Sea cucumber polysaccharide capable of preventing and treating helicobacter pylori infection as well as preparation method and application of sea cucumber polysaccharide

Abstract

The invention provides sea cucumber polysaccharide capable of preventing and treating helicobacter pylori infection and a preparation method and application thereof, and belongs to the technical field of marine organism polysaccharide preparation. The sea cucumber polysaccharide comprises the following components: 35-44% of total sugar, 19-25% of sulfate group, 4.5-5.0% of protein, 3.0-3.5% of uronic acid, 4.0-4.7% of amino sugar, 7.0-9.0% of water and 20-25% of ash content, has the molecular weight of 16-1916 kDa, mainly contains fucose, mannose and galactose, and also contains a small amount of glucosamine, galactosamine, glucuronic acid and glucose. The sea cucumber polysaccharide has good anti-helicobacter pylori activity in vitro, and the minimum effective dose is 20 mg / mL; in vivo, the sea cucumber polysaccharide can reduce stomach inflammation response and helicobacter pylori colonization amount in the helicobacter pylori infection process, reduce the infection rate, and also can reduce the stomach inflammation level and the helicobacter pylori colonization amount after infection, that is, the obtained sea cucumber polysaccharide has prevention and treatment effects on helicobacter pylori infection. When the sea cucumber polysaccharide with good helicobacter pylori inhibitory activity is applied to the fields of food nutrition and pharmacy, the industrial additional value of sea cucumbers can be greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine biological polysaccharide preparation, and particularly relates to a sea cucumber polysaccharide with the function of preventing and treating Helicobacter pylori infection, and a preparation method and application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Helicobacter pylori is a Gram-negative, microaerophilic bacterium that can colonize the mucosal layer of the human stomach, inducing chronic gastritis, peptic ulcer and gastric cancer. Its infection rate reaches about 50% globally, and it has currently been classified as a Group I biological carcinogen by the World Health Organization and the International Agency for Research on Cancer. Clinical studies have shown that the recurrence rate of chronic gastritis, peptic ulcer, and the development of precancerous conditions are directly related to the degree of Helicobacter pylori colonization in the gastric mucosa. Helicobacter pylori is the most important controllable risk factor for preventing gastric cancer. Eradicating Helicobacter pylori can reduce the risk of gastric cancer and effectively prevent gastric cancer. Currently, the traditional treatment method is quadruple therapy, which includes antibiotics. Long-term use will rapidly increase the drug resistance of Helicobacter pylori, reduce the eradication rate, and increase the recurrence rate.

[0004] Polysaccharides have the function of clearing Helicobacter pylori, can effectively prevent the adhesion of Helicobacter pylori to gastric mucosal epithelial cells, and can also correct the intestinal flora imbalance caused by the standard quadruple therapy, protect the gastric mucosa, inhibit gastric inflammation, prevent peptic ulcer, resist gastric cancer, etc. In particular, fucoidan has been increasingly studied in recent years. Polysaccharides have no toxic or side effects on a variety of normal cells and animals, and are a safe biological raw material. Their derivatives have been used as dietary supplements, health products, nutritional cosmetics, etc. Investigating the anti-Helicobacter pylori activity of polysaccharides is of great significance for the subsequent development of anti-Helicobacter pylori drugs and functional foods based on polysaccharides.

