A kind of wax gourd peel polysaccharide and its application

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a kind of wax gourd peel polysaccharide and its application. For the first time, the crude wax gourd peel polysaccharide obtained by the water extraction and alcohol precipitation method is decontaminated, separated and purified to obtain the wax gourd peel polysaccharide with a repeating unit structure. The said wax gourd peel polysaccharide is mainly composed of galactose, galacturonic acid, rhamnose, arabinose and glucose, and the total sugar content is ≥90%. In vitro and in vivo experiments prove that the wax gourd peel polysaccharide of the present invention has excellent gastric mucosa protection effect, and has good application prospects in the preparation of drugs for preventing and treating gastric ulcers or health products or feeds with the function of assisting in protecting the gastric mucosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to a class of wax gourd peel polysaccharides and their applications. Background Art

[0002] Wax gourd peel, as the dried outer pericarp of the wax gourd Benincasa hispida (Thunb.) Cogn. of the Cucurbitaceae family, is obtained by washing the wax gourd, scraping off the outer pericarp, and drying it in the sun. Its physical properties are characterized by being light in weight, brittle in texture, having a faint odor and taste, being in the form of irregular fragments, and often showing a curled-in state. The outer surface of the wax gourd peel is grayish-green or yellowish-white, covered with hoarfrost, while the inner surface is relatively rough. Modern pharmacological studies have shown that wax gourd peel is rich in chemical components such as flavonoids, alkaloids, tannins, trace elements, and vitamins, and exhibits pharmacological activities such as blood pressure lowering, diuresis, blood sugar lowering, and antioxidant activity.

[0003] Polysaccharides, as a class of high-molecular compounds composed of more than 10 monosaccharides polymerized, have highly diverse structures due to the complexity of the main chain, side chain, and connection methods, which to a certain extent limits the in-depth study of them. However, the rapid development of analytical instruments in recent years has greatly promoted the in-depth study of polysaccharides, thus revealing their extensive biological activities in aspects such as antioxidant, immunomodulatory, antitumor, blood sugar lowering, and antibacterial. Polysaccharides are widely distributed in natural plants, animals, and microorganisms in the biological world, but their efficacy is significantly affected by factors such as source and structure, specifically including chain length, glycosidic bond type, connection method, and molecular weight.

[0004] Specifically for wax gourd peel polysaccharides, existing studies have shown that they have blood sugar lowering and antibacterial effects. However, it should be noted that the wax gourd peel polysaccharides obtained by different extraction methods have differences in component content and function. This indicates that the specific efficacy of wax gourd peel polysaccharides is not only related to their chemical composition but also closely related to their structural characteristics.

[0005] Although certain progress has been made in the current research on polysaccharides, regarding wax gourd peel polysaccharides, especially those with specific structures and molecular weights, their applications in other aspects still lack in-depth exploration. The existing technology has not fully revealed the potential uses of these wax gourd peel polysaccharides with specific structures and molecular weights, which provides a broad space for future research. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the current existing technology's lack of in-depth exploration of wax gourd peel polysaccharides, and to provide a wax gourd peel polysaccharide BEP-1a prepared by sequentially using water extraction and alcohol precipitation, deproteinization and decolorization, and separation and purification.

[0007] The object of the present invention is to provide a wax gourd peel polysaccharide with a repeating unit structure.

[0008] Another object of the present invention is to provide the application of the wax gourd peel polysaccharide in the preparation of a drug for preventing and treating gastric ulcers.

[0009] Another object of the present invention is to provide the application of the wax gourd peel polysaccharide in the preparation of a health product or feed with the function of assisting in protecting the gastric mucosa.

[0010] Another object of the present invention is to provide a drug.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The present invention protects a wax gourd peel polysaccharide BEP-1a, which is obtained by a preparation method including the following steps:

[0013] S1. Extraction of crude wax gourd peel polysaccharide: Extract the crude wax gourd peel polysaccharide from wax gourd peel by the water extraction and alcohol precipitation method;

[0014] S2. Deproteinization and decolorization treatment: Perform deproteinization and decolorization treatment on the crude wax gourd peel polysaccharide obtained in step S1 to obtain wax gourd peel polysaccharide BEP;

[0015] S3. After the wax gourd peel polysaccharide BEP obtained in step S2 is formulated into an aqueous solution, it is eluted with a DEAE-Cellulose52 type anion chromatography column, and water, 0.1M NaCl, 0.5M NaCl, and 0.5M NaOH solutions are successively used as eluents for stepwise elution. Collect the fractions eluted with 0.1M NaCl, and perform post-treatment to obtain wax gourd peel polysaccharide BEP-1;

[0016] S4. After the wax gourd peel polysaccharide BEP-1 obtained in step S3 is formulated into an aqueous solution, it is purified with a Sephadex G-25 chromatography column, eluted with 0.1M NaCl as the eluent, and according to the elution curve, collect the eluent of the first single symmetric peak, and perform post-treatment to obtain wax gourd peel polysaccharide BEP-1a.

[0017] The present invention first further removes impurities, separates and purifies the crude wax gourd peel polysaccharide obtained by the water extraction and alcohol precipitation method to obtain a homogeneous polysaccharide - wax gourd peel polysaccharide BEP-1a. In vitro and in vivo experiments have proved that the wax gourd peel polysaccharide BEP-1a obtained by the above preparation method has excellent gastric mucosa protection effects and has good application prospects in the preparation of drugs for preventing and treating gastric ulcers or health products or feeds with the function of assisting in protecting the gastric mucosa.

[0018] Furthermore, the specifications of the DEAE-Cellulose 52 type anion chromatography column are Φ2.5cm×60cm. Among them, Φ2.5cm represents the diameter of the exchange column, and 60cm represents the height of the chromatography column.

[0019] Further, the specifications of the Sephadex G-25 chromatography column are Φ2.5 cm × 60 cm.

[0020] Further, in step S2, the order of the protein removal and decolorization treatments is not specified. As an alternative, the protein removal and decolorization treatments are as follows: the crude wax gourd peel polysaccharide obtained in step S1 is subjected to protein removal to obtain a protein-free solution, and then the protein-free solution is decolorized and post-treated to obtain wax gourd peel polysaccharide BEP.

[0021] Further, the post-treatment includes concentration, dialysis, and drying.

[0022] Preferably, the concentration is carried out under reduced pressure, and the temperature for the reduced pressure concentration is 50 - 70°C, more preferably 60°C.

[0023] Preferably, the drying is carried out by freeze-drying.

[0024] Preferably, the cut-off molecular weight of the dialysis bag for dialysis is 3000 - 5000 Da, more preferably 3500 Da.

[0025] Specifically, the post-treatment includes concentration, dialysis, and drying, and the specific operations are as follows: the product after protein removal and decolorization is concentrated under reduced pressure (60°C) by rotary evaporation, then dialyzed for 3 days using a dialysis bag with a cut-off molecular weight of 3500 Da, the retained solution after dialysis is concentrated again under reduced pressure (60°C), and then freeze-dried to obtain wax gourd peel polysaccharide BEP.

[0026] Further, as a common protein removal method, the protein removal is carried out by the Sevag method.

[0027] Further, as a common implementation method, the method for protein removal by the Sevag method includes the following steps: the wax gourd peel polysaccharide BEP is dissolved in water to prepare a polysaccharide solution, an equal volume of chloroform-n-butanol (4:1) solution is added thereto, shaken well, and then centrifuged. The chloroform phase is discarded, and an equal volume of chloroform-n-butanol (4:1) solution is added to the aqueous phase again. The operation is repeated until no protein is detected.

