Ovomucoid glycopeptide and use thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses ovomucin glycopeptide and application. The application of the ovomucin glycopeptide in the preparation of a drug for preventing or treating alcohol-induced acute gastric mucosal injury is disclosed, and when used for protection, the percentage of the gastric mucosal injury area can be reduced to below 1.0%. The application creatively discovers and proves that the glycopeptide derived from ovomucin has a significant protective effect on alcohol-induced acute gastric mucosal injury. The discovery of the application explicitly expands the application field of the natural active ingredient ovomucin to the protection of acute gastric mucosal injury for the first time, solves the technical problem that there is a lack of efficient and natural gastric mucosal protective ingredients in the prior art, and provides a new material basis and a clear direction for developing a new gastric mucosal protective agent.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to ovomucoid glycopeptides and their applications. Background Technology

[0002] Injury to the digestive tract mucosa, especially acute gastric mucosal injury, is a common pathological basis for many gastrointestinal diseases. The global incidence of gastric mucosal injury is approximately 5%-10%, with a high recurrence rate. Alcohol (ethanol) intake is a common exogenous factor inducing such injury, directly damaging gastric mucosal epithelial cells and barrier function, leading to inflammation, edema, ulceration, and even bleeding. Currently used mucosal protectants such as sucralfate, while having some efficacy, have limitations such as metal accumulation and interference with the absorption of other nutrients with long-term use. Therefore, developing highly effective and safe mucosal protective ingredients derived from natural foods is of great value.

[0003] Ovomucin (OVM) is a highly glycosylated macromolecule glycoprotein found in egg white. It is known to possess anti-inflammatory and immunomodulatory biological activities and is structurally homologous to mucin in gastrointestinal mucus, suggesting a positive effect on gut health. However, to date, no studies have reported the application of ovomucin or its enzymatic hydrolysates in the protection of gastric mucosal injury, nor have any studies demonstrated that its protective efficacy is comparable to that of commonly used clinical mucosal protectants.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to solve at least any of the above technical problems, and provides ovomucin glycopeptide (OVMG) and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Application of ovomucin glycopeptide in the preparation of drugs for the prevention or treatment of alcohol-induced acute gastric mucosal injury.

[0008] Preferably, when used for protection, the ovomucoid can reduce the percentage of gastric mucosal damage area to below 1.0%.

[0009] Preferably, the ovomucoid glycopeptide is prepared from ovomucoid, which is separated from egg white through steps including polyethylene glycol precipitation and washing with sodium chloride solution. Preferably, the ovomucoid glycopeptide is prepared using a process simulating gastrointestinal digestion and agglutinin affinity chromatography.

[0010] Preferably, the simulated gastrointestinal digestion includes: first digesting with pepsin at pH 1.5-2.5 and 32-38°C for 1-3 hours; then digesting with trypsin and α-chymotrypsin at pH 7.5-8.5 and 32-38°C for 4-8 hours.

[0011] Preferably, the ratio of pepsin to substrate is 1:(15-35) U / mg.

[0012] Preferably, the ratio of trypsin to substrate is 1:(15-35) U / mg.

[0013] Preferably, the ratio of the α-chymotrypsin to the substrate is 1:(80-120) U / mg.

[0014] Preferably, in the prevention or treatment, the dosage of the ovomucoid glycopeptide is 50 mg to 150 mg per kilogram of body weight per administration.

[0015] Preferably, the drug is formulated as an oral preparation.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention creatively discovers and confirms that glycopeptides derived from ovomucin have a significant protective effect against alcohol-induced acute gastric mucosal injury. This discovery, for the first time, explicitly expands the application of ovomucin, a natural active ingredient, to the protection against acute gastric mucosal injury, solving the technical problem of the lack of highly effective, natural gastric mucosal protective ingredients in existing technologies, and providing a new material basis and clear direction for the development of novel gastric mucosal protectants.

[0018] This invention reveals that ovomucoid glycopeptides exhibit protective effects comparable to those of sucralfate, a first-line clinical drug. Animal experiments show that after administration of high-dose OVMG, the percentage of gastric mucosal damage area can be reduced to 0.87%, which is not statistically significantly different from the positive control group (sucralfate) (0.80%), and is significantly better than the model group (20.10%). This protective effect, reaching the level of classic drug efficacy, is completely unpredictable by existing technologies.

