Preparation method and application of morchella polysaccharide

Abstract

The invention relates to the field of fungal polysaccharide application, and discloses a preparation method and application of morchella polysaccharide. The preparation method comprises the following steps: step 1) leaching dry morchella esculenta powder with water to obtain a leaching solution; carrying out alcohol precipitation on the leach liquor to obtain a precipitate; 2) adding water into the precipitate for redissolving, removing protein, and then dialyzing and freeze-drying to obtain a morchella esculenta crude polysaccharide extract; 3, the morchella esculenta crude polysaccharide extract is dissolved in water and purified through adsorption chromatography, and the morchella esculenta polysaccharide is obtained.The morchella esculenta polysaccharide has the protection effect on alcoholic stomach injury in vivo and in vitro, stomach tissue inflammatory response is adjusted by adjusting an NF-kb channel, and reference value is provided for development application of the morchella esculenta polysaccharide.

Description

Field of Invention: This invention belongs to the field of polysaccharide preparation and application, specifically relating to a method for preparing morel polysaccharide and its application. Background technology: Gastric ulcers, a prevalent digestive system disease worldwide, severely impact people's quality of life. External factors such as Helicobacter pylori (HP) infection, alcohol consumption, smoking, and nonsteroidal anti-inflammatory drugs (NSAIDs) can all contribute to ulcer formation. Alcohol consumption, a common factor, frequently affects multiple parts of the gastrointestinal tract, directly or indirectly causing gastric lesions (such as edema, erosion, bleeding, and necrosis). Clinically, treatment for alcoholic gastric ulcers primarily involves proton pump inhibitors, acid neutralizers, and digestive enzyme preparations. However, long-term use of these medications often leads to a series of complications, including acute kidney injury, changes in gastric mucosal structure and function, and drug resistance. Therefore, there is significant interest in using natural product alternatives to treat gastric mucosal damage.

[0001] Morels belong to the subphylum Ascomycota in taxonomy ( Ascomycotina Morel family ( Morchell- aceae Morel genus Morchella Dill.ex Pers. Modern pharmacological studies have shown that morel mushrooms possess immunomodulatory and physiological activities. The medicinal value of morel mushrooms was first recorded in the *Compendium of Materia Medica*, where it was described as cold in nature and non-toxic, possessing the effects of improving gastrointestinal health and resolving phlegm and regulating qi. The *Dietary Materia Medica* describes the medicinal value of morel mushrooms as "sweet, cold, and non-toxic, resolving phlegm, regulating qi, and benefiting the stomach and intestines." Currently, although some studies have disclosed the immunomodulatory and antitumor activities of morel mushroom polysaccharides, the protective effect and specific mechanism of its polysaccharide components with clearly defined structural characteristics on alcoholic gastric mucosal damage have not yet been reported. This invention is the first to isolate and purify a homogeneous polysaccharide MEP-1 from morel mushrooms, possessing both anti-inflammatory and antioxidant effects. It prevents alcoholic gastric damage through the NF-κB pathway, providing a new candidate drug for gastric mucosal protection. Summary of the Invention

[0002] To address the challenge of extracting polysaccharides from morel mushrooms for protecting the gastric mucosa, this invention provides a method for preparing morel mushroom polysaccharides and their applications.

[0003] A method for preparing morel polysaccharide includes the following steps: Step 1) Extract the dried morel mushroom powder with water to obtain an extract; The extract was precipitated with alcohol to obtain a precipitate; Step 2) The precipitate is reconstituted with water to remove protein, and then dialyzed and freeze-dried to obtain morel crude polysaccharide extract; Step 3) Dissolve the crude polysaccharide extract of morel mushroom in water and purify it by adsorption chromatography to obtain the morel mushroom polysaccharide. Optionally, in step 1), the dried morel powder is ultrasonically extracted with water; the solid-liquid ratio of the ultrasonic extraction is 1:10~50g / ml; the extraction temperature is 30-70℃; the extraction time is 20-60min; and the extraction power is 100-400W.

[0004] Optionally, in step 1), the extract is concentrated by rotary evaporation; then, it is precipitated with three times its volume of ethanol. The alcohol precipitation temperature is 1-8℃, and the alcohol precipitation time is 10-16h; After alcohol precipitation, centrifugation is performed; the centrifugation conditions are 3000-5000 r / min and the centrifugation time is 5-15 min.

