Composition for preventing alcoholic gastric mucosal damage and liver damage and preparation method thereof

Abstract

The present invention provides a composition for preventing alcoholic gastric mucosal damage and liver damage and a preparation method thereof, belonging to the technical field of protecting gastric mucosa and liver. The core material of the composition is theaflavin-3,3'-digallate monomer (TFDG), and the wall material is nanoparticles prepared from zein and sodium carboxymethyl cellulose. The present invention uses TFDG from natural substances as a raw material, provides a composition for preventing alcoholic gastric mucosal damage and liver damage and a preparation method thereof, the preparation method is simple, and the effect of TFDG in protecting alcoholic gastric mucosal damage and liver damage is improved through the embedding effect of zein and sodium carboxymethyl cellulose, which can solve a series of problems such as gastric mucosal or liver damage after people drink alcohol, and has a good market prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of protecting the gastric mucosa and liver, specifically relating to a composition and preparation method for preventing alcoholic gastric mucosal damage and liver damage. Background Technology

[0002] Alcohol consumption is widespread globally, causing approximately 3 million deaths annually. Alcohol is highly addictive, and excessive drinking is one of the top ten global disease risk factors. Moderate drinking can have warming and sleep-aiding effects. However, excessive drinking can impair brain development and bodily functions. Long-term alcohol abuse affects mental state and judgment, damaging organs such as the brain, heart, gastrointestinal tract, liver, and kidneys, leading to reduced quality of life and shorter lifespan. There is a close relationship between alcohol consumption and the risk of diseases such as stomach damage, cancer, and cirrhosis. Approximately 6% of deaths worldwide are attributable to alcohol-related injuries. Excessive drinking also threatens family members and public safety. Adults who drink alcohol are more dangerous when driving, increasing the risk of traffic accidents and adding to the social burden. Intoxication leads to disorientation, slowed thinking, and a higher risk of falls, resulting in disturbances, fights, and other serious social incidents. The main components of alcohol are ethanol and water. Ethanol, as a polar molecule, is hydrophilic, lipid-soluble, and has widespread systemic toxicity. Approximately 20% of alcohol is slowly absorbed from the stomach through passive diffusion. Blood alcohol concentration is determined by factors such as alcohol intake, gastric emptying rate, and alcohol oxidation rate.

[0003] After alcohol enters the body, it is absorbed through the gastrointestinal tract, enters the bloodstream, and then reaches the liver. The liver is the main organ for breaking down and metabolizing alcohol in the human body. Human liver cells contain two enzymes: alcohol dehydrogenase and aldehyde dehydrogenase. Alcohol dehydrogenase converts alcohol into acetaldehyde, and aldehyde dehydrogenase converts acetaldehyde into acetic acid, which is then broken down into carbon dioxide and water, and excreted from the body as waste products. Different people have different abilities to break down and metabolize alcohol. For example, people with liver disease have a weakened ability to break down and metabolize alcohol, and long-term excessive drinking can lead to alcoholic fatty liver disease and other serious liver damage.

[0004] Gastric dysfunction, including gastritis, peptic ulcers, and gastroesophageal reflux disease, has become a global health problem. Among its complex pathogenic factors, excessive alcohol intake is widely considered a major contributing factor. The pathogenesis of alcoholic gastric injury stems from an imbalance between invasive and protective factors in the stomach. The gastric mucosa represents a dynamic barrier, counteracting the effects of harmful drugs through a series of endogenous antioxidant defense systems. Alcohol directly attacks the gastric mucosa, damaging its protective layer by consuming mucus and bicarbonate. During alcohol metabolism, alcohol is absorbed by the gastrointestinal tract and, under the catalysis of alcohol dehydrogenase and xanthine oxidase, produces acetaldehyde, which is then metabolized to generate free radicals. This leads to impaired antioxidant enzyme activity, resulting in lipid peroxidation, oxidative stress, and inflammatory responses. Reactive oxygen species (ROS) are naturally occurring free radicals generated during metabolic reactions, including superoxide anion radicals, hydrogen peroxide (H₂O₂), highly reactive hydroxyl radicals (OH·), and nitric oxide (NO). Antioxidants include superoxide dismutase, catalase, glutathione (GSH), and glutathione peroxidase (GSH-Px). Both acute and chronic ethanol exposure lead to the production of reactive oxygen species (ROS) and a decrease in antioxidant activity. Furthermore, ethanol can increase edema and clear epithelial cells by increasing microvascular and vascular permeability, resulting in severe damage to the gastrointestinal mucosa and increasing the risk of upper gastrointestinal bleeding, gastric mucosal inflammation, ulcers, and even gastric cancer.