[0019] The OVMG provided by this invention has unique structural features, with mannose-type glycans making up the largest proportion. This unique glycan composition is highly likely to enhance the specific adhesion of glycopeptides to the gastric mucosa surface, thereby exerting a physical barrier protection mechanism similar to sucralfate. Attached Figure Description

[0020] Figure 1A represents the SDS-PAGE electrophoresis analysis results of different samples in Example 1. In the figure, M represents the molecular weight standard, EW represents egg white, A represents ovomucoid, B represents ovomucoid hydrolysate simulating gastric digestion, and C represents ovomucoid hydrolysate simulating gastrointestinal digestion. Figure 1 B represents the statistical analysis of the number of N- / O-glycosylations in ovomucoid glycopeptides; Figure 1 C represents the number of glycan chains in the N-glycoform of ovomucoid glycopeptide; Figure 1 D represents the statistical count of the number of glycan chains in the O-glycan form of ovomucoid glycopeptide;

[0021] Figure 2 A shows the macroscopic morphology of the gastric mucosa in different groups of mice; Figure 2 B shows HE staining images of gastric tissue from different groups of mice. The magnification of the upper and lower images is ×2 and ×20, respectively. Blue polygons indicate mucosal hemorrhage, green polygons indicate mucosal edema, yellow polygons indicate epithelial cell loss, and cyan arrows indicate inflammatory cell infiltration. Figure 2 C represents the statistical data on the area of ​​gastric mucosal damage in different groups of mice; Figure 2 D represents the histopathological assessment of different groups of mice; different lowercase letters indicate significant differences between different groups (p<0.05). Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention provides the use of ovomucin glycopeptide in the preparation of a medicament for the prevention or treatment of alcohol-induced acute gastric mucosal injury.

[0024] This embodiment clarifies the use of ovomucin glycopeptide for the prevention or mitigation of acute gastric mucosal tissue damage caused by direct stimulation of alcohol (ethanol). This ovomucin glycopeptide is suitable for scenarios requiring prevention or improvement of alcohol-related gastric mucosal damage, such as as a gastric mucosal protectant for drinkers and as an adjunct treatment for alcoholic acute gastritis.

[0025] The active ingredient of ovomucin glycopeptides is originally derived from ovomucin in egg white; that is, the preparation source of ovomucin glycopeptides is ovomucin. In this invention, ovomucin glycopeptides specifically refer to peptide fragments that retain their glycan structure after appropriate treatment (preferably enzymatic hydrolysis) of ovomucin.

[0026] The ovomucoid glycopeptide provided in this invention has the highest proportion of mannose-type glycans. Mannose-type glycans refer to glycans composed of N-acetylglucosamine and mannose, with mannose residues at the ends. This structural feature is closely related to the mechanism of action of the ovomucoid glycopeptide. Studies have shown that mannose-rich glycans can interact with certain specific receptors (such as mannose receptors) or proteins on the surface of gastric mucosal epithelial cells or in the mucus layer, thereby enhancing the adhesion and retention of the glycopeptide at the site of injury, forming a stable biological protective film, mimicking the physical barrier effect of sucralfate.

[0027] In some preferred embodiments, the ovomucin glycopeptides are prepared by a process simulating gastrointestinal digestion and lectin affinity chromatography. Simulating gastrointestinal digestion aims to process large ovomucin molecules into a mixture of smaller glycopeptides that are more readily active and have more fully exposed glycans by mimicking the physiological digestive environment. Lectin affinity chromatography aims to specifically enrich the glycan-modified glycopeptide components from the aforementioned complex digestive products.

[0028] The simulated gastrointestinal digestion includes: first, digestion with pepsin at pH 1.5-2.5 and 32-38°C for 1-3 hours; then, digestion with trypsin and α-chymotrypsin at pH 7.5-8.5 and 32-38°C for 4-8 hours. In some preferred embodiments, the ratio of pepsin to substrate is 1:(15-35) U / mg, the ratio of trypsin to substrate is 1:(15-35) U / mg, and the ratio of α-chymotrypsin to substrate is 1:(80-120) U / mg.

[0029] The specific operation method of the lectin affinity chromatography is as follows: the digest solution is loaded onto the lectin affinity chromatography column, the unbound components are washed away, and then the specifically bound glycopeptides are eluted using a buffer containing a competitive sugar (such as 0.1-0.3 M α-D-methylmannoside).

[0030] This invention verifies through specific experimental data that ovomucoid glycopeptides can reduce the percentage of gastric mucosal damage area to below 1.0%. In a preferred embodiment, high-dose ovomucoid glycopeptides show even better results, for example, reducing the percentage of gastric mucosal damage area to approximately 0.87%. This effect is not statistically significantly different from that of sucralfate, a widely used positive control drug in clinical practice (approximately 0.80%), and both are significantly superior to the model control group (damage area exceeding 20%).