[0005] Optionally, in step 2), a dialysis bag with a capacity of 3000-8000 Da is used, and the dialysis time is 48-72 hours.

[0006] 5. The preparation method according to claim 1, characterized in that, in step 3), the crude polysaccharide extract of morel mushroom is dissolved in water, filtered through a 0.45 μm microporous membrane, then passed through an ion exchange column and eluted with a gradient of sodium chloride solution. The eluent is collected, dialyzed, and freeze-dried to obtain a preliminarily purified polysaccharide; the preliminarily purified morel mushroom polysaccharide is dissolved in water, passed through a gel column and eluted with distilled water. The eluent is collected, freeze-dried, and the purified morel mushroom polysaccharide is obtained.

[0007] Optionally, in step 2), the solution gradient elution uses a solution containing 0-0.5 mol / L sodium chloride as the eluent, with an elution rate of 3-5 ml / min; the distilled water elution uses an elution rate of 0.9-1.1 ml / min.

[0008] Optionally, the morel polysaccharide has a weight-average molecular weight of 24.759 kDa, a number-average molecular weight of 38.634 kDa, and a polydispersity index (Mw / Mn) of 1.560. The morel polysaccharide is composed of glucose, mannose, and galactose in a molar ratio of 61.61:26.37:12.02.

[0009] The present invention also proposes the application of the morel polysaccharide, specifically its use in the preparation of drugs for preventing and protecting against gastric mucosal damage.

[0010] Optional gastric mucosal damage includes ethanol-induced gastric mucosal bleeding, edema, and erosion.

[0011] Optionally, the prevention and protection against gastric mucosal damage is achieved by alleviating inflammatory symptoms and regulating oxidative stress.

[0012] By employing the above-described technology, the beneficial effects of the present invention compared to the prior art are as follows: (1) In the method for preparing morel polysaccharides of the present invention, the ultrasonic-assisted hot water extraction method is used. Ultrasonic-assisted extraction can promote cell wall rupture and release of intracellular polysaccharides through cavitation and micro-jet effects, which can shorten the extraction time. Ultrasonic extraction is more likely to obtain polysaccharide components with low molecular weight or more easily exposed active groups, while taking into account the stable maintenance of the physicochemical properties and biological activity of the effective components of morel.

