Edible bird's nest sialoglycan peptide capable of improving lung inflammation and application thereof

Abstract

The application discloses a bird's nest sialic acid polysaccharide peptide capable of improving lung inflammation and application thereof. The bird's nest sialic acid polysaccharide peptide has a sugar chain, wherein the proportion of the sugar chain modified by fucose is 30-60%, and the proportion of the sugar chain modified by sialic acid is 20-40%; the core structure of the fucose type is (HexNAc)3(Man)3(Fuc)1; the core structure of the sialic acid sugar type is (HexNAc)3(Man)3(Gal)1(Fuc)1(NeuAc)1; the bird's nest sialic acid polysaccharide peptide has a high neutral sugar dissolution rate; and the glycopeptide shown in SEQ ID NO 2 has a good effect of improving inflammation.

Description

Technical Field

[0001] This invention belongs to the field of glycoprotein engineering technology, specifically relating to a bird's nest sialic acid polysaccharide peptide that can improve lung inflammation and its application. Background Technology

[0002] Bird's nest, a precious natural tonic, is primarily composed of nests built by swiftlets and other related species using secretions from their sublingual glands. In Traditional Chinese Medicine, bird's nest is believed to possess various medicinal properties, including nourishing yin and lungs, beautifying the skin, and improving brain function. The main components of bird's nest include water-soluble proteins, carbohydrates, trace elements, and amino acids. Sialic acid (bird's nest acid) is its most nutritious component, accounting for up to 10%, and plays an important role in the nervous and immune systems.

[0003] Edible bird's nest contains approximately 50% protein and 30% carbohydrates, which are the main components and play a crucial role in its nutritional value and physiological functions. The proteins and carbohydrates in bird's nest do not exist independently but are combined through glycosylation to form bird's nest glycoproteins, including N-glycosylated proteins and O-glycosylated proteins, with N-glycosylated proteins being the predominant component. The N-glycan structure in glycoproteins refers to a complex formed by the covalent bonding of a sugar chain to asparagine residues on the protein. The structure of N-glycans is complex and diverse, significantly influencing protein stability, biological activity, and immune recognition. The N-glycans in bird's nest are composed of various monosaccharides, including sialic acid, N-acetylglucosamine, and N-acetylglucosamine. Studies have shown that the highly branched sialylated structure of the N-glycans in bird's nest helps inhibit influenza virus infection. Furthermore, advances in the study of the structure and function of N-glycans indicate that they play an important role in the anti-inflammatory and neuroprotective effects of bird's nest.

[0004] The sugar chains in bird's nest mostly exist in the form of proteins bound together. Studies have shown that bird's nest has a high expansion rate after absorbing water and is not easily digested by pepsin and trypsin, resulting in limited bioavailability. Patent CN110257459B discloses a method for preparing bird's nest peptides by denaturing and enzymatically hydrolyzing bird's nest.

[0005] Patent CN107974479A discloses a method for preparing bird's nest oligopeptides using a combination of microwave and membrane technology.

[0006] Patent CN114848791A discloses a method for preparing small-molecule bird's nest glycopeptides that can prevent and improve skin inflammation. However, the bird's nest peptides prepared by these methods all neglect the functionality of the glycan chains in bird's nest glycoproteins, resulting in relatively low sugar and sialic acid content, and the glycan structure of bird's nest is not characterized. Furthermore, there is currently limited research on the anti-inflammatory effects of bird's nest sialic acid polysaccharide peptides.

[0007] Therefore, this invention employs a stepwise enzymatic hydrolysis method combined with acetone enrichment to prepare bird's nest sialic acid polysaccharide peptides. The analysis is performed using liquid chromatography-mass spectrometry tandem analysis and by searching the MSfragger database. Apodiformes A search was conducted on the sialic acid polysaccharide chain glycoprotein of bird's nest (Apocynaceae) to obtain the fine structure of the N-glycan chain of the product. Animal experiments were designed, using lipopolysaccharide (LPS) to create a mouse inflammation model, verifying that the bird's nest sialic acid polysaccharide peptide has ameliorative effects on inflammation in the blood, nerves, liver, and lungs of mice, thus verifying the anti-inflammatory efficacy of the product of this invention. Summary of the Invention

[0008] To address the existing problems, this patent employs a stepwise enzymatic hydrolysis method combined with acetone enrichment to isolate bird's nest sialic acid polysaccharide peptides with anti-inflammatory effects. Furthermore, it utilizes liquid chromatography-mass spectrometry tandem analysis and the MSfragger database for searching... Apodiformes (Apodiformes) A ​​search was conducted on the sialic acid polysaccharide chains of bird's nest to obtain the fine structure of the N-glycan chains in the bird's nest sialic acid polysaccharide peptides. This invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a bird's nest glycopeptide with anti-inflammatory activity, characterized in that the amino acid sequence of the glycopeptide is as shown in SEQ ID NO 2.

[0010] The bird's nest glycopeptide, which exhibits anti-inflammatory activity as described above in some respects, has a sugar chain linked to it in the form of Hex(4)HexNAc(5), and the structural unit of the sugar chain is as follows:

[0011] .

[0012] Secondly, the present invention provides a method for preparing the glycopeptide mixture described in the first aspect, specifically comprising the following steps:

[0013] Includes the following steps:

[0014] (1) Soak the bird's nest, crush it, hydrolyze it with protease, inactivate the enzyme, centrifuge and collect the supernatant; enrich and concentrate it, and separate it by liquid chromatography. Preferably, soaking involves adding the bird's nest to water and then mixing it thoroughly.

