Application of a natural polysaccharide Riclin in the preparation of drugs for preventing and treating gastric ulcer

Abstract

The present invention discloses the use of a natural polysaccharide Riclin in the preparation of a drug for preventing and treating gastric ulcers. The present invention can reduce gastric ulcer damage induced by ethanol; inhibit mast cell activation to reduce gastric acid secretion, promote the production of Muc6 mucin, and promote the formation of gastric protective mucus barrier; increase CAT levels to enhance the first-pass metabolism of ethanol, and reduce oxidative stress and inflammatory response induced by ethanol.

Description

Technical Field

[0001] The invention relates to the technical field of natural polysaccharides, and in particular to application of a natural polysaccharide, Riclin, in preparing medicines for preventing and treating gastric ulcers. Background Art

[0002] Gastric ulcer (GU) is a peptic ulcer that mainly occurs in the gastric angle, gastric antrum and cardia. It has attracted more and more attention due to its increasing incidence, mild clinical symptoms and great potential harm. It is a chronic disease that seriously endangers human health. The pathogenesis of gastric ulcer varies, such as non-steroidal anti-inflammatory drugs, ethanol, hydrochloric acid, Helicobacter pylori infection, etc. Young people drink excessively due to stress, and the prevalence of acute alcoholic gastric ulcer is showing a trend of younger age. The mechanism of ethanol-induced acute gastric ulcer varies. As far as we know, ethanol concentration ≥12.5% ​​will cause changes in the gastric mucosa, destroy the apical cell membrane, cause the intrinsic membrane to fall off, and release histamine. At the same time, pro-inflammatory mediators such as leukocyte product reactive oxygen, neutrophil lysosomal components, cytokines, platelet activation factor, etc. are one of the known causes of gastric ulcer, and the degree of ulcer lesions is closely related to alcohol concentration.

[0003] At present, receptor antagonists and proton pump inhibitors are commonly used in the clinic to treat gastric ulcers, but long-term use of these drugs can lead to side effects, poor healing quality, intestinal microbial flora imbalance and other problems. On the other hand, the treatment of gastrointestinal diseases mainly relies on the use of synthetic drugs. Excessive use of synthetic drugs often causes serious complications, and some are expensive. Therefore, we urgently need a natural, non-toxic and long-term drug for the prevention and treatment of gastric ulcers to maintain human health. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes the use of a natural polysaccharide Riclin in the preparation of a drug for preventing and treating gastric ulcers. The present invention can reduce ethanol-induced gastric ulcer damage; inhibit mast cell activation to reduce gastric acid secretion, promote the production of Muc6 mucin, and promote the formation of a gastric protective mucus barrier; increase CAT levels to enhance the first-pass metabolism of ethanol, and reduce ethanol-induced oxidative stress and inflammatory responses.

[0005] The invention provides application of natural polysaccharide Riclin in preparing medicine for preventing and treating gastric ulcer.

[0006] Preferably, the gastric ulcer is an acute alcoholic gastric ulcer.

[0007] Preferably, the dosage form of the drug is an oral dosage form.

[0008] Preferably, the drug is natural polysaccharide Riclin oral solution.

[0009] Preferably, the content of natural polysaccharide Riclin in the natural polysaccharide Riclin oral liquid is 0.5-2 mg / ml.

[0010] Preferably, the solvent of the natural polysaccharide Riclin oral solution is physiological saline.

[0011] Preferably, the natural polysaccharide Riclin is an exopolysaccharide extracted from Agrobacterium sp. ZCC3656.

[0012] The Agrobacterium sp. ZCC3656 has a deposit number of CCTCC NO: M2018797, which has been disclosed in the patent "Agrobacterium and its anti-inflammatory exopolysaccharide" with application number 201811493131.2.

[0013] Preferably, the average molecular weight of the natural polysaccharide Riclin is 2×10 6 -3×10 6 Da.

[0014] The natural polysaccharide Riclin is a polysaccharide extracted from Agrobacterium ZCC3656. It is a repeating unit of glucose residues formed by modifying a galactose residue with succinic acid, pyruvic acid and acetic acid. Its molecular chain contains a large number of hydroxyl groups and has good biocompatibility and biodegradability.