[0031] The percentage of gastric mucosal damage area is determined through standard animal pharmacodynamic experiments. A typical method involves establishing an alcohol-induced acute gastric mucosal injury model in mice (e.g., by gavage with 75% ethanol at 10 mL / kg), euthanizing the animals, removing the entire stomach, cutting it open along the greater curvature, washing it with physiological saline, and then flattening it for photographing. Professional image analysis software is used to delineate and calculate the total area of ​​all bleeding, erosion, and other damaged areas on the gastric mucosal surface. This area is then divided by the total visible area of ​​the gastric mucosa and multiplied by 100% to obtain the percentage.

[0032] Based on animal equivalent dose conversion and safety considerations, the recommended dosage of the ovomucoid glycopeptide for prevention or treatment is 50 to 150 mg per kilogram of body weight per dose. Within this range, especially at the high-dose end (150 mg per kilogram of body weight per dose), efficacy comparable to positive control drugs has been verified in models. Those skilled in the art will understand that when developing the ovomucoid glycopeptide provided by this invention for human use, further clinical dosage studies must be conducted based on such animal experimental data, following standard practices and regulatory requirements in the field of drug development (e.g., through body surface area conversion), to determine a safe and effective dose suitable for humans.

[0033] Those skilled in the art can use conventional pharmaceutical techniques to mix the ovomucoid glycopeptide of the present invention with suitable pharmaceutical excipients (such as diluents, disintegrants, flavoring agents, binders, etc.) to prepare common dosage forms such as tablets, capsules, granules, powders, or oral solutions. Dosage form preparation itself is a known technology. Among these, oral preparations are the most suitable route of administration for local action on the gastric mucosa.

[0034] The application and effects of ovomucoid glycopeptides are described in detail below through multiple examples.

[0035] Example 1: Preparation and structural identification of ovomucoid glycopeptides

[0036] Preparation of ovomucoid: Egg white from fresh eggs was added to 0.1 mol / L NaCl solution at a volume ratio of 1:1 (v / v) and stirred at 4 ℃ for 1 h. The pH of the diluted egg white solution was adjusted to 6.5, and 3% polyethylene glycol 8000 was added. The mixture was stirred thoroughly at 4 ℃ for 2 h. The egg white mixture was centrifuged (4 ℃, 15000×g, 15 min) to remove the supernatant, and the precipitate was collected as crude ovomucoid. Then, 0.5 mol / L NaCl solution was added to the crude ovomucoid at a ratio of 1:2 (g:g), and the mixture was treated at 4 ℃ and 300 rpm for 4 h. The precipitate was collected by centrifugation (15000×g, 15 min), and the process was repeated twice. The precipitate was then washed twice with ultrapure water at a ratio of 1:2 (g:g) under the same conditions, and the precipitate was collected as purified ovomucoid. The ovomucoid precipitate was freeze-dried under vacuum to obtain lyophilized powder, which was stored at -80 ℃ for later use.

[0037] Preparation of ovomucin glycopeptides: A certain amount of ovomucin was weighed into a beaker and dissolved in PBS (1×). The pH of the protein solution was adjusted to 2.0 with 1 mol / L hydrochloric acid, and pepsin (1:25 U / mg) was added. The solution was then placed in a preheated 37℃ constant temperature shaking incubator for 2 h of digestion. Next, the pH of the digestion solution was adjusted to 7.8, and trypsin (1:25 U / mg) and α-chymotrypsin (1:100 U / mg) were added. The solution was then placed in a 37℃ constant temperature shaking incubator for another 6 h of digestion to obtain the ovomucin polypeptide digestion solution. Electrophoretic analysis was performed on the pre-digestion, gastric digestion, and intestinal digestion samples. The final gastrointestinal digestion solution was collected, filtered through a 0.22 μm filter membrane, and stored at -20℃.

[0038] Ovalbumin hydrolysate was enriched with ovomucin glycopeptides (OVMG) using affinity chromatography and packed into a ConA Sepharose™ 4B column. The column was eluted with 5 column volumes of equilibration buffer. The ovomucin hydrolysate was slowly added to the column. After gently shaking the column and allowing it to stand for 2 min, the column was eluted: first, unbound hydrolyzed peptides were eluted with equilibration buffer and the absorbance was measured at 280 nm to ensure it was close to 0. Then, the glycopeptides attached to the column were eluted with 0.2 M α-D-methylmannoside, and the eluent was collected as OVMG. The absorbance was continuously measured at 280 nm during collection until it approached 0. The collected OVMG was dialyzed, freeze-dried, and stored at -80 °C for later use.

