White gourd peel polysaccharide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to exocarpium benincasae polysaccharide and application thereof. The wax gourd peel polysaccharide with a repetitive unit structure is obtained by performing impurity removal, separation and purification on wax gourd peel crude polysaccharide obtained by a water extraction and alcohol precipitation method for the first time. The exocarpium benincasae polysaccharide is mainly composed of galactose, galacturonic acid, rhamnose, arabinose and glucose, and the total sugar content is larger than or equal to 90%. In-vivo and in-vitro experiments prove that the exocarpium benincasae polysaccharide disclosed by the invention has an excellent gastric mucosa protection effect and has a good application prospect in the aspect of preparing medicines for preventing and treating gastric ulcer or health-care products or feeds with an auxiliary gastric mucosa protection function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a wax gourd peel polysaccharide and application thereof. Background Art

[0002] Winter melon peel, as the dried outer peel of the Cucurbitaceae plant Benincasa hispida (Thunb.) Cogn., is obtained by washing the winter melon, peeling the outer peel and drying it. Its physical characteristics are light weight, crisp texture, faint smell and light taste, irregular fragments, and often curled inward. The outer surface of the winter melon peel is gray-green or yellow-white, covered with white frost, while the inner surface is relatively rough. Modern pharmacological studies have shown that winter melon peel is rich in chemical components such as flavonoids, alkaloids, tannins, trace elements and vitamins, and exhibits pharmacological activities such as antihypertensive, diuretic, hypoglycemic and antioxidant.

[0003] Polysaccharides, as a class of high-molecular compounds composed of more than 10 monosaccharides, have highly diverse structures due to the complexity of the main chain, side chains and connection methods, which to a certain extent limits in-depth research on them. However, the rapid development of analytical instruments in recent years has greatly promoted the in-depth research of polysaccharides, thus revealing the wide range of biological activities of polysaccharides in anti-oxidation, immunomodulation, anti-tumor, hypoglycemic and antibacterial aspects. 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, including chain length, glycosidic bond type, connection method and molecular weight.

[0004] Specifically for wax gourd peel polysaccharides, studies have shown that it has hypoglycemic and antibacterial effects. However, it is worth noting that wax gourd peel polysaccharides obtained by different extraction methods have differences in component content and function. This shows that the specific efficacy of wax gourd peel polysaccharides is not only related to its chemical composition, but also closely related to its structural characteristics.

[0005] Although some progress has been made in the current research on polysaccharides, there is still a lack of in-depth exploration of the application of wax gourd peel polysaccharides, especially wax gourd peel polysaccharides with specific structures and molecular weights, in other aspects. The existing technology has not yet fully revealed the potential uses of wax gourd peel polysaccharides with these 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 shortcomings of the current prior art that wax gourd peel polysaccharide still lacks in-depth exploration, and 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 purpose of the 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 use of the wax gourd peel polysaccharide in the preparation of a medicament for preventing and treating gastric ulcer.

[0009] Another object of the present invention is to provide the use of wax gourd peel polysaccharide in the preparation of health products or feeds with the function of assisting in protecting gastric mucosa.

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

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

[0013] S1. Extraction of crude polysaccharides from wax gourd peel: crude polysaccharides from wax gourd peel were extracted by water extraction and alcohol precipitation method;

[0014] S2. Deproteinization and decolorization: The crude wax gourd peel polysaccharide obtained in step S1 was deproteinized and decolorized to obtain wax gourd peel polysaccharide BEP;

[0015] S3. After preparing the wax gourd peel polysaccharide BEP obtained in step S2 into an aqueous solution, eluting it using a DEAE-Cellulose52 anion chromatography column, using water, 0.1M NaCl, 0.5M NaCl, and 0.5M NaOH solution as eluents for stepwise elution, collecting the fraction eluted with 0.1M NaCl, and post-treating it to obtain wax gourd peel polysaccharide BEP-1;

[0016] S4. After the wax gourd peel polysaccharide BEP-1 obtained in step S3 is prepared into an aqueous solution, it is purified using a polysaccharide G-25 chromatography column, using 0.1M NaCl as an eluent, and the eluent of the first single symmetrical peak is collected according to the elution curve, and post-processed to obtain wax gourd peel polysaccharide BEP-1a.