[0015] Preferably, the method for extracting the natural polysaccharide Riclin comprises the following steps: taking the fermentation liquid of Agrobacterium sp. ZCC3656, adding an alcohol solvent for alcohol precipitation, taking the precipitate and drying it to obtain the crude sugar Riclin; taking the aqueous solution of the crude sugar Riclin, performing a hydrolysis reaction with an inorganic base, then taking the supernatant after the reaction, performing alcohol precipitation with an alcohol solvent, and taking the precipitate to obtain the natural polysaccharide Riclin.

[0016] Agrobacterium sp. ZCC3656 is activated, seed cultured and fermented to obtain a fermentation liquid; preferably, the fermentation temperature is 28-32° C. and the fermentation time is ≥3 days.

[0017] Agrobacterium sp. ZCC3656 was grown in a medium containing 3 wt% sucrose and 0.1 wt% NH 4 The fermentation was carried out in M9 medium (pH 7.2) containing 3 wt % mannitol and 0.1 wt % glutamic acid.

[0018] Preferably, the alcohol solvent is at least one of isopropanol and ethanol.

[0019] Preferably, the volume ratio of the fermentation broth to the alcohol solvent is 1:2.5-5.

[0020] Preferably, the volume ratio of the supernatant after the reaction to the alcohol solvent is 1:2.5-5.

[0021] Preferably, the temperature of the hydrolysis reaction is 100-110° C., and the time of the hydrolysis reaction is 15-30 min.

[0022] Preferably, the pH of the hydrolysis reaction is 8.8-9.2.

[0023] Preferably, the pressure of the hydrolysis reaction is 5-45 kPa.

[0024] The crude sugar Riclin contains a large amount of protein and other impurities, and the protein can be removed by adding an inorganic base for reaction; the extraction method can quickly extract the natural polysaccharide Riclin, which is suitable for industrial production.

[0025] Beneficial effects:

[0026] It has been found that the natural polysaccharide Riclin has biological functional activities such as inhibiting tumor growth, anti-oxidation, and improving hypercholesterolemia. However, no research has been found on the prevention and treatment of gastric ulcers by the natural polysaccharide Riclin. Due to the wide variety of polysaccharides, different types of polysaccharides have different structural formulas, and even the structures of many polysaccharides have not been determined. Not all polysaccharides have the effect of preventing and treating gastric ulcers. Their mechanisms of action and targets in the body are different. In addition, polysaccharides have large molecular weights and complex structures. They are not easily absorbed when taken orally, and have low bioavailability. Therefore, those skilled in the art do not know which polysaccharides have the effect of preventing and treating gastric ulcers.

[0027] The inventors surprisingly found through research that the natural polysaccharide Riclin has a good effect in preventing and treating gastric ulcers. The present invention dissolves the natural polysaccharide Riclin in physiological saline, which can maintain its own anti-inflammatory and antioxidant effects to the greatest extent. It is used to prevent and treat gastric ulcer diseases, which can further inhibit gastric ulcer surface lesions and reduce ulcer damage, and its bioavailability is high.

[0028] In addition, the present invention uses the natural polysaccharide Riclin as an oral preparation, which can inhibit mast cell activity, reduce gastric acid secretion, promote the production of protective mucus, and improve the protective effect on gastric mucosa; at the same time, it can promote the first-pass metabolism of ethanol, and further prevent and treat gastric ulcer disease. The preparation method of the present invention is simple, suitable for industrial mass production, and low in price. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1These are the gastric tissue appearance pictures and ulcer evaluations obtained from each group in Example 2.

[0030] Figure 2 Optical microscopic images of HE sections of gastric tissues in each group.

[0031] Figure 3 The optical microscopic images of PAS staining of gastric tissues in each group.

[0032] Figure 4 Fluorescence microscopy images of gastric tissues and quantitative results of gastric acid secretion-related proteins of each group, where a is a fluorescence microscopy image of gastric tissues, and bd are the quantitative results of Chymase, Muc6, and CAT, respectively.

