Application of rhizoma bletillae polysaccharide in preparation of medicine for treating gastric mucosal lesion and preparation method of rhizoma bletillae polysaccharide

Abstract

The invention relates to application of rhizoma bletillae polysaccharide in preparation of a medicine for treating gastric mucosal lesion and a preparation method of the rhizoma bletillae polysaccharide, the rhizoma bletillae polysaccharide is prepared, and when an organism intakes excessive ethanol, the rhizoma bletillae polysaccharide can achieve the effect of preventing acute alcoholic gastric mucosal lesion by protecting the integrity of gastric mucosa. By regulating expression disorder of inflammatory factors, the inflammatory factors can be maintained at a normal level, so that a preventive protection effect on gastric mucosal lesion is achieved; a foundation is laid for further exploration of a repair mechanism of the rhizoma bletillae polysaccharide on gastric mucosal lesion, and an experimental basis is provided for further development and application of rhizoma bletillae polysaccharide health food or medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of drug development and analysis, and specifically relates to an application of Bletilla striata polysaccharide in preparing a drug for treating gastric mucosal injury and a preparation method thereof. Background Art

[0002] The stomach is an organ that performs digestive functions in the human body, and the main component of the stomach wall is the mucosal layer. With the rapid development of the economy, people's pace of life has gradually accelerated, and their lifestyle and diet have become irregular, resulting in an increasing incidence of gastric diseases in recent years. Most gastric diseases are related to gastric mucosal damage. According to epidemiological statistics, the incidence of chronic gastritis is as high as 80%-90%, the incidence of peptic ulcer accounts for 10%-12% of the population, and more than 160,000 patients die of gastric cancer each year. Gastric diseases have become a common disease in daily life, so it is particularly important to study the damage mechanism and protection principle of the gastric mucosa.

[0003] Bletilla striata is the dried tuber of the orchid plant Bletilla striata, which is naturally distributed in the Yangtze River Basin and the provinces and regions to the south of it. The main production areas of wild Bletilla striata are believed to be Guizhou, Guangxi Zhuang Autonomous Region, and Anhui. With the scarcity of wild resources, the planting area of ​​Bletilla striata has gradually expanded. Currently, Guizhou, Yunnan, Sichuan and other places are the main planting areas.

[0004] The pseudobulbus of the genus Bletilla is used as medicine. At present, the pharmacological research of this genus is mainly focused on Bletilla. Bletilla has been a traditional Chinese medicine for thousands of years. Its main functions include astringent hemostasis, swelling and muscle regeneration, immunity enhancement, antibacterial and anti-inflammatory, anti-tumor, etc. Coagulation and hemostasis: Bletilla can be widely used to treat vomiting blood, hemoptysis and traumatic bleeding. Studies have shown that Bletilla extract can significantly shorten the coagulation time. Treatment of ulcers: Bletilla polysaccharide can effectively treat ulcerative colitis, promote mucosal repair, inhibit inflammation, and balance the body's immunity. Studies have shown that the treatment of ulcerative colitis by Bletilla polysaccharide may be related to the promotion of TNF-α, IL-5, IL-10 and IL-13 secretion, and promote the proliferation of spleen lymphocytes in colitis mice. Studies have found that Bletilla polysaccharide can improve the body's immunity while treating colitis mice, thus concluding that Bletilla polysaccharide can inhibit inflammation and restore immune balance. Anti-tumor: Studies have shown that although the anti-tumor effect of Bletilla striata polysaccharide in vitro is not obvious, it has obvious anti-tumor effect in vivo and can enhance the immunity of mice. Some researchers have established an S180 tumor-bearing mouse model and observed the in vivo therapeutic effect of Bletilla striata polysaccharide. They found that Bletilla striata polysaccharide can slow down the growth rate of tumors and activate macrophages to secrete NO. Some researchers have found that Bletilla striata polysaccharide can significantly inhibit the growth of liver cancer cells through interventional treatment of experimental rat liver cancer cells. It is a natural and effective anti-cancer drug. Improve immunity: Studies have shown that Bletilla striata polysaccharide can improve the immune function of tumor-bearing mice and has an immunomodulatory effect. Research results show that Bletilla striata polysaccharide can significantly improve the phagocytic ability of macrophages and the proliferation ability of spleen lymphocytes in immunosuppressed mice. The experiment of Bletilla striata polysaccharide on the immune function of rats with cervical erosion showed that Bletilla striata can significantly restore the erosion of rat cervical epithelial tissue and balance the immune disorder caused by inflammation.

[0005] At present, the research on Bletilla striata mainly focuses on the treatment of immune regulation and anti-tumor, while the research on the functionality and preventive effect of Bletilla striata polysaccharide is rare. There are reports on the repair of gastric mucosa and gastric ulcer by Bletilla striata polysaccharide, but its repair mechanism is still unknown. Therefore, the research on the protective mechanism of Bletilla striata polysaccharide on gastric mucosa provides experimental basis for the development and research of Bletilla striata polysaccharide health food or medicine.

[0006] In view of the above problems, the present invention team adopts the extraction method of Bletilla striata polysaccharide optimized in the early stage, designs Bletilla striata polysaccharide of different dosage groups, and gavages SD rats for 28 days, fasts for 24 hours (water is not allowed), and then gavages SD rats with ethanol to establish an acute gastric mucosal injury model, and removes various tissue organs. A detailed pathological examination of the gastrointestinal tract is performed, and the length and width of the bleeding points or bleeding of the gastric tissue are observed according to the general observation and scoring criteria of anhydrous ethanol injury. The gastric tissue is also subjected to histopathological observation and scoring to obtain a pathological diagnosis. The contents of SOD, MDA, ET-1, TNF-α, PGE2, and EGF in gastric mucosal tissue are determined. The results show that Bletilla striata polysaccharide has a good preventive effect on acute alcoholic gastric mucosal injury, and the possible mechanism of action includes protecting the integrity of the gastric mucosa of the body, improving the oxidative stress state of the tissue, and inhibiting the expression of inflammatory factors, which lays the foundation for the next step of exploring the repair mechanism of Bletilla striata polysaccharide on gastric mucosal injury, and provides an experimental basis for the further development and application of Bletilla striata polysaccharide health food or medicine. Summary of the invention

[0007] The purpose of the present invention is to provide an application of Bletilla striata polysaccharide in the preparation of a drug for treating gastric mucosal damage;

[0008] Another object of the present invention is to provide a method for preparing Bletilla striata polysaccharide;

[0009] The invention discloses an application of the bletilla striata polysaccharide in preparing a medicine for treating gastric mucosal oxidative stress.

[0010] Preferably, the Bletilla striata polysaccharide of the present invention is used in the preparation of a drug for treating gastric mucosal inflammatory factors.

[0011] Further preferably, the gastric mucosal injury of the present invention is alcoholic gastric mucosal injury.

[0012] The Bletilla striata polysaccharide of the present invention is added with pharmaceutically acceptable adjuvants to prepare a pharmaceutically acceptable preparation; the preparation is freeze-dried powder, granules, capsules, tablets, pills, sprays, lozenges, dropping pills, and creams.