[0015] Furthermore, the mass of water is 20 to 50 times the dry weight of the bird's nest, for example: 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or 50 times.

[0016] Preferably, the soaked bird's nest is pulverized using a colloid mill with a tooth gap of 50-120 μm. This ensures that the particles in the bird's nest paste are at the micron level.

[0017] Preferably, the grinding time of the colloid mill is 20 to 40 minutes, for example: 20 minutes, 25 minutes, 30 minutes, 35 minutes, or 40 minutes.

[0018] Preferably, the protease in step (2) is selected from any one or a combination of hydrolytic protease, papain, and alkaline protease.

[0019] Furthermore, when the protease is an alkaline protease, the pH of the bird's nest paste is adjusted to 9.5~10.5, and alkaline protease is added at an enzyme-to-protein ratio of 6000~12000 U / mg (enzyme activity / dry weight of bird's nest). After mixing, the mixture is placed in an enzymatic hydrolysis reaction tank and treated for 10~14 h at 35~50℃ and 80~150 r / min.

[0020] Furthermore, when the protease is papain, the pH of the bird's nest paste is adjusted to 5.5~7.5, and papain is added at an enzyme-to-base ratio of 6000~12000 U / mg (enzyme activity / dry weight of bird's nest). After mixing, the mixture is placed in an enzymatic hydrolysis reaction tank and treated for 10~14 h at 37~60℃ and 80~150 r / min.

[0021] Furthermore, when the protease is a hydrolytic protease, the pH of the bird's nest paste is adjusted to 7.5~10.5, and the hydrolytic protease is added at an enzyme-to-protein ratio of 6000~12000 U / mg (enzyme activity / dry weight of bird's nest). After mixing, the mixture is placed in an enzymatic reaction tank and treated for 10~14 h at 50~70℃ and 80~150 r / min.

[0022] Furthermore, when the protease is a combination of hydrolytic protease and papain, the pH of the bird's nest paste is adjusted to 7.5-10.5, and hydrolytic protease is added at an enzyme-to-protein ratio of 6000-12000 U / mg (enzyme activity / dry weight of bird's nest). After mixing, the mixture is placed in an enzymatic hydrolysis reactor and treated at 50-70 ℃ and 80-150 r / min for 10-14 h to inactivate the enzyme. The pH of the bird's nest paste is then adjusted to 5.5-7.5, and papain is added at an enzyme-to-protein ratio of 6000-12000 U / mg (enzyme activity / dry weight of bird's nest). After mixing, the mixture is placed in an enzymatic hydrolysis reactor and treated at 37-60 ℃ and 80-150 r / min for 1-5 h.

[0023] Preferably, the enzyme inactivation temperature in step (2) is 90~100℃, for example: 90℃, 92℃, 95℃, 97℃, 100℃. The enzyme inactivation time is 10~30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0024] Preferably, the centrifugal speed in step (2) is 5000~10000 r / min, for example: 5000 r / min, 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min.

[0025] Preferably, the centrifugation time in step (2) is 15 to 30 minutes, for example: 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0026] Preferably, during the acetone enrichment in step (3), the volume fraction of anhydrous acetone is 40-80% of the total system, for example: 40%, 50%, 60%, 70%, 80%.

[0027] Preferably, the enrichment time in step (3) is 10 to 30 minutes, for example: 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0028] Preferably, the centrifugation speed in step (3) is 3000~5000 r / min, for example: 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min, 5000 r / min.

[0029] Preferably, the centrifugation time in step (3) is 15~30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0030] Preferably, the rotary evaporation speed in step (3) is 100~200 r / min, for example: 100 r / min, 120 r / min, 150 r / min, 180 r / min, 200 r / min.

[0031] Preferably, the rotary evaporation temperature in step (3) is 40~60℃, for example: 40℃, 45℃, 50℃, 55℃, 60℃.

[0032] Preferably, the drying in step (3) is performed by freeze drying or low-temperature drying, and more preferably by freeze drying.

[0033] Furthermore, the freeze-drying process involves first rapidly freezing the concentrated liquid after rotary evaporation, and then freeze-drying it.

[0034] Furthermore, the freeze-drying temperature is -90 to -60°C, for example: -90°C, -85°C, -80°C, -75°C, -70°C, -65°C, -60°C.

[0035] Furthermore, the quick-freezing time is 10-12 hours, for example: 10 hours, 10.5 hours, 11 hours, 11.5 hours, or 12 hours.

[0036] Furthermore, the freeze-drying time is 12 to 24 hours, for example: 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours.

[0037] Furthermore, the protease hydrolysis includes the following steps: adjusting the pH value to 8.0-8.5, adding hydrolyzing protease, inactivating the enzyme after 12 h of enzymatic hydrolysis, adjusting the pH value of the hydrolysate to 7.0-7.4, adding papain for 2 h of enzymatic hydrolysis.

[0038] Furthermore, the enrichment and concentration process includes: washing the amide-bonded silica gel packing material with 100% acetonitrile, activating it with equilibration buffer, and uniformly packing it into a chromatography column; centrifuging and filtering the enzymatic hydrolysate of bird's nest through a filter membrane, removing small molecule impurities through an ultrafiltration tube, replacing the solvent with equilibration buffer and concentrating it to below 20 mL, followed by secondary filtration; loading the sample, rinsing with equilibration buffer to remove impurities, performing gradient elution, detecting the fractions by LC-MS / MS, matching the retention time of the target glycopeptide, and combining the fractions containing the target product after identifying characteristic ion pairs.