[0013] (2) The morel polysaccharide proposed in this invention can reduce the level of ROS and the apoptosis rate of ethanol-induced human gastric mucosal cells GES-1. It reduces the ulceration index of ethanol-induced gastric mucosa in mice, restores the activity of antioxidant enzymes in damaged mice, alleviates inflammation, and regulates the inflammatory damage of mouse gastric tissue through the NF-κB pathway. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 DEAE-52 elution curve for MEP; Figure 2 The Sephadex G-100 elution curve for MEP; Figure 3 UV spectrum of MEP Figure 4 The graph shows the molecular weight determination of MEP. Figure 5 The HPLC chromatogram of the monosaccharide composition of MEP; Figure 6 Effects of morel polysaccharides on GES-1 cell proliferation Figure 7 Effects of morel polysaccharides on ROS content in GES-1 cells Figure 8 Effects of morel polysaccharides on GES-1 cell apoptosis Figure 9 Figures showing weight changes and organ indices in mice with acute gastric injury. Figure 10 The image shows the morphology and HE staining of tissues from acute gastric injury in mice. Figure 11 The effect of MEP on the activity of antioxidant enzymes in mice with gastric injury; Figure 12 The effect of MEP on the levels of inflammatory factors in mice with gastric injury; Figure 13 The effect of MEP on PEG2 and NO levels in mice with gastric injury; Figure 14 The effect of MEP on the expression levels of NF-κB pathway proteins in mice with gastric injury. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0017] Furthermore, the numerical ranges in this invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the equipment, apparatus, materials, reagents, etc., used are all commercially available. The purpose of this invention is to provide a morel polysaccharide and its preparation method. Morel polysaccharide is extracted using dried morel mushrooms as raw material by an ultrasonic-assisted extraction-ethanol precipitation method. A homogeneous morel polysaccharide component, named MEP, is obtained after purification. The first aspect provided by this invention is a method for preparing morel polysaccharide, which is as follows: Step 1) Extract the dried morel mushroom powder with water to obtain an extract; The extract was precipitated with alcohol to obtain a precipitate; Step 2) The precipitate is reconstituted with water to remove protein, and then dialyzed and freeze-dried to obtain morel crude polysaccharide extract; Step 3) Dissolve the crude polysaccharide extract of morel mushroom in water and purify it by adsorption chromatography to obtain the morel mushroom polysaccharide. Further, in step 1), the extraction ratio of morel powder to water is 1:20-1:40; in one embodiment of the present invention, the material-liquid ratio is 1:30. Further, in step 1), the ultrasonic extraction temperature is 30-70℃; the extraction time is 20-60 min; and the extraction power is 100-400 W. In one embodiment of the present invention, the extraction temperature is 60℃; the extraction time is 40 min; and the extraction power is 200 W. Furthermore, the concentration mentioned in step 1) refers to concentrating the volume to 1 / 3 of its original volume using a rotary evaporator at 70°C; Further, the alcohol precipitation described in step 1) is performed by using 3 times the volume of 95% ethanol, placing it in a refrigerator at 4°C for more than 12 hours, and then centrifuging to collect the precipitate; Furthermore, in step 2), the protein removal process uses the Sevage method, in which chloroform and n-butanol are mixed evenly at a volume ratio of 1:4, and then mixed and stirred with the polysaccharide solution at a volume ratio of 1:4. Further, the freeze-drying described in step 2) is to obtain morel crude polysaccharide by freeze-drying at -80°C and 0.01 Pa using a vacuum freeze dryer; Further, in step 3), the crude morel polysaccharide was dissolved in water, filtered through a 0.45 μm microporous membrane, and purified by elution using a DEAE-52 ion exchange column and a Sephadex G-100 gel column to obtain a homogeneous morel polysaccharide fraction. Furthermore, the polysaccharide described in step 3) is soluble in water, and the concentration of the polysaccharide solution is 10 mg / ml; Furthermore, the DEAE-52 ion exchange column described in step 3) is eluted with sodium chloride of different concentrations at a rate of 4 ml / min; Furthermore, the Sephadex G-100 gel column described in step 3) is eluted with distilled water at a rate of 1 ml / min.

[0020] Another objective of this invention is to provide an application of morel polysaccharide as a preventive and therapeutic agent for alcohol-induced gastric mucosal injury. An alcohol-induced gastric injury model was established, and its potential in the prevention and treatment of alcoholic gastric injury was demonstrated through in vivo and in vitro experiments and by examining anti-inflammatory and antioxidant indicators.

[0021] Furthermore, this invention uses a KM mouse acute gastric injury model to study the in vivo prevention and treatment effects on gastric mucosa. The results showed that MEP has a good effect in reducing gastric mucosal bleeding and swelling.

[0022] Example 1: Extraction and purification of morel polysaccharides (1) Extraction of morel polysaccharides After drying, the morel mushroom fruiting bodies were pulverized and passed through a 60-mesh sieve. Polysaccharide extraction was performed using ultrasound-assisted extraction. 100g of morel mushroom powder was dissolved in 3000ml of distilled water, and extraction was carried out at 60℃ for 40min using an ultrasonic extractor with a power of 200W. After filtration, the residue was subjected to a second extraction. The filtrates were combined and concentrated to 1 / 3 of their volume using a rotary evaporator. Three times the volume of ethanol was added, and the mixture was placed at 4℃ for 12h to allow the polysaccharides to precipitate completely. The precipitate was collected by centrifugation, reconstituted with water, and then freeze-dried to obtain crude MEP.

[0023] (2) Morel polysaccharide protein removal Morel mushroom crude polysaccharide sample was thoroughly dissolved in distilled water to prepare a polysaccharide solution. Sevage reagent (a mixture of chloroform and n-butanol in a 4:1 ratio) was added. The mixture was stirred at 600 rpm for 30 min, then centrifuged at 8000 rpm for 5 min. Clear layering was observed. The white protein layer in the middle was removed using a pipette, and this process was repeated 3-4 times until the protein layer disappeared. After the final centrifugation, the upper polysaccharide layer was collected. All polysaccharide solutions were combined and dialyzed against 3000 da / L in distilled water for 72 h, with the distilled water changed every 4 h. Finally, the obtained polysaccharide solution was freeze-dried to obtain morel mushroom crude polysaccharide.

