0200] After successful modeling, zebrafish were randomly divided into nine groups: a control group, a high-fat diet group, low, medium, and high dose groups of LBGP-Ib, and a positive control group (Orlistat). Before administration, zebrafish were anesthetized with tricaine thiosulfate (MS-222) solution. Drug intervention was administered to each zebrafish via gavage, with a gavage volume of 10 μL. The control group received an equal volume of 0.9% NaCl solution, the positive control group received 25 mg / kg of Orlistat, and the LBGP-Ib groups received 5 mg / kg, 25 mg / kg, and 125 mg / kg of the corresponding test drug, respectively. The administration period was 14 days.

[0201] 1.5 Growth Indicator Monitoring

[0202] Record weekly changes in the weight and body length of the zebrafish. After anesthetizing, remove the zebrafish from the anesthetic solution, pat dry any excess water from their body surface, weigh them, and record the data. Then, use a ruler to measure their standard body length (the straight-line distance from the snout to the tip of the caudal fin) along the zebrafish's body axis. Calculate the Body Mass Index (BMI) using the following formula: BMI = weight / body length 2 Zebrafish were sacrificed 29 days after modeling. Fresh livers were removed, dried with filter paper, and weighed. The liver weight was recorded. The liver-to-body ratio (LBR) was calculated using the following formula: LBR = (liver weight / body weight) × 100%.

[0203] 1.6 Determination of Biochemical Indicators

[0204] Weigh an appropriate amount of liver tissue and add it to ice-cold PBS (pH 7.4) at a solid-liquid ratio of 1:9. Homogenize the mixture at low temperature. After homogenization, centrifuge at 12,000 rpm for 15 min at 4°C. Collect the supernatant and determine the protein concentration using a BCA protein assay kit. The specific procedure is as follows: Dilute 0.5 mg / mL BCA standard protein solution with PBS to concentrations of 0, 1, 2, 3, 4, 6, 8, and 10 mg / mL to plot a standard curve. Mix the protein supernatant obtained by centrifugation with PBS at a ratio of 1:20 and add it to a 96-well plate. Then, mix reagent A and reagent B at a ratio of 50:1 to prepare the working solution. Add 200 μL of the mixed working solution to each well and incubate at 37°C with a shaker for 30 min. Measure the absorbance at a wavelength of 562 nm and calculate the sample protein concentration based on the BCA standard protein curve. Perform the detection of indicators such as TG, TC, LDL-C, HDL-C, ALT, AST, SOD and MDA according to the operating procedures in the instructions of the biochemical reagent kit.

[0205] 1.7 Data Analysis

[0206] Independent samples t-tests were used to analyze the differences in the means of the two groups, while one-way ANOVA was used to compare multiple groups. A p-value < 0.05 indicated statistical significance. # or * The value of p < 0.01 indicates that the difference between groups is highly statistically significant. ## or ** express.

[0207] 2. Experimental Results

[0208] 2.1 Establishment of the Zebrafish MASLD Model

[0209] (1) Monitoring of growth indicators

[0210] Zebrafish were induced to mature using a high-fat diet for 28 days. Weekly body weight and length were monitored to calculate BMI. Liver weight was measured and liver circulatory rate (LBR) was calculated after modeling. The experimental results are as follows: Figure 20 As shown in Table 8.

[0211] Table 8. Zebrafish body weight, body length, BMI, and LBR value

[0212]

[0213] Compared with the normal (ND) group, the high-fat diet (HFD) group showed a significant increase in both body weight and body mass index (BMI) (p < 0.01), while the body length did not differ significantly. In addition, the LBR of the HFD group was also significantly increased (p < 0.01).

[0214] (2) Biochemical indicators

[0215] The effects of a high-fat diet on lipid metabolism-related biochemical indicators in zebrafish liver, such as Figure 21 As shown, compared with the control group (ND) fed with normal diet, the levels of TG, TC, and LDL-C in the liver tissue of HFD zebrafish were significantly increased (p < 0.01), while the level of HDL-C was significantly decreased (p < 0.01), and the levels of AST and ALT were significantly increased (p < 0.01). This indicates that a high-fat diet induces lipid metabolism disorders in the liver of zebrafish.