[0039] In some embodiments, the steps of the method for preparing the bird's nest glycopeptides are:

[0040] (1) Soaking bird's nest involves soaking the bird's nest (dry weight) in 20-50 times its weight of water at a water temperature of 25℃ for 2 hours.

[0041] (2) The pulverization is colloid mill pulverization, preferably with a colloid mill tooth gap of 80 μm, and the liquid obtained in step (1) is subjected to colloid mill treatment for 20-40 min;

[0042] (3) The protease hydrolysis is performed by adjusting the pH of the solution obtained in step (2) to 8.0-8.5 using HCl and NaOH, adding hydrolytic protease (6000 U / mg), mixing and placing it in an enzymatic fermentation tank, and treating it at 55-60℃ and 150 r / min for 12 h. After treatment, the enzyme is inactivated, and the pH of the hydrolysate is adjusted to 7.0-7.4 using HCl and NaOH, adding papain, mixing and placing it in an enzymatic fermentation tank, and treating it at 50℃ and 150 r / min for 2 h.

[0043] (4) First, the amide-bonded silica packing was washed with 100% acetonitrile and activated with equilibration buffer. It was then uniformly packed into the chromatography column and washed at a flow rate of 2 mL / min until the column pressure stabilized, ensuring no air bubbles. The bird's nest enzymatic hydrolysate was centrifuged, filtered through a 0.22 μm filter membrane, and then filtered through a 3 kDa ultrafiltration tube to remove small molecule impurities. The solvent was replaced with equilibration buffer and concentrated to below 20 mL before a second filtration. The sample was loaded at a flow rate of 1 mL / min and washed with equilibration buffer for 5 column volumes to remove impurities. Gradient elution was then performed: acetonitrile decreased from 80% to 70% in 0-10 min, from 70% to 40% in 10-50 min, and from 40% to 20% in 50-60 min, while maintaining a flow rate of 2 mL / min. The fractions were detected by LC-MS / MS. The retention time of the target glycopeptide was matched to 47.94 min ± 2%, and the fractions containing the target product were combined after matching the characteristic ion pairs. The enrichment solution was then purified with 10 kDa ultrafiltration buffer. The solution was concentrated using an ultrafiltration tube, washed three times with ultrapure water, transferred to a 10 kDa dialysis bag, dialyzed at 4°C for 24 hours, and then freeze-dried to obtain glycopeptide powder. Finally, the column was washed with 10 column volumes of 100% acetonitrile and equilibration buffer, and then sealed and stored in 80% acetonitrile at 4°C.

[0044] Fourthly, the present invention provides a composition comprising the bird's nest glycopeptide described in the first aspect, said composition being prepared as a cosmetic or pharmaceutical product, and permitted excipients thereof.

[0045] Fifthly, the present invention provides the use of the bird's nest sialic acid polysaccharide peptide described in the first aspect or the composition described in the fourth aspect in the preparation of anti-inflammatory products.

[0046] In some embodiments, as described above, the anti-inflammatory product is a cosmetic or pharmaceutical product.

[0047] In some implementations, as described above, the anti-inflammatory product is used to prevent or treat inflammation-related diseases.

[0048] Furthermore, the dosage of the bird's nest glycopeptide is 100 mg / kg body weight / day to 300 mg / kg body weight / day, for example: 100 mg / kg body weight / day, 150 mg / kg body weight / day, 200 mg / kg body weight / day, 250 mg / kg body weight / day, 300 mg / kg body weight / day.

[0049] The beneficial effects of this invention are:

[0050] This invention yielded the complete N-glycan structure of bird's nest sialic acid polysaccharide peptide, innovatively identifying the core structure of bird's nest glycans. Furthermore, the prepared bird's nest sialic acid polysaccharide peptide possesses anti-inflammatory effects. Glycopeptide mixtures containing bird's nest sialic acid polysaccharide peptide have broad application prospects in cosmetics, pharmaceuticals, and other fields, promoting the further development of bird's nest products and enhancing the economic and utilization value of bird's nest. Attached Figure Description

[0051] Figure 1 The diagram shows the process flow for the preparation, separation, structural determination, and functional study of bird's nest sialic acid polysaccharide peptides in this invention.

[0052] Figure 2 The image shown is the mass spectrum of the glycopeptide represented by SEQ ID NO 2.

[0053] Figure 3 The diagram shows the glycan structure of the glycopeptide shown in SEQ ID NO 2. Detailed Implementation

[0054] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are all conventional experimental methods and reagents in the art.

[0056] The main reagents used in the examples are as follows:

[0057] Hydrolytic protease (Alcalase® 2.4 L FG, Novozymes Biotechnology Co., Ltd.), papain (S10011, Shanghai Yuanye Biotechnology Co., Ltd.), alkaline protease (S10154, Shanghai Yuanye Biotechnology Co., Ltd.).

[0058] Example 1 Preparation of a glycopeptide mixture

[0059] 1.1 Preparation and application of glycopeptide mixtures

[0060] (1) Soaking bird's nest: Soak bird's nest (dry weight) in 30 times the amount of water at 25°C for 2 hours.

[0061] (2) Colloid milling: Set the gap between the grinding teeth of the colloid mill to 80 μm, and process the liquid obtained in step (1) with the colloid mill for 30 min.

