Biological preparation containing N-acetyl-D-glucosamine and preparation method thereof

Abstract

The invention belongs to the field of biological medicine manufacturing, and particularly relates to a biological preparation containing N-acetyl-D-glucosamine and a preparation method of the biological preparation. The antibacterial and anti-inflammatory drug carrier comprises N-acetyl-D-glucosamine loaded particles, an antibacterial and anti-inflammatory compound and an ascorbic acid solution, the N-acetyl-D-glucosamine loaded particles are obtained by encapsulating paeoniflorin with sunflower phospholipid and the like, modifying with polyglycine and connecting with N-acetyl-D-glucosamine, and the antibacterial and anti-inflammatory drug carrier has relatively good targeting property; the antibacterial and anti-inflammatory compound can effectively relieve osteoarthritis and other problems, the antibacterial compound is short peptide with the C terminal modified by N-acetyl-D-glucosamine, the short peptide sequence is TKKRCK, T is threonine benzyl ester, and the antibacterial and anti-inflammatory compound can well inhibit inflammation. The biological preparation containing the N-acetyl-D-glucosamine can promote proliferation of rat osteoblasts, relieve inflammatory response related to arthritis and promote tissue remodeling at the same time, and is wide in application range.

Description

Technical Field

[0001] The invention belongs to the field of biological medicine manufacturing, and specifically relates to a biological preparation containing N-acetyl-D-glucosamine and a preparation method thereof. Background Art

[0002] N-Acetyl-D-glucosamine, also known as 2-acetylamino-2-deoxy-D-glucose, GlcNAc, has a chemical formula of C8H 15 NO6, CAS number 7512-17-6, molecular weight 221.21, white or off-white powder, easily soluble in water. N-acetyl-D-glucosamine is an acetylated derivative of glucosamine (GlcN), an important functional monosaccharide, and the main component of different human connective tissues, crustacean shells, fungal cell walls, etc. Its content in the environment is second only to cellulose, making it the second most abundant polysaccharide. N-acetyl-D-glucosamine is easily absorbed and utilized by microorganisms, and is an important source of nutrients and signaling molecules. It is used for the catabolism and anabolism of Bacillus, Streptomyces and Candida species. It is also a basic component of various heterologous biopolymers, such as hyaluronic acid and chondroitin sulfate, and plays an important role in cartilage and joints. In addition, it is often observed in glycoproteins and mammalian growth factors, and is widely involved in various physiological activities. Due to the unique properties of N-acetyl-D-glucosamine, it is used in the food, cosmetics and pharmaceutical industries, for example, as a food antioxidant, in the preparation of cartilage health products, etc., or added to cosmetics to play moisturizing and whitening effects, or used as a competitive inhibitory sugar for lectin histochemistry, a component of BSK culture medium for culturing Borrelia burgdorferi, a component of binding buffer for resuspending Streptococcus pneumoniae, etc.

[0003] N-acetyl-D-glucosamine is also a component of polyacetyl glucosamine in the cartilage matrix and synovial fluid in the human body. When the articular cartilage is damaged to a certain extent, cracks will appear, causing the proliferation of granulation tissue, thereby worsening the condition and making the damaged cartilage unable to recover. At this time, supplementing exogenous N-acetyl-D-glucosamine can stimulate chondrocytes to synthesize proteoglycans, thereby stabilizing the cell membrane and enhancing intercellular connections, and has a certain effect on subacute osteoarthritis. In addition, N-acetyl-D-glucosamine can also inhibit the release of hydrolases such as elastase and lysozyme, reducing their hydrolysis and damage to the cartilage matrix, thereby hindering the continuous destruction of cartilage due to the vicious cycle of immune response. N-acetyl-D-glucosamine can also enhance the expression of bone morphogenetic protein in the early stage of fracture, thereby promoting healing.

[0004] Based on the above-mentioned interaction between N-acetyl-D-glucosamine and tissues such as cartilage, it is necessary to further optimize the drug regimen. Summary of the invention

[0005] Based on the above problems, the present invention provides a biological preparation containing N-acetyl-D-glucosamine, comprising N-acetyl-D-glucosamine loaded microparticles, antibacterial and anti-inflammatory complexes and ascorbic acid solution, wherein the N-acetyl-D-glucosamine loaded microparticles are obtained by encapsulating paeoniflorin with sunflower lecithin, polyglycine modification, and connection with N-acetyl-D-glucosamine, and have good targeting, and can effectively alleviate problems such as osteoarthritis inflammation, and the antibacterial complex is a short peptide modified with N-acetyl-D-glucosamine at the C-terminus, and the short peptide sequence is TKKRCK, where T is threonine benzyl ester, and the antibacterial and anti-inflammatory complex can better inhibit the occurrence of inflammation. The biological preparation containing N-acetyl-D-glucosamine obtained in the present application can promote the proliferation of rat osteoblasts, reduce the inflammatory response associated with arthritis, and promote tissue remodeling at the same time, and has a wide range of applications.

[0006] The invention provides a biological preparation containing N-acetyl-D-glucosamine, which comprises the following raw materials in parts by weight: 3-5 parts of N-acetyl-D-glucosamine loaded microparticles, 2-6 parts of antibacterial and anti-inflammatory complexes, and 5-15 parts of ascorbic acid solution.

[0007] The preparation method of the N-acetyl-D-glucosamine loaded microparticles is as follows: L1. Weigh sunflower lecithin, cholesterol, Tween-80, and anhydrous ethanol according to the mass volume ratio of 3-5 mg: 1-2 mg: 0.05-0.1 mL: 2-4 mL, mix and heat to 50-60 ° C, stir at a speed of 300-400 rpm for 10-15 min, and then obtain solution 1. Add paeoniflorin to solution 1, stir until it is evenly dispersed, and then rotary evaporate at 30-40 ° C until a thin film is obtained; L2. Take out the film obtained in step L1, weigh the film and polyglycine solution according to the mass volume ratio of 1-2 mg: 30-50 mL, heat to 30-40 ° C, and ultrasonically treat at a power of 300-400 W for 5-8 minutes. After the end, naturally cool to room temperature, centrifuge at a centrifugal force of 7000-8000g for 15-20 minutes, wash the obtained precipitate with distilled water, and obtain substance 1 after vacuum freeze drying; L3. Weigh N-acetyl-D-glucosamine and sodium acetate buffer according to a mass volume ratio of 1.5-2 mg: 0.8-1 L, stir until uniformly dispersed to obtain solution 2, add substance 1 and EDC hydrochloride obtained in step L2 to solution 2, stir at a speed of 800-1000 rpm at room temperature for 6-8 hours, and then centrifuge at a centrifugal force of 8000-9000g for 10-15 minutes. The obtained precipitate is washed with distilled water, pre-cooled at -80℃ for 10-12 hours, and then vacuum freeze-dried to obtain N-acetyl-D-glucosamine loaded microparticles.