[0028] Further, as a common decolorization method, the decolorization is carried out by the static adsorption decolorization method.

[0029] Further, as a common implementation method, the static adsorption decolorization method uses D101 macroporous adsorption resin for decolorization, and the specific conditions for decolorization are as follows: the protein-free solution is decolorized by the static adsorption decolorization method, the dosage of D101 macroporous adsorption resin is 1 g / 10 mL, the loading concentration of the polysaccharide solution is 3 mg / mL, pH = 7, and the treatment time is 2 h.

[0030] Furthermore, the specific preparation method of the crude wax gourd peel polysaccharide includes the following steps: soaking the wax gourd peel in water sufficiently and then decocting it, filtering, taking the filtrate and concentrating it to obtain a crude extract, adding an alcohol solvent to the obtained crude extract, fully precipitating, and taking the precipitate and drying it to obtain the crude wax gourd peel polysaccharide.

[0031] Furthermore, the alcohol solvent includes ethanol or methanol.

[0032] Preferably, the alcohol solvent includes ethanol or methanol. Among them, the advantages of ethanol compared to methanol are as follows: ① Its toxicity is relatively lower than that of methanol, and its safety is higher; ② It has a certain bactericidal effect and can reduce the pollution of polysaccharides by microorganisms; ③ The solubility of polysaccharides in ethanol is usually lower than that in methanol. Therefore, the alcohol reagent is preferably ethanol.

[0033] Furthermore, after adding the alcohol reagent to the obtained crude extract, the final concentration of the alcohol reagent is 70% - 90%, preferably 80%.

[0034] Furthermore, the mass - volume ratio of the wax gourd peel sample to water is 1:(8 - 20) g / mL, preferably 1:(8 - 10) g / mL.

[0035] Preferably, the time for sufficient soaking is 20 - 40 min, preferably 30 min.

[0036] Furthermore, the conditions for decocting are: first boiling, and then continuing to decoct for 25 - 30 min.

[0037] Specifically, the conditions for decocting are: first boiling at the temperature of 100 °C, and then continuing to decoct at this temperature for 25 - 30 min.

[0038] As an alternative implementation, the conditions for decocting are: first boiling at the temperature of 100 °C, and then continuing to decoct at this temperature for 30 min.

[0039] Furthermore, the filter cloth for filtering is 150 - 250 mesh, preferably 200 mesh.

[0040] Furthermore, the filter residue obtained from the above - mentioned filtering can be repeatedly added with water and decocted again, and the two filtrates are combined, filtered, and the filtrate is concentrated to obtain a crude extract.

[0041] Preferably, the concentration is vacuum concentration, and the temperature of the vacuum concentration is 50 - 70 °C, preferably 60 °C.

[0042] Furthermore, the concentration is carried out until the ratio of the material to the liquid (g / mL) is close to 1:1 to obtain a crude extract.

[0043] Furthermore, as an alternative form, the wax gourd peel is wax gourd peel slices.

[0044] As a preferred embodiment, the method for preparing crude wax gourd peel polysaccharide comprises the following steps:

[0045] Take about 100 g of wax gourd peel decoction pieces, add 1000 mL of water, soak for 30 min, first boil at 100 °C, then decoct at 100 °C for 30 min, filter while it is hot with a 200-mesh filter cloth, take the filter residue, add another 800 mL of water, first boil at 100 °C, then decoct at 100 °C for 25 min, filter while it is hot with a 200-mesh filter cloth, combine the two filtrates, quickly cool the filtrate to room temperature, concentrate under reduced pressure (60 °C) until the ratio of material to liquid is close to 1:1 to obtain a thick extract, then add absolute ethanol to a final concentration of 80%, precipitate at 4 °C overnight, centrifuge to obtain the precipitate, remove the alcohol smell and then freeze-dry to obtain the crude wax gourd peel polysaccharide.

[0046] Further, in step S3 or step S4, the flow rate of the elution is preferably 2 - 4 mL / min, more preferably 3 mL / min.

[0047] Further, in steps S3 - S4, the post-treatment includes concentration and drying.

[0048] Preferably, the concentration is concentration under reduced pressure, and the temperature of the concentration under reduced pressure is 50 - 70 °C, more preferably 60 °C.

[0049] Preferably, the drying is freeze-drying.

[0050] Further, in step S3, the post-treatment further includes dialysis. After dialysis, take the retention solution in the dialysis bag for concentration and drying.

[0051] Preferably, the cut-off molecular weight of the dialysis bag for dialysis is 3000 - 5000 Da, more preferably 3500 Da.

[0052] Specifically, in step S3, the post-treatment includes concentration, dialysis and drying, and the specific operations are as follows: Concentrate the collected fractions by rotary evaporation under reduced pressure (60 °C), then dialyze with a dialysis bag with a cut-off molecular weight of 3500 Da for 3 days, take the retention solution after dialysis and concentrate it again by rotary evaporation under reduced pressure (60 °C), and then freeze-dry to obtain wax gourd peel polysaccharide BEP-1.

[0053] Specifically, in step S4, the post-treatment includes concentration and drying, and the specific operations are as follows: Concentrate the collected eluate by rotary evaporation under reduced pressure (60 °C), and then freeze-dry to obtain wax gourd peel polysaccharide BEP-1a.

[0054] As a preferred scheme, the following method can be used for steps S3 and S4:

[0055] Step S3: After preparing the obtained wax gourd peel polysaccharide BEP into an aqueous solution, it was loaded onto a DEAE-Cellulose 52 type anion exchange resin glass chromatography column (Φ2.5 cm × 60 cm) that had been equilibrated, and separated by successively eluting with 20 column volumes of water, 0.1 M NaCl solution, 0.5 M NaCl solution, and 0.5 M NaOH solution at a flow rate of 3 mL / min. One tube was collected every 100 mL. The elution curve was plotted using the phenol-sulfuric acid method. According to the elution curve, the fractions eluted with 0.1 M NaCl solution were collected, concentrated under reduced pressure by rotary evaporation, dialyzed (the dialysis bag specification was 3500 Da) for 3 days, concentrated under reduced pressure (60 °C), and freeze-dried to obtain wax gourd peel polysaccharide BEP-1.

[0056] Step S4: After preparing the wax gourd peel polysaccharide BEP-1 obtained in Step S3 into an aqueous solution, it was further purified by loading onto a Sephadex G-25 glass chromatography column (Φ2.5 cm × 60 cm) that had been equilibrated, and eluted with 0.1 M NaCl solution at an elution flow rate of 3 mL / min. The polysaccharide was tracked and detected using the sulfuric acid-phenol method. The elution curve was plotted based on the elution volume and absorbance. According to the elution curve, the eluate of the first single symmetric peak was collected and freeze-dried to obtain wax gourd peel polysaccharide BEP-1a.

[0057] The present invention also protects a wax gourd peel polysaccharide with a repeating unit structure, and the relative molecular mass of the wax gourd peel polysaccharide is 4.68×10 4 Da, and the structural formula of the repeating unit of the wax gourd peel polysaccharide is as follows:

[0058]

[0059] Among them, Gal represents galactose, GalA represents galacturonic acid, Rha represents rhamnose, Ara represents arabinose, and Glc represents glucose; f represents the configuration of furanose, and p represents the configuration of pyranose.

[0060] Currently, certain progress has been made in the research on polysaccharides. However, regarding wax gourd peel polysaccharides, especially those with specific structures and molecular weights, there is still a lack of in-depth exploration. The present invention for the first time obtains a wax gourd peel polysaccharide with a brand-new structure. The wax gourd peel polysaccharide is mainly composed of galactose, galacturonic acid, rhamnose, arabinose, and glucose, and the relative molecular mass is 4.68×10 4 Da. In vitro and in vivo experiments have proved that the wax gourd peel polysaccharide described in the present invention has excellent gastric mucosa protection effects.