[0005] Sea cucumbers have been a great nutritional supplement since ancient times. The sea cucumber industry has developed rapidly. However, generally speaking, it is still in the traditional processing stage, and the deep processing is seriously insufficient. Polysaccharides are important active components in sea cucumbers, mainly divided into two types: sea cucumber mucopolysaccharides and sea cucumber fucosyl polysaccharides. Sea cucumber fucosyl polysaccharides have been proven to have anti-Helicobacter pylori activity. However, the preparation process of sea cucumber fucosyl polysaccharides is complicated and the yield is extremely low. The cost of sea cucumbers themselves is high. Developing products with sea cucumber fucosyl polysaccharides as the main raw material, the complex separation and production process and the extremely low yield will greatly increase the cost of the products and make them more expensive. The production process of sea cucumber polysaccharides is simple and the yield is high. Developing products with sea cucumber polysaccharides as the raw material can reduce the cost. Therefore, compared with fucosyl polysaccharides, the industrialization difficulty of sea cucumber polysaccharides is lower and the industrialization prospect is higher. There are few reports on the Helicobacter pylori inhibitory activity of sea cucumber polysaccharides. Finding sea cucumber polysaccharides with good Helicobacter pylori inhibitory activity and applying them to the fields of food nutrition and pharmacy will greatly increase the added value of the sea cucumber industry. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a sea cucumber polysaccharide with the prevention and treatment of Helicobacter pylori infection, its preparation method and application. The sea cucumber polysaccharide provided by the present invention has direct anti-Helicobacter pylori activity in vitro. In vivo, it can not only reduce the gastric inflammation reaction and the colonization amount of Helicobacter pylori during the infection process, reduce the infection rate, but also reduce the gastric inflammation level and the colonization amount of Helicobacter pylori after infection. That is, the sea cucumber polysaccharide obtained by the present invention has both preventive and therapeutic effects on Helicobacter pylori infection.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a sea cucumber polysaccharide with the prevention and treatment of Helicobacter pylori infection is provided. The polysaccharide is composed of fucose, mannose, galactose, glucosamine, galactosamine, glucuronic acid and glucose, and its molecular weight is 16-1916 kDa.

[0009] In the second aspect of the present invention, a preparation method of a sea cucumber polysaccharide with the prevention and treatment of Helicobacter pylori infection is provided, including:

[0010] Drying and pulverizing fresh sea cucumbers to obtain sea cucumber powder, and defatting to obtain defatted sea cucumber powder;

[0011] Adding sodium acetate buffer solution, papain, EDTA and cysteine to the defatted sea cucumber powder for enzymatic extraction, separating the solid and liquid after extraction, and collecting the supernatant;

[0012] Adding cetylpyridinium chloride solution to the supernatant for reaction, and collecting the first precipitate after the reaction is completed;

[0013] Dissolve the first precipitate in a mixed solution of NaCl solution - ethanol, mix well, then mix with ethanol, let it stand, and collect the second precipitate;

[0014] Wash, dry, dialyze and desalt the second precipitate, concentrate and freeze-dry it to obtain sea cucumber polysaccharide.

[0015] In the third aspect of the present invention, there is provided the use of the above-mentioned sea cucumber polysaccharide or the sea cucumber polysaccharide prepared by the above-mentioned method in the preparation of functional foods and / or drugs for preventing and treating Helicobacter pylori.

[0016] Advantages of the present invention

[0017] (1) The sea cucumber polysaccharide provided by the present invention has direct anti-Helicobacter pylori activity in vitro, and the lowest effective dose is 20 mg / mL; in vivo, it can not only reduce the gastric inflammation reaction and the colonization amount of Helicobacter pylori during the infection process, reduce the infection rate, but also reduce the gastric inflammation level and the colonization amount of Helicobacter pylori after infection. That is, the sea cucumber polysaccharide obtained by the present invention has both preventive and therapeutic effects on Helicobacter pylori infection.

[0018] (2) There are few reports on the Helicobacter pylori inhibitory activity of sea cucumber polysaccharide. The present invention first finds a sea cucumber polysaccharide with good Helicobacter pylori inhibitory activity, which is superior to seaweed polysaccharide and has the same Helicobacter pylori inhibitory activity as sea cucumber fucan polysaccharide. Applying it to the fields of food nutrition and pharmacy will greatly increase the added value of the sea cucumber industry.

[0019] (3) The preparation method of the present invention is simple, highly practical and easy to popularize. Brief description of the drawings

[0020] The attached drawings forming a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0021] Figure 1 It is the determination result of the monosaccharide composition of the sea cucumber polysaccharide in Example 1 of the present invention.

[0022] Figure 2 It is the determination result of the monosaccharide composition of the sea cucumber fucan polysaccharide in Example 2 of the present invention.

[0023] Figure 3 It is the experimental result of the Helicobacter pylori inhibition zone of the sea cucumber polysaccharide in Example 1 of the present invention (the left and right figures are the results of 2 repeated experiments).

[0024] Figure 4 It is the experimental result of the Helicobacter pylori inhibition zone of the sea cucumber fucan polysaccharide in Example 2 of the present invention (the left and right figures are the results of 2 repeated experiments).

[0025] Figure 5Morphological observation of the gastric tissue of the mouse in Example 1 of the present invention.

[0026] Figure 6 Results of the rapid urease test on the gastric tissue of the mouse in Example 1 of the present invention.