[0017] The present invention further removes impurities, separates and purifies the crude wax gourd peel polysaccharide obtained by water extraction and alcohol precipitation for the first time, and obtains a uniform polysaccharide, wax gourd peel polysaccharide BEP-1a. In vivo and in vitro experiments have shown that the wax gourd peel polysaccharide BEP-1a obtained by the above preparation method has excellent gastric mucosal protective effect, and has a good application prospect in the preparation of drugs for preventing and treating gastric ulcers or health products or feeds with auxiliary gastric mucosal protection function.

[0018] Furthermore, the specification of the DEAE-Cellulose 52 anion chromatography column is Φ2.5 cm×60 cm, wherein Φ2.5 cm represents the diameter of the exchange column, and 60 cm represents the height of the chromatography column.

[0019] Furthermore, the specification of the dextran gel G-25 chromatography column is Φ2.5cm×60cm.

[0020] Furthermore, in step S2, the deproteinization and decolorization treatments are performed in no particular order. As an optional method, the deproteinization and decolorization treatments are performed by deproteinizing the crude wax gourd peel polysaccharide obtained in step S1 to obtain a deproteinized solution, and then decolorizing and post-treating the deproteinized solution to obtain wax gourd peel polysaccharide BEP.

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

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

[0023] Preferably, the drying is freeze-drying.

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

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

[0026] Furthermore, as a common deproteinization method, the deproteinization is deproteinization by using the Sevag method.

[0027] Furthermore, as a common implementation method, the Sevag method for deproteinization includes the following steps: adding water to wax gourd peel polysaccharide BEP to prepare a polysaccharide solution, adding an equal volume of chloroform-n-butanol (4:1) solution thereto, shaking thoroughly and then centrifuging, discarding the chloroform phase, and continuing to add an equal volume of chloroform-n-butanol (4:1) solution to the aqueous phase, and repeating the operation until no protein is detected.

[0028] Furthermore, as a common decolorization method, the decolorization is performed by static adsorption decolorization.

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

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

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

[0032] Preferably, the alcohol solvent includes ethanol or methanol. Among them, ethanol has the following advantages over methanol: ① It is less toxic than methanol and safer; ② It has a certain bactericidal effect and can reduce the contamination 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 an alcohol reagent to the obtained crude extract, the final concentration of the alcohol reagent is 70% to 90%, preferably 80%.

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

[0035] Preferably, the sufficient soaking time is 20 to 40 minutes, preferably 30 minutes.

[0036] Furthermore, the decoction is performed under the following conditions: first boiling, and then continuing to decoct for 25 to 30 minutes.

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

[0038] As an optional implementation, the decoction condition is: first boil at 100°C, and then continue to decoct at this temperature for 30 minutes.

[0039] Furthermore, the filter cloth for filtration has a mesh size of 150 to 250, preferably 200.

[0040] Furthermore, the filter residue obtained by the above filtration can be repeatedly added with water, decocted again, the two filtrates can be combined, filtered, and the filtrate can be concentrated to obtain a crude extract.

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

[0042] Furthermore, the concentration is performed until the solid-liquid ratio (g / mL) is close to 1:1 to obtain a crude extract.

[0043] Further, as an optional form, the wax gourd peel is wax gourd peel medicinal slices.

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

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

[0046] Furthermore, in step S3 or step S4, the elution flow rate is preferably 2 to 4 mL / min, preferably 3 mL / min.

[0047] Furthermore, in steps S3 to S4, the post-processing includes concentration and drying.

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

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

[0050] Furthermore, in step S3, the post-treatment further comprises dialysis. After dialysis, the retained liquid in the dialysis bag is concentrated and dried.

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

[0052] Specifically, in step S3, the post-treatment includes concentration, dialysis and drying, and the specific operation is as follows: the collected fraction is concentrated under reduced pressure (60°C) by rotary evaporation, and then dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 3500Da. The retained liquid after dialysis is concentrated under reduced pressure (60°C) again, and freeze-dried to obtain wax gourd peel polysaccharide BEP-1.

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

[0054] As a preferred solution, the following method can be used to perform steps S3 and S4:

[0055] Step S3: After the obtained wax gourd peel polysaccharide BEP is prepared into an aqueous solution, it is loaded onto a balanced DEAE-Cellulose52 anion exchange resin glass chromatography column (Φ2.5cm×60cm) for separation, and 20 times the column volume of water, 0.1M NaCl solution, 0.5M NaCl solution and 0.5M NaOH solution are used in sequence for gradual elution at a flow rate of 3mL / min, and one tube is collected for every 100mL. The elution curve is drawn using the phenol-sulfuric acid method. According to the elution curve, the fraction eluted with the 0.1M NaCl solution is collected, and after reduced pressure concentration by rotary evaporation, it is dialyzed (dialysis bag specification is 3500Da) for 3 days, concentrated under reduced pressure (60°C), and freeze-dried to obtain wax gourd peel polysaccharide BEP-1.