[0005] Different concentrations of alcohol and varying durations of exposure cause varying degrees of damage to the gastric mucosa. High concentrations of alcohol easily cause acute gastric mucosal damage; the higher the alcohol concentration and the more severe the damage, especially when ingested on an empty stomach. After gavage administration of anhydrous ethanol to mice / large mice, degeneration and necrosis of gastric epithelial cells, vasodilation, and gastric mucosal hemorrhage are observed. Alcohol-induced damage is distributed throughout the gastric body; in severe cases, the surface shows clots and punctate or linear erosions.

[0006] Drinking alcohol is inevitable in people's daily lives, and more and more people hope to protect their gastric mucosa and liver after drinking. Therefore, there is a need for a functional substance that can prevent alcohol from damaging the gastric mucosa and liver after drinking, so as to protect the health of people who drink alcohol. Summary of the Invention

[0007] To address the problem of alcoholic gastric mucosal damage and liver damage in humans or animals after drinking alcohol, this invention provides a composition and preparation method for preventing alcoholic gastric mucosal damage and liver damage, which can effectively reduce alcoholic gastric mucosal damage and liver damage.

[0008] A composition for preventing alcoholic gastric mucosal damage and liver damage, the composition comprising solid particles prepared from theaflavins-3,3'-bisgallate monomer as the core material and zein and sodium carboxymethyl cellulose as the wall material.

[0009] Meanwhile, the present invention provides a method for preparing a composition for alcoholic gastric mucosal injury and liver injury, comprising the following steps: (1) Weigh out zein powder and dissolve it in an ethanol aqueous solution with a concentration of 60-90% to prepare a zein solution of 3-10 mg / mL; (2) Weigh out sodium carboxymethyl cellulose powder and dissolve it in ultrapure water to prepare a sodium carboxymethyl cellulose aqueous solution of 0.5~2.5 mg / mL; (3) Weigh 10~30 mg of TFDG and dissolve it in the zein solution prepared in step (1) to obtain TFDG-zein ethanol solution; (4) Add the TFDG-zeadin ethanol solution obtained in step (3) to the sodium carboxymethyl cellulose aqueous solution prepared in step (2) and stir to form a suspension of TFDG-loaded zeadin-sodium carboxymethyl cellulose composite nanoparticles. (5) Centrifuge the suspension obtained in step (4) to obtain solid particles.

[0010] In step (3), the volume-to-mass ratio of zein solution to TFDG is 1:1 to 1:3.

[0011] In step (4), the volume ratio of TFDG-zein ethanol solution to sodium carboxymethyl cellulose aqueous solution is 3:1 to 1:1.

[0012] In step (4), the stirring temperature is 15~30℃, the stirring speed is 400~600 rpm, and the stirring time lasts for 30~60 min.

[0013] In step (5), the centrifugation conditions are 1~4℃ and 8000~12000 r / min for 20~30 min. Beneficial effects