[0013] The preparation method of the Bletilla striata polysaccharide of the present invention is:

[0014] 1) Degreasing of Bletilla striata: taking Bletilla striata tubers, crushing them into powder, passing through a 100-mesh sieve to obtain Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, adding 5-7 times the amount of 70-90% ethanol, heating and refluxing extraction at 60-90° C. for degreasing, the extraction time is 1.5-2.5 hours, and filtering; the filter residue is subjected to a second degreasing treatment in the same manner, filtered, and the filter residue is placed in a blast drying oven and dried at 38-42° C. to obtain defatted Bletilla striata powder;

[0015] 2) Extraction of Bletilla striata polysaccharide: Take the defatted Bletilla striata powder, extract twice with Bletilla striata powder: water at a solid-liquid ratio of 1:30-40 g / ml and 1:20-25 g / ml respectively, stir for 50-70 seconds with a flash extractor each time, then heat in a water bath at 80-100°C for 2-4 hours, centrifuge the extract for 10-20 minutes at a speed of 4000 rpm, take the supernatant, concentrate it to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrates to obtain Bletilla striata concentrate;

[0016] 3) Deproteinization of Bletilla striata polysaccharide: freeze the Bletilla striata concentrated solution in a -80°C refrigerator, take it out after 6-10 hours to dissolve, centrifuge it in a centrifuge for 10-20 minutes at a speed of 4000 rpm, take out the supernatant and freeze-thaw it repeatedly 4-6 times to obtain the Bletilla striata concentrated solution with protein removed;

[0017] 4) Alcohol precipitation of Bletilla striata polysaccharide: accurately measure the deproteinized concentrate, mix the concentrate and 80-90% ethanol in a volume ratio of 1:6-9, slowly add 80-90% ethanol to the concentrate while stirring until white flocs appear, let stand for 5-7 hours, filter with suction, take the filter residue and dry it in a freeze dryer for 45-50 hours to obtain a dry product of Bletilla striata polysaccharide, grind it through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

[0018] Preferably, the defatting treatment of the Bletilla striata described in the present invention is: take the Bletilla striata tuber and crush it into powder, pass it through a 100-mesh sieve, take an appropriate amount of Bletilla striata powder, add 6 times the amount of 90% ethanol, heat and reflux to extract at 80°C for defatting treatment, the extraction time is 2h, and filter; the filter residue is defatted for the second time in the same way, filtered, and the filter residue is placed in a forced air drying oven and dried at 40°C to obtain defatted Bletilla striata powder.

[0019] Preferably, the extraction of Bletilla striata polysaccharide described in the present invention is as follows: take defatted Bletilla striata powder, take Bletilla striata powder: water with a solid-liquid ratio of 1:35 g / ml and 1:25 g / ml respectively, extract twice in sequence, stir for 60 seconds with a flash extractor each time, heat in a 90°C water bath for 3 hours, centrifuge the extract for 15 minutes at a speed of 4000 rpm, take the supernatant, and concentrate it to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrated solutions to obtain Bletilla striata concentrated solution.

[0020] Preferably, the deproteinization treatment of the Bletilla striata polysaccharide described in the present invention is: placing the Bletilla striata concentrated solution in a -80°C refrigerator for freezing, taking it out after 8 hours to dissolve, placing it in a centrifuge for centrifugation for 15 minutes at a speed of 4000 rpm, taking the supernatant and repeatedly freezing and thawing it 5 times to obtain the Bletilla striata concentrated solution with the protein removed.

[0021] Preferably, the alcohol precipitation treatment of the Bletilla striata polysaccharide described in the present invention is: accurately measure the deproteinized concentrated solution, mix the concentrated solution and 85% ethanol in a volume ratio of 1:8, slowly add 85% ethanol to the concentrated solution while stirring until white flocs appear, let it stand for 6 hours, filter with suction, take the filter residue and place it in a freeze dryer for 48 hours to obtain a dry product of Bletilla striata polysaccharide, and grind it through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

[0022] Beneficial effects:

[0023] 1. The Bletilla striata polysaccharide of the present invention has a good preventive effect on acute alcoholic gastric mucosal injury. The possible mechanisms of action include protecting the integrity of the gastric mucosa, improving the oxidative stress state of the tissue and inhibiting the expression of inflammatory factors. This lays the foundation for the next step of exploring the repair mechanism of Bletilla striata polysaccharide on gastric mucosal injury and provides an experimental basis for the further development and application of Bletilla striata polysaccharide health foods or medicines.

[0024] 2. The present invention uses a morphological observation experiment on the protective effect of Bletilla striata polysaccharide on ethanol-induced gastric mucosal damage in mice. The results show that the gastric mucosa of mice in the normal group is light yellow, without damage, smooth surface, many and neatly arranged gastric folds, and the stomach area is small after dissection. The positive control group (OMe) has visible gastric wall wrinkles, light yellow, with dot-shaped and linear lesions, and the degree of lesions is mild. Bletilla striata polysaccharide treatment group: The gastric wall of the high-dose group (500mg / kg) is light yellow, with a little erosion and bleeding, and linear and dot-shaped lesions can be seen. The surface is slightly shiny and the folds are clearer; the gastric wall of the medium-dose group (250mg / kg) has bleeding, which is spotted, linear and dot-shaped, and the degree of damage is serious; the gastric wall bleeding in the low-dose group (125mg / kg) is in the form of flakes, black and red in color, and interrupted folds. After giving ethanol, the inner wall of the stomach is severely damaged, dark red, with a large number of blood spots and few folds.

[0025] 2. The present invention conducted an experiment on the effect of Bletilla striata polysaccharide on the gastric injury index of each group of rats. The results showed that the gastric mucosal injury index of rats after ethanol treatment was 66.00±8.93, the highest among the six groups. This indicates that ethanol caused serious damage to the gastric mucosa. Compared with the model group, the gastric mucosal injury index of each drug-treated group was reduced. The high-dose protective effect was the best, with a significant difference (P<0.01), indicating that BSP showed a protective effect on ethanol-type gastric mucosal injury.

[0026] 3. The present invention conducted H&E staining experiments, and the results showed that compared with the model group, the inflammatory cells in the high-dose group of Bletilla striata polysaccharide were significantly reduced, the cells were arranged more neatly, and the bleeding was reduced; compared with the model group, the bleeding in the medium-dose group and the low-dose group was less, but the gastric mucosal cells in the low-dose group were seen to fall off, and there was obvious inflammatory cell infiltration. The positive control group was accompanied by a small amount of bleeding, cell damage, etc., and the inflammatory response was reduced. The gastric mucosal structure of the model group was disordered and unclear, the gastric mucosal epithelial cells were severely damaged, the epithelial cells fell off, there was a large amount of inflammatory cell infiltration, and the superficial layer of the gastric mucosa was congested, accompanied by edema.

[0027] 4. The present invention conducted an antioxidant factor experiment, and the results showed that compared with the control group, the SOD activity in the gastric tissue of the model group rats was significantly reduced (P < 0.01), and at the same time, the content of MDA lipid peroxide was significantly increased (P < 0.01). This shows that ethanol caused oxidative damage to the gastric mucosa. Compared with the model group, the group administered with Bletilla striata polysaccharide had improved SOD activity and inhibited MDA content, and the high-dose group had the most significant effect. The results showed that pre-administration of Bletilla striata polysaccharide to rats had a protective effect on gastric mucosal damage caused by ethanol. Compared with the model group, pre-administration of Bletilla striata polysaccharide increased the activity of SOD to varying degrees, and the high-dose group, medium-dose group, and low-dose group had differences (P < 0.01). Compared with the model group, the MDA content of the high, medium, and low-dose groups pretreated with Bletilla striata polysaccharide was lower than that of the model group (P < 0.01), which was significantly different. It can be seen that pre-administration of Bletilla striata polysaccharide can reduce the MDA content of rats with ethanol-type gastric mucosal damage.