[0024] (3) Purification of morel polysaccharides Morel mushroom crude polysaccharide purification was performed using DEAE-cellulose 52 and Sephadex G-100 column chromatography. MEP was dissolved in distilled water, and the supernatant was obtained after centrifugation (8000 rpm, 8 min). The supernatant was loaded into a DEAE-cellulose 52 column and eluted with NaCl solutions (0, 0.1, 0.2, 0.3, and 0.5 mol / L). The flow rate was maintained at 4 mL / min, and the eluent was continuously collected, with 8 mL collected per tube, until each concentration gradient elution was complete. The polysaccharide content in each tube of eluent was determined using the phenol-sulfuric acid method. Distilled water was used as a blank control, and the absorbance (A value) of each solution was measured at a wavelength of 490 nm. Simultaneously, the polysaccharide concentration in each eluent was calculated according to the standard curve. An elution curve was plotted with the tube number as the x-axis and the absorbance of each tube as the y-axis, as shown below. Figure 1 Based on the elution curve, the elution solutions from the test tubes corresponding to 0.1 mol / L NaCl were combined, concentrated under reduced pressure by rotary evaporation, and then desalted by dialysis. Preliminary purified morel polysaccharides were obtained.

[0025] The preliminarily purified morel polysaccharide was prepared into a 10 mg / ml polysaccharide solution and slowly injected into a Sephadex G-100 chromatography column. The sample volume did not exceed 5% of the column bed volume to ensure separation efficiency. After sample loading, distilled water was used as the mobile phase, and elution was performed at a flow rate of 1 mL / min. 8 mL of eluent was collected from each tube, for a total of 50 tubes. The absorbance of each tube was determined using the phenol-sulfuric acid method. The number of tubes was plotted on the x-axis, and the absorbance value on the y-axis to create an elution curve, as shown below. Figure 2 Collect the eluent corresponding to the number of tubes with the elution peaks, and finally freeze-dry to obtain the purified polysaccharide MEP.

[0026] Example 2 (1) Extraction of morel polysaccharides After drying, the morel mushroom fruiting bodies were pulverized and passed through a 60-mesh sieve. Polysaccharide extraction was performed using ultrasound-assisted extraction. 100g of morel mushroom powder was dissolved in 1000ml of distilled water, and extraction was carried out at 30℃ for 60min using an ultrasonic extractor with a power of 200W. After filtration, the residue was subjected to a second extraction. The filtrates were combined and concentrated to 1 / 3 of their volume using a rotary evaporator. Three times the volume of ethanol was added, and the mixture was placed at 4℃ for 12h to allow the polysaccharides to precipitate completely. The precipitate was collected by centrifugation, reconstituted with water, and then freeze-dried to obtain crude MEP.

[0027] (2) Morel polysaccharide protein removal Morel mushroom crude polysaccharide sample was thoroughly dissolved in distilled water to prepare a polysaccharide solution. Sevage reagent (a mixture of chloroform and n-butanol in a 4:1 ratio) was added. The mixture was stirred at 600 rpm for 30 min, then centrifuged at 3000 rpm for 15 min. Clear layering was observed. The white protein layer in the middle was removed using a pipette, and this process was repeated 3-4 times until the protein layer disappeared. After the final centrifugation, the upper polysaccharide layer was collected. All polysaccharide solutions were combined and dialyzed against distilled water at 6000 da / L for 72 h, with the distilled water changed every 4 h. Finally, the obtained polysaccharide solution was freeze-dried to obtain morel mushroom crude polysaccharide.

[0028] (3) Purification of morel polysaccharides Same as Example 1.