[0033] Figure 5 Are the detection results of antioxidant-related markers in each group, where a is MPO, b is CAT, c is SOD, d is MDA, e is GSH, and f is NO.

[0034] Figure 6 These are the detection results of inflammatory factors in each group, where a is TNF-α, b is IL-6, c is IL-1β, and d is IFN-γ. DETAILED DESCRIPTION

[0035] The technical solutions of the present invention are described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.

[0036] Example 1 (Extraction of natural polysaccharide Riclin)

[0037] The extraction method of natural polysaccharide Riclin comprises the following steps:

[0038] Take Agrobacterium sp. ZCC3656, with a collection number of CCTCC NO: M2018797, and place its colonies on an activation medium (the formula of which is: containing 2.5 mM -1 MgSO 4 and 2.5 mM -1 CaCl 2 The colonies were then inoculated into LB liquid medium and seed cultured at 30°C for 48 h.

[0039] 2% (v / v) of the seed culture was transferred to the fermentation broth (containing 3 wt% sucrose and 0.1 wt% NH 4Cl M9 medium or M9 medium containing 3wt% mannitol and 0.1wt% glutamic acid, pH 7.2), and then incubated in a rotary shaker incubator at 30°C and 250rpm for 3 days; taking the fermentation broth, adding 3 times the volume of isopropanol for alcohol precipitation, and taking the precipitate and drying it to obtain crude sugar Riclin;

[0040] The crude sugar Riclin was prepared into an aqueous solution with a concentration of 0.01 g / mL, and the pH was adjusted to 9.0 with a NaOH aqueous solution. The solution was treated with high-pressure steam at 105°C for 20 min, and then centrifuged at 9000 rpm for 30 min to obtain a supernatant. Three times the volume of ethanol was added at room temperature for alcohol precipitation. The precipitate was dried to obtain the natural polysaccharide Riclin, whose average molecular weight was 2.5×10 6 Da.

[0041] Example 2 (Pharmacological Experiment on the Effect of Natural Polysaccharide Riclin on Preventing Gastric Ulcer)

[0042] Reagents: Omeprazole (OME) was purchased from Mailin Biochemical Technology Co., Ltd. (Shanghai, China); anhydrous ethanol was purchased from Ante Food Co., Ltd. (Anhui, China); TNF-α, IL-6, IL-1β, and TNF-γ enzyme-linked immunosorbent assay (ELISA) kits were provided by Lianke Biotechnology Co., Ltd. (Hangzhou, China); superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), glutathione (GSH), nitric oxide (NO), and catalase (CAT) detection kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0043] Animals: 60 Kunming mice, male, weighing 20-22 g, were randomly divided into groups.

[0044] Animal experiment: After the mice adapted to the environment, they were randomly divided into 6 groups, with 10 mice in each group, namely Control, Model, OME (20 mg / kg) group, Riclin5 (5 mg / kg) group, Riclin10 (10 mg / kg) group, and Riclin20 (20 mg / kg) group.

[0045] Experimental process:

[0046] The mice in the Control group were gavaged with normal saline at 10 am every day. The dose of normal saline was 0.01 ml / mg according to the body weight of the mice, and the gavage lasted for 14 days.

[0047] The mice in the Model group were gavaged with normal saline at 10 am every day. The dose of normal saline was 0.01 ml / mg according to the body weight of the mice, and the gavage lasted for 14 days.

[0048] Omeprazole (OME) was dissolved in normal saline at a concentration of 2 mg / ml, and vortexed for 3 min until evenly mixed to obtain the omeprazole solution. The OMEprazole solution was given to the mice in the OME group by oral gavage at 10 am every day. The dose of omeprazole solution was 0.01 ml / mg according to the mouse body weight, and the gavage was continued for 14 days.

[0049] The natural polysaccharide Riclin prepared in Example 1 was taken and dissolved in physiological saline to prepare natural polysaccharide Riclin solutions with concentrations of 0.5, 1, and 2 mg / ml, respectively. The 0.5, 1, and 2 mg / ml natural polysaccharide Riclin solutions were gavaged to the mice in the Riclin 5, Riclin 10, and Riclin 20 groups at 10 am every day. The dose was 0.01 ml / mg according to the weight of the mice, and the gavage was continued for 14 days.