[0028] 5. The present invention has passed the experiment of the effect of Bletilla striata polysaccharide on gastric mucosal protective factors and attack factors. The results show that compared with the control group, the PGE2 level in the serum of rats in the model group was significantly reduced (P < 0.01); compared with the model group, the PGE2 level in the serum of rats in the positive group was significantly increased (P < 0.01), and the PGE2 level in the serum of rats in the high-dose Bletilla striata polysaccharide group was increased, but the difference was not statistically significant; compared with the control group, the EGF content in the serum of rats in the model group was significantly reduced (P < 0.01); compared with the model group, the EGF content in the serum of rats in the Bletilla striata polysaccharide treatment group: high-dose group and medium-dose group was significantly increased (P < 0.05). It is proved that Bletilla striata polysaccharide components can protect gastric mucosa by increasing the content of gastric mucosal protective factors; compared with the control group, the ET-1 level in the serum of rats in the model group was significantly reduced (P < 0.05); compared with the model group, the ET-1 level in the serum of rats in the positive group was significantly increased (P < 0.05). The ET-1 levels in the serum of rats in the high-dose and medium-dose groups of Bletilla striata polysaccharide were increased, but the difference was not statistically significant.

[0029] 6. The present invention conducted an experiment on the effect of Bletilla striata polysaccharide on the serum NO and pepsin contents of rats in each group. The results showed that compared with the control group, the serum NO and gastric mucosal lesion tissue pepsin contents of rats in the model group were significantly increased; compared with the model group, the serum NO content of rats in the Bletilla striata polysaccharide high-dose group was decreased, and the pepsin contents of gastric mucosal lesion tissues in the Bletilla striata polysaccharide high-dose group and the medium-dose group were decreased; the serum NO content of rats in the positive group and the Bletilla striata polysaccharide high-dose group was significantly decreased, and the difference was statistically significant (P < 0.05); the serum NO and gastric tissue pepsin contents of rats in the Bletilla striata polysaccharide medium-dose group and the low-dose group were decreased, and the difference was not statistically significant (P > 0.05). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Glucose standard curve graph;

[0031] Figure 2 Animal growth charts;

[0032] Figure 3 Animal food intake chart;

[0033] Figure 4 Organ weight chart;

[0034] Figure 5 Diagram of gastric lesion measurement;

[0035] Figure 6 Gastric damage of Bletilla striata polysaccharide in rats of each group;

[0036] Figure 7 Gastric mucosal staining results;

[0037] Figure 8 Antioxidant factor comparison chart;

[0038] Fig. 9 Comparison of PGE2 in rat serum;

[0039] Fig.10 Comparison of EGF in rat serum;

[0040] Fig.11 Comparison of ET-1 in rat serum;

[0041] Fig.12 Comparison of rat serum NO;

[0042] Fig.13 Comparison of pepsin content in rats. DETAILED DESCRIPTION

[0043] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0044] Example 1 Preparation method of Bletilla striata polysaccharide

[0045] 1) Degreasing of Bletilla striata: taking Bletilla striata tubers, crushing them into powder, passing through a 100-mesh sieve to obtain Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, adding 6 times the amount of 90% ethanol, heating and refluxing at 80° C. for degreasing, the extraction time is 2 hours, and filtering; the filter residue is subjected to a second degreasing treatment in the same manner, filtered, and the filter residue is placed in a blast drying oven and dried at 40° C. to obtain defatted Bletilla striata powder;

[0046] 2) Extraction of Bletilla striata polysaccharide: Take the defatted Bletilla striata powder, extract twice with Bletilla striata powder: water at a solid-liquid ratio of 1:35 g / ml and 1:25 g / ml respectively, stir for 60 seconds with a flash extractor each time, then heat in a 90°C water bath for 3 hours, centrifuge the extract for 15 minutes at a speed of 4000 rpm, take the supernatant, and concentrate it to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrates to obtain Bletilla striata concentrate;

[0047] 3) Deproteinization of Bletilla striata polysaccharide: freeze the Bletilla striata concentrate in a -80°C refrigerator, take it out after 8 hours to dissolve, centrifuge it in a centrifuge for 15 minutes at a speed of 4000 rpm, take out the supernatant and freeze and thaw it repeatedly for 5 times to obtain the Bletilla striata concentrate with protein removed;

[0048] 4) Alcohol precipitation of Bletilla striata polysaccharide: Accurately measure the deproteinized concentrate, mix the concentrate and 85% ethanol in a volume ratio of 1:8, slowly add 85% ethanol to the concentrate while stirring until white flocs appear, let stand for 6 hours, filter with suction, take the filter residue and dry it in a freeze dryer for 48 hours to obtain the dry product of Bletilla striata polysaccharide, grind it through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

[0049] Example 2 Preparation method of Bletilla striata polysaccharide

[0050] 1) Degreasing of Bletilla striata: taking Bletilla striata tubers, crushing them into powder, passing through a 100-mesh sieve to obtain Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, adding 5 times the amount of 70% ethanol, heating and refluxing at 60° C. for degreasing, the extraction time is 1.5 h, and filtering; the filter residue is subjected to a second degreasing treatment in the same manner, filtered, and the filter residue is placed in a blast drying oven and dried at 38° C. to obtain defatted Bletilla striata powder;

[0051] 2) Extraction of Bletilla striata polysaccharide: Take the defatted Bletilla striata powder, extract twice with Bletilla striata powder: water at a solid-liquid ratio of 1:30 g / ml and 1:20 g / ml respectively, stir for 50 seconds each time with a flash extractor, heat in a water bath at 80°C for 2 hours, centrifuge the extract for 10 minutes at a speed of 4000 rpm, take the supernatant, concentrate to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrates to obtain Bletilla striata concentrate;

[0052] 3) Deproteinization of Bletilla striata polysaccharide: freeze the Bletilla striata concentrated solution in a -80°C refrigerator, take it out after 6 hours to dissolve, centrifuge it in a centrifuge for 10 minutes at a speed of 4000 rpm, take out the supernatant and freeze-thaw it 4 times to obtain the Bletilla striata concentrated solution with protein removed;

[0053] 4) Alcohol precipitation of Bletilla striata polysaccharide: Accurately measure the deproteinized concentrate and mix the concentrate: 80% ethanol in a volume ratio of 1:8. Slowly add 80% ethanol to the concentrate while stirring until white flocs appear. Let stand for 5 hours, filter with suction, and dry the residue in a freeze dryer for 45 hours to obtain a dry product of Bletilla striata polysaccharide. Grind and pass through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

[0054] Example 3 Preparation method of Bletilla striata polysaccharide

[0055] 1) Degreasing of Bletilla striata: taking Bletilla striata tubers, crushing them into powder, passing through a 100-mesh sieve to obtain Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, adding 7 times the amount of 90% ethanol, heating and refluxing at 90° C. for degreasing, the extraction time is 2.5 hours, and filtering; the filter residue is subjected to a second degreasing treatment in the same manner, filtered, and the filter residue is placed in a blast drying oven and dried at 42° C. to obtain defatted Bletilla striata powder;

[0056] 2) Extraction of Bletilla striata polysaccharide: Take the defatted Bletilla striata powder, extract twice with Bletilla striata powder: water at a solid-liquid ratio of 1:40 g / ml and 1:23 g / ml respectively, stir for 70 seconds each time with a flash extractor, heat in a water bath at 100°C for 4 hours, centrifuge the extract for 20 minutes at a speed of 4000 rpm, take the supernatant, concentrate to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrates to obtain Bletilla striata concentrate;