[0029] Example 3 (1) Extraction of morel polysaccharides After drying, the morel mushroom fruiting bodies were pulverized and passed through a 60-mesh sieve. Polysaccharide extraction was performed using ultrasound-assisted extraction. 100g of morel mushroom powder was dissolved in 5000ml of distilled water, and extraction was carried out at 70℃ for 20min using an ultrasonic extractor with a power of 200W. After filtration, the residue was subjected to a second extraction. The filtrates were combined and concentrated to 1 / 3 of their volume using a rotary evaporator. Three times the volume of ethanol was added, and the mixture was placed at 4℃ for 12h to allow the polysaccharides to precipitate completely. The precipitate was collected by centrifugation, reconstituted with water, and then freeze-dried to obtain crude MEP.

[0030] (2) Morel polysaccharide protein removal Morel mushroom crude polysaccharide sample was thoroughly dissolved in distilled water to prepare a polysaccharide solution. Sevage reagent (a mixture of chloroform and n-butanol in a 4:1 ratio) was added at a ratio of 4:1. The mixture was stirred at 600 rpm for 30 min, then centrifuged at 5000 rpm for 8 min. Clear layering was observed. The white protein layer in the middle was removed using a pipette, and this process was repeated 3-4 times until the protein layer disappeared. After the final centrifugation, the upper polysaccharide layer was collected. All polysaccharide solutions were combined and dialyzed against distilled water at 8000 da / L for 72 h, with the distilled water changed every 4 h. Finally, the obtained polysaccharide solution was freeze-dried to obtain morel mushroom crude polysaccharide.

[0031] (3) Purification of morel polysaccharides Same as Example 1.

[0032] Example 4 1) Morel polysaccharide extraction After drying, the morel mushroom fruiting bodies were pulverized and passed through a 60-mesh sieve. Polysaccharide extraction was performed using ultrasound-assisted extraction. 100g of morel mushroom powder was dissolved in 3000ml of distilled water, and extraction was carried out at 60℃ for 40min using an ultrasonic extractor with a power of 200W. After filtration, the residue was subjected to a second extraction. The filtrates were combined and concentrated to 1 / 3 of their volume using a rotary evaporator. Three times the volume of ethanol was added, and the mixture was placed at 4℃ for 12h to allow the polysaccharides to precipitate completely. The precipitate was collected by centrifugation, reconstituted with water, and then freeze-dried to obtain crude MEP.

[0033] (2) Morel polysaccharide protein removal Morel mushroom crude polysaccharide sample was thoroughly dissolved in distilled water to prepare a polysaccharide solution. Sevage reagent (a mixture of chloroform and n-butanol in a 4:1 ratio) was added. The mixture was stirred at 600 rpm for 30 min, then centrifuged at 8000 rpm for 5 min. Clear layering was observed. The white protein layer in the middle was removed using a pipette, and this process was repeated 3-4 times until the protein layer disappeared. After the final centrifugation, the upper polysaccharide layer was collected. All polysaccharide solutions were combined and dialyzed against 3000 da / L in distilled water for 72 h, with the distilled water changed every 4 h. Finally, the obtained polysaccharide solution was freeze-dried to obtain morel mushroom crude polysaccharide.

[0034] Example 5: Ultraviolet Spectroscopy and Molecular Weight Determination of Morel Polysaccharides (1) Prepare a 1 mg / ml MEP solution, and use a UV spectrophotometer to perform UV scanning in 2 nm segments within the wavelength range of 200-600 nm. Plot the scanning curve and observe whether there are any special absorption peaks at 260 nm and 280 nm to detect the content of nucleic acid and protein in MEP, which is used to reflect the purity of polysaccharides. like Figure 3 The image shows the ultraviolet spectrum analysis of MEP-1, which is a smooth curve. The absence of characteristic absorption peaks at wavelengths of 260 nm and 280 nm indicates that MEP does not contain proteins or nucleic acids.

[0035] (2) Weigh an appropriate amount of MEP and dissolve it in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w). Adjust the MEP concentration to 1 mg / ml. Before loading the sample, filter it through a 0.45 μm filter. Measure the MEP using high performance gel size exclusion chromatography (HGEC). Use Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) gel size exclusion columns connected in series. Column temperature: 45℃; injection volume: 100 μL; mobile phase: A (0.02% NaN3, 0.1M NaNO3); flow rate: 0.6 mL / min; elution gradient: isocratic for 75 min.