[0056] Step S4: After the wax gourd peel polysaccharide BEP-1 obtained in step S3 is prepared into an aqueous solution, it is further purified by loading and balancing the polysaccharide gel G-25 glass chromatography column (Φ2.5cm×60cm), eluted with 0.1M NaCl solution, and the elution flow rate is 3mL / min. The polysaccharide is tracked and detected using the sulfuric acid phenol method, and an elution curve is drawn according to the elution volume and absorbance. According to the elution curve, the eluate of the first single symmetrical peak is collected, and after freeze-drying, the wax gourd peel polysaccharide BEP-1a is obtained.

[0057] The present invention also protects a wax gourd peel polysaccharide having a repeating unit structure, wherein the relative molecular mass of the wax gourd peel polysaccharide is 4.68×10 4 Da, the repeating unit structural formula 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] At present, the research on polysaccharides has made some progress, but there is still a lack of in-depth exploration of wax gourd peel polysaccharides, especially wax gourd peel polysaccharides with specific structure and molecular weight. The present invention obtains a wax gourd peel polysaccharide with a new structure for the first time. The wax gourd peel polysaccharide is mainly composed of galactose, galacturonic acid, rhamnose, arabinose and glucose, with a relative molecular mass of 4.68×10 4 Da. In vivo and in vitro experiments have shown that the wax gourd peel polysaccharide of the present invention has an excellent gastric mucosal protective effect.

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

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

[0063] Furthermore, the monosaccharide composition of 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 connection 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 proportions 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 a specific molecular weight and a repeating unit structure in the preparation of a drug for preventing and treating gastric ulcer.

[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 a specific molecular weight and a repeating unit structure in the preparation of health products or feeds with the function of assisting in protecting gastric mucosa.

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

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

[0070] The present invention is the first to remove impurities, separate and purify the crude wax gourd peel polysaccharide obtained by water extraction and alcohol precipitation to obtain 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 vivo and in vitro experiments have shown that the wax gourd peel polysaccharide of the present invention has an excellent gastric mucosal protective effect, and has a good application prospect in the preparation of drugs for preventing and treating gastric ulcers or health products or feeds with auxiliary gastric mucosal protection function. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0072] Figure 2 This is the HPGPC diagram of homogeneous polysaccharides from wax gourd peel, where Figure A represents BEP-1a and Figure B represents BEP-1b.

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

[0074] Figure 4 This is the FT-IR image of the uniform polysaccharide BEP-1a from wax gourd peel.

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

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

[0077] Figure 7 This is the structural unit diagram of wax gourd peel uniform polysaccharide BEP-1a.

[0078] Figure 8 These are scanning electron micrographs of the homogeneous polysaccharide BEP-1a from wax gourd peel, where Image A is taken at 100× and Image B is taken at 1000×.

[0079] Fig. 9 The figures are statistical diagrams of the protective effect of BEP-1a on ethanol-induced acute gastric ulcer damage in mice; Figure A is a macroscopic representative image of gastric tissue in mice in different treatment groups; Figure B is a statistical diagram of gastric ulcer area; Figure C is a statistical diagram of gastric ulcer index; and Figure D is a statistical diagram of gastric ulcer inhibition rate.

[0080] Fig.10 The figures are statistical diagrams showing that BEP-1a improves ethanol-induced gastric tissue pathological damage in mice; Figure A is the H&E staining image of mouse gastric tissue; Figure B is the H&E staining score image; Figure C is the PAS staining image of mouse gastric tissue; and Figure D is the statistical diagram of the PAS positive staining rate.

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

[0082] Fig.12 Statistical graph showing the effect of different concentrations of BEP-1b on the survival rate of GES-1 cells induced by ethanol. DETAILED DESCRIPTION

[0083] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples 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 art.

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

[0085] Figure 1 A means Figure 1 Figure A in Figure 2 A stands for Figure 2 This is Figure A in the figure. The meanings of the other figures are similar.