[0062] (3) Stepwise enzymatic hydrolysis in a hydrolysis tank: The pH of the liquid obtained in step (2) was adjusted to 8.5 using HCl and NaOH. Hydrolytic protease (6000 U / mg) was added, mixed, and placed in an enzymatic hydrolysis fermenter. The mixture was treated at 60℃ and 150 r / min for 12 h. After treatment, the enzyme was inactivated. The pH of the hydrolysate was adjusted to 7.0 using HCl and NaOH. Papain (10000 U / mg) was added, mixed, and placed in an enzymatic hydrolysis fermenter. The mixture was treated at 50℃ and 150 r / min for 2 h.

[0063] (4) Inactivation of enzyme and centrifugation: Inactivate the enzyme in the hydrolysate obtained in step (3) at 95°C for 20 min, cool to room temperature, centrifuge at 5000 r / min for 20 min, and take the supernatant.

[0064] (5) Acetone enrichment and rotary evaporation concentration: Add anhydrous acetone to the supernatant obtained in step (4) to make the volume fraction of acetone in the final system reach 60%, and stir well. Let stand for more than 10 min, centrifuge at 3500 r / min for 20 min to separate the precipitate and supernatant. Transfer all the supernatant to a rotary evaporation flask, set the water bath temperature to 60℃, and rotary evaporate at 100 r / min to remove ethanol until the volume in the flask remains basically unchanged. Pour out the concentrate, add a small amount of water to rinse the rotary evaporation flask to recover the concentrate, and measure the total volume of the concentrate. Based on the measured volume of the concentrate, add anhydrous acetone again to make the volume fraction of acetone in the final system reach 70%, and stir well. Let stand for more than 10 min, centrifuge at 3500 r / min for 20 min to separate the precipitate and supernatant. Take the supernatant again and transfer it to a rotary evaporation flask for rotary evaporation to remove acetone.

[0065] (6) Separation and preparation: The concentrated solution obtained in step (5) is dispensed into glass petri dishes, about 25~30 mL per plate, and then frozen at -80℃ for 12 h. After freezing, the mixture is placed in a freeze dryer and freeze-dried for 24 h to obtain a powdered glycopeptide mixture.

[0066] (7) Sialic acid content determination: The enzymatic hydrolysis product powder was added to 1% phosphoric acid solution and hydrolyzed in a boiling water bath. The supernatant was obtained by centrifugation. An o-phenylenediamine hydrochloride solution was added and mixed with an equal volume of the supernatant. The mixture was derivatized in a water bath at 80°C in the dark for 40 min. After cooling, the mixture was filtered through a 0.45 mm filter membrane. The sialic acid content was determined by high performance liquid chromatography. The sialic acid content of the enzymatic hydrolysis product was found to be 17.84 wt%.

[0067] (8) Neutral sugar dissolution rate determination: The neutral sugar content in dried bird's nest and bird's nest sialic acid polysaccharide peptide was determined by phenol-sulfuric acid method. Dried bird's nest powder and enzymatic hydrolysis product powder were weighed, 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid were added, vortexed and mixed, and placed at room temperature for 20 min. The absorbance was measured at 490 nm. The neutral sugar dissolution rate of the enzymatic hydrolysis product was 75%.

[0068] (9) Determination of glycopeptide ratio: The peptide content in the enzymatic hydrolysis product was determined by the biuret method. After the enzymatic hydrolysis sample was prepared to an appropriate concentration, biuret (sample to be tested: biuret reagent = 3:2, v / v) was added and mixed evenly on a vortex mixer. The absorbance of the supernatant was measured at 540 nm. The glycopeptide ratio of the enzymatic hydrolysis product was found to be 0.78.

[0069] Example 2: Determination of the N-glycan structure of bird's nest glycopeptides

[0070] 2.1 Procedure for Determining the N-Glycan Structure of Bird's Nest Glycopeptides

[0071] (1) Protein extraction: Add 4 times the volume of lysis buffer (1% SDS, 1% protease inhibitor) to the enzymatic hydrolysate and sonicate. Centrifuge at 12000 r / min for 10 min at 4℃, transfer the supernatant to a new centrifuge tube, and determine the protein concentration using a BCA kit.

[0072] (2) Trypsin hydrolysis: The protein extracted in step (1) was hydrolyzed. The volume was adjusted to be uniform with lysis buffer, and 20% TCA was slowly added to a final concentration. The mixture was vortexed and precipitated at 4°C for 2 h. The mixture was centrifuged at 4500 r / min for 5 min, the supernatant was discarded, and the precipitate was washed 2-3 times with pre-cooled acetone. After drying the precipitate, TEAB was added to a final concentration of 200 mM, and the precipitate was sonicated to disperse it. Trypsin was added at a ratio of 1:50 (protease: protein, m / m), and the mixture was hydrolyzed overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mM, and the mixture was reduced at 56°C for 30 min. Then iodoacetamide (IAA) was added to a final concentration of 11 mM, and the mixture was incubated at room temperature in the dark for 15 min.

[0073] (3) Modification and Enrichment: The peptides obtained from enzymatic digestion in step (2) were dissolved in 200 mL of enrichment buffer (80% acetonitrile / 5% trifluoroacetic acid). The supernatant was transferred to a hydrophilic microcolumn and centrifuged at 1000 r / min for about 15 min to complete the enrichment. The hydrophilic microcolumn was then washed three times with enrichment buffer. The glycopeptides were then eluted with 0.1% trifluoroacetic acid, 50 mM ammonium bicarbonate solution, and 50% acetonitrile, respectively. The eluents were collected and combined and then freeze-dried under vacuum. Finally, the peptides were desalted according to the C18 ZipTips manual, freeze-dried under vacuum, and then used for LC-MS analysis.

