CN-120518733-B - Bird's nest sialoglycopeptide capable of improving lung inflammation and application thereof

CN120518733BCN 120518733 BCN120518733 BCN 120518733BCN-120518733-B

Abstract

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

Inventors

WANG DONGLIANG
QIU SHUANG
QIAN NAN
CHENG XIANGRONG
JU MENGMENG
LIN XIAOXIAN
MIAO SHU

Assignees

北京榕树堂生物科技有限公司
北京小仙炖生物科技有限公司

Dates

Publication Date: 20260512
Application Date: 20250430

Claims (5)

1. A cubilose glycopeptide capable of improving pulmonary inflammation, characterized in that the glycopeptide consists of a peptide and an N-sugar chain, the amino acid sequence of the peptide is shown as SEQ ID NO 2, the glycoform of the N-sugar chain connected with the glycopeptide is Hex (4) HexNAc (5), and the structural unit of the N-sugar chain is as follows: 。
2. A method for preparing the bird's nest glycopeptide according to claim 1, comprising the following steps in order: Soaking nidus Collocaliae, pulverizing, hydrolyzing with protease, inactivating enzyme, centrifuging to obtain supernatant, concentrating, separating and purifying; The protease hydrolysis comprises the steps of adjusting the pH value to 8.0-8.5, adding Alcalase 2.4L FG, inactivating enzyme after enzymolysis of 12 h, adjusting the pH value of the enzymolysis solution to 7.0-7.4, and adding papain for enzymolysis of 2 h; the enrichment concentration comprises the steps of washing an amido bond silica gel filler with 100% acetonitrile, activating the amido bond silica gel filler through a balance buffer solution, uniformly loading the mixture into a chromatographic column, centrifuging an edible bird's nest enzymatic hydrolysate, filtering the mixture through a filter membrane, removing small molecular impurities through an ultrafiltration tube, replacing a solvent with the balance buffer solution, concentrating the mixture to be below 20 mL, performing secondary filtration, loading the sample, washing the mixture with the balance buffer solution to remove the impurities, performing gradient elution, detecting fractions through LC-MS/MS, matching the retention time of a target glycopeptide and a characteristic ion pair, and combining the fractions containing the target product.
3. The method of manufacturing according to claim 2, characterized in that the steps of the method comprise: (1) The edible bird's nest is fully foamed by 20-50 times of water, the water temperature is 25 ℃, and the time is 2 h; (2) The crushing is colloid mill crushing; (3) The protease is hydrolyzed to adjust the pH value of the feed liquid obtained in the step (2) to 8.0-8.5 by utilizing HCL and NaOH, alcalase 2.4L FG is added with the enzyme amount of 6000U/mg, the mixture is put into an enzymolysis fermentation tank, 12h is treated under the conditions of 55-60 ℃ and the rotating speed of 150 r/min, enzyme is deactivated after the treatment, the pH value of the enzymolysis liquid is adjusted to 7.0-7.4 by utilizing HCL and NaOH, papain is added, the mixture is put into the enzymolysis fermentation tank, and 2h is treated under the conditions of 50 ℃ and the rotating speed of 150 r/min; (4) The enrichment concentration comprises the steps of washing an amido bond silica gel filler with 100% acetonitrile, activating the filler by using an equilibrium buffer solution, uniformly loading the filler into a chromatographic column, flushing the chromatographic column at a flow rate of 2 mL/min until the column pressure is stable, ensuring no bubble, centrifuging an edible bird's nest enzymatic hydrolysate, filtering the solution by using a 0.22 mu m filter membrane, removing small molecular impurities by using a 3 kDa ultrafiltration tube, replacing a solvent by using the equilibrium buffer solution, concentrating the solution to below 20mL, carrying out secondary filtration, loading the sample at a flow rate of 1 mL/min, flushing the solution by using the equilibrium buffer solution for 5 times of column volume to remove the impurities, carrying out gradient elution, reducing 0-10 min acetonitrile from 80% to 70%, reducing 10-50 min acetonitrile from 70% to 40%, reducing 50-60 min acetonitrile from 40% to 20% at a flow rate of 2 mL/min, detecting fractions by using LC-MS/MS, matching target glycopeptide retention time 47.94 min +/-2%, and merging fractions containing target products after characteristic ion pairs; (5) The separation and purification are that the enrichment solution is concentrated by a 10 kDa ultrafiltration tube and washed and filtered by ultrapure water for 3 times, the enrichment solution is transferred into a 10 kDa dialysis bag for 4 ℃ dialysis for 24 hours, the glycopeptide powder is obtained by freeze drying, and finally, the column is washed by 100 percent acetonitrile and balanced buffer solution with 10 times of column volume, and the glycopeptide powder is stored in 80 percent acetonitrile in a sealing way at 4 ℃.
4. A composition comprising the bird's nest glycopeptide of claim 1 and pharmaceutically acceptable excipients.
5. Use of the cubilose glycopeptide of claim 1 or the composition of claim 4 for the preparation of an anti-inflammatory product, wherein the anti-inflammatory product is a pharmaceutical product, the use comprising at least one of: (1) Use in the preparation of a product capable of ameliorating neuroinflammation; (2) Use in the preparation of a product capable of ameliorating pulmonary inflammation; (3) Use in the preparation of a product capable of ameliorating liver inflammation.