[0050] After the gavage on the 14th day, all mice were fasted and allowed to drink water freely. After fasting for 24 hours, all mice except the control group were gavaged with 0.15 ml / 10 mg of anhydrous ethanol. After 3 hours of induction, the mice were killed by cervical dislocation.

[0051] After the mice were killed, the stomach tissue was immediately opened along the greater curvature of the stomach, the stomach surface was carefully rinsed with ice PBS, and the stomach tissue was fixed with paraformaldehyde. The gross changes in the appearance of the stomach tissue were observed, and the appearance pictures were recorded with a digital camera (Nikon Z50). The ulcer conditions on the stomach surface were graded and scored.

[0052] The evaluation criteria of the ulcer index (UI) are as follows: no damage; 1-mild erythema on the mucosal surface; 2-small ecchymosis (<1mm); 3-moderate ecchymosis (1mm-2mm); 4-large ecchymosis (2mm-3mm); 5-extensive damage (≥3mm); 3-ulcer streak length (<1mm); 4-ulcer streak length (1mm-2mm); 5-ulcer streak length (≥2mm); if the streak width is greater than 1mm, the score is doubled. The ulcer index was used to evaluate the preventive effect of polysaccharide Riclin on gastric ulcers; the results are as follows Figure 1 shown.

[0053] Figure 1 These are the gastric tissue appearance pictures and ulcer evaluations obtained from each group in Example 2.

[0054] Remark: Figure 1 middle # Indicates significant difference compared with the Control group (P<0.05), among which, # indicates P < 0.05, ## indicates P < 0.01, ###Indicates P<0.001; * indicates significant difference compared with the Model group (P<0.05), where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; & Compared with the OME group, the difference was significant (P<0.05). & indicates P < 0.05, && indicates P < 0.01, &&& Indicates P<0.001.

[0055] Depend on Figure 1 a It can be seen that there was no obvious morphological change on the surface of gastric tissue in the Contorl group, the mucosa was uniform and undamaged, and the surface was pink; the Model group showed morphological changes after ethanol treatment, accompanied by severe mucosal damage, hemorrhagic erythema, extensive ulcer erosion and submucosal edema; in the OME group, after pretreatment in the OME group, compared with the Model group, the ulcer formation area was significantly reduced, and the hemorrhagic erythema stripes were reduced. In the Riclin5, 10, and 20 groups, the gastric surface lesions were significantly reduced after treatment with the polysaccharide Riclin, with almost no severe hemorrhagic erythema, and the submucosal edema was reduced, and it showed a certain dose dependence.

[0056] Depend on Figure 1 b It can be seen that: the Contorl group had no ulcer lesions. According to the above ulcer index method, the ulcer index of the Contorl group was 0; the ulcer index of the Model group was 8.67; the ulcer index of the OME group was 5.67, which was significantly lower than that of the Model group; the ulcer index of the Riclin5 group was 6.17, which showed a downward trend compared with the ulcer index of the Model group (P < 0.05); the ulcer index of the Riclin10 and 20 groups was 4.67 and 3.5, respectively, which was significantly lower than that of the Model group; Figure 1 As shown in b, the ulcer index of Riclin 5, 10, and 20 groups showed dose dependence.

[0057] Example 3

[0058] After the mice in Example 2 were killed, the stomach tissue was immediately opened along the greater curvature of the stomach, and the stomach surface was carefully rinsed with ice PBS. The tissue was fixed with paraformaldehyde, and alcohol was used as a dehydrating agent from low to high concentrations to gradually remove the water in the tissue block. The tissue block was then placed in xylene, a transparent agent that is soluble in both alcohol and paraffin, to make it transparent. The alcohol in the tissue block was replaced with xylene before it was immersed in wax and embedded. The transparent tissue block was placed in the melted paraffin and placed in a wax melting box for insulation. After the paraffin has completely immersed in the tissue block, the embedded wax block was fixed on a microtome, cut into thin slices, generally 5-8mm thick, and dried in a constant temperature box at 45°C. The paraffin in the slice was removed with xylene, and then it was sequentially passed through high to low concentrations of alcohol, and finally put into distilled water for HE staining. Observe and scan under an optical microscope. The results are as follows Figure 2 shown.