[0086] Experimental materials and equipment

[0087] (1) Experimental animals

[0088] This experiment used 4-week-old SPF male Kunming mice purchased from the Guangdong Medical Experimental Animal Center. The mice were placed in the SPF laboratory of the Experimental Animal Center of Guangdong Pharmaceutical University for feeding. The laboratory environment temperature was about 24.0±2.0℃ and the relative humidity was about 50±10%. The light and dark were alternated every 12 hours, and the animals were free to eat and drink 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 Guide" of Guangdong Pharmaceutical University.

[0089] (2) Experimental cells

[0090] GES-1 cells were purchased from Wuhan Sun 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 separation of wax gourd peel polysaccharides

[0100] 1. Experimental methods

[0101] Take about 100g wax gourd peel slices, put them in a casserole, add 1000mL water and soak for 30min, boil them at 100℃ first, then continue to decoct them at 100℃ for 30min, and filter them through a 200-mesh filter cloth while hot; add 800mL water to the residue, boil them at 100℃, then continue to decoct them at 100℃ for 25min, filter them through a 200-mesh filter cloth while hot, combine the two filtrates, quickly cool the filtrate to room temperature, repeat the operation to treat a total of 5.0Kg wax gourd peel medicinal materials, concentrate the combined filtrates under reduced pressure (60℃) to a concentrated extract with a solid-liquid ratio close to 1:1, then add anhydrous ethanol to a final concentration of 80%, precipitate them at 4℃ overnight, centrifuge to obtain the precipitate, remove the alcohol taste and freeze-dry to obtain wax gourd peel crude polysaccharide.

[0102] The obtained wax gourd peel crude polysaccharide is added with distilled water to prepare a sugar solution, the protein is removed by the Sevag method, the deproteinized sugar solution is depigmented by D101 macroporous adsorption resin, and then the solution is concentrated under reduced pressure by rotary evaporation (60°C), dialyzed (dialysis bag specification is 3500Da) for 3 days, and then concentrated under reduced pressure (60°C), and freeze-dried to obtain the deproteinized and decolorized wax gourd peel crude polysaccharide.

[0103] The crude wax gourd peel polysaccharide after protein removal and decolorization was dissolved with an appropriate amount of distilled water, and the equilibrated DEAE-Cellulose 52 anion exchange resin glass chromatography column (Φ2.5cm×60cm) was loaded for separation, and 20 column volumes of distilled water, 0.1M NaCl solution, 0.5M NaCl solution and 0.5M NaOH solution were used in sequence for stepwise elution at a flow rate of 3mL / min, and one tube was collected for every 100mL. The elution curve was drawn using the phenol-sulfuric acid method, and the eluted sample was collected. The polysaccharide was collected according to the elution curve, and after reduced pressure concentration (60°C) by rotary evaporation, dialyzed (dialysis bag specification was 3500Da) for 3 days, reduced pressure concentration (60°C), and freeze-dried to obtain wax gourd peel polysaccharide BEP-1 (see Figure 1 A, obtained by collecting the fraction 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 by loading a balanced dextran gel G-25 glass chromatography column (Φ2.5cm×60cm), eluted with 0.1M NaCl solution, the elution flow rate was 3mL / min, the polysaccharide was tracked and detected using the sulfuric acid phenol method, and the elution curve was drawn according to the elution volume and absorbance. Different components were combined and collected according to the elution curve, freeze-dried and stored, and wax gourd peel polysaccharide BEP-1a (see Figure 1 B, obtained by collecting the eluate of the first single symmetrical 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 symmetrical peak through the elution curve).

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

[0106] Sevag method for protein removal: Add an equal volume of chloroform-n-butanol (4:1) solution to the polysaccharide solution, shake thoroughly, and centrifuge. Discard the chloroform phase and continue to add an equal volume of chloroform-n-butanol (4:1) solution to the aqueous phase. Repeat the operation until no protein is detected.

[0107] Depigmentation treatment with D-101 macroporous adsorption resin: The polysaccharide sample after protein removal was decolorized by static adsorption decolorization method. The dosage of D-101 resin was 1g / 10mL, the polysaccharide concentration on the sample was 3mg / mL, pH=7, and the treatment was performed for 2h.

[0108] 2. Experimental results

[0109] The crude polysaccharide of wax gourd peel was extracted from wax gourd peel by water extraction and alcohol precipitation, with a yield of (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) The BEP-1 fraction was purified using a Sephadex G-25 glass column. The elution curve is shown in Figure 1 B, further concentrated to obtain two polysaccharides, BEP-1a and BEP-1b, with yields of (21.75±0.01)% and (9.88±0.05)%, respectively.