[0074] (4) Liquid Chromatography-Mass Spectrometry (LC-MS / MS): The peptides obtained by modification and enrichment in step (3) were dissolved in mobile phase A of the liquid chromatography system and then separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient settings: 0~2 min, 4%~7% B; 2~42 min, 7%~20% B; 42~52 min, 20%~30% B; 52~56 min, 30%~80% B; 56~60 min, 80% B, with the flow rate maintained at 500 nl / min. After separation by the ultra-high performance liquid chromatography system, the peptides were injected into an NSI ion source for ionization and then analyzed by an Orbitrap Exploris 480 mass spectrometer. The ion source voltage was set to 2100 V, and the FAIMS compensation voltage (CV) was set to [value missing]. High-resolution Orbitrap was used for detection and analysis of both the peptide precursor ion and its secondary fragments. The primary mass spectrometry scan range was set to 700–2000 m / z, and the scan resolution was set to 60,000 m / s. The secondary mass spectrometry scan range had a fixed starting point of 100 m / z, and the secondary scan resolution was set to 30,000 m / s. TurboTMT was set to off. Data-dependent scanning (DDA) was used for data acquisition. After the primary scan, the precursor ion with the highest signal intensity was selected and sequentially entered into the HCD collision cell for fragmentation at 40% of the fragmentation energy, followed by secondary mass spectrometry analysis. To improve the efficiency of mass spectrometry, automatic gain control (AGC) was set to 200%, the signal threshold was set to 50,000 ions / s, the maximum injection time was set to 75 ms, and the dynamic exclusion time for tandem mass spectrometry scans was set to 30 s to avoid duplicate scans of the precursor ion.

[0075] 2.2 Results of N-glycan structure determination of glycopeptides

[0076] (1) Basic sugar type classification: According to the composition and distribution of sugars in the sugar chain, the sugar chain is divided into five types: ① Paucimannose ② High mannose ③ Fucosylated ④ Sialylated ⑤ Complex / hybrid.

[0077] (2) Identification results of intact glycopeptides and statistical analysis of glycoforms: Secondary mass spectrometry data were retrieved using MSFragger (v3.4) and the Glygen database. Retrieval parameter settings: Database: Apodiformes (Apodiformes) A ​​reverse library was added to calculate the false positive rate (FDR) caused by random matching, and common contamination libraries were added to the database to eliminate the influence of contaminating proteins in the identification results. The enzyme digestion method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum peptide length was set to 7 amino acid residues; the maximum number of peptide modifications was set to 3; and the quality error tolerance for primary precursor ions and secondary fragment ions was set to 20 ppm. Cysteine ​​alkylation with Carbamidomethyl (C) was set as a fixed modification, with variable modifications including methionine oxidation and N-terminal acetylation of proteins. The FDR for protein identification and PSM identification was set to 1%. To obtain high-quality analytical results, further data filtering was performed on the library search analysis results.

[0078] The overall situation of the identified complete glycopeptides after data filtering is shown in Table 1. A total of 92 glycopeptides, 63 glycans, and 9 glycoproteins were identified in the enzymatic hydrolysis products. In addition, 8 glycoproteins contained only one glycosylation site, and each modification site had two or more glycans distributed on it.

[0079] Table 1. Results of identification of the number of intact glycopeptides

[0080]

[0081] As shown in Table 2, among the 92 glycopeptides identified, 36 glycopeptides were modified by sialylated glycoforms, 29 by fucose, 18 by complex glycoforms, 7 by high-mannose, and 2 by oligomannose.

[0082] Table 2. Statistics on Sugar Type Classification

[0083]

[0084] N-acetylglucosamine and mannose form the basic framework of the sugar chain, while special monosaccharides such as fucose and sialic acid can be considered as modifications that occur on the basic framework of the sugar chain. Different special monosaccharides modify the sugar chain to obtain different special sugar chain structures, thereby enabling the sugar chain to perform different functions. For example, fucose modification has been found to be related to tumor migration in studies, and terminal sialic acid modification is related to adhesion or recognition, antiviral and other functions.

[0085] The proportion of glycopeptides modified with fucose was 52.38%, of which 50.79% were modified by a single fucose. The proportion modified with sialic acid was 36.51%, of which 26.98% were modified by a single sialic acid. In addition, statistical analysis of the detected glycans revealed four glycosylation sites for the (HexNAc)5(Man)3(Gal)3(Fuc)1(NeuAc)1 glycan composition. Furthermore, three other glycan compositions, (HexNAc)5(Man)3(Gal)2(Fuc)1, (HexNAc)5(Man)3(Gal)3(Fuc)1, and (HexNAc)5(Man)3(Gal)3(Fuc)1(NeuAc)2, were each identified at three glycosylation sites.

[0086] (3) Glycopeptide glycan structure composition: Based on the Glygen database, the structural images corresponding to the glycans are extracted. Due to the complexity and diversity of sugar structures, a glycan may have multiple matches in the database. We prioritize outputting the glycan structure images that are classified as N-linked in the database and have the highest scores. Ten glycan structures are listed according to their matching scores as shown in Table 3.

[0087] Table 3. Partial glycan structure of sialic acid polysaccharide peptides from bird's nest

[0088]

[0089] Note: Blue square: N-acetylglucosamine (HexNAc); Yellow square: N-acetylglucosamine (GalNAc); Green circle: Mannose; Yellow circle: Galactose; Red triangle: Fucose (Fuc); Purple rhombus: Sialic acid (NeuAc)

[0090] Of the 63 glycan structures identified, 2 were oligomannose, 6 were high-mannose, 12 were complex, 20 were fucose, and 23 were sialylated. Among these, 12 glycans contained both sialic acid and fucose. Based on the identified structures, the core composition of the fucose glycan was (HexNAc)3(Man)3(Fuc)1, and the core composition of the sialylated glycan was (HexNAc)3(Man)3(Gal)1(Fuc)1(NeuAc)1, as shown in Table 4.