[0059] Figure 2 Optical microscopic images of HE sections of gastric tissues in each group.

[0060] Figure 2 The results reflected the differences in gastric histology between the different groups. The gastric tissue results of the Control group were good in morphology, with intact mucosa, no loss of epithelial cells, and no hemorrhagic damage. Compared with the Control group, the Model group showed obvious tissue damage, severe epithelial cell loss (black arrow), hemorrhagic damage, and mucosal edema (arrow in the middle of the Model group), and a large number of inflammatory cells were found (arrow on the far right of the Model). The OME group, Riclin5, 10, and 20 groups showed that OME and Riclin intervention could effectively improve the histological damage induced by anhydrous ethanol, and compared with the Model group, epithelial cell loss and inflammatory cells were reduced. No mucosal edema, no hemorrhagic damage, and intact tissue mucosa were found in the Riclin20 group.

[0061] Example 4 (Determination of gastric juice secretion and gastric acid concentration in mice)

[0062] After the mice in Example 2 were killed, the stomach was opened along the greater curvature to collect the stomach contents, centrifuged at 3000 rpm for 10 min, and the pH and mucus weight were measured. The test results are shown in Table 1.

[0063] Table 1 Results of the measurement of pH value and mucus quality of ethanol-induced gastric ulcer in mice by Riclin

[0064] Experimentgroup pH Mucus weight (mg) Control 6.50±0.47 278.02±28.55 Model <![CDATA[2.50±0.42 # ]]> <![CDATA[103.63±13.83 # ]]> OME 4.63±0.29* 184.89±16.12* Riclin 5mg / kg 4.47±0.38* 174.04±17.48* Riclin 10mg / kg <![CDATA[5.49±0.54* & ]]> 177.25±15.26* Riclin 20mg / kg <![CDATA[5.89±0.47* & ]]> 213.89±13.61*

[0065] Note: In the table # Indicates significant difference compared with the Control group (P<0.05); *Indicates significant difference compared with the Model group (P<0.05); &It indicated that the difference was significant compared with the OME group (P<0.05).

[0066] As can be seen from Table 1: The gastric juice pH of the Control group was 6.50±0.47, which was consistent with the acid-base environment of the normal mouse stomach, and the gastric environment was stable; after ethanol treatment, the gastric acid secretion in the Model group increased and the pH value decreased, and the gastric juice pH of the OME group was 2.50±0.42 (P<0.001); after treatment with OME and Riclin homogenized solution, the acidic environment in the stomach was improved, and the gastric pH value was increased. The pH values ​​of the OME group, Riclin5, 10, and 20 groups were 4.63±0.29, 4.47±0.38, 5.49±0.54, and 5.89±0.47, respectively (P<0.05).

[0067] The large amount of gastric acid secretion will destroy the balance of the gastric environment, and the mucus in the stomach forms a "mucus-bicarbonate" barrier, which can protect the mucosa from gastric acid damage. The weight of mucus collected in the Control group was 278.02±28.55mg, which was consistent with the gastric mucus level of normal mice; due to the increased gastric acid secretion, the gastric mucus consumption increased and the gastric mucus production decreased, and the gastric mucus weight of the Model group was 103.63±13.83mg (P<0.001); in the OME group, Riclin5, 10, and 20 groups, due to the treatment intervention of OME and Riclin, the gastric mucus loss was improved, and the gastric mucus levels were 184.89±16.12mg, 174.04±17.48mg, 177.25±15.26mg, and 213.89±13.61mg, respectively (P<0.05).