[0008] Preferably, the amount of paeoniflorin added in step L1 is 0.5-1 mg / mL.

[0009] Preferably, the polyglycine in step L2 has a molecular weight of 500-1000, and the mass percentage concentration of the polyglycine solution is 3-5%.

[0010] Preferably, the sodium acetate buffer in step L3 has a concentration of 0.2M and a pH of 4.0, the amount of substance 1 added is 2-3 mg / L, the amount of EDC hydrochloride added is 0.2-0.5 mg / L, the CAS number of EDC hydrochloride is 25952-53-8, and the product with a purity of ≥99% is preferred.

[0011] The preparation method of the antibacterial and anti-inflammatory compound is as follows: S1. Weigh chloro-2-acetylamino-2-deoxy-3,4,6-tri-O-acetyl-α-D-pyranose, N-(9-fluorenylmethoxycarbonyl)-L-threonine benzyl ester and 1,2-dichloroethane according to a mass volume ratio of 90 mg:54 mg:1 mL, stir until uniformly dispersed to obtain solution 3, add mercuric bromide to solution 3, heat to 60-70°C and reflux for 5-6 hours to obtain a mixture, vacuum concentrate the mixture, and purify it by silica gel column chromatography to obtain substance 2; S2. Prepare a short peptide by Fmoc solid phase synthesis at room temperature, add the substance 2 obtained in step S1 into a peptide synthesis tube, remove the Fmoc protecting group, couple the amino acids one by one in the order of KKRCK, remove the Fmoc protecting group after the last amino acid is coupled, then wash the peptide synthesis tube alternately with N,N-dimethylformamide and anhydrous methanol, vacuum filter, and blow dry with nitrogen to obtain the product; S3. Weigh the product obtained in step S2 and the cleavage reagent according to a mass-to-volume ratio of 3-5 mg:10-20 mL, mix and stir at a speed of 400-500 rpm for 2-3 hours in a light-proof environment, vacuum filter to obtain a filtrate, weigh the filtrate and ether according to a volume ratio of 1:5-8, keep them in a low-temperature environment until precipitation is complete, centrifuge at a centrifugal force of 8000-10000 g for 8-10 minutes to obtain precipitate 2, wash precipitate 2 with ether, blow dry with nitrogen, and obtain substance 3 after vacuum freeze-drying; S4. Weigh the substance 3 obtained in step S3 and anhydrous methanol according to a mass-to-volume ratio of 1-3 mg: 5-8 mL, stir until uniformly dispersed, drop sodium methoxide solution into it until the pH is 10, stir at a speed of 200-400 rpm for 8-10 min in the presence of nitrogen, and then add dry ice until the dry ice is completely sublimated. The obtained liquid is purified by chromatography and then freeze-dried to obtain the antibacterial and anti-inflammatory complex.

[0012] Preferably, in step S1, the CAS number of chloro-2-acetylamino-2-deoxy-3,4,6-tri-O-acetyl-α-D-pyranose is 3068-34-6, the CAS number of N-(9-fluorenylmethoxycarbonyl)-L-threonine benzyl ester is 73724-48-8, the CAS number of 1,2-dichloroethane is 107-06-2, the amount of mercuric bromide added is 100 mg / mL, and the eluent used in the chromatography purification is preferably prepared from ethyl acetate and hexane in a volume ratio of 7:3.

[0013] Preferably, the cutting reagent in step S3 is prepared from trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 35-40:1:1, the CAS number of N,N-dimethylformamide is 68-12-2, and the low temperature environment is preferably a temperature that can be achieved under experimental conditions and is higher than the freezing point of ether, such as -20°C, -80°C.

[0014] Preferably, the concentration of sodium methoxide in step S4 is preferably 0.1-0.15M.

[0015] The mass percentage concentration of the ascorbic acid solution is 3-6%.

[0016] The present invention also provides a method for preparing a biological preparation containing N-acetyl-D-glucosamine, and the specific steps are as follows: V1. Obtain N-acetyl-D-glucosamine loaded microparticles, antibacterial and anti-inflammatory complexes and ascorbic acid solution according to mass fractions, mix the antibacterial and anti-inflammatory complexes with the ascorbic acid solution, and stir until uniformly dispersed to obtain solution 3; V2. Prepare it before use. Add the N-acetyl-D-glucosamine loaded microparticles into the solution 3 obtained in step V1 and shake until it is uniform to obtain a biological preparation containing N-acetyl-D-glucosamine.

[0017] The beneficial effects of the present invention are as follows: The present invention prepares a biological preparation containing N-acetyl-D-glucosamine, including N-acetyl-D-glucosamine loaded microparticles and antibacterial and anti-inflammatory complexes. Peony glycosides are encapsulated by sunflower lecithin, cholesterol and Tween-80 to obtain a nanoliposome film, and the film is further modified by polyglycine, and then the substance 1 is connected to N-acetyl-D-glucosamine by the action of EDC hydrochloride. The prepared N-acetyl-D-glucosamine loaded microparticles have good targeting, and the N-acetyl-D-glucosamine loaded microparticles can play a role inside abnormal cells by means of endocytosis of lectin receptors, thereby effectively alleviating problems such as osteoarthritis. The antibacterial and anti-inflammatory complex is a short peptide modified by N-acetyl-D-glucosamine at the C-terminus, and the short peptide sequence is TKKRCK, where T is threonine benzyl ester. The antibacterial and anti-inflammatory complex can well inhibit the occurrence of inflammation, thereby supporting the improvement of aseptic arthritis, and can be used for arthritis associated with infection because of its good antibacterial effect. The ascorbic acid solution mainly plays a role of solvent and stabilizes the antibacterial and anti-inflammatory complex. The biological preparation containing N-acetyl-D-glucosamine obtained in the present application can promote the proliferation of rat osteoblasts, reduce the inflammatory response related to arthritis, and promote tissue remodeling, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 This is the result of MTT experiment at a concentration of 0.1 mg / mL; Figure 2 This is the result of MTT experiment at a concentration of 0.2 mg / mL; Figure 3 This is the result of MTT experiment at a concentration of 0.5 mg / mL; Figure 4 is the structural formula of substance 2; Figure 5 It is the structural formula of the antibacterial and anti-inflammatory complex; Figure 6 This is a diagram showing the results of animal experiments related to osteoarthritis. DETAILED DESCRIPTION