[0014] Firstly, our experiments demonstrated that TFDG corn gluten nanoparticles can effectively reduce liver indices and decrease the levels of ALT, AST, TC, TG, and MDA in mouse plasma. TFs significantly reduced GSH depletion and improved the survival rate of mice after alcohol injury. They also alleviated the infiltration of F4 / 80 inflammatory cells and collagen fiber formation in the livers of mice with alcoholic liver injury, thereby inhibiting liver inflammation and fibrosis. Furthermore, they protected the structural integrity of the gastric mucosa, submucosa, and muscularis propria, reduced gastric mucosal glycoprotein loss, and may prevent alcohol-induced liver and gastric injury in mice by regulating the AMPK / mTOR / Beclin1 / LC3 II signaling pathway to restore the autophagy state of gastric cells. Zeadin and sodium carboxymethyl cellulose showed some protective effects against TFDG, thus enhancing its protective effect against liver and gastric injury in mice. Empty corn gluten nanoparticles, however, showed no protective effect, indicating that the main active ingredient is derived from TFDG. The effect of TFDG alone was less than that of TFDG corn gluten nanoparticles, demonstrating that corn gluten nanoparticles have a protective effect against gastric and liver injury. Meanwhile, our research group also prepared chitosan-gelatin-TFDG nanoparticles. The results showed that their drug delivery efficacy was similar to TFDG, without any significant advantage. Therefore, we speculate that due to the complex gastrointestinal environment and its acidic pH, although the nanoparticles encapsulated TFDG, the nanoparticle wall material was easily digested and absorbed by various enzymes in the gastrointestinal tract after entering, leading to the disintegration of the nanoparticle structure and failing to achieve controlled or sustained release. Thus, the experiments showed that TFDG and chitosan-gelatin-TFDG nanoparticles had comparable effects.

[0015] Secondly, this invention uses TFDG from natural substances as a raw material to prepare a composition that prevents alcoholic gastric mucosal damage and liver damage. The preparation method is simple, and the cost of zein and sodium carboxymethyl cellulose is low, with significantly better effects than other nanoparticles. Therefore, this invention enhances the protective effect of TFDG against alcoholic gastric mucosal and liver damage, addressing a range of problems caused by alcohol consumption and demonstrating excellent market potential. Attached Figure Description

[0016] Figure 1 Morphological images of gastric mucosal epithelial cells of each group of mice observed under a scanning electron microscope (1000x magnification).

[0017] Figure 2 HE staining images of stomach tissue from mice in each group (100x and 200x magnification).

[0018] Figure 3 PAS staining images of gastric tissue from mice in each group (50x and 100x magnification).

[0019] Figure 4Immunohistochemical staining images of liver tissue from mice in each group (200x magnification).

[0020] Figure 5 Masson staining images of livers from mice in each group (100x and 200x magnification). Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims. Example

[0022] In this embodiment, the mouse modeling method is as follows: Sixty ICR mice were acclimatized in the SPF animal facility for one week before the experiment. During the acclimatization period, each mouse was acclimatized by gavage with physiological saline to prevent the mice from struggling and choking on the trachea during the administration of alcohol, which could lead to injury or death. The mice were randomly divided into 6 groups: blank control group (Control, n=10), model group (Model, n=10), TFs group (TFs, 200 mg / kg / d, n=10), TFDG group (TFDG, 100 mg / kg / d, n=10), TFDG corn protein nanoparticle group (TFDGN, 100 mg / kg / d, n=10), and empty corn protein nanoparticle group (KN, n=10). Every morning, the gavage volume of each experimental group was 5 mL / kg, and the blank control group and model group mice were gavaged with the same volume of physiological saline. One hour after daily administration, mice in the blank control group were administered an equal volume of physiological saline by gavage according to their body weight. The model group and all experimental groups were administered 65° liquor by gavage at the following doses: 5 mL / kg / day for 2 days; 6 mL / kg / day for 2 days; 7 mL / kg / day for 3 days; 8 mL / kg / day for 20 days; 9 mL / kg / day for 10 days; and 10 mL / kg / day for 19 days. This gavage continued for 8 weeks (56 days). Mice had free access to food and water throughout the experimental period, and their food and water intake, body weight, and overall condition were recorded daily. After the 8th week of gavage, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized with ether and dissected. Blood was collected from the enucleated eyeballs into heparin sodium anticoagulant tubes, centrifuged at 3500 r / min for 10 min at 4°C, and the supernatant was transferred to 1.5 mL centrifuge tubes and aliquoted for storage at -80°C for later use. The mouse stomach tissue was dissected and removed, cut along the greater curvature, and cleaned of its contents with ice-cold saline. The tissue was then flattened and photographed. One half of the stomach tissue was removed, fixed with aluminum foil, and placed in formalin solution for pathological staining analysis. A sample block (length, width, and height no more than 5 mm) was taken from the same location in the other half of the stomach tissue and immersed in ice-cold 2.5% glutaraldehyde solution, stored at 4°C for scanning electron microscopy observation. The remaining stomach tissue was flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0023] Preparation method of TFDG corn protein nanoparticles.