[0068] Example 5

[0069] After the mice in Example 2 were killed, the stomach tissue was immediately opened along the greater curvature of the stomach, and the stomach surface was carefully rinsed with ice PBS, fixed with paraformaldehyde, and paraffin sections were prepared according to Example 3. The sections were dewaxed until the end and washed with distilled water, and then rinsed with periodic acid solution and tap water for 10 minutes, Schiff's solution for 10 minutes, and running water. The nuclei were stained with Harry's hematoxylin or Mayer's hematoxylin, and after running water rinsing, they were routinely dehydrated, transparentized, and sealed. They were used to detect carbohydrates in gastric tissue. The results are shown in Figure 3 shown.

[0070] Figure 3 The optical microscopic images of PAS staining of gastric tissues in each group.

[0071] Figure 3The results reflect the PAS staining analysis of gastric tissues in different groups; the mucosal epithelium of the Control group was intact, and the PAS-stained gastric mucosal epithelial cytoplasm was purple-red; PAS staining of the Model group showed that the mucosal epithelial cytoplasm was mostly blue, and the purple was significantly reduced; the purple reaction of the mucosal epithelial cells in the OME group and the Riclin5 group did not change significantly compared with the Model group; the mucosal epithelial cells in the Riclin10 and 20 groups had an increase in mucous cells, a significant increase in purple expression, and an increase in the secretion of neutral mucus; the anterior gastric mucosal epithelium is composed of columnar cells and mucous cells, and the cytoplasm of the anterior gastric mucosal mucous cells and gastric glandular type I cells is purple-red, indicating that it contains mixed neutral and acidic mucous substances, while the cytoplasm of the cells is blue, indicating that it contains acidic mucous substances. The arrows point to the purple reaction of PAS glycogen.

[0072] From this we can see that the natural polysaccharide Riclin can promote the production of protective mucus.

[0073] Example 6

[0074] 5mm thick sections were prepared from gastric tissues of different groups. Paraffin sections were washed with distilled water and placed in a repair box filled with EDTA antigen repair buffer (PH8.0) for antigen repair in a microwave oven. Medium heat for 8 minutes, stop for 8 minutes and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the sections should not be dried. After natural cooling, the slides were placed in PBS (PH7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time. After the slices were slightly dried, a circle was drawn around the tissue with a tissue pen, and BSA was added to the circle and incubated for 30 minutes. The blocking solution was gently shaken off, and the primary antibodies (Chymase, Muc6, CAT) prepared in a certain proportion in PBS were added to the slices. The slices were placed flat in a wet box and incubated overnight at 4°C. The slides were placed in PBS (pH 7.4) and shaken on a decolorizing shaker for 3 times, 5 minutes each time. The slices were slightly dried, covered with secondary antibodies in a certain proportion, and incubated at room temperature for 50 minutes. The cell nuclei were re-stained with DAPI, the autofluorescence was quenched, and the anti-fluorescence quenching sealant was used to seal. The fluorescence was placed, and the slices were photographed under a fluorescence microscope. Fluorescence quantitative analysis was performed using ImageJ software. The results are shown in Figure 4 shown.

[0075] Figure 4 Fluorescence microscopy images of gastric tissues and quantitative results of gastric acid secretion-related proteins of each group, where a is a fluorescence microscopy image of gastric tissues, and bd are the quantitative results of Chymase, Muc6, and CAT, respectively.

[0076] Note: In the figure # Compared with the Control group, the difference was significant (P<0.05). # indicates P < 0.05,## indicates P < 0.01, ### Indicates P<0.001; * indicates significant difference compared with the Model group (P<0.05), where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; & Compared with the OME group, the difference was significant (P<0.05). & indicates P < 0.05, && indicates P < 0.01, &&& Indicates P<0.001.

[0077] Increased Chymase expression indicates increased mast cell activation. On the tissue surface, mast cell activation will produce a large amount of hydrochloric acid, damaging the surface of gastric tissue.

[0078] like Figure 4 As shown in a, in the Contorl group, Chymase was basically not expressed on the surface of gastric tissue, and the fluorescence expression was low; Figure 4 As shown in b, the expression of Chymase in the Model group (193.56) was significantly increased compared with the Contorl group (34.78); the expression of Chymase in the OME group (74.39) and the Riclin 20 group (31.25) was significantly decreased compared with the Model group, inhibiting ethanol-induced mast cell activation.