Description

Bird's nest sialoglycopeptide capable of improving lung inflammation and application thereof Technical Field The invention belongs to the technical field of glycoprotein engineering, and particularly relates to a bird's nest sialoglycan peptide capable of improving lung inflammation and application thereof. Background The bird's nest is a kind of precious natural tonic, and is mainly made up by using swiftlet and several bird's nest of the same genus through the process of bonding them together by using sublingual gland secretion. In the theory of traditional Chinese medicine, the bird's nest is considered to have various medical effects of nourishing yin, tonifying lung, beautifying, nourishing brain, improving intelligence and the like. The main components of nidus Collocaliae comprise water-soluble protein, carbohydrate, microelements and amino acid. Sialic acid (nidus Collocaliae acid) in nidus Collocaliae is the most nutritious component, and has content up to 10%, and has important effects on nerve and immune system. The edible bird's nest contains protein in 50% and carbohydrate in 30%, and is the main component of bird's nest, and has important functions in nourishing and physiological functions. The protein and carbohydrate present in the bird's nest do not exist independently, but are combined with each other through glycosylation to generate bird's nest glycoprotein, including N-glycosylated protein and O-glycosylated protein, and N-glycosylated protein is the main component, wherein the N-sugar structure in the glycoprotein refers to a complex formed by covalent combination of sugar chains and asparagine residues on the protein. The structure of the N-sugar chain is complex and various, and has important influence on the stability, biological activity, immune recognition and the like of the protein. The N-sugar chain in nidus Collocaliae is composed of various monosaccharides including sialic acid, N-acetamido galactose, N-acetamido glucose, etc. Studies have shown that sialylated hyperbranched structures in the N-sugar chain of bird's nest help to inhibit influenza virus infection. In addition, the structural and functional studies of N-sugar chains have progressed to show that N-sugar chains play an important role in anti-inflammatory and neuroprotective effects of bird's nest. The sugar chains in the bird's nest exist in a form of being combined with protein, and researches show that the bird's nest has the characteristics of high expansion degree after water absorption and difficult digestion by pepsin and trypsin, so that the bioavailability of the bird's nest is limited. Patent CN110257459B discloses a method for preparing nidus Collocaliae peptide by denaturing and enzymolysis nidus Collocaliae, Patent CN107974479A discloses a method for preparing bird's nest oligopeptide by combining microwave and membrane technologies, Patent CN114848791a discloses a method for preparing small molecule cubilose glycopeptide for preventing and improving skin inflammation. However, the functionality of sugar chains in the bird's nest glycoprotein is ignored in the bird's nest peptides prepared by the methods, the sugar content in the obtained bird's nest peptides is relatively low, the sialic acid content is relatively low, and the sugar chain structure of the bird's nest is not characterized. In addition, less research is currently being conducted on the anti-body inflammation efficacy of bird's nest sialoglycan peptides. Therefore, the invention adopts a step enzymolysis method and an acetone enrichment method to prepare the bird's nest sialoglycan peptide, and utilizes MSFRAGGER database search Apodiformes (of the order of the rainbird's nest) to search the bird's nest sialoglycan chain glycoprotein through liquid chromatography-mass spectrum tandem analysis to obtain the product N-sugar chain fine structure. And designing animal experiments, utilizing Lipopolysaccharide (LPS) to make a mouse inflammation model, verifying that the bird's nest sialoglycan peptide has an improvement effect on mouse blood, nerve, liver and lung inflammation, and verifying that the product has an anti-inflammatory effect. Disclosure of Invention Aiming at the existing problems, the patent adopts a method of combining a step-by-step enzymolysis method with acetone enrichment to separate and obtain the bird's nest sialoglycose peptide with anti-inflammatory effect. And searching the bird's nest sialoglycan chain glycoprotein by utilizing a liquid chromatography-mass spectrometry tandem analysis and utilizing MSFRAGGER database searching Apodiformes (of the order of the Rauyan) to obtain the fine structure of the N-sugar chain in the bird's nest sialoglycan peptide. The invention provides the following technical scheme: in a first aspect, the invention provides a bird's nest glycopeptide with anti-inflammatory activity, which is characterized in that the amino acid sequence of the glycopeptide is shown in SEQ ID NO 2. In some