[0024] This embodiment provides a method for preparing sustained-release TFDG encapsulated in zein-sodium carboxymethyl cellulose nanoparticles. The TFDG-zein-sodium carboxymethyl cellulose composite nanoparticles are prepared by anti-solvent precipitation (ASP). The specific steps are as follows: (1) Weigh out zein powder and dissolve it in a 75% ethanol aqueous solution to prepare a 5 mg / mL zein solution; (2) Weigh out sodium carboxymethyl cellulose powder and dissolve it in ultrapure water to prepare a sodium carboxymethyl cellulose aqueous solution of 1.5 mg / mL; (3) Weigh 20 mg of TFDG and dissolve it in 10 mL of the zein solution prepared in step (1), so that the volume-to-mass ratio of zein solution to TFDG is 1:2. (4) Under magnetic stirring, the TFDG-zeadin ethanol solution obtained in step (3) is added dropwise to the sodium carboxymethyl cellulose aqueous solution prepared in step (2) using a syringe, so that the volume ratio of TFDG-zeadin ethanol solution to sodium carboxymethyl cellulose aqueous solution is 2:1. Stirring forms a suspension of TFDG-loaded zeadin-sodium carboxymethyl cellulose composite nanoparticles; the stirring temperature is 25℃, the stirring speed is 500 rmp, and the stirring time lasts for 40 min.

[0025] (5) Centrifuge the suspension obtained in step (4) at 4℃ and 12000 r / min for 25 min and collect the solid particles.

[0026] The method for observing mouse gastric tissue using scanning electron microscopy is as follows.

[0027] (1) Sampling: Use clean, sharp scissors to quickly cut the stomach tissue without damaging the sample. Each sample should be taken from the same location.

[0028] (2) Fixation: Quickly place the tissue into 2.5% glutaraldehyde fixative at 4℃, so that the sample is completely immersed in the fixative for 4~12 h.

[0029] (3) Dehydration: Rinse with PBS at room temperature 3 times, 20 min each time. After discarding the PBS solution, elute with an ethanol gradient: 30%→50%→70%→80%→90%→100% (4℃, 20 min each time), and finally replace with 100% acetone (4℃, 2 times, 20 min each time).

[0030] (4) Drying: The sample soaked in acetone is sent to the instrument sample chamber at low temperature → liquid CO2 is injected to replace acetone phosphate → CO2 vaporization → exhaust → drying → scanning electron microscopy observation the next day.

[0031] (5) Observation: Fix the sample on the sample tray → spray gold → load the sample → observe and take pictures under a scanning electron microscope.

[0032] Figure 1 The following are the observations of the surface epithelial cells of the mouse gastric mucosa under a scanning electron microscope.

[0033] Blank control group (Control): The upper layer of gastric mucosal cells protruded in an oval shape, with intact cell structure, basically uniform shape and size, and covered with mucus; Model control group: The gastric mucosa surface is rough and eroded, with broken and sloughed cells of different shapes, unclear intercellular spaces, and no mucus covering the surface; Theaflavins crude group (TFs): The gastric mucosal epithelial cells are distributed in a relatively regular manner, appearing as pebbles. The cells are relatively intact in morphology and arranged in a relatively neat manner. The surface is covered with a small amount of mucus, and the cells are not obviously damaged. Theaflavins monomer TFDG group (TFDG): The gastric mucosal epithelial cells are arranged more tightly and regularly, with a flattened and round shape, a small amount of epithelial cell erosion, and a small amount of mucus; TFDG corn protein nanoparticles (TFDGN): The gastric mucosal epithelial cells are tightly and regularly arranged, with a round and flat shape, no obvious cell damage, and a relatively intact surface.

[0034] Empty corn protein nanoparticle group (KN): Erosion of gastric mucosa surface, epithelial cell rupture, irregular, incomplete cell morphology, no mucus observed.

[0035] Scanning electron microscopy revealed that alcohol can cause damage to gastric mucosal epithelial cells. TFDG corn protein nanoparticles can alleviate this damage, but the effect of empty-loaded corn protein nanoparticles was not significant.