[0079] Muc6 is a major mucin in gastric mucus and has the function of protecting gastric tissue from damage.

[0080] like Figure 4 As shown in c, the expression of Muc6 in the Model group (417.48) was decreased compared with that in the Contorl group (1002.01); compared with the Model group, the expression of Muc6 in the OME group (642.32) and the Riclin 20 group (797.76) was increased after treatment with OME and Riclin solutions.

[0081] CAT plays an important role in the first-pass metabolism of alcohol.

[0082] like Figure 4 As shown in Figure d, the CAT content in the Model group decreased significantly (1420.08→242.28), while the CAT content in the OME group (1321.98) and the Riclin 20 group (1809.36) increased significantly compared with the Model group. The increase in CAT in gastric tissue indicates a stronger first-pass metabolism of alcohol, which further weakens the damaging effect of alcohol on the stomach.

[0083] From this we can see that the natural polysaccharide Riclin can inhibit mast cell activation, reduce gastric acid secretion, promote the production of protective mucus, and improve the protective effect on the gastric mucosa; at the same time, it can promote the first-pass metabolism of ethanol, and further prevent and treat gastric ulcer disease.

[0084] Example 7

[0085] The gastric tissue samples of each group of mice were accurately weighed, and the gastric tissue weight (g): saline volume (mL) = 1:9, and saline was added to the tissue for homogenization. The tissue was centrifuged at 2500rpm / min for 10 minutes, and the supernatant was taken to measure oxidative stress and antioxidant related markers. Superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), glutathione (GSH), nitric oxide (NO), and catalase (CAT) detection kits were provided by Nanjing Jiancheng Bioengineering Institute. The results are shown in Figure 5 shown.

[0086] Figure 5 Are the detection results of antioxidant-related markers in each group, where a is MPO, b is CAT, c is SOD, d is MDA, e is GSH, and f is NO.

[0087] Note: In the figure # Compared with the Control group, the difference was significant (P<0.05). # indicates P < 0.05, ## indicates P < 0.01, ### Indicates P<0.001; * indicates significant difference compared with the Model group (P<0.05), where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; & Compared with the OME group, the difference was significant (P<0.05). & indicates P < 0.05, && indicates P < 0.01, &&& Indicates P<0.001.

[0088] Ethanol injury is often accompanied by local ischemia and hypoxia of the gastric mucosa. The gastric mucosa produces many reactive oxygen free radicals (ROS). Excessive ROS can cause oxidative stress, including lipid peroxidation, protein oxidation and DNA damage. In addition, ROS can act as second messengers to activate different redox-sensitive signal transduction pathways, leading to tissue inflammation and damage.

[0089] like Figure 5The results of a, d, and f showed that due to the damage caused by ethanol, the levels of MPO (2.83→8.11), MDA (3.76→8.82), and NO (2.20→5.14) in the Model group were significantly increased, while in the Riclin 20 group, the levels of the above three oxidative stress factors were reduced.

[0090] like Figure 5 As shown in b, ethanol injury reduced the CAT level in gastric tissue, Model group (18.96), Control group (63.75); Riclin 20 group (56.52) improved the damaging effect of ethanol on the stomach and weakened the reduction of CAT level.

[0091] like Figure 5 As shown in c and e, compared with the Model group, the Riclin 20 group also weakened the gastric ulcer damage, and the levels of SOD and GSH were significantly increased.

[0092] Example 8

[0093] After collecting blood from the orbits of mice in each group, the orbital blood was placed in a 1.5 mL centrifuge tube and allowed to stand at room temperature for 30-60 min. The blood samples were centrifuged at a relative centrifugal force of 14000 g for 10-15 minutes, and the serum was separated by a pipette for the detection of inflammatory factors (IL-1β, IL-6, TNF-α, IFN-γ). Inflammatory indicators were detected by ELISA kits. TNF-α, IL-6, IL-1β and TNF-γ enzyme-linked immunosorbent assay (ELISA) kits were provided by Lianke Biotechnology Co., Ltd. (Hangzhou, China). The results are shown in the figure. Figure 6 shown.