[0036] Measurement results: such as Figure 4 As shown, the weight-average molecular weight (Mw) of MEP is 38.634 kDa; the number-average molecular weight (Mn) is 24.7594 kDa; and the polydispersity index Mw / Mn is 1.159, indicating that MEP is a medium molecular weight polysaccharide.

[0037] Example 6: Determination of Monosaccharide Composition of Morel Polysaccharides (1) Weigh an appropriate amount of MEP into a clean chromatographic vial, add 1 ml of 2M TFA acid solution to the vial, and heat at 121 °C for 2 hours. After drying with nitrogen, add methanol to clean, and then dry again. Repeat the cleaning process 2-3 times. Dissolve in sterile water, transfer to a chromatographic vial, and perform analysis. The chromatographic system used is a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), which uses an electrochemical detector to analyze and detect monosaccharide components. The parameters are as follows: Dionex™ CarboPac™ PA20 (150*3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1 M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0038] The results are as follows Figure 5 The monosaccharide components and their molar percentages in MEP are as follows: N-acetylglucosamine 0.69%, galactose 27.07%, glucose 6.23%, and mannose 66.01%.

[0039] Example 7: Protective effect of morel polysaccharides on GES-1 cells (1) GES-1 cell proliferation experiment Cells in the logarithmic growth phase were subjected to morel polysaccharide, which was filtered through a 0.22 μm filter and diluted with culture medium to concentrations of 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg / ml. 100 μL of each concentration was added to each well of a 96-well plate, with three replicates per well. The plates were incubated at 37 °C and 5% CO2 for 24 h, followed by 2 h of further incubation with CCK-8. The absorbance was then measured.

[0040] (2) Determination of ROS content in GES-1 cells Logarithmic growth phase cells were induced with 6% ethanol for 2 hours, followed by treatment with different concentrations of morel polysaccharides for 24 hours. ROS levels were detected using a ROS assay kit, followed by staining with DCHF-DA dye. Fluorescence intensity was then analyzed using flow cytometry. (3) Determination of apoptosis rate in GES-1 cells Logarithmic growth phase cells were induced with 6% ethanol for 2 hours, then treated with different concentrations of morel polysaccharide for 24 hours. Apoptosis was detected using Annexin V-FITC and PI double staining reagents.

[0041] (4) Results and Analysis The results are as follows Figure 6 As shown, within the concentration range of 0.25–4 mg / mL, MEP significantly promoted the proliferation of GES-1 cells in a dose-dependent manner. At a concentration of 4 mg / mL, morel polysaccharide significantly increased cell viability (P<0.05), with a cell viability of 117.11 ± 2.46%.

[0042] ROS content structure as follows Figure 7 Compared with the Model group, the ROS levels decreased after polysaccharide intervention, with 18.9% in the 1 mg / mL group, 17.0% in the 2 mg / mL group, and 10.9% in the 4 mg / mL group. The results indicate that polysaccharide treatment can significantly inhibit the excessive production of ROS in GES-1 cells, and the effect is dose-dependent, with higher doses showing a more significant reduction.

[0043] Apoptosis results as follows Figure 8 As shown, after treatment with 6% ethanol for 2 h, the apoptosis rate in the model group was significantly higher than that in the normal group. After pretreatment with low, medium, and high concentrations of MEP for 24 hours, the apoptosis rate was significantly lower than that in the model group (P<0.05), showing a dose-dependent relationship. This indicates that MEP treatment can effectively prevent ethanol-induced apoptosis and increase the survival rate of GES-1 cells.

[0044] Example 8: Application of Morel Polysaccharide in the Prevention and Treatment of Gastric Mucosal Damage (1) Laboratory animals and conditions Sixty SPF-grade male KM mice (4W) were acclimatized for one week under conditions of 65% relative humidity, 25℃, and 12-hour light-dark cycles before being randomly assigned to six groups: ① Control group (NC); ② Model group (MC); ③ Omeprazole group (PC): omeprazole 20 mg / kg / day by gavage; ④ Low-dose morel polysaccharide group (LMEP): morel polysaccharide 100 mg / kg / day by gavage; ⑤ Medium-dose morel polysaccharide group (MMEP): morel polysaccharide 200 mg / kg / day by gavage; ⑥ High-dose morel polysaccharide group (HMEP): morel polysaccharide 400 mg / kg / day by gavage. The mice were fed for a total of 21 days, and their weight was recorded every three days. On the last day of feeding, the mice were fasted but allowed to drink water. One hour after each group was given the drug by gavage, except for the control group, the other groups were given 0.5 ml / kg of anhydrous ethanol by gavage. One hour after gavage, the mice were anesthetized with ether and their stomach tissue was taken.