[0061] Furthermore, the total sugar content of the wax gourd peel polysaccharide ≥ 90%.

[0062] Furthermore, the total sugar content of the wax gourd peel polysaccharide is 93.06%.

[0063] Furthermore, the monosaccharide composition of the wax gourd peel polysaccharide consists of 88.55% galactose, 4.02% galacturonic acid, 3.89% rhamnose, 1.93% arabinose, and 1.61% glucose.

[0064] Furthermore, the linkage modes of the wax gourd peel polysaccharide include: T-Ara, 1,4-Rha, 1,2,4-Rha, 1,4-Glc, and 1,4-Gal, and their mass percentages are 0.98%, 1.92%, 1.10%, 1.34%, and 94.66% respectively.

[0065] The present invention also protects the use of the wax gourd peel polysaccharide BEP-1a or the wax gourd peel polysaccharide with the specific molecular weight and repeating unit structure as described above in the preparation of drugs for preventing and treating gastric ulcers.

[0066] Furthermore, the gastric ulcer includes alcoholic gastric ulcer.

[0067] The present invention also protects the use of the wax gourd peel polysaccharide BEP-1a or the wax gourd peel polysaccharide with the specific molecular weight and repeating unit structure as described above in the preparation of health products or feeds with the function of assisting in protecting gastric mucosa.

[0068] The present invention protects a drug, and the drug contains the wax gourd peel polysaccharide BEP-1a or the wax gourd peel polysaccharide with the specific molecular weight and repeating unit structure as described above.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention first obtains the wax gourd peel crude polysaccharide by water extraction and alcohol precipitation, and through impurity removal, separation, and purification, obtains the wax gourd peel polysaccharide with a repeating unit structure. The wax gourd peel polysaccharide is mainly composed of galactose, galacturonic acid, rhamnose, arabinose, and glucose, and the total sugar content is ≥90%. In vitro and in vivo experiments prove that the wax gourd peel polysaccharide of the present invention has excellent gastric mucosa protection effects and has good application prospects in the preparation of drugs for preventing and treating gastric ulcers or health products or feeds with the function of assisting in protecting gastric mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 In FIG. A, it is the gradient elution curve of the wax gourd peel crude polysaccharide on the DEAE-52 column; in FIG. B, it is the elution curve of the wax gourd peel crude polysaccharide BEP-1 fraction on the Sephadex G-100 column.

[0072] Figure 2 It is the HPGPC diagram of the wax gourd peel homogeneous polysaccharide, wherein FIG. A represents BEP-1a and FIG. B represents BEP-1b.

[0073] Figure 3The monosaccharide composition of the homogeneous polysaccharide BEP-1a from wax gourd peel. Figure A is the HPLC chromatogram of the mixed standard, and Figure B is the HPLC chromatogram of the sample.

[0074] Figure 4 The FT-IR spectrum of the homogeneous polysaccharide BEP-1a from wax gourd peel.

[0075] Figure 5 The total ion chromatogram of the methylated homogeneous polysaccharide BEP-1a from wax gourd peel.

[0076] Figure 6 The nuclear magnetic resonance spectrum of the homogeneous polysaccharide BEP-1a from wax gourd peel.

[0077] Figure 7 The structural unit diagram of the homogeneous polysaccharide BEP-1a from wax gourd peel.

[0078] Figure 8 The scanning electron micrograph of the homogeneous polysaccharide BEP-1a from wax gourd peel. Figure A is at 100× magnification under the microscope, and Figure B is at 1000× magnification under the microscope.

[0079] Figure 9 The statistical chart of the protective effect of BEP-1a on ethanol-induced acute gastric ulcer injury in mice; among them, Figure A is the macroscopic representative diagram of the gastric tissues of mice in different treatment groups; Figure B is the statistical chart of the gastric ulcer area; Figure C is the statistical chart of the gastric ulcer index; Figure D is the statistical chart of the gastric ulcer inhibition rate.

[0080] Figure 10 The statistical chart of the improvement of ethanol-induced pathological injury of gastric tissues in mice by BEP-1a; among them, Figure A is the H&E staining diagram of the gastric tissues of mice; Figure B is the H&E staining score diagram; Figure C is the PAS staining diagram of the gastric tissues of mice; Figure D is the statistical chart of the PAS positive staining rate.

[0081] Figure 11Statistical chart of the protective effect of BEP-1a on ethanol-induced GES-1 cell damage; among them, Figure A is the statistical chart of the effect of different concentrations of ethanol on the survival rate of GES-1 cells; Figure B is the statistical chart of the toxicity of different concentrations of BEP to GES-1 cells; Figure C is the statistical chart of the effect of different concentrations of BEP-1a on the survival rate of ethanol-induced GES-1 cells; Figure D is the statistical chart of the detection of GES-1 cell mortality by flow cytometry; Figure E is the morphological diagram of GES-1 cells in different treatment groups under an inverted microscope; Figure F is the statistical chart of the apoptosis of GES-1 cells in different treatment groups detected by Annexin V-FITC / Pi; Figure G is the statistical chart of the activity of GES-1 cells in different treatment groups detected by Calcein / PI; Figure H is the statistical chart of the number of Calcein-positive cells; Figure I is the statistical chart of the number of PI-positive cells; Figure J is the statistical chart of the Calcein fluorescence area; Figure K is the statistical chart of the PI fluorescence area; Figure L is the statistical chart of the Calcein fluorescence intensity; M is the statistical chart of the PI fluorescence intensity.

[0082] Figure 12 Statistical chart of the effect of different concentrations of BEP-1b on the survival rate of ethanol-induced GES-1 cells. Detailed implementation mode

[0083] The present invention will be further described below in conjunction with the specification drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0084] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0085] Figure 1 A represents Figure 1 Figure A in Figure 2 A represents and represents Figure 2 Figure A in

[0086] Experimental materials and equipment

[0087] (1) Experimental animals

[0088] In this experiment, 4-week-old SPF male Kunming mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center. The mice were placed in the SPF laboratory of the Laboratory Animal Center of Guangdong Pharmaceutical University for feeding. The laboratory environmental temperature was about 24.0 ± 2.0 °C, and the relative humidity was about 50 ± 10%. The light and dark cycles alternated every 12 hours, and the animals had free access to food and water during the feeding period. In addition, all animal experiments were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University and were conducted in accordance with the "Animal Experiment Guidelines" of Guangdong Pharmaceutical University.

[0089] (2) Experimental cells

[0090] GES-1 cells were purchased from Wuhan Shang'en Biotechnology Co., Ltd.

[0091] (3) Experimental materials and reagents

[0092] Table 1 Experimental materials and reagents

[0093]

[0094]

[0095] (4) Experimental instruments

[0096] Table 2 Experimental instruments

[0097]

[0098]

[0099] Example 1 Extraction and isolation of wax gourd peel polysaccharide

[0100] 1. Experimental method

[0101] Take about 100 g of wax gourd peel slices, place them in a casserole, add 1000 mL of water and soak for 30 min. First, boil at 100 °C, then continue to boil at 100 °C for 30 min, and filter while it is hot with a 200-mesh filter cloth; add 800 mL of water to the residue, boil at 100 °C, and then continue to boil at 100 °C for 25 min, and filter while it is hot with a 200-mesh filter cloth. Combine the two filtrates, quickly cool the filtrate to room temperature, repeat the operation to process a total of 5.0 Kg of wax gourd peel medicinal materials, combine the filtrates and concentrate them under reduced pressure (60 °C) until the ratio of material to liquid is close to 1:1 to obtain a thick extract, then add absolute ethanol to a final concentration of 80%, precipitate overnight at 4 °C, centrifuge to obtain the precipitate, remove the alcohol smell and then freeze-dry to obtain the crude wax gourd peel polysaccharide.