[0094] Figure 6 These are the detection results of inflammatory factors in each group, where a is TNF-α, b is IL-6, c is IL-1β, and d is IFN-γ.

[0095] Note: In the figure # Compared with the Control group, the difference was significant (P<0.05). # indicates P < 0.05, ## indicates P < 0.01, ### Indicates P<0.001; * indicates significant difference compared with the Model group (P<0.05), where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001; & Compared with the OME group, the difference was significant (P<0.05). & indicates P < 0.05, && indicates P < 0.01, &&& Indicates P<0.001.

[0096] like Figure 6 The results showed that compared with the Model group, the Riclin 20 group reduced the level of inflammation induced by ethanol damage, and the levels of IL-1β, IL-6, TNF-α, and IFN-γ were significantly decreased. Ethanol-induced gastric mucosal damage can lead to severe inflammatory reactions and increase the levels of inflammatory factors.

[0097] In summary, the natural polysaccharide Riclin has the effect of preventing and treating gastric ulcers. The natural polysaccharide Riclin can be prepared into an oral preparation, which can reduce the damage of gastric ulcers induced by ethanol; inhibit mast cell activation to reduce gastric acid secretion, promote the production of Muc6 mucin, and promote the formation of gastric protective mucus barrier; increase CAT levels to enhance the first-pass metabolism of ethanol, and reduce ethanol-induced oxidative stress and inflammatory response.

[0098] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of a natural polysaccharide Riclin in the preparation of drugs for preventing and treating gastric ulcers by inhibiting mast cell activation, promoting Muc6 mucin production, and increasing CAT levels; Gastric ulcer is acute alcoholic gastric ulcer; Riclin is a natural polysaccharide isolated from Agrobacterium Agrobacterium Exopolysaccharides extracted from sp. ZCC3656; The average molecular weight of natural polysaccharide Riclin is 2×10 6 -3×10 6 Da; The method for extracting natural polysaccharide Riclin comprises the following steps: taking Agrobacterium Agrobacterium sp. ZCC3656 fermentation liquid, add alcohol solvent to carry out alcohol precipitation, take the precipitate and dry to obtain crude sugar Riclin; take the aqueous solution of crude sugar Riclin, carry out hydrolysis reaction with inorganic base, then take the supernatant after the reaction, carry out alcohol precipitation with alcohol solvent, take the precipitate to obtain natural polysaccharide Riclin.

2. The use of the natural polysaccharide Riclin according to claim 1 in the preparation of a drug for preventing and treating gastric ulcer, characterized in that: The dosage form of the drug is an oral dosage form.

3. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: The medicine is natural polysaccharide Riclin oral solution.

4. The use of the natural polysaccharide Riclin according to claim 3 in the preparation of a drug for preventing and treating gastric ulcer, characterized in that: The content of natural polysaccharide Riclin in the natural polysaccharide Riclin oral solution is 0.5-2 mg / ml.

5. The use of the natural polysaccharide Riclin according to claim 3 in preparing a drug for preventing and treating gastric ulcer, characterized in that: The solvent of natural polysaccharide Riclin oral solution is normal saline.

6. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: The alcohol solvent is at least one of isopropanol and ethanol.

7. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: Agrobacterium Agrobacterium When an alcohol solvent is added to the fermentation broth of sp. ZCC3656 for alcohol precipitation, the volume ratio of the fermentation broth to the alcohol solvent is 1: 2.5-5.

8. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: When the supernatant after the reaction is taken and alcohol precipitation is performed with an alcohol solvent, the volume ratio of the supernatant after the reaction to the alcohol solvent is 1: 2.5-5.

9. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: The temperature of the hydrolysis reaction is 100-110°C, and the time of the hydrolysis reaction is 15-30 min.

10. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: The pH of the hydrolysis reaction is 8.8-9.

2.

11. The use of the natural polysaccharide Riclin according to claim 1 in preparing a drug for preventing and treating gastric ulcer, characterized in that: The pressure of the hydrolysis reaction is 5-45 kPa.

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