[0091] Table 4 Core Structure of Fucose-type and Sialidized Sugar-type

[0092]

[0093] Database analysis identified eight glycopeptides that may have anti-inflammatory and anti-inflammatory effects. Cellular testing was performed on each glycopeptide, and three glycopeptides with strong anti-inflammatory effects were identified. Their amino acid sequences and glycoforms are as follows:

[0094] Table 5. Sequences and glycoforms of bird's nest glycopeptides with anti-inflammatory effects.

[0095]

[0096] Example 3: Purification of bird's nest sialic acid polysaccharide peptides

[0097] Materials: Amide-Sepharose 4B amide-bonded silica gel packing material (particle size 50-100 μm, pore size 100 Å, laboratory glass column (inner diameter 1.6 cm, column length 20 cm, packing volume 40 mL) was used. The mobile phase included equilibration / starting buffer (80% acetonitrile (ACN) + 20% 10 mM ammonium formate solution (containing 0.1% formic acid, pH 4.5)), elution buffer (20% acetonitrile + 80% 10 mM ammonium formate solution), regeneration buffer (100% acetonitrile, all filtered and degassed using a 0.22 μm filter membrane), peristaltic pump, UV detector (214 nm), automatic fraction collector, 10 kDa ultrafiltration tube, dialysis bag (molecular weight cutoff 10 kDa), and freeze dryer.

[0098] First, the amide-bonded silica packing material was washed with 100% acetonitrile and activated with equilibration buffer. It was then uniformly packed into the chromatography column and washed at a flow rate of 2 mL / min until the column pressure stabilized (0.3-0.5 MPa), ensuring no air bubbles. The bird's nest enzymatic hydrolysate was centrifuged (12,000×g, 30 min), filtered through a 0.22 μm membrane, and then passed through a 3 kDa ultrafiltration tube to remove small molecule impurities. The solvent was replaced with equilibration buffer and concentrated to below 20 mL, followed by a second filtration. The sample was loaded at a flow rate of 1 mL / min (not exceeding one column volume). It was washed with equilibration buffer for 5 column volumes to remove impurities, followed by gradient elution: acetonitrile decreased from 80% to 70% (discarded) at 0-10 min, from 70% to 40% (collected at 1 mL / tube) at 10-50 min, and from 40% to 20% (discarded) at 50-60 min, maintaining a flow rate of 2 mL / min. The final product was analyzed by LC-MS / MS. The fractions were analyzed, and the fractions containing the target product were combined after matching the retention time (47.94 min ± 2%) and characteristic ion pairs of the target glycopeptide. The enriched solution was concentrated by a 10 kDa ultrafiltration tube, washed three times with ultrapure water, transferred to a dialysis bag (10 kDa), dialyzed at 4°C for 24 hours (with three water changes), and freeze-dried (pre-frozen at -80°C, vacuum-dried at -55°C for 24 hours) to obtain glycopeptide powder. Finally, the column was washed with 10 column volumes of 100% acetonitrile and equilibration buffer, and stored in 80% acetonitrile at 4°C.

[0099] Example 4: Improvement of blood inflammation in mice by bird's nest sialic acid polysaccharide peptides

[0100] 4.1 Establishment of animal models

[0101] Fifty-six 8-week-old male C57BL / 6J mice were selected. They were randomly divided into four groups (n=8 per group): a blank control group, a model group, a bird's nest group, and a bird's nest glycopeptide group (groups I-III). All mice underwent a one-week acclimatization period. From week 2 onwards, the control and model groups were administered 0.2 mL of 0.9% saline solution daily by gavage. The bird's nest group was administered an equal volume of bird's nest homogenate by gavage, and the bird's nest sialic acid polysaccharide peptide group was administered an equal volume of glycopeptide (200 mg / kg / day) by gavage, for seven consecutive weeks. In the final week of feeding, the control group received an intraperitoneal injection of 0.2 mL of 0.9% saline solution daily, while the other groups received an equal volume of LPS solution (2 mg / kg) intraperitoneally, for one week. Mice were fasted for 12 hours and then sacrificed.

[0102] 4.2 Blood index measurement

[0103] Blood cell counts, including inflammatory cells such as leukocytes, neutrophils, and lymphocytes, were performed using a fully automated hematology analyzer with 50 mL of fresh whole blood. A suitable amount of fresh blood was collected, allowed to stand at room temperature for 30 min, and then centrifuged at 4 ℃ and 3000 r / min for 15 min. The supernatant was collected to prepare serum. The levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum were measured using a commercially available ELISA kit. The procedure was performed according to the kit instructions, and the absorbance of a 96-well plate was measured at 490 nm. The inflammatory factors were quantified using a standard curve. The effects of bird's nest peptide on mouse blood count indicators are shown in Table 6, and the effects on mouse blood inflammatory factors are shown in Table 3.

[0104] Table 6. Effects of bird's nest sialic acid polysaccharide peptides on blood count parameters in mice.

[0105]

[0106] Note: Groups I to III represent bird's nest glycopeptides with serial numbers 1, 2, and 3 in Table 5, respectively.