[0102] The obtained crude wax gourd peel polysaccharide was formulated into a sugar solution with distilled water. The protein was removed by the Sevag method. After the protein-free sugar solution was decolorized with D101 macroporous adsorption resin, it was concentrated under reduced pressure by rotary evaporation (60 °C), dialyzed (dialysis bag size: 3500 Da) for 3 days, then concentrated under reduced pressure again (60 °C), and freeze-dried to obtain the crude wax gourd peel polysaccharide after protein removal and decolorization.

[0103] The crude wax gourd peel polysaccharide after protein removal and decolorization was dissolved with an appropriate amount of distilled water and separated on a DEAE-Cellulose 52 anion exchange resin glass chromatography column (Φ2.5 cm × 60 cm) after equilibration. It was gradually eluted with 20 column volumes of distilled water, 0.1 M NaCl solution, 0.5 M NaCl solution, and 0.5 M NaOH solution at a flow rate of 3 mL / min. One tube was collected every 100 mL. The elution curve was plotted using the phenol-sulfuric acid method, and the eluted samples were collected. According to the elution curve, the polysaccharide was collected. After concentration under reduced pressure by rotary evaporation (60 °C), it was dialyzed (dialysis bag size: 3500 Da) for 3 days, concentrated under reduced pressure (60 °C), and freeze-dried to obtain wax gourd peel polysaccharide BEP-1 (see Figure 1 A, obtained by collecting the fractions eluted with 0.1 M NaCl solution through the elution curve) and wax gourd peel polysaccharide BEP-2 (see Figure 1 A, obtained by collecting the fractions eluted with 0.5 M NaCl solution through the elution curve).

[0104] The obtained wax gourd peel polysaccharide BEP-1 was further purified on a Sephadex G-25 glass chromatography column (Φ2.5 cm × 60 cm) after equilibration. It was eluted with 0.1 M NaCl solution at an elution flow rate of 3 mL / min. The polysaccharide was tracked and detected using the sulfuric acid-phenol method. The elution curve was plotted based on the elution volume and absorbance. Different components were collected by combining according to the elution curve, freeze-dried, and stored to obtain wax gourd peel polysaccharide BEP-1a (see Figure 1 B, obtained by collecting the eluate of the first single symmetric peak through the elution curve) and wax gourd peel polysaccharide BEP-1b (see Figure 1 B, obtained by collecting the eluate of the second single symmetric peak through the elution curve).

[0105] The specific steps for protein removal and decolorization are as follows:

[0106] Protein removal by the Sevag method: An equal volume of chloroform-n-butanol (4:1) solution was added to the polysaccharide solution, shaken well, and centrifuged. The chloroform phase was discarded. An equal volume of chloroform-n-butanol (4:1) solution was continued to be added to the aqueous phase, and the operation was repeated until no protein was detected.

[0107] The decolorization treatment was carried out with D-101 macroporous adsorption resin: The static adsorption decolorization method was used to decolorize the polysaccharide sample after protein removal. The amount of D-101 resin used was 1 g / 10 mL, the polysaccharide concentration of the sample loaded was 3 mg / mL, pH = 7, and the treatment time was 2 h.

[0108] 2. Experimental results

[0109] The crude polysaccharide of wax gourd peel was extracted by the water extraction and alcohol precipitation method, and the yield was (2.24 ± 0.15)%. After decolorization and removal of impurities such as protein, two polysaccharide components, namely BEP-1 and BEP-2 ( Figure 1 A), were obtained by purification through a DEAE-Cellulose 52 anion exchange resin glass chromatography column. The BEP-1 component was further purified by a Sephadex G-25 glass chromatography column, and the elution curve is shown in Figure 1 B. Two polysaccharides, BEP-1a and BEP-1b, were further concentrated, and the yields were (21.75 ± 0.01)% and (9.88 ± 0.05)%, respectively.

[0110] Example 2 Determination of the relative molecular weight of the homogeneous polysaccharide from wax gourd peel

[0111] 1. Experimental method

[0112] The high performance gel permeation chromatography (HPGPC) was used to determine the relative molecular weights of the wax gourd peel polysaccharides BEP-1a and BEP-1b. The instrument was equipped with three Ultrahydrogel (Waters, American) chromatographic columns in series (the specifications were 250, 1000 and 2000; 30 cm × 7.8 mm; 6 μm), the column oven temperature was 40 °C, the flow rate was 0.5 mL / min, and the sample loading volume was 20 μL. The mobile phase was 0.02 M KH2PO4 solution. Standard curves were plotted with T-dextran standards of different molecular weights (5.2×10 3 Da, 4.86×10 4 Da, 6.68×10 5 Da).

[0113] 2. Experimental results

[0114] The HPGPC results are shown in Figure 2 , and the figures all show a single symmetric peak, indicating that BEP-1a ( Figure 2 A) and BEP-1b ( Figure 2 B) are homogeneous polysaccharides, that is, pure polysaccharides. According to the molecular weight - retention time standard curve measured with the standard dextran standard, it is lg(Mw) = -0.1863T + 12.13, R 2= 0.9876. The relative molecular masses of BEP-1a and BEP-1b calculated based on the peak time of HPGPC were 4.68×10 4 Da and 3.19×10 6 Da. During the in vitro bioactivity screening (Example 9), it was found that BEP-1a had certain bioactivity, and no bioactivity of BEP-1b was found. Therefore, BEP-1a was mainly explored in the following.

[0115] Example 3 Chemical Composition Analysis of Homogeneous Polysaccharide from Wax Gourd Peel

[0116] (1) Chemical Composition Analysis of Homogeneous Polysaccharide from Wax Gourd Peel

[0117] The phenol-sulfuric acid method was used to measure the neutral sugar content in the polysaccharide, with α-D-glucose as the standard; the Coomassie brilliant blue method was used to determine the protein content, with bovine serum albumin as the standard; and the m-hydroxybiphenyl method was used to determine the uronic acid content, with galacturonic acid as the standard.

[0118] (2) Monosaccharide Composition Analysis of Polysaccharide from Wax Gourd Peel

[0119] The monosaccharide composition of the wax gourd peel polysaccharide BEP-1a was determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization HPLC method. 5 mg of each of L-Ara, D-Gal, α-D-Glc, D-Xyl, D-Man, L-Rha, D-Rib, D-GalA and D-GlcA were quantitatively dissolved in a 10 mL volumetric flask with ultrapure water to prepare a mixed standard solution. 100 μL of the mixed standard solution was added with 100 μL of 0.6 mol / L sodium hydroxide solution and 200 μL of 0.5 mol / L PMP methanol solution and mixed well. The reaction was carried out in a constant temperature water bath at 70 °C for 100 min, then taken out and cooled to room temperature. 200 μL of 0.3 mol / L hydrochloric acid solution was added, shaken well, and ultrapure water was added to 2 mL. Chloroform was added in an equal volume, vortexed and shaken, and then allowed to stand for layer separation. The upper aqueous phase solution was taken, and extraction was repeated twice with chloroform. The aqueous phase solution was filtered through a 0.22 μm microporous filter membrane for HPLC analysis.