[0057] 3) Deproteinization of Bletilla striata polysaccharide: The Bletilla striata concentrated solution was placed in a -80°C refrigerator for freezing, and after 10 hours, it was taken out and dissolved, and centrifuged in a centrifuge for 20 minutes at a speed of 4000 rpm. The supernatant was taken and repeatedly frozen and thawed for 6 times to obtain the Bletilla striata concentrated solution with protein removed;

[0058] 4) Alcohol precipitation of Bletilla striata polysaccharide: Accurately measure the deproteinized concentrate, mix the concentrate and 90% ethanol in a volume ratio of 1:8, slowly add 90% ethanol to the concentrate while stirring until white flocs appear, let stand for 7 hours, filter with suction, take the filter residue and dry it in a freeze dryer for 50 hours to obtain the dry product of Bletilla striata polysaccharide, grind it through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

[0059] The polysaccharide of Bletilla striata prepared by any preparation method of Examples 1-3 is used in the preparation of the preparations of Examples 4-11

[0060] Example 4 Preparation

[0061] Take an appropriate amount of Bletilla striata polysaccharide, add 6 times the amount of water for injection and 0.05% sodium carboxymethyl starch according to the conventional preparation method of solid powder preparation, filter and freeze-dry to obtain freeze-dried powder.

[0062] Example 5 Preparation

[0063] Take an appropriate amount of Bletilla striata polysaccharide, add 0.3% silicon dioxide and 0.2% magnesium stearate and mix well according to the conventional preparation method of solid powder preparation, dry granulate, sieve the whole granules, mix well, and obtain granules.

[0064] Example 6 Preparation

[0065] Take an appropriate amount of Bletilla striata polysaccharide, add 6% pregelatinized starch, 5% microcrystalline cellulose and 2% talcum powder according to the conventional preparation method of solid powder preparation, mix well, put into capsules, and obtain capsules.

[0066] Example 7 Preparation

[0067] Take an appropriate amount of Bletilla striata polysaccharide, add 0.5% starch and 0.1% talcum powder according to the conventional preparation method of solid powder preparation, mix and granulate, and press to obtain tablets.

[0068] Example 8 Preparation

[0069] Take an appropriate amount of Bletilla striata polysaccharide, add 0.6% starch and 20% ethanol according to the conventional preparation method of solid powder preparations, and go through the process of demoulding → forming → covering → drying → pill selection → coating → obtaining pills.

[0070] Example 9 Preparation

[0071] Take an appropriate amount of Bletilla striata polysaccharide, add an appropriate amount of menthol, flavor, citric acid, sodium benzoate, ethanol and water according to the conventional preparation method of solid powder preparations, and prepare a spray.

[0072] Example 10 Preparation

[0073] Take an appropriate amount of Bletilla striata polysaccharide, spray 0.3% peppermint oil and 1% magnesium stearate into it according to the conventional preparation method of solid powder preparation, mix well, and prepare lozenges.

[0074] Example 11 Preparation

[0075] Take an appropriate amount of Bletilla striata polysaccharide, and according to the conventional method for preparing solid powder preparations, take an appropriate amount of polyethylene glycol 6000, heat to melt, mix it with Bletilla striata polysaccharide, drip it into pills, and remove the oil marks on the surface to obtain the product.

[0076] Example 12 Preparation

[0077] Take an appropriate amount of Bletilla striata polysaccharide, add the above-mentioned borneol and menthol eutectic according to the conventional preparation method of solid powder preparation, mix well, and prepare a cream.

[0078] Example 13 Application

[0079] Application of Bletilla striata polysaccharide in the preparation of drugs for treating gastric mucosal injury;

[0080] Application of Bletilla striata polysaccharide in the preparation of drugs for treating gastric mucosal oxidative stress;

[0081] Application of Bletilla striata polysaccharide in the preparation of drugs for treating gastric mucosal inflammatory factors.

[0082] Experimental example: In order to prove the scientificity and rationality of the present invention, the following methodological experimental studies were conducted:

[0083] 1 Materials

[0084] 1.1 Instrument

[0085]

[0086]

[0087] 1.2 Reagents

[0088]

[0089] 1.3 Experimental animals

[0090] Animal source: Hunan Slake Jingda Experimental Animal Co., Ltd., experimental animal production license number: SCXK (Xiang) 2019-0004, animal qualification certificate number: 430727230102332161. At the beginning of the experiment, the weight difference of the animals did not exceed 20% of the average weight of the same sex. Specific experimental animal information is shown in Table 1.

[0091] Table 1 Experimental animal information

[0092]

[0093] Experimental animal grouping and identification: Appropriate animals were selected according to the health status and weight of the animals during the adaptation period, and randomly grouped according to weight before the start of the experiment. Each animal had a unique animal number. Experimental animal breeding facilities and feeding management: The animals were raised in independent ventilated cages (IVC), with 5 animals per cage, free access to animal feed and drinking water, artificial lighting, 12 hours of light and 12 hours of no light alternating. The feed was Co60 feed for mice and rats provided by Beijing Keao Xieli Feed Co., Ltd., the animal drinking water was bottled water, and the irradiated sterilized corn cob bedding was produced by Xietong Biotechnology Co., Ltd.

[0094] 2 Preparation of Bletilla striata polysaccharide

[0095] 2.1 Degreasing treatment of Bletilla striata

[0096] The tuber of Bletilla striata was crushed into powder and passed through a 100-mesh sieve to obtain 2 kg of Bletilla striata powder. Extraction was carried out in 2 batches, and 1 kg of Bletilla striata powder was weighed in a 20L round-bottom flask for each batch, and 6L of 90% ethanol solution was added to the round-bottom flask, and the extraction was heated to reflux at 80°C for degreasing. The extraction time was 2h, and finally filtered with gauze. The filter residue was degreased for the second time in the same way. After filtering with gauze, the filter residue was used for standby. The second batch of Bletilla striata was degreased twice in the same way, and finally the two batches of filter residues were combined and placed in a forced air drying oven to dry at 40°C to obtain 1.8 kg of defatted Bletilla striata powder for the next step of extracting Bletilla striata polysaccharides.

[0097] 2.2 Extraction of Bletilla striata polysaccharides

[0098] After defatting, the Bletilla striata powder was extracted in 18 batches, 100 g of Bletilla striata powder was weighed for each batch, and each batch was extracted twice, with the ratio of Bletilla striata powder to water being 1:35 and 1:25, respectively. Each time, the Bletilla striata powder was stirred for 60 seconds with a flash extractor, and then heated in a 90°C water bath for 3 hours. Finally, the extract was centrifuged for 15 minutes at a speed of 4000 rpm, and the supernatant was taken and concentrated to 0.9-1.1 g / ml with a rotary evaporator, and the two concentrates were combined. The same method was used to extract Bletilla striata polysaccharides from the remaining batches of Bletilla striata powder, and all the concentrates were finally combined.

[0099] 2.3 Deproteinization of polysaccharides

[0100] The combined concentrated solution was placed in a -80°C refrigerator for freezing, taken out after 8 hours to dissolve, placed in a centrifuge for 15 minutes at 4000 rpm, and the supernatant was taken and repeatedly frozen and thawed 5 times to obtain a Bletilla striata concentrated solution with protein removed.

[0101] 2.4 Alcohol precipitation of Bletilla striata polysaccharide

[0102] Accurately measure the volume of the deproteinized concentrate, mix the concentrate and 85% ethanol at a volume ratio of 1:8, slowly add 85% ethanol to the concentrate while stirring until white floccules appear, let it stand for 6 hours, filter it with a suction filter, and take the residue. The residue is placed in a freeze dryer and dried for 48 hours to obtain the dried product of Bletilla striata polysaccharide, which is crushed to a certain particle size with a pulverizer and passed through a 100-mesh sieve to obtain 250g of Bletilla striata polysaccharide.