[0036] The method for hematoxylin and eosin (HE) staining is as follows: Gastric tissue paraffin sections were prepared as follows: xylene I (10 min) → xylene II (10 min) → xylene III (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 100% ethanol III (5 min) → 95% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → hematoxylin staining (3 min) → rinse with tap water and return to blue (10 min) → eosin staining (20 s) → anhydrous ethanol (30 s) → 95% ethanol (30 s) → 80% ethanol (30 s) → ethanol and xylene 1:1 (30 s) → xylene (30 s) → mounting for observation.

[0037] The stomach tissue structure of a mouse consists of four parts: the mucosa, submucosa, muscularis propria, and adventitia. Mouse stomach tissue sections stained with hematoxylin and eosin (HE) are shown below. Figure 2As shown in the images, under 100x and 200x microscopes, the stomach structure of mice in the Control group was neatly arranged, smooth, and flat, with intact and clearly defined layers of the mucosa, submucosa, muscularis propria, and adventitia. No obvious lesions were observed in the submucosa. Excessive alcohol intake caused widening of the interstitial spaces and edema in the submucosa of mice. In contrast, the Model group showed cell separation and detachment in the mucosa, disordered cell arrangement, significant submucosa edema, and muscularis propria rupture. The theaflavins-treated groups showed more neatly arranged gastric mucosal cells and improved submucosa edema. The KN group showed an irregular stomach structure, significant submucosa edema, and a small number of cell detachments. These results indicate that TFDG corn protein nanoparticles have a good protective effect against gastric tissue damage in mice after alcohol intake.

[0038] The method for periodic acid-Schiff (PAS) staining is as follows.

[0039] (1) Processing of paraffin sections of gastric tissue: Xylene I (10 min) → Xylene II (10 min) → Anhydrous ethanol (5 min) → 90% ethanol (2 min) → 80% ethanol (2 min) → 70% ethanol (2 min) → Wash with distilled water (2 min).

[0040] (2) Oxidation with periodic acid solution: Add periodic acid solution to the sample, react in a humidified box in the dark for 10 min, remove the periodic acid solution, soak in distilled water and wash on a shaker for 5 min.

[0041] (3) Schiff reagent staining: Add Schiff reagent to the sample, place it in a humidified chamber, and stain in a 37°C oven in the dark for 1 h. Remove the staining solution, soak in distilled water, and wash on a shaker for 5 min.

[0042] (4) Hematoxylin staining: Add hematoxylin staining solution to the sample, stain for 30 seconds, remove the staining solution, and rinse with distilled water more than twice until the floating color is washed away.

[0043] (5) Dehydration and clearing: 90% ethanol (2 min) → anhydrous ethanol (2 min) → xylene I (5 min) → xylene II (5 min) → mounting.

[0044] The mucus secreted by the upper layer of the gastric mucosa connects with gastric tissue cells to form a protective barrier, which is an important defense factor for gastric epithelial tissue against external damage (such as alcohol and acid). PAS staining of mouse gastric tissue was performed to examine the content of glycogen, glycoproteins, and other polysaccharides in the gastric mucosal surface layer; purple areas indicate the distribution areas of gastric mucosal glycoproteins. Mouse gastric tissue sections after PAS staining... Figure 3As shown, the gastric tissue of mice in the Control group showed a deeper purple hue, and the distribution of glycoproteins in the upper gastric mucosa was more obvious and continuous. Long-term alcohol intake depletes the glycoproteins in the gastric mucosa of mice, resulting in indistinct PAS staining in the gastric tissue of mice in the Model group. Cell shedding from the mucosal layer led to very faint overall PAS staining, or even a lack of purple in the upper gastric mucosa, indicating severe gastric mucosal damage. The PAS staining intensity and overall structural integrity of the gastric tissue of mice in each theaflavin-treated group were significantly increased. The integrity and continuity of the gastric mucosal protective barrier in the TFs and TFDG groups were closer to those in the Control group, with the TFDG corn gluten nanoparticle group showing the best protective effect. The PAS staining in the KN group was similar to that in the Model group, lacking purple, with epithelial cell shedding and gastric mucosal damage. PAS staining results indicate that alcohol-induced gastric mucosal damage may severely impair gastric mucosal glycoproteins in mice, disrupting the gastric mucosal protective barrier and ultimately leading to gastric damage. The TFDG corn gluten nanoparticle treatment group was able to protect the integrity of gastric tissue by preventing the loss of gastric mucosal glycoproteins and maintaining the protective barrier between gastric mucus and epithelial cells.