[0107] As shown in Table 7, there were significant differences in the levels of white blood cells, neutrophils, monocytes, and eosinophils between the model group and the normal group. p The value <0.05 indicates successful modeling. After intervention in mice, each intervention group (bird's nest group and bird's nest glycopeptide group) showed a significant reduction in the levels of white blood cells, neutrophils, monocytes, and eosinophils in the mouse blood compared to the model group. The bird's nest group showed no significant difference in its intervention effect on inflammatory cells in the blood, while the bird's nest glycopeptide groups I-III, obtained through stepwise enzymatic hydrolysis, showed significant differences compared to the bird's nest group, indicating that the bird's nest glycopeptides prepared in this invention have a significant ameliorative effect on blood inflammation in mice.

[0108] Table 7. Effects of bird's nest sialic acid polysaccharide peptides on blood inflammatory factors in mice.

[0109]

[0110] Note: Groups I to III represent bird's nest glycopeptides with serial numbers 1, 2, and 3 in Table 5, respectively.

[0111] As shown in Table 7, there were significant differences in the levels of the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group. pThe value <0.05 indicates successful modeling. After intervention in mice, each intervention group (bird's nest group, bird's nest glycopeptide group) showed a significant reduction in the levels of TNF-α, IL-1β, and IL-6 in the blood of mice compared to the model group. There was no significant difference in the intervention effect on inflammatory factors in the blood compared to the bird's nest group, while the bird's nest glycopeptide groups I-III, obtained by stepwise enzymatic hydrolysis, showed significant differences compared to other groups. This indicates that the bird's nest glycopeptides prepared in this invention have a significant ameliorative effect on blood inflammation in mice.

[0112] Example 5: Improvement of neuroinflammation in mice by bird's nest peptides

[0113] 5.1 Animal model establishment: The same method as in 3.1 of Example 3 for establishing an animal model.

[0114] 5.2 Measurement of neuroinflammatory factors: After obtaining mouse cerebral cortex, the cerebral cortex was homogenized, and the serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were measured using a commercially available ELISA kit. The procedure was performed according to the kit instructions, and the absorbance of the 96-well plate was measured at 490 nm. The inflammatory factors were quantified according to the standard curve. The results are shown in Table 8.

[0115] Table 8 Effects of bird's nest glycopeptides on neuroinflammatory factors in mice

[0116]

[0117] Note: Groups I to III represent bird's nest glycopeptides with serial numbers 1, 2, and 3 in Table 5, respectively.

[0118] As shown in Table 8, there were significant differences in the levels of the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group. p The value <0.05 indicates successful modeling. After intervention in mice, the intervention groups (bird's nest group and bird's nest glycopeptide group) showed a significant reduction in the levels of TNF-α, IL-1β, and IL-6 in the mouse cerebral cortex compared to the model group. The bird's nest group showed no significant difference in its intervention effect on inflammatory factors in the mouse cerebral cortex. Bird's nest glycopeptide groups I and II showed some improvement in neuroinflammatory factors, while bird's nest glycopeptide group III, obtained through stepwise enzymatic hydrolysis, showed a significant difference compared to other groups. This indicates that the bird's nest glycopeptides prepared in this invention have a significant ameliorative effect on neuroinflammation in mice.

[0119] Example 6: Improvement of liver inflammation in mice by bird's nest glycopeptides

[0120] 6.1 Animal model establishment: The same method as in 4.1 of Example 4 for establishing an animal model.

[0121] 6.2 Measurement of Liver Inflammatory Factors: After obtaining mouse livers, the livers were homogenized, and the levels of serum inflammatory factors TNF-α, IL-1β, and IL-6 were measured using a commercially available ELISA kit. The procedure was performed according to the kit instructions. The absorbance of the 96-well plate was measured at 490 nm, and the inflammatory factors were quantified based on the standard curve. The results are shown in Table 9.

[0122] Table 9. Effects of bird's nest glycopeptides on inflammatory factors in mouse liver

[0123]

[0124] Note: Groups I to III represent bird's nest glycopeptides with serial numbers 1, 2, and 3 in Table 5, respectively.

[0125] As shown in Table 9, there were significant differences in the levels of the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group. p The value <0.05 indicates successful modeling. After intervention in mice, each intervention group (bird's nest group, bird's nest peptide group) showed a significant reduction in the levels of TNF-α, IL-1β, and IL-6 in the mouse liver compared to the model group. Bird's nest glycopeptide groups I and II showed some improvement in liver inflammatory factors, while group III showed significant differences compared to other groups. This indicates that the bird's nest glycopeptide prepared in this invention has a significant effect on improving liver inflammation in mice.

[0126] Example 7: Improvement of Lung Inflammation in Mice by Bird's Nest Peptides

[0127] 7.1 Animal model establishment: The same method as in Example 3.1 for establishing the animal model.

[0128] 7.2 Measurement of Lung Inflammatory Factors: After obtaining mouse lungs, the lungs were homogenized, and the levels of serum inflammatory factors TNF-α, IL-1β, and IL-6 were measured using a commercially available ELISA kit. The procedure was performed according to the kit instructions. The absorbance of the 96-well plate was measured at 490 nm, and the inflammatory factors were quantified based on the standard curve. The results are shown in Table 10.

[0129] Table 10 Effects of bird's nest peptides on inflammatory factors in mouse lungs

[0130]

[0131] Note: Groups I to III represent bird's nest glycopeptides with serial numbers 1, 2, and 3 in Table 5, respectively.