[0027] Figure 7 Scores of the rapid urease test on the gastric tissue of the mouse in Example 1 of the present invention.

[0028] Figure 8 HE staining map of the gastric tissue of the mouse in Example 1 of the present invention.

[0029] Figure 9 Giemsa staining map of the gastric tissue of the mouse in Example 1 of the present invention. Detailed implementation manners

[0030] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0031] Currently, the infection rate of Helicobacter pylori at home and abroad is high and it has been recognized as a risk factor for inducing gastric diseases. The market for drugs or functional foods targeting the prevention and treatment of Helicobacter pylori is huge. The sea cucumber industry has developed rapidly. However, overall, it is in the traditional processing stage and the deep processing is seriously insufficient. Polysaccharides are important active components in sea cucumbers, mainly divided into two types, sea cucumber mucopolysaccharides and sea cucumber fucoidan. Sea cucumber fucoidan has been proven to have anti-Helicobacter pylori activity. However, the preparation process of sea cucumber fucoidan is complicated and the yield is extremely low. The cost of sea cucumbers themselves is high. Developing products with sea cucumber fucoidan as the main raw material, the complex separation and production process and the extremely low yield will greatly increase the cost of the products and make them more expensive. The production process of sea cucumber polysaccharides is simple and the yield is high. Developing products with sea cucumber polysaccharides as the raw material can reduce the cost. Therefore, compared with fucoidan, the industrialization difficulty of sea cucumber polysaccharides is lower and the industrialization prospect is higher. There are few reports on the Helicobacter pylori inhibitory activity of sea cucumber polysaccharides. Finding sea cucumber polysaccharides with good Helicobacter pylori inhibitory activity and applying them to the fields of food nutrition and pharmacy will greatly increase the added value of the sea cucumber industry.

[0032] In view of this, the present invention proposes a sea cucumber polysaccharide with the prevention and treatment of Helicobacter pylori infection, its preparation method and application.

[0033] A typical embodiment of the present invention is a sea cucumber polysaccharide with the function of preventing and treating Helicobacter pylori infection. The composition of the sea cucumber polysaccharide is as follows: total sugar 35 - 44%, sulfate group 19 - 25%, protein 4.5 - 5.0%, uronic acid content 3.0 - 3.5%, amino sugar content 4.0 - 4.7%, moisture 7.0 - 9.0%, ash content 20 - 25%, and the molecular weight is 16 - 1916 kDa. It mainly contains fucose, mannose, galactose, and also contains a small amount of glucosamine, galactosamine, glucuronic acid, and glucose.

[0034] The sea cucumber polysaccharide provided by the present invention has good anti - Helicobacter pylori activity in vitro, and the minimum effective dose is 20 mg / mL. In vivo, it can not only reduce the gastric inflammatory response and the colonization amount of Helicobacter pylori during the infection process, reduce the infection rate, but also reduce the gastric inflammatory level and the colonization amount of Helicobacter pylori after infection. That is, the sea cucumber polysaccharide obtained in the present invention has both preventive and therapeutic effects on Helicobacter pylori infection.

[0035] Another typical embodiment of the present invention provides a preparation method of the above - mentioned sea cucumber polysaccharide with the function of preventing and treating Helicobacter pylori infection, which includes the following steps:

[0036] S1. Dry and crush fresh sea cucumbers and defat them.

[0037] S2. Add sodium acetate buffer solution, papain, EDTA and cysteine to the defatted sea cucumber powder for enzymatic extraction. After extraction, perform solid - liquid separation and collect the supernatant.

[0038] S3. Add cetylpyridinium chloride (CPC) solution to the supernatant and react at room temperature for 24 h, then collect the precipitate.

[0039] S4. Dissolve the precipitate in a mixed solution of NaCl solution - ethanol, stir evenly, then mix with ethanol and let it stand, and collect the precipitate part.

[0040] S5. Wash the precipitate obtained in step S4 with ethanol, dry it, then perform dialysis for desalting, concentrate and freeze - dry to obtain the sea cucumber polysaccharide.

[0041] In some embodiments of this embodiment, the drying method of sea cucumbers is freeze - drying.

[0042] In some embodiments of this embodiment, the defatting is specifically as follows: take the crushed sea cucumber powder, add acetone with a volume 5 - 10 times that of the powder for soaking and defatting.