[0019] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0020] Example 1: This example provides a method for preparing N-acetyl-D-glucosamine loaded microparticles, and the specific steps are as follows: L1. Weigh sunflower lecithin, cholesterol, Tween-80, and anhydrous ethanol according to a mass volume ratio of 3mg:1mg:0.05mL:2mL, mix, heat to 50°C, and stir at 300rpm for 10min to obtain solution 1. Add paeoniflorin to solution 1 in an amount of 0.5mg / mL, stir until evenly dispersed, and then rotary evaporate at 30°C until a thin film is obtained; L2. Take out the film obtained in step L1, weigh the film and polyglycine solution according to the mass volume ratio of 1 mg:30 mL, the mass percentage concentration of the polyglycine solution is 3%, and the polyglycine used is a product with a molecular weight of 500, heat to 30°C, and ultrasonically treat at a power of 300 W for 5 minutes. After the end, naturally cool to room temperature, centrifuge at a centrifugal force of 7000g for 15 minutes, wash the obtained precipitate with distilled water, and obtain substance 1 after vacuum freeze-drying; L3. Weigh N-acetyl-D-glucosamine and sodium acetate buffer at a mass volume ratio of 1.5 mg:0.8 L. The sodium acetate buffer has a concentration of 0.2 M and a pH of 4.0. Stir until uniformly dispersed to obtain solution 2. Add substance 1 and EDC hydrochloride obtained in step L2 to solution 2. The amount of substance 1 added is 2 mg / L, and the amount of EDC hydrochloride added is 0.2 mg / L. Stir at 800 rpm at room temperature for 6 hours. Centrifuge at 8000 g for 10 minutes. Wash the precipitate with distilled water, pre-cool at -80°C for 10 hours, and then freeze-dry under vacuum to obtain N-acetyl-D-glucosamine loaded microparticles.

[0021] Example 2: This example provides a method for preparing N-acetyl-D-glucosamine loaded microparticles, and the specific steps are as follows: L1. Weigh sunflower lecithin, cholesterol, Tween-80, and anhydrous ethanol according to a mass volume ratio of 4 mg: 1.5 mg: 0.08 mL: 3 mL, mix, heat to 55 ° C, and stir at a speed of 350 rpm for 12 minutes. After the end, obtain solution 1, add paeoniflorin to solution 1, the amount of paeoniflorin added is 0.8 mg / mL, stir until uniformly dispersed, and rotary evaporate at 35 ° C until a thin film is obtained; L2. The film obtained in step L1 was taken out, and the film and polyglycine solution were weighed according to a mass volume ratio of 1.5 mg:40 mL, the mass percentage concentration of the polyglycine solution was 4%, and the polyglycine used was a product with a molecular weight of 800. The film was heated to 35°C, and ultrasonically treated at a power of 350 W for 7 minutes. After the end, it was naturally cooled to room temperature, and centrifuged at a centrifugal force of 7600 g for 18 minutes. The obtained precipitate was washed with distilled water, and vacuum freeze-dried to obtain substance 1; L3. Weigh N-acetyl-D-glucosamine and sodium acetate buffer at a mass volume ratio of 1.8 mg:0.9 L. The sodium acetate buffer has a concentration of 0.2 M and a pH of 4.0. Stir until uniformly dispersed to obtain solution 2. Add substance 1 and EDC hydrochloride obtained in step L2 to solution 2. The amount of substance 1 added is 2.5 mg / L, and the amount of EDC hydrochloride added is 0.35 mg / L. Stir at 900 rpm at room temperature for 7 hours. Centrifuge at 8500 g for 12 minutes. Wash the precipitate with distilled water, pre-cool at -80°C for 11 hours, and then freeze-dry under vacuum to obtain N-acetyl-D-glucosamine loaded microparticles.

[0022] Example 3: This example provides a method for preparing N-acetyl-D-glucosamine loaded microparticles, and the specific steps are as follows: L1. Weigh sunflower lecithin, cholesterol, Tween-80, and anhydrous ethanol according to a mass volume ratio of 5 mg: 2 mg: 0.1 mL: 4 mL, mix, heat to 60°C, and stir at 400 rpm for 15 min. After the mixture is finished, obtain solution 1. Add paeoniflorin to solution 1 in an amount of 1 mg / mL. Stir until evenly dispersed, and then rotary evaporate at 40°C until a thin film is obtained. L2. The film obtained in step L1 was taken out, and the film and polyglycine solution were weighed according to a mass volume ratio of 2 mg:50 mL, the mass percentage concentration of the polyglycine solution was 5%, and the polyglycine used was a product with a molecular weight of 1000. The film was heated to 40°C, and ultrasonically treated at a power of 400 W for 8 minutes. After the end, it was naturally cooled to room temperature, and centrifuged at a centrifugal force of 8000 g for 20 minutes. The obtained precipitate was washed with distilled water, and vacuum freeze-dried to obtain substance 1; L3. Weigh N-acetyl-D-glucosamine and sodium acetate buffer at a mass volume ratio of 2mg:1L. Sodium acetate buffer is a product with a concentration of 0.2M and pH=4.0. After stirring until uniformly dispersed, obtain solution 2. Add substance 1 and EDC hydrochloride obtained in step L2 to solution 2. The amount of substance 1 added is 3mg / L, and the amount of EDC hydrochloride added is 0.5mg / L. Stir at room temperature at a speed of 1000rpm for 8h. After the end, centrifuge at a centrifugal force of 9000g for 15min. Wash the obtained precipitate with distilled water, pre-cool at -80℃ for 12h, and then vacuum freeze-dry to obtain N-acetyl-D-glucosamine loaded microparticles.

[0023] Example 4: This example provides a method for preparing an antibacterial and anti-inflammatory compound, and the specific steps are as follows: S1. Weigh chloro-2-acetylamino-2-deoxy-3,4,6-tri-O-acetyl-α-D-pyranose glucose, N-(9-fluorenylmethoxycarbonyl)-L-threonine benzyl ester and 1,2-dichloroethane according to a mass volume ratio of 90 mg:54 mg:1 mL, stir until uniformly dispersed, and obtain solution 3, add mercuric bromide to solution 3, the amount of mercuric bromide added is 100 mg / mL, heat to 60°C and reflux for 5 h to obtain a mixture, vacuum concentrate the mixture, and purify it through C18 silica gel column chromatography, the eluent used is prepared by ethyl acetate and hexane in a volume ratio of 7:3, and obtain substance 2; S2. Prepare short peptides by Fmoc solid phase synthesis at room temperature. Add 1g polystyrene resin (loading capacity 0.8mmol / g) to a 25mL peptide synthesis tube, add 22mL N,N-dimethylformamide, swell the polystyrene resin in the presence of nitrogen, and then remove N,N-dimethylformamide by vacuum filtration. Add substance 2 (0.2mmol) obtained in step S1 to the peptide synthesis tube, and then add 3 times the volume of the resin piperidine solution. The volume concentration of the piperidine solution is 20%, which is prepared from N,N-dimethylformamide. Blow nitrogen for 15min to remove the Fmoc protecting group. Vacuum filtration to remove the piperidine solution, and then use N,N-dimethylformamide and anhydrous methanol to wash the peptide synthesis tube alternately at a molar ratio of 0.8:0.8:0.8. Weigh Fmoc-K, HATU, and HOBt, add them to the peptide synthesis tube, then add N,N-dimethylformamide, react for 3 hours at room temperature in the presence of nitrogen under light-proof conditions, vacuum filter to remove N,N-dimethylformamide after the reaction, then add piperidine solution and remove the Fmoc protecting group, continue to repeatedly add the amino acid to be coupled, remove the Fmoc protecting group after the reaction is completed, and remove the Fmoc protecting group after the last amino acid is coupled, then use N,N-dimethylformamide and anhydrous methanol to wash the peptide synthesis tube alternately, vacuum filter, and blow dry with nitrogen to obtain the product; S3. Weigh the product obtained in step S2 and the cutting reagent according to a mass volume ratio of 3 mg:10 mL, where the cutting reagent is prepared from trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 35:1:1, mix and stir at a speed of 400 rpm for 2 hours in a light-proof environment, vacuum filter to obtain the filtrate, weigh the filtrate and ether according to a volume ratio of 1:5, keep them in a low temperature environment of -20°C for 36 hours until the precipitation is complete, centrifuge at a centrifugal force of 8000 g for 8 minutes to obtain precipitate 2, wash precipitate 2 with ether, blow dry with nitrogen, and obtain substance 3 after vacuum freeze-drying; S4. Weigh the substance 3 obtained in step S3 and anhydrous methanol according to a mass-to-volume ratio of 1 mg:5 mL, stir until uniformly dispersed, drop sodium methoxide solution into it until the pH is 10, the concentration of the sodium methoxide solution is 0.1 M, stir at 200 rpm for 8 min in the presence of nitrogen, add dry ice after the end, until the dry ice is completely sublimated, and the obtained liquid is purified by chromatography and freeze-dried to obtain the antibacterial and anti-inflammatory complex.