[0132] As shown in Table 10, there were significant differences in the levels of the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group. pThe value <0.05 indicates successful modeling. After intervention in mice, each intervention group (bird's nest group, bird's nest peptide group) showed a significant reduction in the levels of TNF-α, IL-1β, and IL-6 in mouse lung tissue compared to the model group. There was no significant difference in the intervention effect on lung inflammatory factors between the bird's nest group and the model group. Bird's nest glycopeptide groups I and II showed some improvement in lung inflammatory factors, while group III showed a significant difference compared to other groups. This indicates that the bird's nest glycopeptide prepared in this invention has a significant ameliorative effect on lung inflammation in mice.

[0133] The above experiments show that the bird's nest sialic acid polysaccharide peptide prepared by the stepwise enzymatic hydrolysis combined with acetone enrichment method of the present invention has a significant effect on improving inflammation in the blood, nerves, liver and lungs of mice, and the improvement effect is higher than that of the group that took bird's nest. This indicates that breaking down bird's nest into glycopeptides with a higher sugar content can better exert its anti-inflammatory effect and can be applied to the production of pharmaceuticals.

[0134] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A bird's nest glycopeptide capable of improving lung inflammation, characterized in that, The glycopeptide is composed of a peptide and an N-glycan chain. The amino acid sequence of the peptide is shown in SEQ ID NO 2. The glycoform of the N-glycan chain to which the glycopeptide is attached is Hex(4)HexNAc(5). The structural unit of the N-glycan chain is as follows: 。 2. A method for preparing bird's nest glycopeptides as described in claim 1, characterized in that, The steps are as follows: The bird's nest is soaked, pulverized, hydrolyzed with protease, enzyme-inactivated, centrifuged to obtain the supernatant, enriched, concentrated, and purified. The protease hydrolysis includes the following steps: adjusting the pH to 8.0-8.5, adding 2.4 L of Alcalase FG, hydrolyzing for 12 h and then inactivating the enzyme, adjusting the pH of the hydrolysate to 7.0-7.4, adding papain and hydrolyzing for 2 h; The enrichment and concentration process includes: washing the amide-bonded silica gel packing material with 100% acetonitrile, activating it with equilibration buffer, and uniformly packing it into a chromatography column; centrifuging and filtering the enzymatic hydrolysate of bird's nest through a filter membrane, removing small molecule impurities through an ultrafiltration tube, replacing the solvent with equilibration buffer and concentrating it to below 20 mL, followed by secondary filtration; loading the sample, rinsing with equilibration buffer to remove impurities, performing gradient elution, detecting the fractions by LC-MS / MS, matching the retention time and characteristic ion pairs of the target glycopeptide, and combining the fractions containing the target product.

3. The preparation method according to claim 2, characterized in that, The steps of the method include: (1) Soaking bird's nest involves soaking the bird's nest (dry weight) in 20-50 times its weight of water at a water temperature of 25℃ for 2 hours. (2) The pulverization is performed using a colloid mill; (3) The protease hydrolysis is performed by adjusting the pH of the solution obtained in step (2) to 8.0-8.5 using HCl and NaOH, adding Alcalase 2.4L FG at an enzyme dosage of 6000 U / mg, mixing and placing it in an enzymatic fermentation tank, and treating it at 55-60℃ and 150 r / min for 12 h. After treatment, the enzyme is inactivated, and the pH of the hydrolysate is adjusted to 7.0-7.4 using HCl and NaOH. Papain is added, mixed and placed in an enzymatic fermentation tank, and treated at 50℃ and 150 r / min for 2 h. (4) The enrichment and concentration process involves first washing the amide-bonded silica filler with 100% acetonitrile, then activating it with equilibration buffer, uniformly loading it into the chromatography column, and rinsing it at a flow rate of 2 mL / min until the column pressure stabilizes and no air bubbles are present. After centrifugation and filtration through a 0.22 μm filter membrane, the bird's nest enzymatic hydrolysate is passed through a 3 kDa ultrafiltration tube to remove small molecule impurities. The solvent is replaced with equilibration buffer and concentrated to below 20 mL before secondary filtration. The sample is loaded at a flow rate of 1 mL / min, and the column is rinsed with equilibration buffer for 5 column volumes to remove impurities. Gradient elution is then performed: acetonitrile decreases from 80% to 70% in 0-10 min, from 70% to 40% in 10-50 min, and from 40% to 20% in 50-60 min, while maintaining a flow rate of 2 mL / min. The fractions are detected by LC-MS / MS, and the fractions containing the target product are combined after matching the retention time of the target glycopeptide (47.94 min ± 2%) and the characteristic ion pairs. (5) The separation and purification process involves concentrating the enriched solution through a 10 kDa ultrafiltration tube, washing it three times with ultrapure water, transferring it to a 10 kDa dialysis bag, dialyzing it at 4°C for 24 hours, and freeze-drying it to obtain glycopeptide powder. Finally, the column is washed with 10 column volumes of 100% acetonitrile and equilibration buffer, and then sealed and stored in 80% acetonitrile at 4°C.

4. A composition, characterized in that, The composition comprises the bird's nest glycopeptide of claim 1 and excipients permitted to be added to the pharmaceutical product.

5. The use of the bird's nest glycopeptide of claim 1 or the composition of claim 4 in the preparation of anti-inflammatory products, characterized in that, The anti-inflammatory product is a pharmaceutical product, and the application includes at least one of the following: (1) Application in the preparation of products that can improve neuroinflammation; (2) Application in the preparation of products that can improve lung inflammation; (3) Application in the preparation of products that can improve liver inflammation.

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