[0039] Electrophoresis results showed that polyethylene glycol 8000 precipitation combined with NaCl solution washing could separate ovomucoid samples from egg white, effectively removing major egg white proteins such as ovalbumin and ovotransferrin. Figure 1 A). Following in vitro simulated digestion in the gastrointestinal tract, ovomucoid was hydrolyzed into small polypeptides with a molecular weight of less than 37 kDa. OVMG was obtained by enriching the ovomucoid polypeptide solution using affinity chromatography.

[0040] The complete glycopeptide structure of OVMG was identified using liquid chromatography-mass spectrometry. Specifically, the enriched OVMG was separated by chromatography using an Easy nLC 1200 system with a flow rate of nanoliters. Mobile phase A was a solution containing 0.1% formic acid, and mobile phase B was a solution containing 80% acetonitrile and 0.1% formic acid. Samples were injected into a trap column and then subjected to gradient separation using a C18 chromatographic column at a flow rate of 300 nL / min. The separated peptides were analyzed by DDA mass spectrometry using a Q-Exactive HF-X mass spectrometer for 120 min. The secondary mass spectrometry data were retrieved and glycopeptides were quantified using pGlyco, pGlyconovo, and pGlycoQuant software. The retrieval parameters were: trypsin as the enzyme digestion method, a maximum number of missed cleavage sites of 2, and a maximum number of peptide modifications of 3. Cysteine ​​alkylation (Carbamidomethyl) was set as a fixed modification, methionine oxidation and N-terminal acetylation of proteins were set as variable modifications, and variable glycan modification was set to pH (phosphoHex) (N-glycan / O-glycan database), with a maximum variable glycan modification of 1.

[0041] The identification results showed that a total of 200 complete N-glycopeptide structures were identified, involving 15 glycosylation sites and 17 peptide sequences; and 174 complete O-glycopeptide structures were identified, involving 15 glycosylation sites and 16 peptide sequences. After removing repetitive structures, 84 and 85 unique N-glycans and O-glycans were identified, respectively. Figure 1 B). The N-glycan structures of OVMG are classified into four types: mannose-type, fucose-type, sialylated type, and others. The O-glycan structures of OVMG are classified into four types: hexose-type, fucose-type, N-acetylhexamine-type, and sialylated type. Among them, mannose-type N-glycans and hexose-type O-glycans account for the highest proportion, with 188 and 150 types respectively. Figure 1 C Figure 1 D).

[0042] Example 2: Protective effect of ovomucin glycopeptides against acute gastric mucosal injury

[0043] Animal experiments were conducted to verify the protective effect of ovomucoid glycopeptides against acute gastric mucosal injury. All procedures were reviewed and approved by the Animal Experiment Ethics Committee of Chengdu University, and the experimental protocols complied with relevant national regulations on laboratory animal welfare and ethics.

[0044] Animal grouping: Forty-eight male KM mice were housed in an environment with controlled temperature and humidity (22±2 ℃ and 50±10%, respectively), and were allowed free access to drinking water and food. After a one-week acclimatization period, the mice were randomly divided into 6 groups (n=8 per group): normal control group, model group, sucralfate positive control group, and low, medium, and high dose OVMG groups (L-, M-, and H-OVMG).

[0045] Animal drug administration and model establishment: Gavage administration was performed at 9:00 AM. Mice in the L-, M-, and H-OVMG groups were administered OVMG solution at concentrations of 50, 100, and 150 mg / kg bw, respectively; mice in the Sucralfate group were administered sucralfate solution at a concentration of 165 mg / kg bw; mice in the Normal and Model groups were administered the same volume of physiological saline. One hour after gavage, all groups except the Normal group were administered 75% ethanol (10 mL / kg bw) by gavage, while the control group received the same volume of physiological saline. The criteria for successful establishment of the acute gastric mucosal injury model were visible gastric mucosal hemorrhage and linear blood spots.

[0046] Sample collection: Two hours after gavage, all mice were anesthetized with 10% chloral hydrate (0.3 mL / 100 g) and euthanized by cervical dislocation. All mice were dissected, and the stomach tissue was removed intact. The stomach was cut along the greater curvature, and the gastric mucosa contents were rinsed with pre-cooled physiological saline. The moisture was blotted dry with filter paper, and the extent of gastric mucosal damage was observed and photographed. The gastric mucosal tissue was stored in 4% formalin for histological analysis.