[0103] 3 Determination of the content of polysaccharides in Bletilla striata

[0104] 3.1 Preparation of standards

[0105] Accurately weigh 0.0100 g of glucose standard in a 100 mL volumetric flask, add water to the mark to obtain a standard solution with a final concentration of 0.100 mg / mL for later use.

[0106] 3.2 Drawing of standard curve

[0107] Accurately pipette 0, 2.0, 4.0, 6.0, 8.0, 10.0 mL of the prepared standard solution into a 10 mL volumetric flask, add water to the mark, and obtain concentrations of 0, 20, 40, 60, 80, 100 μg / mL, respectively. Then accurately pipette 1 mL of the standard solution of different concentrations into a 10 mL colorimetric tube, add 1 mL of 5% phenol solution in sequence, and quickly drop 5 mL of concentrated sulfuric acid (add vertically to the liquid surface, do not touch the wall of the test tube, so as to fully mix with the reaction solution, and slowly add along the inner wall of the colorimetric tube to avoid splashing and burning), shake well and place for 10 minutes, place in a 30°C water bath to react for 20 minutes, and measure absorbance at 490 nm. Use glucose mass concentration as the horizontal axis and absorbance as the vertical axis to develop a standard curve.

[0108] 3.3 Sample determination

[0109] Weigh 50 mg of Guizhou Meitan Bletilla striata polysaccharides (MT-1, MT-2, MT-3) prepared according to the method under "2", purchased Guizhou Bletilla striata polysaccharides (GZ-1, GZ-2, GZ-3), and purchased Shaanxi Bletilla striata polysaccharides (SX-1, SX-2, SX-3) respectively, and put them into a 100 mL volumetric flask, add water to make up to the scale, then pipette 1 mL of the above samples into a 10 mL volumetric flask, add water to make up to the scale, and then accurately pipette 1 mL of the sample solution into a 10 mL colorimetric tube, add 1 mL of 5% phenol solution in turn, and quickly add 5 mL of concentrated sulfuric acid dropwise (add vertically to the liquid surface, do not touch the wall of the test tube, so as to fully mix with the reaction solution, instead slowly add along the inner wall of the colorimetric tube to avoid splashing and burns), shake well and let stand for 10 minutes, place in a 30°C water bath to react for 20 minutes, and measure the absorbance at 490 nm.

[0110] 3.4 Calculation

[0111] Calculation formula for crude polysaccharides in test samples:

[0112]

[0113] Where:

[0114] X——Crude polysaccharide content (g / 100g)

[0115] w——the content of glucose in the sample solution to be measured (μg / mL)

[0116] m——Sample mass (g)

[0117] V1——The volume of the test solution at the last settling (mL)

[0118] V2——Volume of the test solution absorbed into the colorimetric tube (mL)

[0119] V3——Volume of sample during preparation (mL)

[0120] 0.9 - Correction factor for conversion of glucose to dextran

[0121] The absolute difference between two independent test results obtained under repeated conditions shall not exceed 10% of the arithmetic mean.

[0122] 3.5 Results of Bletilla striata polysaccharide content

[0123] A glucose standard curve was established, with the glucose concentration of each diluted group as the horizontal axis and the absorbance (A) value of each group measured as the vertical axis. The obtained curve is shown in the figure. Figure 1 .

[0124] Depend on Figure 1 It can be seen that the regression equation is: y = 92.982x-0.7156, R 2 =0.9992. The results showed that the linear range was good in the range of 0-100 μg / ml. The polysaccharide content was finally calculated to be 87.10% according to the glucose standard curve. The specific data are shown in Table 2.

[0125] Table 2 Bletilla striata polysaccharide content

[0126]

[0127] 4 Animal testing

[0128] 60 SD rats were randomly divided into six groups. The normal control group was not given any treatment, and was fed and drunk normally. The model control group was given pure water (10 ml / kg body weight). The positive control group was given omeprazole. The low-concentration drug treatment group: 125 mg / kg·WB / day of Bletilla striata polysaccharide. The medium-concentration drug treatment group: 250 mg / kg·WB / day of Bletilla striata polysaccharide. The high-concentration drug treatment group: 500 mg / kg·WB / day of Bletilla striata polysaccharide. After each oral gavage, the rats were fasted for 2 hours but not water. Different concentrations of Bletilla striata polysaccharide were gavaged continuously for 28 days, as shown in Table 3.

[0129] Table 3 Animal numbers and doses

[0130]

[0131] 4.1 Model Building

[0132] All experimental animals were fasted for 18 hours before the last oral gavage, but not water. 30 minutes after oral gavage, the model control group, positive control group, low-dose group, medium-dose group, and high-dose group (5 groups except the normal control group) were given oral gavage of anhydrous ethanol (1 ml / rat). One hour later, the rats in the normal control group were euthanized by carbon dioxide. After blood was collected from the abdominal aorta, organs were collected: heart, liver, spleen, lung, kidney, gonad, and stomach were weighed. The stomach, liver, and spleen were retained. The pylorus and cardia were ligated and placed in 10% neutral formalin solution for 10 minutes. Then, the stomach was cut open along the greater curvature, and the contents were washed away with 0.09% sodium chloride solution / PBS. The gastric mucosa was unfolded for observation, and the injury index and inhibition rate were measured and calculated and photographed. Tissue samples were taken from the stomach and liver (the stomach was divided into 4 parts: pathology, WB, ELISA, PCR; the liver was divided into 3 parts: -1, -2), and the tissues were frozen in cryopreservation tubes (the indicators for PCR detection were stored in enzyme-free cryopreservation tubes). The spleen, part of the stomach and liver tissues were fixed with 10% neutral formalin and set aside.

[0133] 4.2 Clinical observation and testing

[0134] 4.2.1 General clinical observation: Cage-side observation, including the time, degree and duration of changes in the skin, eyes, mucous membranes, respiratory system, circulatory system, nervous system, limb movements, behavioral patterns, etc.

[0135] 4.2.2 Animal body weight: Animals were weighed once before grouping and once a week during the experiment.

[0136] 4.2.3 Food intake: Food intake was measured once a week during the experiment. The method for measuring food intake was to add feed twice a week, weigh the remaining amount before each addition, and not add extra feed in the middle. The food intake was calculated based on the amount of food added and the amount of food left over. Food intake = (amount of food added - amount of food left over) / number of days between additions / number of animals. The number of animals in each cage was based on the number of animals alive when the remaining feed was measured.

[0137] 4.2.4 Results

[0138] 4.2.4.1 General observation and growth of animals:

[0139] During the experiment, the SD rats in each group were in good spirits, responsive, had a normal diet, had thick and shiny fur, and had brown stools. Since the normal control group was not given any treatment, and had normal drinking water and feeding, and no gavage, their weight increased rapidly after the second week. In the second, third, and fourth weeks, the weight of the control group increased significantly compared with the model group and the Bletilla striata treatment group, and the difference was statistically significant (P < 0.05). No significant differences were found in the other groups. Growth is shown in Figure 2 , specific weight data are shown in Table 4.

[0140] Table 4 Experimental animal weight record

[0141]

[0142] 4.2.4.2 Food intake:

[0143] There was no significant difference in the daily food intake of the control group, model group and Bletilla striata polysaccharide treatment group on days 0-6 and 7-13. The daily food intake of the control group, model group and Bletilla striata polysaccharide treatment group increased significantly on days 14-20, and the difference was statistically significant (P < 0.05). No significant difference was found in the other groups. For specific food intake, see Figure 3 .