[0043] In some embodiments of this embodiment, the concentration of the sodium acetate buffer solution is 0.1 - 0.3 mol / L, preferably 0.1 mol / L; preferably, the material - liquid ratio of the crushed sea cucumber powder to the sodium acetate buffer solution is 1:20 - 30 g / mL.

[0044] In some embodiments of this embodiment, the papain enzyme activity is 700 - 900 U / mg, preferably 800 U / mg; preferably, 0.04 - 0.08 g of papain is used to treat each gram of the pulverized sea cucumber powder.

[0045] In some embodiments of this embodiment, the final concentrations of both EDTA and cysteine are 4 - 6 mmol / L, preferably 5 mmol / L.

[0046] In some embodiments of this embodiment, for enzymatic hydrolysis extraction, the extraction temperature is 50 - 70 °C and the time is 20 - 30 h.

[0047] In some embodiments of this embodiment, the mass - volume ratio of the CPC solution is 10%; the material - liquid ratio of the pulverized sea cucumber powder to the CPC solution is 1:1.6 - 2 g / mL.

[0048] In some embodiments of this embodiment, in the NaCl solution - ethanol mixed solution, the volume ratio of the NaCl solution to ethanol is 100:15; preferably, the concentration of the NaCl solution is 2 - 3 mol / L and the concentration of ethanol is 95% (volume percentage).

[0049] In some embodiments of this embodiment, when the mixed solution is mixed with ethanol and allowed to stand, the volume ratio of the two is mixed solution:ethanol = 1:3 - 6; preferably, for the standing, the standing temperature is 4 - 8 °C and the time is 12 - 24 h.

[0050] The third typical embodiment of the present invention provides the application of the above - mentioned sea cucumber polysaccharide with the prevention and treatment of Helicobacter pylori infection in the preparation of functional foods and / or drugs for preventing and treating Helicobacter pylori.

[0051] The following combines specific embodiments to further elaborate on the present invention. It should be noted that the specific embodiments are interpretations rather than limitations of the present invention.

[0052] Example 1

[0053] A preparation method of a sea cucumber polysaccharide with the prevention and treatment of Helicobacter pylori infection includes the following steps:

[0054] S1. Freeze - dry and pulverize fresh sea cucumbers to obtain sea cucumber powder; take an appropriate amount of sea cucumber powder, add 5 - fold volume of acetone for soaking and defatting, repeat 2 times; after standing and centrifuging, the sea cucumber powder is naturally air - dried.

[0055] S2. Add 0.1 mol / L sodium acetate buffer solution to the sea cucumber powder, and the material - liquid ratio of the pulverized sea cucumber powder to the sodium acetate buffer solution is 1:20 g / mL; add 8 g of papain (800 U / mg), stir and react at 60 °C for 20 h, then centrifuge to collect the supernatant.

[0056] S3. Add a 10% (w / v) cetylpyridinium chloride (CPC) solution to the supernatant. The material-liquid ratio of the pulverized sea cucumber powder to the CPC solution is 1:1.6 g / mL. Stir and let stand for 24 h, then centrifuge and collect the precipitate.

[0057] S4. Dissolve the precipitate obtained in S3 thoroughly in a mixed solution of 3 mol / L NaCl solution: 95% (v / v) ethanol = 100:15 (v / v). Add 95% (v / v) ethanol solution with a volume three times that of the mixed solution, let stand at 4°C for 24 hours, centrifuge, collect the precipitate, dialyze the precipitate to remove salts, and concentrate the desalted solution under reduced pressure and freeze-dry it to obtain sea cucumber polysaccharide.

[0058] Example 2

[0059] Separate the sea cucumber polysaccharide obtained in Example 1 by Q Sepharose Fast Flow ion exchange chromatography, elute with 0 - 3 mol / L aqueous sodium chloride solution, collect the eluate fraction with high-concentration sodium chloride solution, concentrate it under reduced pressure and dialyze to remove salts, and then concentrate the desalted solution under reduced pressure and freeze-dry it to obtain sea cucumber fucan.

[0060] Experimental Example 1

[0061] The following are the results of the component analysis and activity determination of the sea cucumber polysaccharide in Example 1 and the sea cucumber fucan in Example 2, including the determination of the polysaccharide composition, monosaccharide composition analysis, and in vitro and in vivo anti-Helicobacter pylori activity analysis.