[0045] In the control group, the gastric mucosa of mice showed a deeper purple hue, with a more pronounced and continuous distribution. Long-term alcohol intake depletes glycoproteins in the gastric mucosa of mice, resulting in less pronounced purple PAS staining in the gastric tissue of the model group mice. Cell shedding from the mucosal layer led to very light overall PAS staining, indicating severe gastric mucosal damage. Compared to the model group, the PAS staining depth and overall structural integrity of the gastric tissue in each theaflavin-treated group were significantly increased. The TFDG corn gluten nanoparticle group (TFDGN group) showed a purple hue depth closer to that of the control group. The KN group showed a lighter purple PAS staining, similar to the model group. PAS staining results indicate that alcohol-induced gastric mucosal damage may severely impair gastric mucosal glycoproteins, disrupting the gastric mucosal protective barrier and ultimately leading to gastric injury. TFDG corn gluten nanoparticle administration (TFDGN) can protect the integrity of gastric tissue by preventing the loss of gastric mucosal glycoproteins and maintaining the protective barrier between gastric mucus and epithelial cells.

[0046] The method for immunohistochemistry (IHC) staining is as follows.

[0047] (1) Dewaxing of liver tissue sections (adhesive glass slides): Xylene I (10 min) → Xylene II (10 min) → Xylene III (10 min) → Anhydrous ethanol I (5 min) → Anhydrous ethanol II (5 min) → Anhydrous ethanol III (5 min) → 95% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → PBS (5 min) (2) Antigen retrieval: Antigen retrieval was performed on the dewaxed liver tissue sections to unfold the proteins in the tissue and expose the antigens, which facilitated their binding with antibodies. The sections were immersed in antigen retrieval solution (pH=6), microwaved for 5 min on high and 15 min on medium, and then allowed to cool to room temperature. The sections were then washed three times with PBS buffer for 5 min each time.

[0048] (3) Remove endogenous peroxidase: After wiping the PBS buffer off the slice, add endogenous peroxidase inhibitor (HRP) to the sample, incubate at 4°C in the dark for 15 min, and wash with PBS as above.

[0049] (4) Serum blocking: Add goat serum working solution to the sample, cover it gently with sealing film to avoid the generation of air bubbles, and keep it moist. Incubate at room temperature in the dark for 1 hour.

[0050] (5) Primary antibody (F4 / 80) incubation: Carefully remove the sealing film, gently wipe away excess liquid, and take care to protect the sample. Dilute the F4 / 80 antibody according to the ratio of goat plasma working solution: F4 / 80 antibody = 250:1, mix well, and drop it onto the tissue sample. Cover with sealing film and place in a refrigerator at 4°C overnight (keep moist). The next day, wash three times with PBST, 5 min each time.

[0051] (6) Secondary antibody incubation: Wipe the glass slide dry, add secondary antibody IgG polymer, cover with sealing film, incubate at room temperature for 20 min, and wash with PBST as above.

[0052] (7) DAB staining: Dilute DAB staining solution according to the ratio of solution A: solution B = 1:50, and use immediately after preparation. Observe under a microscope while staining with DAB staining solution for 8 min. After the staining reaction is completed, place the slide in PBST to stop staining.

[0053] (8) Counterstaining: Dilute the hematoxylin staining solution with pure water: hematoxylin staining solution = 1:3, then stain the sections for 3 minutes. After that, rinse the sections in tap water for 10 minutes to regain blue color.

[0054] (9) Tissue dehydration: 70% ethanol (5 min) → 80% ethanol (5 min) → anhydrous ethanol (5 min) → xylene (5 min) → mounting.

[0055] (10) Microscopic examination and image acquisition: Observe and photograph under a microscope, and use ImageJ to perform quantitative analysis of the brown-yellow positive areas. Analyze the area of ​​the immunohistochemical staining positive areas: Use ImageJ software to change the image mode to RGB Stack mode, adjust the threshold of the positive response area, and statistically analyze the area ratio of each group.