[0216] 2.2 Effects of Lycium barbarum glycopeptide LBGP-Ib on growth and biochemical parameters of MASLD zebrafish

[0217] (1) Growth indicators

[0218] The effect of Lycium barbarum glycopeptide LBGP-Ib component on the growth of MASLD zebrafish is as follows: Figure 22 As shown in the figure. Compared with the high-fat diet HFD group, LBGP-Ib of Lycium barbarum reduced the body weight of MASLD zebrafish, but had no significant effect on their body length. BMI and LBR are important indicators for measuring the growth and liver health of zebrafish. Compared with the high-fat diet HFD group, high-dose LBGP-Ib significantly reduced (p < 0.01) the LBR of MASLD zebrafish, but had no significant effect on BMI.

[0219] (2) Biochemical indicators

[0220] The effects of Lycium barbarum glycopeptide LBGP-Ib fraction on lipid accumulation biochemical indicators in MASLD zebrafish, such as Figure 23As shown, the levels of TG, TC, LDL-C, ALT, and AST in the liver of zebrafish treated with LBGP-Ib were all decreased in a dose-dependent manner. Simultaneously, HDL-C levels were increased in a dose-dependent manner. Therefore, Lycium barbarum glycopeptides can improve lipid accumulation in the liver of zebrafish induced by a high-fat diet.

[0221] The effects of Lycium barbarum glycopeptide LBGP-Ib component on oxidative stress biochemical indicators in MASLD zebrafish, such as Figure 24 As shown, after intervention with wolfberry glycopeptides, the MDA content in zebrafish liver decreased, while the SOD content increased in a dose-dependent manner.

[0222] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glycopeptide LBGP-Ib, characterized in that, The glycopeptide has the following characteristics: (1) The average molecular weight is 700-1000 Da; (2) The polypeptide content is 70%–80%, and the polysaccharide content is 10%–20%; (3) The sugar chain is composed of monosaccharide residues, and the monosaccharide is glucose; (4) The sugar chain and peptide chain are connected by O-glycopeptide bond.

2. The glycopeptide LBGP-Ib according to claim 1, characterized in that, The average molecular weight of the glycopeptide is 850-900 Da; preferably, the average molecular weight of the glycopeptide is 857.8 Da.

3. The glycopeptide LBGP-Ib according to claim 1, characterized in that, The glycopeptide also contains a uronic acid component, wherein the uronic acid content is 6.0% to 9.0%.

4. The glycopeptide LBGP-Ib according to claim 1, characterized in that, The sugar chain ends contain a T-β-Glcp-(1→) structure.

5. The glycopeptide LBGP-Ib according to claim 1, characterized in that, The polypeptide contains 1 to 10 amino acid residues; preferably, the polypeptide contains 2 to 9 amino acid residues.

6. The glycopeptide LBGP-Ib according to claim 1, characterized in that, The amino acid composition of the polypeptide includes glutamic acid and / or aspartic acid; further, the polypeptide includes the following amino acids: 。 7. The glycopeptide LBGP-Ib according to claim 1, characterized in that, The glycopeptide was isolated and purified from wolfberry.

8. Use of the glycopeptide LBGP-Ib according to any one of claims 1 to 7 in the preparation of a medicament for the prevention and / or treatment of lipid metabolism disorder-related diseases; further, the lipid metabolism disorder-related diseases are selected from at least one of diabetes mellitus, metabolic-associated fatty liver disease, insulin resistance, hyperglycemia, hyperlipidemia, obesity, atherosclerosis, and cardiovascular diseases; preferably, the lipid metabolism disorder-related disease is metabolic-associated fatty liver disease.

9. The use according to claim 8, characterized in that, The drug has at least one of the following effects: (1) Reduce lipid deposition; (2) Lowering blood lipid levels; (3) Improves liver function; (4) Reduce oxidative stress levels. Preferably, The reduction of blood lipid levels includes lowering at least one of the following indicators: TG, TC, and LDL-C; and / or The improvement in liver function includes lowering ALT and / or AST levels; and / or The reduction of oxidative stress levels includes reducing MDA content and / or increasing SOD activity.

10. A pharmaceutical composition, characterized in that, It contains the glycopeptide LBGP-Ib as described in any one of claims 1 to 7.

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