[0144] 4.3 Pathological examination

[0145] 4.3.1 Dissection and macroscopic observation: Perform complete systematic dissection and detailed macroscopic observation on the experimental animals; record the autopsy findings in detail.

[0146] 4.3.2 Organ weighing: The animals planned for dissection were weighed before dissection. The absolute weights of the organs listed in the table were weighed and recorded during the dissection process. The organs were then stored in 10% neutral buffered formalin fixative for fixation. HE staining was used to observe the pathological morphological changes of the stomach in different components. The tissues / organs that were removed, weighed, fixed, and subjected to histopathological examination are shown in Table 5.

[0147] Table 5 Pathological examination tissue table

[0148]

[0149] 4.3.3 Number of inflammatory cell infiltration: The number of infiltrating neutrophils, lymphocytes, plasma cells, etc. in the mucosal lamina propria was counted in different visual fields, and the average was taken as the number of inflammatory cell infiltration in this group.

[0150] 4.3.4 Gross anatomical results

[0151] Results of organ weight: There was no significant difference in heart weight between the control group and the model group, and between the model group and the group treated with Bletilla striata polysaccharide. The liver weight of the model group was significantly reduced compared with that of the control group, and the difference was statistically significant (P < 0.05). The liver weight of the model group and the high-dose Bletilla striata polysaccharide group was significantly reduced, and the difference was statistically significant (P < 0.05). There was no significant difference in spleen weight between the control group and the model group, and between the model group and the group treated with Bletilla striata polysaccharide. There was no significant difference in lung weight between the control group and the model group, and between the model group and the group treated with Bletilla striata polysaccharide. The stomach weight of the high-dose Bletilla striata polysaccharide group, the positive group and the model group was significantly increased, and the difference was statistically significant (P < 0.05). There was no significant difference in gonad weight between the control group and the model group, and between the model group and the group treated with Bletilla striata polysaccharide. Figure 4 , Table 6.

[0152] Table 6 Weights of Organs of Experimental Animals

[0153]

[0154] 4.4 Gastric damage detection

[0155] The removed stomach was ligated at the pylorus and cardia, placed in 10% neutral formalin solution for 10 minutes, cut open along the greater curvature of the stomach, and the contents were washed away with 0.09% sodium chloride solution / PBS. The gastric mucosa was unfolded and observed, and the length and width of the injury were measured with a vernier caliper, the injury index was calculated, and photos were taken. Figure 5 . The injury index / inhibition rate was calculated according to the Guth standard. Guth score measurement standard: The sum of the injury scores is the injury index. Point bleeding, erosion, and injury length <1mm are scored as 1 point; linear bleeding, injury length 1mm are scored as 2 points, 1-2mm are scored as 3 points; 2-4mm are scored as 4 points; >4mm are scored as 5 points. When the above injury width is >1mm, the score is doubled.

[0156] 4.4.1 Morphological observation of the protective effect of Bletilla striata polysaccharide on ethanol-induced gastric mucosal injury in mice

[0157] The gastric mucosa of mice in the normal group was light yellow, without damage, smooth surface, many and neatly arranged gastric folds, and the stomach area was small after dissection. In the positive control group (OMe), the gastric wall folds were visible, light yellow, with dot-shaped and linear lesions, and the degree of lesions was mild. Groups treated with Bletilla striata polysaccharide: The gastric wall of the high-dose group (500 mg / kg) was light yellow, with a little erosion and bleeding, and linear and dot-shaped lesions were visible. The surface was slightly shiny and the folds were clearer; the gastric wall of the medium-dose group (250 mg / kg) had bleeding in spots, lines and dots, and the degree of damage was serious; the gastric wall bleeding in the low-dose group (125 mg / kg) was flake-shaped, black and red, and the folds were interrupted. After the administration of ethanol, the inner wall of the stomach was severely damaged, dark red, with a large number of blood spots, few folds, and intestinal adhesions.

[0158] 4.4.2 Effect of Bletilla striata polysaccharide on gastric injury index of rats in each group

[0159] The ulcer index of rats in the model group was compared with that in the control group, and the ulcer index increased significantly, and the difference was statistically significant (P < 0.01). The ulcer index of rats in the model group and the group treated with Bletilla striata polysaccharide was compared, and the degree of gastric mucosal damage in rats was gradually alleviated, and the ulcer index of rats with laminar glands in the high-dose Bletilla striata polysaccharide group was reduced, and the difference was statistically significant (P < 0.01). The number of ulcer finger scar groups in rats in the medium-dose Bletilla striata polysaccharide group and the low-dose Bletilla striata polysaccharide group was reduced, and the difference was not statistically significant (P > 0.05). Figure 6 .

[0160] The gastric mucosal injury index of rats after ethanol treatment was 66.00±8.93, the highest among the six groups. This indicates that ethanol caused serious damage to the gastric mucosa. Compared with the model group, the gastric mucosal injury index of each Bletilla striata polysaccharide treatment group was reduced. The high dose had the best protective effect, with a significant difference (P<0.01). The specific scores are shown in Table 7. This indicates that BSP has a protective effect on ethanol-induced gastric mucosal injury.

[0161] Table 7 Comparison of gastric mucosal injury index

[0162]

[0163] 4.5 ELISA

[0164] Sample collection: tissue samples were collected from the stomach (the same part was collected from each group), and the tissues were stored in cryopreservation tubes and stored in a -80°C low-temperature refrigerator for testing. The animals were fasted overnight the day before blood collection, and drinking water was not limited. The animals were anesthetized with carbon dioxide and blood was collected from the abdominal aorta. No anticoagulant was added to the whole blood. After standing for 1 hour, the serum was collected in an EP tube at 2000r / min low-temperature ultracentrifuge for 10 minutes and stored in a -80°C low-temperature refrigerator for testing.

[0165] ELISA kits were selected, and the double antibody (antigen) sandwich method was used to determine the content of rat PGE2, EGF, ET-1, TNF-α, NO, MDA, SOD, TNF-α, and IL-10 in the specimens. The purified rat antibody (antigen) was coated on the microplate to make a solid-phase antibody (antigen), and the detection index was added to the microwells coated with the monoclonal antibody in turn, and then combined with the HRP-labeled detection index antibody to form an antibody-antigen-enzyme-labeled antibody complex (antigen-antibody-enzyme-labeled antigen complex). After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color was positively correlated with the detection index in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a full-wavelength scanning microplate reader, and the content of each index in the sample was calculated by the standard curve, as shown in Table 8.

[0166] Table 8 Enzyme-linked immunosorbent assay table

[0167]

[0168] 4.5.1 H&E staining results

[0169] like Figure 7As shown in the figure, under a 400x microscope, it can be seen that the gastric mucosal structure of the normal group is relatively complete, the gastric mucosal cells are neatly arranged, and there is no inflammatory cell infiltration. Compared with the model group, the inflammatory cells in the high-dose group of Bletilla striata polysaccharide were significantly reduced compared with the model group, the cells were neatly arranged, and the bleeding was reduced; compared with the model group, the bleeding in the medium-dose group and the low-dose group was less, but the gastric mucosal cells in the low-dose group were seen to fall off, and there was obvious inflammatory cell infiltration. The positive control group was accompanied by a small amount of bleeding, cell damage, and reduced inflammatory response. The gastric mucosal structure of the model group was disordered and unclear, the gastric mucosal epithelial cells were severely damaged, the epithelial cells fell off, there was a large number of inflammatory cell infiltration, and the superficial layer of the gastric mucosa was congested, accompanied by edema.