[0024] Example 5: This example provides a method for preparing an antibacterial and anti-inflammatory compound, and the specific steps are as follows: S1. Referring to the steps of Example 4, solution 3 was obtained and mercuric bromide was added, and the mixture was heated to 65° C. and refluxed for 5.5 h to obtain a mixture, which was concentrated in vacuo and purified by C18 silica gel column chromatography. The eluent used was consistent with that in Example 4 to obtain substance 2; S2. Prepare a short peptide by Fmoc solid phase synthesis at room temperature. Referring to the steps of Example 4, add the substance 2 obtained in step S1 into a peptide synthesis tube, remove the Fmoc protecting group, couple the amino acids one by one in the order of KKRCK, remove the Fmoc protecting group after the last amino acid is coupled, and then wash the peptide synthesis tube alternately with N,N-dimethylformamide and anhydrous methanol, vacuum filter, and blow dry with nitrogen to obtain the product; S3. Weigh the product obtained in step S2 and the cutting reagent according to a mass volume ratio of 4 mg:15 mL, wherein the cutting reagent is prepared from trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 38:1:1, mix, stir at a speed of 420 rpm for 2.5 h in a light-proof environment, obtain the filtrate by vacuum filtration, weigh the filtrate and ether according to a volume ratio of 1:6, keep them in a low temperature environment of -20°C for 36 h, until the precipitation is complete, centrifuge at a centrifugal force of 9000 g for 9 min, obtain precipitate 2, wash precipitate 2 with ether, blow dry with nitrogen, and obtain substance 3 after vacuum freeze-drying; S4. Weigh the substance 3 obtained in step S3 and anhydrous methanol according to a mass-to-volume ratio of 2 mg:6 mL, stir until uniformly dispersed, drop sodium methoxide solution into it until the pH is 10, the concentration of the sodium methoxide solution is 0.12 M, stir at 300 rpm for 9 minutes in the presence of nitrogen, and then add dry ice until the dry ice is completely sublimated. The obtained liquid is purified by chromatography and then freeze-dried to obtain the antibacterial and anti-inflammatory complex.

[0025] Example 6: This example provides a method for preparing an antibacterial and anti-inflammatory compound, and the specific steps are as follows: S1. Referring to the steps of Example 4, solution 3 was obtained and mercuric bromide was added, and the mixture was heated to 70° C. and refluxed for 6 h to obtain a mixture, which was concentrated in vacuo and purified by C18 silica gel column chromatography. The eluent used was consistent with that in Example 4 to obtain substance 2; S2. Prepare a short peptide by Fmoc solid phase synthesis at room temperature. Referring to the steps of Example 4, add the substance 2 obtained in step S1 into a peptide synthesis tube, remove the Fmoc protecting group, couple the amino acids one by one in the order of KKRCK, remove the Fmoc protecting group after the last amino acid is coupled, and then wash the peptide synthesis tube alternately with N,N-dimethylformamide and anhydrous methanol, vacuum filter, and blow dry with nitrogen to obtain the product; S3. Weigh the product obtained in step S2 and the cutting reagent according to a mass volume ratio of 5 mg:20 mL, wherein the cutting reagent is prepared from trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 40:1:1, mix and stir at a speed of 500 rpm for 3 hours in a light-proof environment, vacuum filter to obtain the filtrate, weigh the filtrate and ether according to a volume ratio of 1:8, keep them in a low temperature environment of -80°C for 8 hours until the precipitation is complete, centrifuge at a centrifugal force of 10000 g for 10 minutes to obtain precipitate 2, wash precipitate 2 with ether, blow dry with nitrogen, and obtain substance 3 after vacuum freeze-drying; S4. Weigh the substance 3 obtained in step S3 and anhydrous methanol according to a mass-to-volume ratio of 3 mg:8 mL, stir until uniformly dispersed, drop sodium methoxide solution into it until the pH is 10, the concentration of the sodium methoxide solution is 0.15 M, stir at 400 rpm for 10 min in the presence of nitrogen, and then add dry ice until the dry ice is completely sublimated. The obtained liquid is purified by chromatography and then freeze-dried to obtain the antibacterial and anti-inflammatory complex.

[0026] The specific contents of the chromatographic purification in step S4 in Example 4-6 are as follows: purification was carried out using a Waters Prep 150 preparative liquid chromatography system, the chromatographic column was an XBridge Prep C18μm OBD (19mm×150mm), the detection wavelength was 215nm, the column temperature was 30°C, phase A was 0.1% TFA acetonitrile solution, phase B was 0.1% TFA aqueous solution, the sample concentration was 25mg / mL, the injection volume was 5mL, the flow rate was 10mL / min, and the initial elution conditions were 0~3min, 50% phase A; 3~8min, 75% phase A; 8~25min, 100% phase A; 25~28min, 40% phase A; 28~30min, 45 phase A.