[0062] The composition of the sea cucumber polysaccharide is shown in Table 1. The total sugar content is 43.05%, the protein content is 4.89%, the sulfate group content is 24.22%, the uronic acid content is 3.44%, the amino sugar content is 4.65%, the moisture content is 8.40%, and the ash content is 20.51%.

[0063] Table 1 Determination of the composition of sea cucumber polysaccharide

[0064]

[0065] The results of the monosaccharide composition determination are as Figure 1 shown. The sea cucumber polysaccharide mainly contains fucose, mannose, and galactose, and also contains a small amount of glucosamine, galactosamine, glucuronic acid, and glucose, with a molar ratio (mol%) of 55.96:9.39:7.07:5.38:8.42:4.22:9.56. The molecular weight of the sea cucumber polysaccharide was determined by high-performance gel permeation chromatography, and its molecular weight was 16 - 1916 kDa.

[0066] In Example 2, the polysaccharide obtained by Q Sepharose Fast Flow ion exchange chromatography mainly consists of fucose, with a molar ratio (mol%) of 83.05%( Figure 2 ), indicating that it is sea cucumber fucan.

[0067] Culture of Helicobacter pylori: Resuscitate Helicobacter pylori in Columbia blood agar medium containing 7% defibrinated sheep blood, and culture it in a triple gas incubator (5% O 2 , 10% CO 2 , 85% N 2 ).

[0068] Helicobacter pylori inhibition zone experiment: Prepare sea cucumber polysaccharide and sea cucumber fucan into 20 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL respectively, and prepare amoxicillin into 5 μg / mL. Filter through a 0.22 μm sterile microporous filter membrane and set aside. Use sterile water as the blank control and amoxicillin as the positive control. Evaluate the Helicobacter pylori inhibitory activity of sea cucumber-derived polysaccharides through the inhibition zone experiment. Scrape an appropriate amount of Helicobacter pylori and place it in Columbia agar medium containing 7% sterile defibrinated sheep blood. Pour into a petri dish. After the medium solidifies, use sterile forceps to stick a 6 mm diameter sterile filter paper onto the petri dish and press gently. Place 6 filter papers on each petri dish, and add sterile water (blank control), amoxicillin solution (positive control), and 4 different concentration polysaccharide solutions (sample group) respectively. Place the petri dish in a triple gas incubator, take it out after culturing for 48 h and observe. Repeat 3 times. The results are as Figure 3 shown. There is no inhibition zone in the blank control group, and the inhibition zone in the positive drug amoxicillin group is larger, showing good inhibitory activity. The sea cucumber polysaccharide in the sample group has an inhibition zone and the inhibition zone is obvious at high doses, indicating that sea cucumber polysaccharide has direct inhibitory activity against Helicobacter pylori in vitro, which is better than seaweed polysaccharide (Patent 2024103876092, an anti-Helicobacter pylori sea cucumber intestine fucan and its preparation method and application) and is equivalent to the Helicobacter pylori inhibitory activity of the sea cucumber fucan prepared in Example 2( Figure 4 ).

[0069] Determination of minimum inhibitory concentration: Scrape an appropriate amount of Helicobacter pylori and place it in brain heart infusion broth medium containing 10% fetal bovine serum. Culture for 2 days, and adjust the concentration of the bacterial suspension to 1×10 8CFU / mL for backup. Dilute sea cucumber polysaccharide to 40, 20, 10 mg / mL by the two-fold method with brain heart infusion broth medium containing 10% fetal bovine serum. Add 50 μL of the above solution and 50 μL of fresh Helicobacter pylori suspension into a 96-well plate. Use 100 μL of medium as the negative control, 50 μL of medium + 50 μL of medium solution containing polysaccharide as the negative control for the sample, 50 μL of suspension + 50 μL of medium as the blank control, and amoxicillin as the positive control group. Incubate at 37 °C for 72 h under microaerophilic conditions. Detect the absorbance value (OD = 600 nm) of each well with an enzyme-linked immunosorbent assay (ELISA) reader and determine the inhibition rate.

[0070]

[0071] According to the literature, when the inhibition rate is greater than 50%, the MIC value can be considered reliable. If the inhibition rate is less than 50%, the MIC value (the previous dilution gradient of the initially determined concentration) is re-determined until the inhibition rate meets the judgment criteria.