[0170] 4.6 Enzyme-linked immunosorbent assay

[0171] 4.6.1 Antioxidant Factors

[0172] Depend on Figure 8 It can be seen that compared with the control group, the SOD activity in the gastric tissue of the rats in the model group was significantly reduced (P < 0.01), and at the same time, the content of MDA lipid peroxide was significantly increased (P < 0.01). This shows that ethanol caused oxidative damage to the gastric mucosa. Compared with the model group, the Bletilla striata polysaccharide administration group increased the SOD activity and inhibited the MDA content, and the high-dose group had the most significant effect. The results showed that the pre-administration of Bletilla striata polysaccharide to rats had a protective effect on gastric mucosal damage caused by ethanol. Compared with the model group, the pre-administration of Bletilla striata polysaccharide increased the SOD activity to varying degrees, and the high-dose group, medium-dose group, and low-dose group were different (P < 0.01). Compared with the model group, the MDA content of the high, medium, and low-dose groups of Bletilla striata polysaccharide pretreatment was significantly lower than that of the model group (P < 0.01). It can be seen that pre-administration of Bletilla striata polysaccharide can reduce the MDA content of rats with ethanol-induced gastric mucosal damage.

[0173] 4.6.2 Effects of Bletilla striata polysaccharide on gastric mucosal protective factors and attack factors

[0174] Depend on Fig. 9 It can be seen that compared with the control group, the PGE2 level in the serum of rats in the model group was significantly decreased (P < 0.01); compared with the model group, the PGE2 level in the serum of rats in the positive group was significantly increased (P < 0.01), and the PGE2 level in the serum of rats in the high-dose Bletilla striata polysaccharide group was increased, but the difference was not statistically significant.

[0175] Depend on Fig.10It can be seen that compared with the control group, the EGF content in the serum of the model group rats was significantly reduced (P < 0.01); compared with the model group, the EGF content in the serum of the positive group rats was significantly increased (P < 0.01), and the EGF content in the serum of the rats in the Bletilla polysaccharide treatment group: the high-dose group and the medium-dose group was significantly increased (P < 0.01). The results suggest that Bletilla polysaccharide components can protect the gastric mucosa by increasing the content of gastric mucosal protective factor.

[0176] Depend on Fig.11 It can be seen that compared with the control group, the ET-1 level in the serum of the rats in the model group was significantly reduced (P < 0.05); compared with the model group, the ET-1 level in the serum of the rats in the positive group was significantly increased (P < 0.05). The ET-1 level in the serum of the rats in the high-dose and medium-dose groups of Bletilla striata polysaccharide was increased, but the difference was not statistically significant.

[0177] 4.6.3 Effects of Bletilla striata polysaccharide on serum NO and pepsin levels in rats of each group

[0178] Depend on Fig.12 , Fig.13 It can be seen that compared with the control group, the serum NO content and gastric mucosal lesion tissue pepsin content of the rats in the model group were significantly increased; compared with the model group, the serum NO content of the rats in the high-dose Bletilla striata polysaccharide group was decreased, and the pepsin content of the gastric mucosal lesion tissue of the high-dose Bletilla striata polysaccharide group and the medium-dose Bletilla striata polysaccharide group was decreased, and the serum NO content of the rats in the high-dose Bletilla striata polysaccharide group was significantly decreased, and the difference was statistically significant (P<0.05). The serum NO and gastric tissue pepsin contents of the rats in the medium-dose Bletilla striata polysaccharide group and the low-dose Bletilla striata polysaccharide group were decreased, and the difference was not statistically significant (P>0.05).

[0179] 5 Discussion and Conclusion

[0180] 5.1 Discussion

[0181] Gastric ulcer is the most common gastrointestinal disease, affecting about 4% to 5% of the world's population. Excessive drinking is one of the main causes of gastric ulcer. [1] .

[0182] Existing studies have shown that alcoholic gastric ulcers are mainly related to damage to the gastric mucosal protective layer, disordered oxidative stress levels in gastric tissue, and infiltration of inflammatory factors. [2] The intact gastric mucosa is the primary barrier to protect gastric tissue from direct damage by ethanol. Some scholars have proposed that the most important step in preventing gastric ulcers is to form a protective layer on the inner wall of the stomach to protect the underlying mucosa from external irritants. [3] In this experiment, after pretreatment with the high-dose group of Bletilla striata polysaccharide, the gastric tissue of rats was effectively protected, and the pathological manifestations and indexes of ulcers were significantly reduced, indicating that when the body ingests excessive ethanol, Bletilla striata polysaccharide can protect the integrity of the gastric mucosa and thus prevent acute alcoholic gastric mucosal damage.

[0183] When the gastric mucosa is stimulated, it will also induce the overexpression of reactive oxygen species, causing oxidative damage and peroxidation of biological macromolecules. The superoxide anions, hydrogen peroxide, hydroxyl free radicals, etc. produced by it will lead to the disorder of the antioxidant defense system of gastric tissue. [4-5] Therefore, rationally improving the excessively stressed oxidative state in gastric tissue is also one of the important ways to prevent and treat gastric ulcers. However, most drugs for treating gastric ulcers, such as omeprazole, a proton pump inhibitor, simply inhibit or neutralize excessive gastric acid secretion and cannot intervene in the oxidative stress of damaged gastric tissue. This is also an important reason why drugs cannot cure gastric ulcers and cause them to recur. [6] .

[0184] Superoxide dismutase (SOD) is the main antioxidant enzyme in the body that inhibits and protects against free radical damage. It catalyzes the dismutation reaction of oxygen free radicals, eliminates the effects of superoxide anion free radicals, and plays an important role in the body's oxidation and antioxidant balance system. [7] Malondialdehyde (MDA) is a substance produced by the oxidative degradation of polyunsaturated fatty acids by oxygen free radicals. It can reflect the degree of lipid peroxidation in the body and the degree of damage to cells by free radicals. [8] . The MDA content in rat gastric tissue was significantly reduced. Gastric mucosal damage is related to the excessive production of oxygen free radicals. If the excessive oxygen free radicals are not removed in time, the polyunsaturated fatty acids in the gastric mucosa will undergo lipid peroxidation, causing changes in the function and structure of the gastric mucosa, thereby leading to necrosis and degeneration of the gastrointestinal epithelial mucosa, increased mucosal permeability, and ultimately inducing or aggravating ulcers. SOD can remove oxygen free radicals in the tissue, which can block and resist the damage caused by oxygen free radicals to cells, and can repair damaged cells in time. Therefore, the increase in SOD content may be the enhancement of the protective effect of the gastric mucosa, while the decrease in MDA content in gastric tissue is the enhancement of the defensive effect of the gastric mucosa. The experimental results show that the high, medium and low dose groups of Bletilla striata polysaccharide can improve the body's oxidative stress state by promoting the activity of the antioxidant enzyme SOD in the body's gastric tissue, reducing the content of MDA and inhibiting the degree of lipid peroxidation after pretreatment, thereby achieving a preventive protective effect on alcoholic gastric mucosal damage.