[0027] Example 7: This example provides a method for preparing a biological preparation containing N-acetyl-D-glucosamine, the specific contents are as follows: The biological preparation containing N-acetyl-D-glucosamine in this embodiment comprises the following raw materials in parts by weight: 3 parts of N-acetyl-D-glucosamine loaded microparticles, 2 parts of antibacterial and anti-inflammatory complexes, and 5 parts of ascorbic acid solution, wherein the mass percentage concentration of the ascorbic acid solution is 3%, the N-acetyl-D-glucosamine loaded microparticles are prepared in Example 1, and the antibacterial and anti-inflammatory complexes are prepared in Example 4; V1. Obtain N-acetyl-D-glucosamine loaded microparticles, antibacterial and anti-inflammatory complexes and ascorbic acid solution according to mass fractions, mix the antibacterial and anti-inflammatory complexes with the ascorbic acid solution, and stir until uniformly dispersed to obtain solution 3; V2. Prepare it before use. Add the N-acetyl-D-glucosamine loaded microparticles into the solution 3 obtained in step V1 and shake until it is uniform to obtain a biological preparation containing N-acetyl-D-glucosamine.

[0028] Example 8: This example provides a method for preparing a biological preparation containing N-acetyl-D-glucosamine, the specific contents are as follows: The biological preparation containing N-acetyl-D-glucosamine in this embodiment comprises the following raw materials in parts by weight: 4 parts of N-acetyl-D-glucosamine loaded microparticles, 5 parts of antibacterial and anti-inflammatory complexes, and 8 parts of ascorbic acid solution, wherein the mass percentage concentration of the ascorbic acid solution is 4%, the N-acetyl-D-glucosamine loaded microparticles are prepared in Example 2, and the antibacterial and anti-inflammatory complexes are prepared in Example 5; V1. Obtain N-acetyl-D-glucosamine loaded microparticles, antibacterial and anti-inflammatory complexes and ascorbic acid solution according to mass fractions, mix the antibacterial and anti-inflammatory complexes with the ascorbic acid solution, and stir until uniformly dispersed to obtain solution 3; V2. Prepare it before use. Add the N-acetyl-D-glucosamine loaded microparticles into the solution 3 obtained in step V1 and shake until it is uniform to obtain a biological preparation containing N-acetyl-D-glucosamine.

[0029] Example 9: This example provides a method for preparing a biological preparation containing N-acetyl-D-glucosamine, the specific contents are as follows: The biological preparation containing N-acetyl-D-glucosamine of this embodiment comprises the following raw materials in parts by weight: 5 parts of N-acetyl-D-glucosamine loaded microparticles, 6 parts of antibacterial and anti-inflammatory complexes, and 15 parts of ascorbic acid solution, wherein the mass percentage concentration of the ascorbic acid solution is 6%, the N-acetyl-D-glucosamine loaded microparticles are prepared by Example 3, and the antibacterial and anti-inflammatory complexes are prepared by Example 6; V1. Obtain N-acetyl-D-glucosamine loaded microparticles, antibacterial and anti-inflammatory complexes and ascorbic acid solution according to mass fractions, mix the antibacterial and anti-inflammatory complexes with the ascorbic acid solution, and stir until uniformly dispersed to obtain solution 3; V2. Prepare it before use. Add the N-acetyl-D-glucosamine loaded microparticles into the solution 3 obtained in step V1 and shake until it is uniform to obtain a biological preparation containing N-acetyl-D-glucosamine.

[0030] The CAS numbers of the reagents involved in Examples 1-9 are listed below:

[0031] Most of the above reagents were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0032] Comparative Example 1: This comparative example provides a biological preparation, the specific contents are as follows: The biological preparation of this comparative example comprises the following raw materials in parts by mass: 4 parts of paeoniflorin, 2 parts of N-acetyl-D-glucosamine, and 15 parts of ascorbic acid solution, wherein the mass percentage concentration of the ascorbic acid solution is 6%. After the above substances are stirred until they are uniformly dispersed, the biological preparation of this comparative example is obtained.

[0033] Experimental test: MTT assay: The substances obtained in Examples 1-6 were subjected to MTT experiments. Taking Example 1 as an example, the specific experimental steps were as follows: the parietal bones of newborn rats were taken, the surface soft tissues were scraped off and cut into pieces, digested with 0.25% pancreatic enzyme at 37°C for 30 minutes, and the digestion solution was discarded. After digestion with 0.1% type I collagenase to form a suspension, it was passed through a 100-mesh metal sieve, washed once with Hank's, and prepared with 1640 culture medium to 1×10 5 / mL cell concentration, inoculate 48-well plates, 0.5ml per well. The experimental group is to dissolve the N-acetyl-D-glucosamine loaded microparticles obtained in Example 1 in water, obtain a solution with a mass volume concentration of 0.1, 0.2, and 0.5mg / mL, and add it to 1640 culture fluids with an addition amount of 10%. The control group does not add the above solution, and the 48-well plates are placed at 37 ° C, 5% CO2 and incubated under the conditions of 37 ° C, 5% CO2, and the solution is changed every other day. From the second day, the experimental group and the control group take 5 holes every day respectively, and after each hole is added with MTT reagent 0.75ml to cultivate 4h, the culture fluid is abandoned, the bottom crystal is retained, and all are taken out on the 10th day of cultivation, washed 3 times with 0.5mL PBS, and each hole is sealed with DMSO 1mL with transparent adhesive tape, and vibrated for 15min. After the crystallization is fully dissolved, 200 μL is taken from each hole in a 96-well plate, and the absorbance value is read at a wavelength of 550nm of an ELISA instrument.

[0034] The results are as follows Figures 1 to 3 As shown, Figures 1 to 3 The corresponding dosages are 0.1, 0.2, and 0.5 mg / mL solutions, respectively. Figures 1 to 3 It can be seen that the substances obtained in Examples 1-6 all have a certain promoting effect on the proliferation of rat osteoblasts.