[0120] Weigh 10 mg of the polysaccharide sample and place it in a stoppered test tube. Add 3 mL of 2 M trifluoroacetic acid (TFA), mix well, and hydrolyze at 110 °C for 4 h. Then, dry the polysaccharide acid hydrolysate using a rotary evaporator and replace it with methanol multiple times until there is no sour taste. Derive the polysaccharide acid hydrolysis product using the method of derivatization with the standard PMP. The liquid phase analysis conditions are as follows: Shimadzu high-performance liquid chromatograph LC-2030C 3D Plus, the chromatographic column is COSMOSIL 5C18-AR-II chromatographic column (4.6 mm I.D.×250 mm, 5 μm), the detection wavelength is 254 nm, the mobile phase is 83% 0.05 M potassium dihydrogen phosphate solution (pH = 6.9) and 17% acetonitrile, the flow rate is 1.0 mL / min, and the injection volume is 10 μL.

[0121] 2. Experimental Results

[0122] Table 3 Chemical Composition of Homogeneous Polysaccharides from Wax Gourd Peel

[0123]

[0124] The results are shown in Table 3. The total sugar content of BEP-1a is 93.06%, the uronic acid content is 3.52%, the protein content is 2.81%, and the monosaccharide composition is composed of galactose (Gal, 88.55%), galacturonic acid (GalA, 4.02%), rhamnose (Rha, 3.89%), arabinose (Ara, 1.93%), and glucose (Glc, 1.61%) ( Figure 3 ).

[0125] Example 4 Fourier Transform Infrared Analysis (FT-IR) of Wax Gourd Peel Polysaccharide

[0126] 1. Experimental Method

[0127] Weigh 1.0 mg of the dried polysaccharide sample BEP-1a, prepare a sample using the potassium bromide tablet pressing method, with a tablet thickness of 1 mm, and use a Fourier transform infrared spectrometer (BRUKER TEMSOR 27, Germany) to measure the infrared spectral characteristics of the polysaccharide. The scanning wavenumber ranges from 400 cm -1 -4000 cm -1 .

[0128] 2. Experimental Results

[0129] The infrared absorption spectrum of wax gourd peel polysaccharide BEP-1a is as Figure 4 shown. Among them, the absorption peak at 3487.5 cm -1 represents the stretching vibration of the O-H bond of carbohydrates, and the absorption peak at 2927.9 cm -1 corresponds to the stretching vibration of the C-H bond of methyl or methylene. In addition, 1200 - 1400 cm -1A group of peaks within a certain range are the angular vibrations of C-H bonds. Based on these three characteristic peaks, it can be determined that the polysaccharide has a carbohydrate structure. In addition, the absorption peak at 1738.2 cm -1 indicates that the polysaccharide contains a carboxyl (COO-) group. The strong absorption peak at 950 - 1200 cm -1 is the characteristic absorption of the pyranose ring, while the weak absorption peak at 902.8 cm-1 corresponds to the absorption of the β-pyranose ring.

[0130] Example 5 Methylation Analysis of Wax Gourd Peel Polysaccharide

[0131] 1. Experimental Method

[0132] Weigh 5 mg of the wax gourd peel polysaccharide sample BEP-1a, add phosphorus pentoxide anhydrous and dry overnight, dissolve it in 200 μL DMSO, fill the system with nitrogen to replace air for protection, add 15 mg of dry sodium hydroxide powder and dissolve it by ultrasonic treatment, place it in an ice-water bath to solidify, then slowly drop 100 μL of KI solution, while slowly heating to 20 °C and ultrasonicating for 30 min. Subsequently, add 1 mL of 4 mmol / L Na2SO3 aqueous solution, mix well, add 500 μL of chloroform, vortex and shake, let it stand and take the chloroform layer, repeat the extraction three times, combine the solutions, add anhydrous Na2SO4 to remove water, centrifuge and filter, then dry it with nitrogen, and analyze its methylation degree by infrared spectroscopy. If the hydroxyl groups in the polysaccharide are not completely substituted, the above process needs to be repeated until the hydroxyl absorption peak disappears in the infrared spectrum. Dissolve the methylation product in 2 mol / L TFA, hydrolyze it at 120 °C for 3 h and reduce it with sodium borohydride in deuterium oxide, then acetylate it to convert the monosaccharides into partially methylated alditol acetates. Analyze the acetylated sample by GC-MS, identify the peaks of the methylated sugars through mass spectrometry, estimate its relative molecular mass by combining the peak area of GC and the corresponding response value, and calculate the response factor of the partially methylated alditol acetates through effective carbon reaction.

[0133] The GC-MS conditions are as follows: the chromatographic column is RXI-5SIL MS (30 m × 0.25 mm, 0.25 μm), the initial temperature is 140 °C, it is heated to 240 °C at a rate of 5 °C / min and held for 10 min; the carrier gas is helium; the split ratio is 1:10; the ion source is an electron impact ion source (EI ion source), the ionization potential is 70 eV, and the injection volume is 1 μL.

[0134] 2. Experimental Results

[0135] Table 4 Methylation Analysis Results of the Homogeneous Wax Gourd Peel Polysaccharide BEP-1a

[0136]

[0137] The methylation results show (see Figure 5And in Table 4), the linkage modes of polysaccharide BEP-1a include: T-Ara (0.98%), 1,4-Rha (1.92%), 1,2,4-Rha (1.10%), 1,4-Glc (1.34%), and 1,4-Gal (94.66%). The contents of branched sugar residues and terminal sugar residues are basically the same, indicating that the methylation of the polysaccharide is complete. It can be seen from this that the polysaccharide is composed of galactan consisting of about 90% 1,4-Gal and about 10% pectin of RG-I type.

[0138] Example 6 Nuclear Magnetic Resonance Analysis of Wax Gourd Peel Polysaccharide

[0139] 1. Experimental Method

[0140] Weigh 20 mg of the wax gourd peel polysaccharide sample and dissolve it in 2 mL of deuterated water. After freeze-drying, redissolve it in 600 μL of deuterated water. Under room temperature conditions, use a Bruker AM 500 nuclear magnetic resonance spectrometer to complete the 1 1H spectrum, 13 13C spectrum, 1 1H- 1 1H COSY spectrum, 1 1H- 13 1H- 1 13C HMBC spectrum and 13 1H-

[0141] 2. Experimental Results

[0142] As shown in Figure 6 Figures 6A and 6B, the chemical shifts of the anomeric carbon and anomeric hydrogen of the galactose residues of BEP-1a are δ107.45 and δ5.03 respectively, indicating that galactose is mainly in the α configuration. Combining the H-H COSY ( Figure 6 Figure 6C) and HSQC spectra ( Figure 6 Figure 6D), it can be inferred that the carbon-hydrogen assignments of the galactose residues are C2 / H2 (δ82.57 and δ4.04), C3 / H3 (δ77.38 and δ3.92), C4 / H4 (δ78.51 and δ4.13), C5 / H5 (δ73.10 and δ3.81), C6 / H6 (δ67.84 and δ3.72), and C6′ / H6′ (δ68.02 and δ3.67). In the HMBC spectrum ( Figure 6 Figure 6E), the cross-peaks of galactose C1 / H4 and C4 / H1 can be observed, further proving that the galactose residues of the polysaccharide are linked to each other through 1,4 bonds.

[0143] To sum up, the present invention speculates that the homogeneous polysaccharide BEP-1a from wax gourd peel has a repeating unit structure as shown in Figure 7 the figure.

[0144] Example 7 Scanning Electron Microscope Detection

[0145] 1. Experimental methods

[0146] The surface of wax gourd peel polysaccharide samples was observed using a Sigma 500 field emission scanning electron microscope. An appropriate amount of polysaccharide powder was placed on a copper cross-section sample stage, sprayed with gold, and then observed under a scanning electron microscope.