[0072] The inhibition rate of sea cucumber polysaccharide is shown in Table 2. Sea cucumber polysaccharide shows good anti-Helicobacter pylori activity. When the concentration is higher than 20 mg / mL, the inhibition rate can reach more than 50%. Determine its MIC 50 to be 20 mg / mL.

[0073] Table 2 Determination results of the inhibition rate of sea cucumber polysaccharide

[0074]

[0075] Determination of the anti-Helicobacter pylori activity of sea cucumber polysaccharide in vivo: Specific pathogen-free (SPF) KM mice weighing about 20 g were adaptively fed for 7 days and randomly divided into 4 groups of 6 mice each. The blank control group (Group A) was given sterile water by gavage; the model group (Group B) was given Helicobacter pylori suspension by gavage until the modeling was successful; the preventive group of sea cucumber polysaccharide was first given the polysaccharide solution for 1 week, and then given sea cucumber polysaccharide by gavage first and then Helicobacter pylori suspension; the treatment group of sea cucumber polysaccharide was given sea cucumber polysaccharide by gavage every day after the modeling was successful. After the experiment, the mice were sacrificed, and the whole stomachs of the mice were dissected under sterile conditions.

[0076] Cut the whole stomach of the mouse along the greater curvature of the stomach, wash the gastric contents with sterile normal saline, and observe the conditions of the gastric mucosa, fundus, and antrum with the naked eye for any histological changes such as bleeding, erosion, and ulcer. Take two portions of the tissue for subsequent experiments: 1) rapid urease test of the antral tissue; 2) HE staining of the antral tissue; 3) Giemsa staining of the antral tissue.

[0077] Rapid urease test: Helicobacter pylori has a large number of active extracellular ureases. The gastric antrum tissue can be quickly diagnosed by putting it into the detection kit. Take out the substrate microplate strip in the kit, gently open the upper cover of the microplate strip, add 100 μl of reaction solution into the microplate. After the drug film is completely dissolved into a yellow transparent solution, immediately use sterile forceps to place the gastric antrum tissue into the microplate, incubate it in an incubator at 25 °C, observe the results and score. Place it against a white background and observe the color change of the liquid medicine in each well under natural light. If the color of the liquid medicine in the microplate is red or purplish red, it is positive; if there is no color change (yellow) in the liquid medicine in the microplate, it is negative.

[0078] Pathological section staining of gastric antrum tissue: Fix the removed gastric tissue with 4% paraformaldehyde fixative for 24 h, rinse it with PBS 2 - 3 times, and then put the tissue specimen into ethanol with different concentrations for dehydration treatment. After blotting the water with filter paper, immediately put it into xylene for transparency treatment. After completion, immediately put it into paraffin solution for 2 h for paraffin embedding. After the paraffin solidifies, cut the specimen into a cube for tissue sectioning. Use a paraffin tissue slicer to cut the tissue specimen into thin slices of 5 - 7 μm, take the thin slices and place them in a 43 °C water bath, sample them with a glass slide and mark them, and put them into an oven at 60 °C for dewaxing treatment. After overnight dewaxing, perform routine HE staining and Giemsa staining, and scan and read the slides.

[0079] Observation of mouse gastric tissue is as Figure 5 shown. The gastric mucosa of the blank group is ruddy and shiny, without bleeding or erosion symptoms. The gastric tissue of the model group shows punctate bleeding and even erosive changes (arrow), indicating that Helicobacter pylori infection has caused gastric lesions such as gastric ulcer and gastric fundus bleeding. Neither the prevention group nor the treatment group of sea cucumber polysaccharide shows bleeding or erosion symptoms, indicating that sea cucumber polysaccharide can improve the gastric lesions caused by Helicobacter pylori infection.

[0080] The infection status of each group of mice was preliminarily evaluated by the rapid urease method, and the results are as Figure 6 shown. The blank group shows yellow, indicating no infection. The model group shows purplish red, indicating the presence of Helicobacter pylori infection. The infection levels of the prevention group and the treatment group are reduced, especially the treatment group. The rapid urease score is as Figure 7 shown. Compared with the blank group, there is a highly significant difference in the model group, indicating that the modeling is successful. Compared with the model group, there are highly significant differences in both the prevention group and the treatment group, indicating that both the prevention group and the treatment group of sea cucumber can reduce the Helicobacter pylori infection level.