[0035] 2. Material composition analysis: In the process of preparing the antibacterial and anti-inflammatory compound, substance 2, substance 3 and the antibacterial and anti-inflammatory compound were used 1 H NMR and 13 CNMR was used for verification and the results are recorded as follows: Substance 2 1 H NMR: δ 1.37 (3H, d, J = 6.6 Hz), 1.90-2.11 (12H, 1.95 (s), 2.02 (s), 2.06 (s), 2.06 (s)), 3.70 (1H, td, J = 4.2, 2.7 Hz), 3.97-4.10 (2H,4.03 (dq, J = 6.8, 6.6 Hz), 4.04 (dd, J = 3.5, 2.7 Hz)), 4.26-4.37 (2H, 4.32 (d, J = 4.2 Hz), 4.32 (d, J = 4.2 Hz)), 4.53-4.67 (5H, 4.58 (d, J = 2.7 Hz), 4.58 (t, J =3.1 Hz), 4.61 (d, J= 6.8 Hz), 4.61 (d, J = 3.1 Hz), 4.61 (d, J = 3.1 Hz)), 4.79(1H, d, J = 2.3 Hz), 4.90-5.06 (3H, 4.95 (d, J = 2.3 Hz), 4.97 (dd, J = 3.5, 2.7Hz), 5.00 (t, J = 3.5 Hz)), 5.11-5.21 (2H, 5.16 (s), 5.16 (s)), 7.27-7.49 (9H,7.34 (ddd, J = 7.7, 6.9, 1.1 Hz), 7.34 (ddd, J = 7.7, 6.9, 1.1 Hz), 7.34 (tt, J =7.5, 1.3 Hz), 7.36 (ddd, J = 8.5, 6.9, 1.4 Hz), 7.36 (ddd, J = 8.5, 6.9, 1.4 Hz),7.37 (dddd, J = 7.8, 7.5, 1.6, 0.6 Hz), 7.43 (dddd, J = 7.8, 1.3, 1.0, 0.6 Hz)),7.60-7.73 (2H, 7.67 (ddd, J = 7.7, 1.4, 0.5 Hz), 7.67 (ddd, J = 7.7, 1.4, 0.5Hz)), 7.74-7.93 (2H, 7.80 (ddd, J = 8.5, 1.1, 0.5 Hz), 7.86 (ddd, J = 8.5, 1.1,0.5 Hz)); For Substance 2 1313C NMR: δ 19.8 (1C, s), 20.6 - 20.9 (3C, 20.7 (s), 20.8 (s), 20.8(s)), 22.6 (1C, s), 47.1 (1C, s), 53.5 (1C, s), 57.6 (1C, s), 62.3 (1C, s), 67.1 (1C, s), 67.3 (1C, s), 68.5 (1C, s), 69.7 (1C, s), 72.0 (1C, s), 73.3 (1C, s), 85.3 (1C, s), 101.7 (1C, s), 119.9 - 120.0 (2C, 120.0 (s), 120.0 (s)), 125.0 - 125.1 (2C, 125.0 (s), 125.0 (s)), 127.0 - 127.1 (2C, 127.0 (s), 127.0(s)), 127.2 (1C, s), 127.7 - 127.8 (2C, 127.7 (s), 127.7 (s)), 128.1 (2C, s), 128.5 (2C, s), 135.0 (1C, s), 141.2 - 141.3 (2C, 141.3 (s), 141.3 (s)), 143.7 - 143.8 (2C, 143.7 (s), 143.7 (s)), 156.4 (1C, s), 157.5 (1C, s), 169.3 (1C, s), 169.4 (1C, s), 171.6 (1C, s), 173.6 (1C, s); of Substance 3 1H NMR:δ 1.26-1.73 (19H, 1.33 (tt, J = 7.7, 7.3 Hz), 1.33 (tt,J = 7.7, 7.3 Hz), 1.34 (tt, J = 7.3, 7.1 Hz), 1.7.7 (tt (d, J = 6.6 Hz), 1.40 (quint, J = 7.4 Hz), 1.40 (quint, J = 7.4 Hz),1.50 (tt, J = 7.4, 7.3 Hz), 1.50 (tt, J = 7.4, 7.5 Hz), Jtt, 1.53 (J = 7.4, 7.5. 7). (tt, J = 7.7, 7.4 Hz), 1.55 (tt, J = 7.3, 7.2 Hz), 1.55 (tt, J= 7.3, 7.2 Hz), 1.56 (quint, J = 7.3 Hz), 1.56 (quint, J = 7.1.6 = 7), 1.67 (quint, J = 7.5 Hz)), 1.79-2.11 (18H, 1.85 (dt, J =7.6, 7.4 Hz), 1.85 (dt, J = 7.6, 7.4 Hz), 1.87 (dt, J = 1.7, 7.6,( 7.7 Hz), 7.4 Hz), 1.90 (dt, J = 7.6, 7.1 Hz), 1.90 (dt, J = 7.6, 7.1Hz), 1.95 (s), 2.02 (s), 2.06 (s), 2.06 (s)), 2.19 (Ht 2,2.25 = 2.19 (t, J = 7.4 Hz)), 2.57–2.69 (2H, 2.63 (t, J = 7.3 Hz), 2.63 (t, J =7.3 Hz)), 2.99–3.25 (8H, 3.05 (d, J = 7.2 = 5), 3.14 (t,J = 7.5 Hz), 3.14 (t, J = 7.5 Hz), 3.17 (t, J = 7.2 Hz), 3.17 (t, J = 7.2Hz), 3.19 (t, J = 7.2 Hz), 3.19 (t, J = 7).2 Hz)), 3.54 - 3.75 (4H, 3.60 (t, J = 7.6 Hz), 3.60 (t, J = 7.6 Hz), 3.60 (t, J = 7.6 Hz), 3.70 (td, J = 4.2, 2.7 Hz)), 3.97 - 4.10 (2H, 4.04 (dq, J = 6.9, 6.6 Hz), 4.03 (dd, J = 3.5, 2.7 Hz)), 4.26 - 4.37 (2H, 4.32 (d, J = 4.2 Hz), 4.32 (d, J = 4.2 Hz)), 4.42 - 4.70 (3H, 4.48 (t, J = 7.2 Hz), 4.58 (d, J = 2.7 Hz), 4.64 (d, J = 6.9 Hz)), 4.79 (1H, d, J = 2.3 Hz), 4.90 - 5.06 (3H, 4.95 (d, J = 2.3 Hz), 4.97 (dd, J = 3.5, 2.7 Hz), 5.00 (t, J = 3.5 Hz)), 5.11 - 5.21 (2H, 5.16 (s), 5.16 (s)), 7.28 - 7.48 (5H, 7.34 (tt, J = 7.5, 1.3 Hz), 7.37 (dddd, J = 7.8, 7.5, 1.6, 0.6 Hz), 7.41 (dddd, J = 7.8, 1.3, 1.0, 0.6 Hz));. of Substance 3 1313C NMR: δ 19.8 (1C, s), 20.6 - 20.9 (3C, 20.7 (s), 20.8 (s), 20.8(s)), 22.5 - 22.7 (3C, 22.5 (s), 22.6 (s), 22.6 (s)), 24.4 (1C, s), 25.6 - 25.8(2C, 25.7 (s), 25.7 (s)), 27.3 (1C, s), 27.7 (1C, s), 29.4 - 29.5 (2C, 29.4(s), 29.5 (s)), 30.4 - 30.6 (3C, 30.5 (s), 30.5 (s), 30.5 (s)), 36.6 (1C, s),39.3 (1C, s), 40.0 (1C, s), 40.3 (1C, s), 40.5 (1C, s), 51.9 - 52.0 (3C, 52.0(s), 52.0 (s), 52.0 (s)), 53.1 (1C, s), 53.5 (1C, s), 57.6 (1C, s), 62.3 (1C,s), 67.3 (1C, s), 68.5 (1C, s), 69.7 (1C, s), 72.0 (1C, s), 73.3 (1C, s),85.3 (1C, s), 101.7 (1C, s), 127.2 (1C, s), 128.1 (2C, s), 128.5 (2C, s),135.0 (1C, s), 156.9 (1C, s), 157.5 (1C, s), 169.3 (1C, s), 169.4 (1C, s),170.1 - 170.2 (3C, 170.1 (s), 170.1 (s), 170.1 (s)), 171.3 (1C, s), 171.6 (1C,s), 172.8 (1C, s), 173.6 (1C, s); of the antibacterial and anti - inflammatory complex 1H NMR:δ 1.26-1.73 (19H, 1.33 (tt, J = 7.7, 7.3 Hz),1.33 (tt, J = 7.7, 7.3 Hz), 1.37 (tt, J = 7.3, 7.1 Hz), 1.1.37 (tt, Hz), J.1.37 (tt, 7.1 Hz). J = 6.6 Hz), 1.40 (quint, J = 7.4 Hz), 1.40 (quint, J = 7.4Hz), 1.50 (tt, J = 7.4, 7.3 Hz), 1.50 (tt, J = 7.4, 7.3 Hz), 1.1.53 (tt, J = 7.4, 7.3 Hz), 1.1.53.7 (tt, 7.3 Hz). = 7.7, 7.4 Hz), 1.55 (tt, J = 7.3, 7.2 Hz), 1.55(tt, J = 7.3, 7.2 Hz), 1.56 (quint, J = 7.3 Hz), 1.56 (quint), J = 7.6 7.7 Hz),1,6 (quint, J = 7.5 Hz)), 1.79-1.99 (9H, 1.85 (dt,J = 7.6, 7.4 Hz), 1.85 (dt, J = 7.6, 7.4 Hz), 1.87 (dt, J = 7.6, J.1.7 = Hz), Hz), 1.90 (dt, J = 7.6, 7.1 Hz), 1.90 (dt, J = 7.6,7.1 Hz), 1.94 (s)), 2.13-2.25 (2H, 2.19 (t, J = 7.4 Hz), 2.2-17 = 2.4 (t, J = 7.4 Hz). (2H, 2.63 (t, J = 7.3 Hz), 2.63 (t, J = 7.3 Hz)), 2.99-3.25(9H, 3.05 (d, J = 7.2 Hz), 3.05 (d, J = 7.2 Hz), 3.75,14 (t, J = 4). Hz), 3.15 (dd, J = 3.5, 2.7 Hz), 3.17 (t, J = 7.2 Hz), 3.17 (t, J = 7.2 Hz), 3.19 (t, J = 7.2 Hz), 3.19 (t, J = 7.2 Hz)), 3.46 (1H, td, J =4.5, 2.7 Hz), 3.54-3.66 (3H, 3.60 (t, J = 7.6 Hz), 3.60 (t, J = 7.6 Hz), 3.60(t, J = 7.6 Hz)), 3.76-3.92 (4H, 3.82 (d, J = 4.5 Hz), 3.82 (d, J = 4.5 Hz), 3.86 (dd, J = 3.5, 2.7 Hz), 3.86 (t, J = 3.5 Hz)), 4.03 (1H, dq, J = 6.9, 6.6Hz), 4.42-4.65 (3H, 4.48 (t, J = 7.2 Hz), 4.55 (d, J = 2.7 Hz), 4.59 (d, J =6.9 Hz)), 5.11-5.21 (2H, 5.16 (s), 5.16 (s)), 7.28-7.49 (5H, 7.34 (tt, J =7.5, 1.3 Hz), 7.37 (dddd, J = 7.8, 7.5, 1.6, 0.6 Hz), 7.43 (dddd, J = 7.8,1.3, 1.0, 0.6 Hz));. Antibacterial and anti-inflammatory complex 13C NMR: δ 19.8 (1C, s), 22.5-22.7 (3C, 22.5 (s), 22.6(s), 22.6 (s)), 24.4 (1C, s), 25.6-25.8 (2C, 25.7 (s), 25.7 (s)), 27.3 (1C,s), 27.7 (1C, s), 29.4-29.5 (2C, 29.4 (s), 29.5 (s)), 30.4-30.6 (3C, 30.5(s), 30.5 (s), 30.5 (s)), 36.6 (1C, s), 39.3 (1C, s), 40.0 (1C, s), 40.3 (1C,s), 40.5 (1C, s), 51.9-52.0 (3C, 52.0 (s), 52.0 (s), 52.0 (s)), 53.1 (1C, s), 53.8 (1C, s), 57.6 (1C, s), 62.6 (1C, s), 67.3 (1C, s), 69.6 (1C, s), 72.0 (1C, s), 74.1 (1C, s), 78.2 (1C, s), 101.7 (1C, s), 127.2 (1C, s), 128.1 (2C,s), 128.5 (2C, s), 135.0 (1C, s), 156.9 (1C, s), 170.1-170.2 (3C, 170.1 (s),170.1 (s), 170.1 (s)), 171.3 (1C, s), 171.6 (1C, s), 172.8 (1C, s), 173.6 (1C, s).