[0045] (2) Sample collection The obtained mouse gastric tissue was cut open along the greater curvature of the stomach, the gastric contents were washed away with physiological saline, the surface moisture was absorbed with filter paper, and the tissue was laid flat on a measuring plate for visual observation and recording of the gastric mucosal damage. (3) Mouse body weight and organ index Throughout the experiment, there were no significant differences in the major organ indices among the mouse groups, such as Figure 9 This indicates that MEP did not exhibit significant toxicity in mice. Mouse body weight was not significantly affected by MEP, with no significant difference between groups (p>0.05). Furthermore, no deaths or abnormal behaviors were observed during the experiment, supporting the good safety profile of MEP.

[0046] (4) Gastric tissue morphology and HE sections like Figure 10 As shown in the figure, the gastric mucosa in the NC group was normal in morphology and color, with no obvious lesions. In contrast, the gastric tissue in the MC group showed obvious petechial hemorrhages and large areas of congestion, erosion, swelling, and other forms of damage. The PC group and the MEP treatment group showed improvement in gastric mucosal swelling and other pathological conditions, with a significant reduction in bleeding points and a significant decrease in the area of ​​swelling and congestion. From a macroscopic perspective, MEP has a certain protective effect on the gastric mucosa. In addition, histological changes induced by ethanol in the mouse stomach were observed by HE staining. The gastric tissue structure of the NC group was normal, the gastric glands were arranged closely, and no phenomena such as congestion and edema, cell exfoliation, and inflammatory cell infiltration were observed. Compared with the NC group, obvious defects in the gastric gland structure and submucosal edema were observed in the stomach of the MC group. Epithelial cells exfoliated, the glands were arranged in a disorderly manner, the blood vessels showed dilation and congestion, and there was inflammatory cell infiltration. However, these phenomena were improved in both the PC group and the MEP group. This indicates that MEP can maintain the integrity of the gastric mucosal structure. Overall, MEP has a protective effect on ethanol-induced gastric lesions in mice.

[0047] (5) Determination of the contents of MDA, SOD, CAT, and GSH in mouse gastric tissue According to the kit instructions, the contents of MDA, SOD, CAT, and GSH in mouse gastric tissue were measured strictly in accordance with the operation steps of the instructions. The results are as Figure 11 shown. Compared with the NC group, the activities of SOD, GSH-Px, and CAT in the gastric tissue of the MC group showed a significant decrease (P<0.05), while the MDA level showed a significant increase (P<0.05). After the intervention of MEP, the activities of antioxidant enzymes in the mouse stomach increased, while the content of lipid peroxides decreased, and this effect showed a dose dependence. The activities of SOD, GSH-Px, and CAT in the HMEP group showed a significant upregulation (P<0.05).

[0048] (6) Determination of the contents of IL-6, IL-1β, and TNF-α in mouse gastric tissue The gastric tissue IL-6, IL-1β, and TNF-α were quantitatively analyzed using an ELISA kit. The results are as Figure 12 shown. Compared with the NC group, the levels of TNF-α, IL-1β, and IL-6 in the gastric tissue of the MC group were significantly increased, confirming the successful induction of gastric mucosal injury. Compared with the MC group, oral administration of MEP and omeprazole significantly reduced the levels of TNF-α, IL-6, and IL-1β (p<0.05). The levels of TNF-α and IL-6 in the model group were significantly higher than those in the normal group. Compared with the model group, the treatment effects were ranked as follows: LMEP < MMEP < HMEP < omeprazole.