[0147] 2. Experimental results

[0148] The surface morphology of wax gourd peel polysaccharide BEP-1a was evaluated by scanning electron microscopy (SEM). Figure 8 As shown, at 100× magnification ( Figure 8 A), wax gourd polysaccharide BEP-1a is in irregular stone shape. Under 1000× magnification ( Figure 8 B), wax gourd polysaccharide BEP-1a has a distinct strip-like lamellar structure.

[0149] Example 8 Evaluation of the efficacy of wax gourd peel polysaccharide BEP-1a on gastric ulcer lesions in mice

[0150] 1. Experimental methods

[0151] (1) Drug preparation

[0152] Preparation of 0.5% CMC-Na solution: Weigh 1.0 g of CMC-Na powder and place it in 200 mL of purified water and heat to dissolve. After complete dissolution, cool the solution to room temperature, add purified water to make up the weight, and place it in a refrigerator at 4°C until ready for use.

[0153] Preparation of wax gourd peel polysaccharide BEP-1a solution: Weigh an appropriate amount of wax gourd peel polysaccharide BEP-1a powder and dissolve it in 0.5% CMC-Na solution. The oral volume for mice is 0.1 mL / 10 g, with a low dose of 25 mg / kg and a high dose of 50 mg / kg.

[0154] Preparation of omeprazole solution: Weigh an appropriate amount of omeprazole powder and dissolve it in 0.5% CMC-Na solution. The oral volume for mice is 0.1 mL / 10 g, and the dose is 20 mg / kg.

[0155] (2) Animal grouping and dosing regimen

[0156] After 1 week of adaptive feeding, all the mice were divided into a normal group (Control), a model group (Model), an omeprazole positive drug group (OME, 20 mg / kg), a low-dose wax gourd peel polysaccharide group (BEP-1a-L, 25 mg / kg), and a high-dose wax gourd peel polysaccharide group (BEP-1a-H, 50 mg / kg) according to the principle of random grouping, with 12 mice in each group. Administration was carried out according to a gavage volume of 0.1 mL / 10 g. The normal group and the model group were given 0.5% CMC-Na solution, once a day by gavage for 3 consecutive days, and once by gavage 1 h before modeling on the 4th day.

[0157] (3) Establishment of an acute gastric ulcer injury model in mice induced by ethanol.

[0158] After the administration ended on the 3rd day, all the mice were fasted for 24 h and water-deprived for 2 h. After administration 1 h before modeling on the 4th day, except for the normal group, all the mice in other groups were gavaged with absolute ethanol according to 0.2 mL / 10 g, and the normal group was given an equal volume of 0.5% CMC-Na solution. After 1 h, the modeling ended, and all the mice were sacrificed by cardiac blood collection after anesthesia with isoflurane.

[0159] (4) Macroscopic examination of the gastric tissue morphology of mice

[0160] After the mice were sacrificed by cardiac blood collection, the gastric tissue was taken out, cut along the greater curvature of the stomach, and the contents of the gastric tissue were washed clean with ice-cold saline. The washed gastric tissue was flattened on filter paper, photographed with a camera and archived to evaluate the macroscopic morphology of the gastric tissue of mice in different treatment groups. Subsequently, the gastric ulcer area was quantified using Image J software, and the ulcer index and ulcer inhibition rate were calculated.

[0161] The formula for calculating the ulcer index is: ulcer index = gastric ulcer area / total gastric area × 100

[0162] The formula for calculating the ulcer inhibition rate is: (ulcer index of the model group - ulcer index of the drug group) / ulcer index of the model group × 100%

[0163] (5) Histopathological examination of the gastric tissue of mice

[0164] ① H&E staining of the gastric tissue of mice

[0165] The taken-out gastric tissue was fixed in 4% paraformaldehyde fixative for 24 h, and then prepared into paraffin sections. The paraffin sections were dewaxed to water, and then hematoxylin staining and eosin staining were carried out in sequence. After the staining ended, dehydration and mounting were carried out. The sections were placed in a scanner imaging system to collect images, and then H&E histological scoring was carried out on the collected images.

[0166] ② PAS staining of the gastric tissue of mice

[0167] The excised gastric tissues were fixed in 4% paraformaldehyde fixative for 24 h, and then prepared into paraffin sections. The paraffin sections were dewaxed to water, and then subjected to periodic acid staining, Schiff staining, and hematoxylin staining in sequence. After staining, dehydration and mounting were carried out. The sections were placed in a scanner imaging system to collect images, and then the PAS positive staining rate was statistically analyzed using Image J software.

[0168] 2. Experimental Results

[0169] The present invention macroscopically examined the gastric tissues of mice to evaluate the protective effect of BEP-1a on ethanol-induced acute gastric ulcer injury. Figure 9 A is the macroscopic picture of the gastric tissues of mice in different treatment groups. It can be seen from the figure that the macroscopic morphology of the gastric tissues of mice in the normal group is normal, without ulceration and bleeding. In the ethanol group, large-area bleeding lesions appeared in the gastric tissues of mice, showing a patchy distribution. Compared with the ethanol group, the bleeding in the gastric tissues of mice in the positive drug OME group and the BEP-1a group was significantly reduced, showing a punctate and small patchy distribution.

[0170] To further evaluate the protective effect of BEP-1a on the ethanol-induced acute gastric ulcer injury model in mice, the present invention used Image J software to quantitatively analyze the gastric ulcer area of different treatment groups, and calculated the ulcer index and ulcer inhibition rate. The results showed that the gastric ulcer area of the model group was 13442±2284 mm 2 . Both the positive drug OME group and the BEP-1a group could significantly reduce the gastric ulcer area of mice, and the BEP-1a group showed a dose-dependent effect. Among them, the gastric ulcer area of the BEP-1a-H group was 3814±1399 mm 2 , with the best effect. The gastric ulcer areas of the BEP-1a-L group and the OME group were 5410±2005 mm 2 and 8260±3892 mm 2 ( Figure 9 B). Further analysis of the ulcer index found that the ulcer index of the model group was 75.48±9.28%, which was significantly increased compared with the normal group. The administration of the OME group and the BEP-1a group both significantly reduced the ulcer index. Among them, the ulcer index of the BEP-1a-H group was 22.56±10.76%, the BEP-1a-L group was 38.60±17.60%, and the OME group was 38.87±19.82%( Figure 9 C). In terms of the ulcer inhibition rate, the administration of the OME group and the BEP-1a group both significantly increased. The ulcer inhibition rate of the BEP-1a-H group was 71.00±10.78%, the BEP-1a-L group was 50.40±16.53%, and the OME group was 50.41±20.70%( Figure 9 D).

[0171] To further evaluate the efficacy of BEP-1a, the present invention performed H&E and PAS staining on the gastric tissues of mice in different treatment groups. The results of H&E staining ( Figure 10 A) showed that the gastric mucosal glands in the normal group were arranged neatly, and the mucosal layer, submucosal layer, and muscular layer were intact. In contrast, severe damage occurred in the gastric tissues of the model group mice, manifested as gastric mucosal erosion and bleeding, inflammatory cell infiltration, and submucosal edema of the gastric mucosa. However, after treatment with OME and BEP-1a, the gastric tissues of the mice were significantly protected, the area of gastric mucosal erosion and bleeding decreased, the phenomenon of submucosal edema of the gastric mucosa was alleviated, and there was no obvious inflammatory cell infiltration. The histological score of H&E staining showed ( Figure 10 B) that the score of the model group was 8.78 ± 0.69, and the degree of gastric mucosal damage was the most severe. The scores of the OME, BEP-1a-H, and BEP-1a-L groups were 4.45 ± 1.07, 3.55 ± 0.39, and 5.78 ± 1.07, respectively, all of which had a significant protective effect on the gastric mucosa, and the BEP-1a-H group had a better effect.