[0036] Among them, the structural formulas of substance 2 and the antibacterial and anti-inflammatory complex are as follows: Figure 4 , Figure 5 shown.

[0037] 3. Animal Experimentation 1) Rat osteoarthritis modeling: SPF-grade SD rats were adaptively raised for one week before the start of the experiment. The weight of the SD rats used was between 180-220 g. The temperature of the breeding environment was maintained at 25±2°C, the relative humidity was maintained at 60±5%, and the light-dark ratio was 12:12 (h); The SD rats were fixed on a sterile operating table and 1 cm around the right knee joint was shaved. 2The fur in the area was disinfected and deiodinated, and the patellar ligament was found in the supine position with 45° flexion and extension. The depression on the outside was slowly pushed with 0.2mL papain solution at the injection point. The papain solution was prepared with normal saline, and the mass percentage concentration of the papain solution was 7%. The injection point was pressed with a cotton swab to prevent the papain solution from oozing out, and the knee joint was repeatedly and gently flexed and extended to fill the joint cavity with the mixed solution. To prevent infection in rats, sodium penicillin was used for intramuscular injection. The above steps were performed once on the 1st day, 4th day, and 7th day of the beginning of modeling, and continued for two weeks. In the injection area of ​​the rat knee joint, no obvious bleeding, suppuration, and ulceration were observed, and the modeling was successful.

[0038] 2) Experimental grouping and dosage: 120 SD rats were used, and each of 10 rats was randomly divided into a group, corresponding to the control group, model group and experimental group, respectively. The model group and the experimental group were treated according to the above-mentioned modeling method, and the SD rats in the control group were managed normally during the period. The experimental group involved a total of 10 groups, and the substances obtained in Examples 1-9 and Comparative Example 1 were used for administration treatment, and the experimental groups were injected with 80 μL. In Example 1, 3 parts by mass of N-acetyl-D-glucosamine loaded microparticles were placed in 5 parts by mass of physiological saline solution, and then injected after being prepared into a liquid. By analogy, the mass ratios of Examples 2 and 3 were 4:8 and 5:15, respectively. Similarly, the mass ratios of the antibacterial and anti-inflammatory complexes and physiological saline solutions in Examples 4-6 were 2:5, 5:8, and 6:15, respectively. Examples 7-9 and Comparative Example 1 were directly measured 80 μL for injection. In addition, the control group and the model group were injected with the same amount of physiological saline solution.