[0185] Acute alcoholic gastric mucosal injury is also associated with inflammatory response. Excessive ethanol intake can stimulate the microcirculatory blood vessels on the gastric mucosa, produce a large number of inflammatory mediators and cytokines, and destroy the normal structure and physiological function of gastric tissue. [9] Prostatic genomic DNA (PGE2) is an important regulator of gastric mucosal integrity, gastric tissue pH environment and gastric mucus secretion level, and is of great significance for the prevention and treatment of gastric ulcers. [10-12]. Epidermal growth factor (EGF) is an important bioactive peptide. Modern research has found that EGF is widely distributed in the digestive tract. It can inhibit gastric acid secretion and promote the synthesis of RNA, DNA and protein in tissues and cells, accelerating the healing of digestive system ulcers. At the same time, EGF has the effects of promoting epithelial proliferation, tissue repair and cell protection. It can protect the gastric mucosa from damage factors and maintain the integrity of the gastrointestinal mucosa. It plays a very important role. Thromboendothelin-1 (ET-1) is a polypeptide substance synthesized by endothelial cells. It exists in the entire digestive system and has a very strong vasoconstriction effect. It can cause continuous microvascular contraction, resulting in a decrease in gastric mucosal blood flow, which leads to necrosis of the gastric mucosa due to lack of normal blood supply.

[13] .

[0186] In this experiment, pretreatment with high-dose / medium-dose Bletilla striata polysaccharide can effectively alleviate the inhibition of PGE2, EGF, and ET-1 production after ethanol intake. NO is a neurotransmitter and messenger molecule released by non-cholinergic and non-adrenergic nerves in the gastrointestinal tract. Ethanol-induced gastric ulcers are usually related to dysregulation of the NO pathway. [14-15] Pretreatment with high-dose Bletilla striata polysaccharide group can improve the NO regulation disorder in rats and maintain it at a normal level.

[0187] In summary, Bletilla striata polysaccharide has a good preventive effect on acute alcoholic gastric mucosal injury. The possible mechanisms of action include protecting the integrity of the gastric mucosa, improving the oxidative stress state of the tissue, and inhibiting the expression of inflammatory factors, which lays a foundation for the next step of exploring the repair mechanism of Bletilla striata polysaccharide on gastric mucosal injury and provides experimental basis for the further development and application of Bletilla striata polysaccharide health food or medicine.

[0188] 5.2 Conclusion

[0189] ① When the body ingests excessive ethanol, Bletilla striata polysaccharide can prevent acute alcoholic gastric mucosal damage by protecting the integrity of the gastric mucosa.

[0190] ②When the body ingests excessive ethanol, Bletilla striata polysaccharides can enhance the antioxidant capacity of gastric tissue, thereby achieving a preventive protective effect against acute alcoholic gastric mucosal damage.

[0191] ③When the body ingests excessive ethanol, Bletilla striata polysaccharide regulates the abnormal expression of inflammatory factors and maintains them at normal levels, thereby playing a preventive protective role against gastric mucosal damage.

[0192] 6 References

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[0208] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An application of Bletilla striata polysaccharide in the preparation of a drug for treating gastric mucosal damage.

2. The use according to claim 1, characterized in that: Application of Bletilla striata polysaccharide in the preparation of drugs for treating gastric mucosal oxidative stress.

3. The use according to claim 1, characterized in that: Application of Bletilla striata polysaccharide in the preparation of drugs for treating gastric mucosal inflammatory factors.

4. The use according to claim 1, characterized in that: The gastric mucosal injury is alcoholic gastric mucosal injury.

5. The use according to claim 1, characterized in that: The Bletilla striata polysaccharide is added with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable preparation; the preparation is freeze-dried powder, granules, capsules, tablets, pills, sprays, lozenges, pellets, and creams.

6. A method for preparing the Bletilla striata polysaccharide according to claim 1, characterized in that: The preparation method of the Bletilla striata polysaccharide is as follows: 1) Degreasing of Bletilla striata: taking Bletilla striata tubers, crushing them into powder, passing through a 100-mesh sieve to obtain Bletilla striata powder, taking an appropriate amount of Bletilla striata powder, adding 5-7 times the amount of 70-90% ethanol, heating and refluxing extraction at 60-90° C. for degreasing, the extraction time is 1.5-2.5 hours, and filtering; the filter residue is subjected to a second degreasing treatment in the same manner, filtered, and the filter residue is placed in a blast drying oven and dried at 38-42° C. to obtain defatted Bletilla striata powder; 2) Extraction of Bletilla striata polysaccharide: Take the defatted Bletilla striata powder, extract twice with Bletilla striata powder: water at a solid-liquid ratio of 1:30-40 g / ml and 1:20-25 g / ml respectively, stir for 50-70 seconds with a flash extractor each time, then heat in a water bath at 80-100°C for 2-4 hours, centrifuge the extract for 10-20 minutes at a speed of 4000 rpm, take the supernatant, concentrate it to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrates to obtain Bletilla striata concentrate; 3) Deproteinization of Bletilla striata polysaccharide: freeze the Bletilla striata concentrated solution in a -80°C refrigerator, take it out after 6-10 hours to dissolve, centrifuge it in a centrifuge for 10-20 minutes at a speed of 4000 rpm, take out the supernatant and freeze-thaw it repeatedly 4-6 times to obtain the Bletilla striata concentrated solution with protein removed; 4) Alcohol precipitation of Bletilla striata polysaccharide: accurately measure the deproteinized concentrate, mix the concentrate and 80-90% ethanol in a volume ratio of 1:6-9, slowly add 80-90% ethanol to the concentrate while stirring until white flocs appear, let stand for 5-7 hours, filter with suction, take the filter residue and dry it in a freeze dryer for 45-50 hours to obtain a dry product of Bletilla striata polysaccharide, grind it through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

7. The preparation method according to claim 6, characterized in that: The degreasing treatment of 1) Bletilla striata comprises: taking Bletilla striata tubers, crushing them into powder, passing them through a 100-mesh sieve to obtain Bletilla striata powder, adding 6 times the amount of 90% ethanol, heating and refluxing at 80° C. for degreasing treatment, the extraction time is 2 hours, and filtering; the filter residue is subjected to a second degreasing treatment in the same manner, filtered, and the filter residue is placed in a forced air drying oven and dried at 40° C. to obtain the defatted Bletilla striata powder.

8. The preparation method according to claim 6, characterized in that: The extraction of Bletilla striata polysaccharide 2) is as follows: take defatted Bletilla striata powder, take Bletilla striata powder: water with a solid-liquid ratio of 1:35 g / ml and 1:25 g / ml respectively, extract twice in sequence, stir for 60 seconds with a flash extractor each time, heat in a 90°C water bath for 3 hours, centrifuge the extract for 15 minutes at a speed of 4000 rpm, take the supernatant, concentrate it to 0.9-1.1 g / ml with a rotary evaporator, combine the two concentrated solutions to obtain Bletilla striata concentrated solution.

9. The preparation method according to claim 6, characterized in that: The deproteinization treatment of the Bletilla striata polysaccharide 3) is as follows: the Bletilla striata concentrated solution is placed in a -80°C refrigerator for freezing, taken out after 8 hours to dissolve, placed in a centrifuge for centrifugation for 15 minutes at a speed of 4000 rpm, and the supernatant is repeatedly frozen and thawed 5 times to obtain the Bletilla striata concentrated solution with protein removed.

10. The preparation method according to claim 6, characterized in that: 4) The alcohol precipitation treatment of Bletilla striata polysaccharide is as follows: Accurately measure the deproteinized concentrate and mix them in a volume ratio of 1:8 concentrate: 85% ethanol. Slowly add 85% ethanol to the concentrate while stirring until white flocs appear. Let it stand for 6 hours, filter it with suction, and dry the residue in a freeze dryer for 48 hours to obtain the dry product of Bletilla striata polysaccharide. Grind it through a 100-mesh sieve to obtain Bletilla striata polysaccharide.

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