[0049] (7) Determination of the contents of PGE2 and NO in mouse gastric tissue The gastric tissue PGE2 and NO were quantitatively analyzed using an ELISA kit, and the results are as Figure 13Compared with the NC group, the levels of prostaglandin E2 (PGE2) and nitric oxide (NO) in the gastric tissue of ulcerated MC were significantly lower (P<0.05), and these levels increased significantly in the mucosa after administration of MEP (100, 200, and 400 mg / kg). These findings suggest that the gastric protective mechanism of MEP may involve the positive regulation of PGE2 and NO synthesis. Furthermore, MEP exhibits a dose-dependent effect on the increase of PGE2 and NO.

[0050] (8) Results of determination of NF-κB pathway protein content in mouse gastric tissue The results are as follows Figure 14 As shown, compared with the MC group, all drug-treated groups (PC group, LMEP group, MMEP group, and HMEP group) showed varying degrees of increased IκBα expression. The MEP group showed a slightly lower expression than the PC group, indicating that the effect of MEP treatment may not be as significant as that of the PC group. However, compared with the MC group, it significantly improved IκBα expression and inhibited the inflammatory response. In the drug-treated groups, MEP was lower than that in the MC group, indicating that MEP can inhibit NF-κB activation to some extent and reduce the body's inflammatory response.

[0051] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing morel polysaccharide, characterized in that, Includes the following steps: Step 1) Extract the dried morel mushroom powder with water to obtain an extract; The extract was precipitated with alcohol to obtain a precipitate; Step 2) The precipitate is reconstituted with water to remove protein, and then dialyzed and freeze-dried to obtain morel crude polysaccharide extract; Step 3) Dissolve the crude polysaccharide extract of morel mushroom in water and purify it by adsorption chromatography to obtain the morel mushroom polysaccharide.

2. The preparation method according to claim 1, characterized in that, In step 1), the dried morel powder is ultrasonically extracted with water; the solid-liquid ratio of ultrasonic extraction is 1:10~50g / ml; the extraction temperature is 30-70℃; the extraction time is 20-60min; and the power is 100-400W.

3. The preparation method according to claim 1, characterized in that, In step 1), the extract is concentrated by rotary evaporation; then, it is precipitated with three times its volume of ethanol. The alcohol precipitation temperature is 1-8℃, and the alcohol precipitation time is 10-16h; After alcohol precipitation, centrifugation is performed; the centrifugation conditions are 3000-5000 r / min and the centrifugation time is 5-15 min.

4. The preparation method according to claim 1, characterized in that, In step 2), a dialysis bag with a capacity of 3000-8000 Da is used, and the dialysis time is 48-72 hours.

5. The preparation method according to claim 1, characterized in that, In step 3), the crude polysaccharide extract of morel mushroom is dissolved in water, filtered through a 0.45 μm microporous membrane, and then eluted with a sodium chloride solution gradient after passing through an ion exchange column. The eluent is collected, dialyzed, and freeze-dried to obtain the preliminarily purified polysaccharide. The preliminarily purified morel polysaccharide was dissolved in water, passed through a gel column, eluted with distilled water, and the eluent was collected, freeze-dried, and the purified morel polysaccharide was obtained.

6. The preparation method according to claim 5, characterized in that, In step 2), the solution gradient elution uses a solution containing 0-0.5 mol / L sodium chloride as the eluent, and the elution rate is 3-5 ml / min. The elution rate of distilled water is 0.9~1.1 ml / min.

7. The morel polysaccharide prepared by any one of claims 1 to 6, characterized in that, The morel polysaccharide has a weight-average molecular weight of 24.759 kDa, a number-average molecular weight of 38.634 kDa, and a polydispersity index (Mw / Mn) of 1.

560. The morel polysaccharide is composed of glucose, mannose, and galactose, with a glucose:mannose:galactose molar ratio of 61.61:26.37:12.

02.

8. The application of the morel polysaccharide according to claim 7, characterized in that, The morel polysaccharide is used in the preparation of drugs for preventing and protecting against gastric mucosal damage.

9. The application in preventing and protecting gastric mucosal damage according to claim 8, characterized in that, The gastric mucosal damage includes gastric mucosal bleeding, edema, and erosion caused by ethanol.

10. The application in preventing and protecting gastric mucosal damage according to claim 8, characterized in that, The aforementioned prevention and protection against gastric mucosal damage is achieved by alleviating inflammatory symptoms and regulating oxidative stress.

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