[0039] 3) Detection indicators: The experiment was carried out for 3 weeks, during which the drug was treated every day. After the experiment, the SD rats were anesthetized and placed in a supine position. The patellar ligament was cut from the proximal end of the knee joint and a small incision was made to access the knee joint cavity. 0.1 mL of normal saline solution was injected into the knee joint cavity, and the synovial fluid was obtained after repeated aspiration. The supernatant was collected after centrifugation at 4000 g for 10 min, and the levels of inflammatory factors TNF-α and matrix metalloproteinase MMP-13 were detected. The above indicators were processed using ELISA kits purchased from Shanghai Yubo Biotechnology Co., Ltd., and the relevant steps were carried out according to the instructions.

[0040] 4) Experimental results: The results are as follows Figure 6 As shown by Figure 6 It can be seen that the biological preparations obtained in Examples 7-9 have the best effect, followed by the N-acetyl-D-glucosamine loaded microparticles obtained in Examples 1-3. The antibacterial and anti-inflammatory complex also has a certain effect of reducing inflammation and promoting tissue remodeling. In comparison, the comparative example 1 involving only paeoniflorin, N-acetyl-D-glucosamine and ascorbic acid solution has a poor effect.

[0041] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A biological preparation containing N-acetyl-D-glucosamine, characterized in that: The invention comprises the following raw materials in parts by weight: 3-5 parts of N-acetyl-D-glucosamine loaded microparticles, 2-6 parts of antibacterial and anti-inflammatory complexes, and 5-15 parts of ascorbic acid solution.

2. The biological preparation containing N-acetyl-D-glucosamine according to claim 1, characterized in that: The preparation method of the N-acetyl-D-glucosamine loaded microparticles is as follows: L1. Weigh sunflower lecithin, cholesterol, Tween-80, and anhydrous ethanol according to the mass volume ratio of 3-5 mg: 1-2 mg: 0.05-0.1 mL: 2-4 mL, mix, heat, and stir to obtain solution 1, add paeoniflorin to solution 1, stir until evenly dispersed, and rotary evaporate until a film is obtained; L2. The film obtained in step L1 was taken out, and the film and polyglycine solution were weighed according to a mass volume ratio of 1-2 mg: 30-50 mL, heated and ultrasonically treated, and then naturally cooled to room temperature, centrifuged, and the precipitate was washed with distilled water and vacuum freeze-dried to obtain substance 1; L3. Weigh N-acetyl-D-glucosamine and sodium acetate buffer according to the mass volume ratio of 1.5-2mg:0.8-1L, stir until uniformly dispersed to obtain solution 2, add substance 1 and EDC hydrochloride obtained in step L2 to solution 2, stir at room temperature, centrifuge after completion, wash the obtained precipitate with distilled water, and vacuum freeze-dry to obtain N-acetyl-D-glucosamine loaded microparticles.

3. The biological preparation containing N-acetyl-D-glucosamine according to claim 2, characterized in that: The amount of paeoniflorin added in step L1 is 0.5-1 mg / mL.

4. The biological preparation containing N-acetyl-D-glucosamine according to claim 3, characterized in that: The mass percentage concentration of the polyglycine solution in step L2 is 3-5%.

5. The biological preparation containing N-acetyl-D-glucosamine according to claim 4, characterized in that: In step L3, the concentration of sodium acetate buffer is 0.2M, pH=4.0, the amount of substance 1 added is 2-3 mg / L, and the amount of EDC hydrochloride added is 0.2-0.5 mg / L.

6. The biological preparation containing N-acetyl-D-glucosamine according to claim 5, characterized in that: The preparation method of the antibacterial and anti-inflammatory compound is as follows: S1. Weigh chloro-2-acetylamino-2-deoxy-3,4,6-tri-O-acetyl-α-D-pyranose, N-(9-fluorenylmethoxycarbonyl)-L-threonine benzyl ester and 1,2-dichloroethane according to a mass volume ratio of 90 mg:54 mg:1 mL, stir until uniformly dispersed to obtain solution 3, add mercuric bromide to solution 3, heat to reflux to obtain a mixture, vacuum concentrate the mixture, and purify it through silica gel column chromatography to obtain substance 2; S2. Prepare a short peptide by Fmoc solid phase synthesis at room temperature, add the substance 2 obtained in step S1 into a peptide synthesis tube, remove the Fmoc protecting group, couple the amino acids one by one in the order of KKRCK, remove the Fmoc protecting group after the last amino acid is coupled, then wash the peptide synthesis tube alternately with N,N-dimethylformamide and anhydrous methanol, vacuum filter, and blow dry with nitrogen to obtain the product; S3. Weigh the product obtained in step S2 and the cleavage reagent according to a mass-to-volume ratio of 3-5 mg:10-20 mL, mix and stir in a light-proof environment, obtain a filtrate by vacuum filtration, weigh the filtrate and ether according to a volume ratio of 1:5-8, keep them in a low-temperature environment until precipitation is complete, centrifuge to obtain precipitate 2, wash precipitate 2 with ether, blow dry with nitrogen, and obtain substance 3 after vacuum freeze-drying; S4. Weigh the substance 3 obtained in step S3 and anhydrous methanol according to a mass-to-volume ratio of 1-3 mg: 5-8 mL, stir until uniformly dispersed, drop sodium methoxide solution into it until the pH is 10, stir under nitrogen, and after completion, add dry ice until the dry ice is completely sublimated. The obtained liquid is purified by chromatography and then freeze-dried to obtain the antibacterial and anti-inflammatory complex.

7. The biological preparation containing N-acetyl-D-glucosamine according to claim 6, characterized in that: The amount of mercuric bromide added was 100 mg / mL, and the eluent used in the chromatography purification was prepared from ethyl acetate and hexane in a volume ratio of 7:

3.

8. The biological preparation containing N-acetyl-D-glucosamine according to claim 7, characterized in that: In step S3, the cutting reagent is prepared from trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 35-40:1:

1.

9. The biological preparation containing N-acetyl-D-glucosamine according to claim 8, characterized in that: The mass percentage concentration of the ascorbic acid solution is 3-6%.

10. A biological preparation containing N-acetyl-D-glucosamine according to any one of claims 1 to 9, characterized in that: The specific steps are as follows: V1. Obtain N-acetyl-D-glucosamine loaded microparticles, antibacterial and anti-inflammatory complexes and ascorbic acid solution according to mass fractions, mix the antibacterial and anti-inflammatory complexes with the ascorbic acid solution, and stir until uniformly dispersed to obtain solution 3; V2. Prepare it before use. Add the N-acetyl-D-glucosamine loaded microparticles into the solution 3 obtained in step V1 and shake until it is uniform to obtain a biological preparation containing N-acetyl-D-glucosamine.

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