[0047] Macroscopic assessment of gastric mucosal damage: The macroscopic morphology of the gastric mucosa in all mice was observed. The area of ​​the damaged region was statistically analyzed using Image-ProPlus software, with bright red areas designated as the damaged areas. The percentage of mucosal damage area was calculated using the following formula: .

[0048] Histopathological analysis: Gastric tissue was fixed with 4% formalin solution, dehydrated with graded ethanol (80-100%), embedded in paraffin, and sectioned (5 μm). Gastric tissue sections were stained with hematoxylin and eosin and observed under an optical microscope. The scoring criteria for histopathological features are as follows: (1) Hemorrhage (blue polygon): 0-4 points; (2) Mucosal edema (green polygon): 0-4 points; (3) Epithelial cell loss (yellow polygon): 0-4 points; (4) Inflammatory cell infiltration (cyan arrow): 0-4 points.

[0049] All data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA with Origin 2021 software. A p-value < 0.05 was considered statistically significant.

[0050] Changes in macroscopic morphology of the gastric mucosa and pathological morphology of the gastric tissue in mice: The gastric tissue of mice in the Normal group was pale red, intact, and showed no congestion or edema. Figure 2 A). The gastric tissue of the Model group mice showed obvious gastric mucosal hemorrhage and erosion, with a damaged area of ​​20.10±0.31% (A). Figure 2 C); Compared with the Model group, the Sucralfate group and the OVMG group showed lower degrees of gastric mucosal damage, and the gastric mucosal congestion and damaged area in the OVMG group decreased in a dose-dependent manner (p<0.05); among them, the gastric mucosal damaged area in the H-OVMG group was 0.87±0.11%, which was not significantly different from that in the Sucralfate group (0.80±0.12%) (p>0.05). This indicates that OVMG (especially H-OVMG) has a physical defense effect against ethanol-induced acute gastric mucosal injury in mice, similar to Sucralfate.

[0051] HE staining of gastric tissue sections ( Figure 2 B) and gastric histopathological features score ( Figure 2 D) Further analysis of the protective effect of OVMG against ethanol-induced gastric mucosal injury. In the Normal group, the gastric mucosa remained morphologically intact, with orderly arrangement of gastric glands and no hemorrhage or inflammatory cell infiltration. Clearly, the Model group exhibited severe ulcer-like changes in the gastric mucosa, with occasional focal erosions, necrosis and sloughing of mucosal and gastric glandular epithelial cells, inflammatory cell infiltration, and hemorrhage, and its histopathological score was the highest (14.75±0.56). OVMG and Sucralfate intervention significantly reduced gastric mucosal injury in mice. The H-OVMG and Sucralfate groups showed almost no epithelial cell sloughing in their gastric tissues, and their histopathological scores were 4.23±0.22 and 4.40±0.25, respectively, significantly lower than those of the Model group (p<0.05). Studies have shown that Sucralfate can form a viscous polymer gel on the mucosal surface and bind to proteins on the damaged mucosal surface through electrostatic interactions or multivalent bridging, forming a durable physical barrier at the site of mucosal damage, preventing further erosion of the mucosa by gastric acid, pepsin, or bile. Furthermore, Sucralfate can increase the hydrophobicity of the mucus layer, promote bicarbonate release, and enhance the release of local prostaglandins and repair factors, synergistically strengthening the mucosal defense function. OVM is a highly glycosylated macromolecular glycoprotein with good gelling properties and is highly homologous to mucins in the gastric mucosa. This invention found that H-OVMG has a protective effect comparable to Sucralfate, suggesting that OVMG may exert a similar defensive effect to Sucralfate, thereby protecting against ethanol-induced gastric mucosal damage.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of ovomucoid glycopeptide in the preparation of a drug for preventing alcohol-induced acute gastric mucosal injury, characterized in that, The ovomucoid glycopeptide has the highest proportion of mannose-type glycans, and the ovomucoid glycopeptide is prepared by a process that simulates gastrointestinal digestion and lectin affinity chromatography. The simulated gastrointestinal digestion process includes: first, digestion with pepsin at pH 2.0 and 37°C for 2 hours; then, digestion with trypsin and α-chymotrypsin at pH 7.8 and 37°C for 6 hours. The ratio of pepsin to ovomucoid is 1:25 U / mg; The ratio of trypsin to ovomucoid is 1:25 U / mg; The ratio of α-chymotrypsin to ovomucoid is 1:100 U / mg.

2. The application as described in claim 1, characterized in that, The ovomucoid glycopeptide is prepared from ovomucoid, which is separated from egg white through steps including polyethylene glycol precipitation and washing with sodium chloride solution.

3. The application as described in claim 1, characterized in that, The drug is formulated as an oral preparation.

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