[0081] The HE staining results of mouse gastric tissue are as Figure 8As shown, the surface of the gastric tissue mucosa layer in the blank group is simple columnar epithelium. The number of gastric glands in the lamina propria is abundant and they are arranged closely. The gastric tissue is intact, the layer boundary is clear, and the morphology is good. The gastric gland epithelial cells in the model group showed vacuolization. There was an inflammatory infiltration mainly composed of lymphocytes at the bottom of the lamina propria and in the submucosa. The blood vessels were slightly dilated, and there was a large range of thickening and edema in the submucosa. Therefore, Helicobacter pylori infection can damage gastric tissue and cause gastric inflammation. Both the prevention group and the treatment group can improve gastric lesions such as gastric cell vacuolization, edema, and inflammatory infiltration.

[0082] The Giemsa staining results of mouse gastric tissue are as Figure 9 shown. Helicobacter pylori appears as strip-shaped dark blue. A large number of Helicobacter pylori appeared on the gastric mucosa in the model group, further indicating the success of the modeling. The colonization amount of Helicobacter pylori in the treatment group and the prevention group was significantly reduced.

[0083] In summary, the sea cucumber polysaccharide provided by the present invention has good anti-Helicobacter pylori activity in vitro; in vivo, it can not only reduce the gastric inflammatory response and the colonization amount of Helicobacter pylori during the infection process, reduce the infection rate, but also reduce the gastric inflammatory level and the colonization amount of Helicobacter pylori after infection. That is, the sea cucumber polysaccharide obtained in the present invention has both preventive and therapeutic effects on Helicobacter pylori infection.

[0084] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection, characterized in that: The sea cucumber polysaccharide is composed of fucose, mannose, galactose, glucosamine, galactosamine, glucuronic acid and glucose, and has a molecular weight of 16-1916 kDa.

2. A method for preparing sea cucumber polysaccharides for preventing and treating Helicobacter pylori infection, characterized in that: include: Drying and crushing fresh sea cucumbers to obtain sea cucumber powder, and defatting to obtain defatted sea cucumber powder; Adding sodium acetate buffer, papain, EDTA and cysteine ​​to the defatted sea cucumber powder for enzymatic extraction, performing solid-liquid separation after extraction, and collecting the supernatant; Adding cetylpyridinium chloride solution to the supernatant to react, and collecting the first precipitate after the reaction is completed; Dissolving the first precipitate in a NaCl solution-ethanol mixed solution, stirring evenly and then mixing with ethanol, allowing to stand, and collecting a second precipitate; The second precipitate is washed, dried, dialyzed for desalination, concentrated, and freeze-dried to obtain sea cucumber polysaccharide.

3. The method for preparing sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: The solid-liquid ratio of the sea cucumber powder to the sodium acetate buffer is 1:20-30 g / mL; Or, the concentration of the sodium acetate buffer is 0.1-0.3 mol / L.

4. The method for preparing sea cucumber polysaccharides for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: Each gram of sea cucumber powder is treated with 0.04-0.08g of papain; Or, the papain activity is 700-900 U / mg, or, is 800 U / mg.

5. The method for preparing sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: The final concentrations of EDTA and cysteine ​​are both 4-6 mmol / L, or 5 mmol / L.

6. The method for preparing sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: The enzymatic extraction temperature is 50-70°C and the time is 20-30h.

7. The method for preparing sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: The material-liquid ratio of sea cucumber powder to CPC solution is 1:1.6-2g / mL; Or, the mass volume ratio of CPC solution is 10%-15%.

8. The method for preparing sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: In the NaCl solution-ethanol mixed solution, the volume ratio of NaCl solution to ethanol is 100:15; Or, the concentration of the NaCl solution is 2-3 mol / L; Or, the concentration of ethanol is 90%-95%, by volume.

9. The method for preparing sea cucumber polysaccharide for preventing and treating Helicobacter pylori infection according to claim 2, characterized in that: When the NaCl solution-ethanol mixed solution is mixed and allowed to stand with ethanol, the volume ratio of the two is mixed solution: ethanol = 1:3-6; Alternatively, the standing temperature is 4-8°C and the standing time is 12-24h.

10. Use of the sea cucumber polysaccharide according to claim 1 or the sea cucumber polysaccharide prepared by the method according to any one of claims 2 to 9 in the preparation of functional food and / or medicine for preventing and treating Helicobacter pylori.

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