[0056] Figure 4Immunohistochemical staining of F4 / 80 macrophages in mouse liver is shown. As indicated, inflammatory cells in the liver immunohistochemical sections appear brownish-yellow and are distributed near the portal vein. The more brownish-yellow positive granules, the more inflammatory cells are present, and the more severe the liver inflammation. The control group contained trace amounts of F4 / 80 positive expression granules. The number of brownish-yellow positive granules in the model and KN groups was significantly higher than that in the control group. The number of brownish-yellow positive granules in the TFs, TFDG, and TFDG corn protein nanoparticle groups was significantly lower than that in the model group, with the TFDG corn protein nanoparticle group showing the fewest inflammatory cells.

[0057] The Masoon staining method is as follows: Dewaxing liver tissue sections: Xylene I (15 min) → Xylene II (15 min) → 95% ethanol (2 min) → 70% ethanol (2 min) → 30% ethanol (2 min) → 40℃ warm water I (1 min) → 40℃ warm water II (1 min) → Stain with prepared Weigert iron hematoxylin staining solution for 5 min → Differentiate with acidic ethanol differentiation solution for 15 s, wash with water for 60 s → Re-blue with Masson blue solution for 5 min, rinse with running water for 30 s, soak in distilled water for 1 min → Stain with Ponceau S and fuchsin staining solution for 5 min → Soak in weak acid working solution for 1 min → Wash with phosphomolybdic acid solution for 2 min, soak in weak acid working solution for 1 min → Stain with aniline blue staining solution for 2 min, soak in weak acid working solution for 1 min → Rapid dehydration with 95% ethanol for 3 s; dehydration with anhydrous ethanol 3 times, 10 s each time → Clearing with xylene 3 times, 2 min each time, and mounting with neutral resin.

[0058] Liver fibrosis refers to the excessive deposition of diffuse extracellular matrix in the liver. It is a repair response of the body to chronic liver injury and a common pathological basis for the development of various chronic liver diseases. Figure 5 As shown in the figure, the blue area represents collagen fibers. The darker and more widely distributed the blue area, the higher the degree of liver fibrosis and the more severe the liver damage. According to Masoon staining, 50% of mice in the Model and KN groups showed liver fibrosis, while the degree of liver fibrosis was not obvious in the TFs, TFDG, and TFDG corn protein nanoparticle groups (TFDGN group), with the TFDG corn protein nanoparticle group showing the mildest degree of liver fibrosis. Example

[0059] This embodiment provides a composition and preparation method for preventing alcoholic gastric mucosal damage and liver damage. The composition for preventing alcoholic gastric mucosal damage and liver damage includes TFDG, zein, and sodium carboxymethyl cellulose, and its preparation method is as follows.

[0060] The TFDG-zein-sodium carboxymethyl cellulose composite nanoparticles were prepared using the anti-solvent precipitation (ASP) method. The specific steps are as follows: (1) Weigh out the zein powder and dissolve it in a 60% ethanol aqueous solution to prepare a 10 mg / mL zein solution; (2) Weigh out sodium carboxymethyl cellulose powder and dissolve it in ultrapure water to prepare a sodium carboxymethyl cellulose aqueous solution of 2.5 mg / mL; (3) Weigh 10 mg of TFDG and dissolve it in 10 mL of the zein solution prepared in step (1), so that the volume-to-mass ratio of zein solution to TFDG is 1:1. (4) Under magnetic stirring, the TFDG-zeadin ethanol solution obtained in step (3) is added dropwise to the sodium carboxymethyl cellulose aqueous solution prepared in step (2) using a syringe, so that the volume ratio of TFDG-zeadin ethanol solution to sodium carboxymethyl cellulose aqueous solution is 3:1. Stirring forms a suspension of TFDG-loaded zeadin-sodium carboxymethyl cellulose composite nanoparticles; the stirring temperature is 15℃, the stirring speed is 600 rmp, and the stirring time lasts for 30 min.