[0172] The PAS staining was used to measure the glycoprotein on the surface layer of the gastric mucosa. After PAS staining, the glycoprotein on the surface layer of the gastric mucosa produced a positive reaction and showed purplish red. The gastric mucosa of the normal group mice was intact, with a large area of positive staining, and the PAS staining rate was 3.52 ± 0.90%, indicating that there was no loss of gastric mucosal protein. In the model group, there was a large area of epithelial cell shedding, and the PAS staining rate was 0.21 ± 0.01%, which was significantly lower than that of the normal group in terms of PAS positive staining. The PAS staining rates of the BEP-1a-H and BEP-1a-L groups were 2.79 ± 0.78% and 1.60 ± 0.06%, respectively, and the PAS staining rates were significantly increased compared with the model group, indicating that after treatment with BEP-1a, the gastric tissues of the mice were significantly protected, reducing the loss of glycoprotein on the surface layer of the gastric mucosa. The above results showed that BEP-1a-H had the best effect on protecting the gastric mucosal injury of mice ( Figure 10 C-D).

[0173] Example 9 Protective effect of wax gourd peel polysaccharide BEP-1a on ethanol-induced GES-1 cell injury

[0174] 1. Experimental method

[0175] (1) Culture of GES-1 cells

[0176] ① Cell resuscitation

[0177] Take out the cryopreservation tube containing GES-1 cells from the liquid nitrogen tank, quickly place it in a 37°C water bath, gently rotate the cryopreservation tube until only a small ice core remains in the tube, immediately transfer it to the laminar flow hood, wipe the sealing area with an alcohol cotton ball, transfer the cryopreservation liquid to an EP tube containing 2 mL of complete medium, centrifuge at 1000 r / min for 4 min, discard the supernatant, add 2 mL of complete medium, pipette and mix well, then inoculate into a culture flask containing 2 mL of complete medium, mix well, and place it in a carbon dioxide cell incubator for culture. After 12 h, when the cells adhere to the wall, replace the fresh complete medium and continue the culture.

[0178] ② Subculture of GES-1 cells

[0179] When observing under the microscope that the growth density of GES-1 cells reaches 80%-90%, subculture can be carried out. Aspirate the original culture medium in the cell culture flask, add 2 mL of PBS buffer and wash twice, then add 500 μL of 0.25% trypsin solution for digestion. When observing under the microscope that the cells are digested until they retract and become round (the time is about 3 min), the trypsin in the culture flask is aspirated, add 3 mL of complete medium to terminate the digestion, pipette the cells at the bottom of the cell culture flask to detach them from the wall, centrifuge the cell suspension at 1000 r / min for 4 min, discard the supernatant, add 3 mL of fresh complete medium, pipette and mix well, and transfer it to a new cell culture flask at a ratio of 1:3 for continuous culture.

[0180] ③ Cryopreservation of GES-1 cells

[0181] GES-1 cells are cultured in a cell culture flask. When observing under the microscope that the cell growth density reaches 80-90%, cryopreservation of the cells can be carried out. After digesting the cells according to the method in "② Subculture of GES-1 cells", centrifuge the cell suspension at 1000 r / min for 4 min, discard the supernatant, add 1 mL of cell cryopreservation solution, resuspend and add it to a cryopreservation tube, place it in a cryopreservation box, and put it in a -80°C refrigerator. After 24 h, transfer it to the liquid nitrogen tank for long-term storage.

[0182] (2) MTT assay for cell viability

[0183] The MTT method is used to determine the viability of GES-1 cells. That is, the cells in the 96-well plate are co-incubated with 10 μl of MTT solution at 37°C for 4 h until purple formazan crystals are formed. Discard the liquid in the wells, add 100 μL of DMSO solution to each well, gently shake and place it in the incubator for 15 min. After the purple formazan crystals are completely dissolved, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 490 nm, and calculate the cell viability.

[0184] (3) Establishment of ethanol-induced GES-1 cell injury model

[0185] Human gastric mucosal epithelial cells GES-1 in the logarithmic growth phase were seeded at 5×10 3 / well in a 96-well cell culture plate and cultured in a cell incubator. After 24 h, they were divided into a blank group and an ethanol group (ethanol concentrations were: 2%, 3%, 4%, 5%, 6%, 7%, 8%). Different groups were treated accordingly. After 2 h, the survival rate of GES-1 cells was detected by the MTT method to determine the optimal ethanol concentration for model establishment.

[0186] (4) Effect of wax gourd polysaccharide BEP-1a on the cytotoxicity of GES-1 cells

[0187] Human gastric mucosal epithelial cells GES-1 in the logarithmic growth phase were seeded at 5×10 3 / well in a 96-well cell culture plate and cultured in a cell incubator. After 24 h, they were divided into a blank group and a drug administration group (concentrations of wax gourd polysaccharide BEP-1a were: 1000, 500, 100, 50, 10, 5, 1, 0.5, 0.1 μg / mL). After different groups were treated accordingly, they were cultured in a 37°C, 5% CO2 cell incubator. After 24 h, the cytotoxic effect of wax gourd polysaccharide BEP-1a on GES-1 cells was detected by the MTT method.

[0188] (5) Effects of wax gourd polysaccharides BEP-1a and BEP-1b on the survival rate and morphology of ethanol-induced GES-1 cell injury

[0189] Human gastric mucosal epithelial cells GES-1 in the logarithmic growth phase were seeded at 5×10 3 / well in a 96-well cell culture plate and cultured in a cell incubator. After 12 h, they were divided into a blank group, a model group, and a drug group (concentrations of wax gourd polysaccharides BEP-1a and BEP-1a were: 200, 100, 50, 25, 12.5 μg / mL). After different groups were pretreated accordingly for 12 h, modeling was carried out under the optimal modeling conditions, and at the same time, different concentrations of BEP-1a were given for co-incubation. After the modeling was completed, the cell morphology was observed under an inverted microscope and images were collected, and the cell survival rate was measured by the MTT method.

[0190] (6) Detection of GES-1 cell apoptosis by flow cytometry

[0191] Human gastric mucosal epithelial cells GES-1 in the logarithmic growth phase were seeded at 25×10 4Inoculate into 96-well cell culture plates, place them in a cell culture incubator for culture. After 24 hours, divide them into a blank group, a model group, a low-dose BEP-1a group (100 μg / mL), and a high-dose BEP-1a group (200 μg / mL). Administer drugs and establish the model under the same time conditions as in step (3) "Establishment of ethanol-induced GES-1 cell injury model". After the model establishment, digest and collect the cells with 0.25% trypsin, stain them according to the Annexin V-FITC / PI apoptosis detection kit instructions, and perform detection using an ultra-high-speed flow cytometer after staining.

[0192] (7) Calcein-AM / PI staining study of GES-1 cells

[0193] After administration and model establishment by the same method (5), stain according to the Calcein-AM / PI cell viability detection kit instructions, and perform fluorescence image acquisition using an intelligent cell imaging system after staining.

[0194] 2. Experimental results

[0195] The present invention has demonstrated in vivo that BEP-1a has a protective effect on ethanol-induced acute gastric ulcer injury in mice. In order to clarify whether BEP-1a inhibits ethanol-induced gastric ulcer injury by protecting gastric mucosal epithelial cell injury, the present invention established an ethanol-induced GES-1 injury model in vitro to clarify the protective effect of BEP-1a on gastric mucosal epithelial cells.