[0061] (5) Centrifuge the suspension obtained in step (4) at 1℃ and 8000 r / min for 20 min and collect the solid particles. Example

[0062] This embodiment provides a composition and preparation method for preventing alcoholic gastric mucosal damage and liver damage. The composition for preventing alcoholic gastric mucosal damage and liver damage includes TFDG, zein, and sodium carboxymethyl cellulose, and its preparation method is as follows.

[0063] The TFDG-zein-sodium carboxymethyl cellulose composite nanoparticles were prepared using the anti-solvent precipitation (ASP) method. The specific steps are as follows: (1) Weigh out zein powder and dissolve it in a 90% ethanol aqueous solution to prepare a 3 mg / mL zein solution; (2) Weigh out sodium carboxymethyl cellulose powder and dissolve it in ultrapure water to prepare a sodium carboxymethyl cellulose aqueous solution of 0.5 mg / mL; (3) Weigh 30 mg of TFDG and dissolve it in 10 mL of the zein solution prepared in step (1), so that the volume-to-mass ratio of zein solution to TFDG is 1:3. (4) Under magnetic stirring, the TFDG-zeadin ethanol solution obtained in step (3) is added dropwise to the sodium carboxymethyl cellulose aqueous solution prepared in step (2) using a syringe, so that the volume ratio of TFDG-zeadin ethanol solution to sodium carboxymethyl cellulose aqueous solution is 1:1. Stirring forms a suspension of TFDG-loaded zeadin-sodium carboxymethyl cellulose composite nanoparticles; the stirring temperature is 30℃, the stirring speed is 400 rmp, and the stirring time lasts for 60 min.

[0064] (5) Centrifuge the suspension obtained in step (4) at 4℃ and 12000 r / min for 30 min and collect the solid particles.

Claims

1. A composition for preventing alcoholic gastric mucosal damage and liver damage, characterized in that: The composition comprises solid particles with theaflavin-3,3'-bisgallate monomer as the core material and zein and sodium carboxymethyl cellulose as the wall material.

2. The method for preparing the composition for alcoholic gastric mucosal injury and liver injury according to claim 1, characterized in that: Includes the following steps: (1) Weigh out zein powder and dissolve it in an ethanol aqueous solution with a concentration of 60-90% to prepare a zein solution of 3-10 mg / mL; (2) Weigh out sodium carboxymethyl cellulose powder and dissolve it in ultrapure water to prepare a sodium carboxymethyl cellulose aqueous solution of 0.5~2.5 mg / mL; (3) Weigh 10~30 mg of TFDG and dissolve it in the zein solution prepared in step (1) to obtain TFDG-zein ethanol solution; (4) Add the TFDG-zeadin ethanol solution obtained in step (3) to the sodium carboxymethyl cellulose aqueous solution prepared in step (2) and stir to form a suspension of TFDG-loaded zeadin-sodium carboxymethyl cellulose composite nanoparticles. (5) Centrifuge the suspension obtained in step (4) to obtain solid particles.

3. The method for preparing the composition for alcoholic gastric mucosal injury and liver injury according to claim 2, characterized in that: In step (3), the volume-to-mass ratio of zein solution to TFDG is 1:1 to 1:

3.

4. The method for preparing the composition for alcoholic gastric mucosal injury and liver injury according to claim 2, characterized in that: In step (4), the volume ratio of TFDG-zein ethanol solution to sodium carboxymethyl cellulose aqueous solution is 3:1 to 1:

1.

5. The method for preparing the composition for alcoholic gastric mucosal injury and liver injury according to claim 2, characterized in that: In step (4), the stirring temperature is 15~30℃, the stirring speed is 400~600 rmp, and the stirring time lasts for 30~60 min.

6. The method for preparing the composition for alcoholic gastric mucosal injury and liver injury according to claim 2, characterized in that: In step (5), the centrifugation conditions are 1~4℃ and 8000~12000 r / min for 20~30 min.

7. The use of the TFDG-zein-sodium carboxymethyl cellulose composition according to claim 1, characterized in that: The TFDG-zein-carboxymethyl cellulose sodium composition is used to prevent alcoholic gastric mucosal damage.

8. The use of the TFDG-zein-sodium carboxymethyl cellulose composition according to claim 1, characterized in that: The TFDG-zein-carboxymethyl cellulose sodium composition is used to prevent alcoholic liver damage.

Images