[0196] To determine the optimal model establishment concentration of ethanol, normal GES-1 cells were treated with different concentrations of ethanol for 2 hours, and the effect of ethanol on the survival rate of GES-1 cells was detected. The results showed that as the ethanol concentration increased, the survival rate of GES-1 cells decreased. When the ethanol concentration was 4%, the survival rate of GES-! cells was 59.36 ± 6.66%, meeting the model requirements for the experiment ( Figure 11 A). Therefore, in subsequent experiments, co-incubation of 4% ethanol with GES-1 cells for 2 hours was used as the optimal model establishment condition.

[0197] The toxicity of the drug needs to be investigated before its biological activity. Therefore, the present invention co-incubated different concentrations of BEP-1a with GES-1 cells to determine the safe concentration range of BEP-1a. From Figure 11As can be seen from Figure B, with the increase in the concentration of BEP-1a, it has certain cytotoxicity to GES-1 cells. When the administration concentration of BEP-1a is 1000 μg / mL and 500 μg / mL, significant cytotoxicity is shown compared with the normal group. Therefore, in the ethanol-induced GES-1 cell injury model, the present invention selects a concentration below 500 μg / mL to explore the efficacy of BEP-1a. The MTT test results show that BEP-1a can increase the survival rate of ethanol-induced GES-1 cells ( Figure 11 C). Compared with the normal group, the cell survival rate of the model group is 33.74 ± 0.71%. BEP-1a at different concentrations all showed varying degrees of protective effects. Among them, when the concentration of BEP-1a is 200 μg / mL and 100 μg / mL, the protection rates are 45.35 ± 2.73% and 42.43 ± 3.35% respectively, showing significant protective effects. Therefore, 200 μg / mL and 100 μg / mL will be used as the high dose and low dose of BEP-1a in subsequent in vitro experiments.

[0198] The MTT test results of BEP-1b show that ( Figure 12 ) BEP-1b cannot increase the cell survival rate of ethanol-induced GES-1 cell injury.

[0199] The effect of BEP-1a on the cell morphology of ethanol-induced GES-1 cell injury was observed under an inverted microscope. The cells in the normal group were flat and fusiform, adhered to the wall and grew, and were arranged closely; a large number of cells in the model group shrank and became round, the cell adhesion ability became weak, the cell gap increased, and the floating cells increased significantly; in the high and low concentration groups of BEP-1a, as the drug concentration increased, the growth state and morphology of the cells gradually approached the normal group ( Figure 11 E).

[0200] Annexin V-FITC / PI apoptosis kit and flow cytometry were used to detect the apoptosis of ethanol-induced GES-1 cells ( Figure 11 F). The number of Annexin V-FITC / PI double-positive cells in the model group increased significantly, indicating that cell apoptosis was advanced. Treatment with BEP-1a decreased the number of double-positive cells in a dose-dependent manner, indicating that BEP-1a may protect GES-1 cells by inhibiting the transition of late apoptosis and necrosis ( Figure 11 D).

[0201] To more intuitively investigate the protective effect of BEP-1a on GES-1 cells, the present invention uses the Calcein / PI cell viability kit for fluorescence staining. Calcein-AM stains live cells with green fluorescence, and PI stains dead cells with red fluorescence. As Figure 11As shown in Figure G, the model group showed a decrease in Calcein-AM positive cells and an increase in PI positive cells, manifested as a reduction in the green fluorescence area and an enhancement in the red fluorescence area, indicating ethanol-induced cell damage. Compared with the model group, the BEP-1a group reversed these trends, and with the increase in the concentration of BEP-1a, the protective effect against ethanol-induced GES-1 cell damage became more significant( Figure 11 H-M).

[0202] In summary, the present invention verified the application of BEP-1a polysaccharide in the preparation of products for the prevention and treatment of gastric ulcers using animal and cell models, especially in the prevention and treatment of ethanol-induced gastric ulcers. However, the application of BEP-1a polysaccharide in the preparation of products for the prevention and treatment of gastric ulcers is applicable not only to humans but also to other animals, including but not limited to mice, pigs, cows, sheep, etc.

[0203] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A wax gourd peel polysaccharide BEP-1a, characterized in that, Obtained by a preparation method including the following steps: S1. Extraction of crude wax gourd peel polysaccharide: Extract crude wax gourd peel polysaccharide from wax gourd peel by the water extraction and alcohol precipitation method; S2. Deproteinization and decolorization treatment: Perform deproteinization and decolorization treatment on the crude wax gourd peel polysaccharide obtained in step S1 to obtain wax gourd peel polysaccharide BEP; S3. After formulating the wax gourd peel polysaccharide BEP obtained in step S2 into an aqueous solution, elute it using a DEAE-Cellulose 52 anion exchange chromatography column, and sequentially use water, 0.1 M NaCl, 0.5 M NaCl, and 0.5 M NaOH solutions as eluents for stepwise elution. Collect the fractions eluted with 0.1 M NaCl, dialyze, and lyophilize to obtain wax gourd peel polysaccharide BEP-1; S4. After formulating the wax gourd peel polysaccharide BEP-1 obtained in step S3 into an aqueous solution, purify it using a Sephadex G-25 chromatography column, elute it with 0.1 M NaCl as the eluent, and according to the elution curve, collect the eluent of the first single symmetric peak, and perform post-treatment to obtain wax gourd peel polysaccharide BEP-1a; The specific preparation method of the crude wax gourd peel polysaccharide includes the following steps: Soak the wax gourd peel in water sufficiently, then decoct it, filter, concentrate the filtrate to obtain a crude extract, add an alcohol solvent to the obtained crude extract, precipitate sufficiently, and take the precipitate and dry it to obtain the crude wax gourd peel polysaccharide; The conditions for the decoction are: First boil, and then continue to decoct for 25 - 30 min.

2. The wax gourd peel polysaccharide BEP-1a according to claim 1, wherein The alcohol solvent includes ethanol or methanol.

3. A wax gourd peel polysaccharide, characterized in that, The relative molecular mass of the wax gourd peel polysaccharide is 4.68×10 4 Da, and the structural formula of the repeating unit of the wax gourd peel polysaccharide is shown as follows: ； Among them, Gal represents galactose, GalA represents galacturonic acid, Rha represents rhamnose, Ara represents arabinose, and Glc represents glucose; f represents the configuration of furanose, p and represents the configuration of pyranose.

4. The wax gourd peel polysaccharide according to claim 3, characterized in that, The total sugar content of the wax gourd peel polysaccharide ≥ 90%.

5. The wax gourd peel polysaccharide according to claim 3, wherein The monosaccharide composition of the wax gourd peel polysaccharide is composed of 88.55% galactose, 4.02% galacturonic acid, 3.89% rhamnose, 1.93% arabinose, and 1.61% glucose.

6. Use of the wax gourd peel polysaccharide BEP-1a as claimed in claim 1 or 2 or the wax gourd peel polysaccharide as claimed in any one of claims 3 - 5 in the preparation of a drug for preventing and treating gastric ulcer.

7. The application according to claim 6, wherein The gastric ulcer includes alcoholic gastric ulcer.

8. Use of the wax gourd peel polysaccharide BEP-1a as claimed in claim 1 or 2 or the wax gourd peel polysaccharide as claimed in any one of claims 3 - 5 in the preparation of a health product or feed having the function of assisting in protecting the gastric mucosa.

9. A drug, characterized in that, The drug contains the wax gourd peel polysaccharide BEP-1a as claimed in claim 1 or 2 or the wax gourd peel polysaccharide as claimed in any one of claims 3 - 5.

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