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

[0111] 1. Experimental methods

[0112] The relative molecular weight of wax gourd peel polysaccharides BEP-1a and BEP-1b was determined by high performance gel permeation chromatography (HPGPC). The instrument was equipped with three series-connected Ultrahydrogel (Waters, American) chromatographic columns (specifications: 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 volume was 20 μL. The mobile phase was 0.02 M KH2PO4 solution. Different molecular weight T-dextran standards (5.2 × 10 3 Da, 4.86×10 4 Da, 6.68×10 5 Da) Draw the standard curve.

[0113] 2. Experimental results

[0114] HPGPC results are shown in Figure 2 , the figure shows a unique symmetrical peak, indicating that BEP-1a( Figure 2 A) and BEP-1b( Figure 2 B) is a homogeneous polysaccharide, i.e., a pure polysaccharide. The molecular weight-retention time standard curve measured according to the standard dextran standard is lg (Mw) = -0.1863T + 12.13, R 2=0.9876. According to the HPGPC peak time, the relative molecular masses of BEP-1a and BEP-1b were calculated to be 4.68×10 4 Da and 3.19×10 6 In the in vitro biological activity screening (Example 9), it was found that BEP-1a has certain biological activity, but no biological activity of BEP-1b has been found, so the subsequent research focuses on BEP-1a.

[0115] Example 3 Chemical composition analysis of homogeneous polysaccharides from wax gourd peel

[0116] (1) Chemical composition analysis of homogeneous polysaccharides from wax gourd peel

[0117] The neutral sugar content in polysaccharides was measured by sulfuric acid phenol method, with α-D-glucose as the standard; the protein content was determined by Coomassie brilliant blue method, with bovine serum albumin as the standard; the uronic acid content was determined by m-hydroxybiphenyl method, with galacturonic acid as the standard.

[0118] (2) Analysis of monosaccharide composition of wax gourd peel polysaccharides

[0119] The monosaccharide composition of wax gourd peel polysaccharide BEP-1a was determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization HPLC method. 5 mg 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. Take 100 μL of the mixed standard solution, add 100 μL of 0.6 mol / L sodium hydroxide solution and 200 μL of 0.5 mol / L PMP methanol solution, mix well, react in a constant temperature water bath at 70°C for 100 min, take out and cool to room temperature, add 200 μL of 0.3 mol / L hydrochloric acid solution, shake well, add ultrapure water to 2 mL, add an equal volume of chloroform, vortex and shake, let stand to separate, take the upper aqueous phase solution, extract it twice with chloroform, filter the aqueous phase solution with a 0.22 μm microporous filter membrane, and wait for HPLC analysis.

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

[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 was 93.06%, the uronic acid content was 3.52%, the protein content was 2.81%, and the monosaccharide composition was 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 methods

[0127] 1.0 mg of the dried polysaccharide sample BEP-1a was weighed and prepared by potassium bromide tableting method with a tablet thickness of 1 mm. The infrared spectrum characteristics of the polysaccharide were determined by Fourier transform infrared spectroscopy (BRUKER TEMSOR 27, Germany) with a scanning wave number from 400 cm -1 -4000cm -1 .

[0128] 2. Experimental results

[0129] The infrared absorption spectrum of wax gourd peel polysaccharide BEP-1a is as follows Figure 4 As shown. Among them, 3487.5cm -1 The absorption peak at 2927.9 cm represents the stretching vibration of the OH bond of sugars. -1 The absorption peak at 1200-1400 cm -1The peaks in the range of 1738.2cm are the angle vibrations of CH bonds. Based on these three characteristic peaks, it can be judged that the polysaccharide has a carbohydrate structure. -1 The absorption peak at 950~1200cm indicates that the polysaccharide contains a carboxyl (COO-) group. -1 The strong absorption peak at 902.8 cm-1 is the characteristic absorption of the pyridine 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 Polysaccharides

[0131] 1. Experimental methods

[0132] Weigh 5 mg of wax gourd peel polysaccharide sample BEP-1a, add anhydrous phosphorus pentoxide and dry overnight, dissolve in 200 μL DMSO, fill the system with nitrogen to replace air for protection, add 15 mg of dry sodium hydroxide powder and ultrasonically dissolve, place in an ice water bath to solidify, then slowly drop 100 μL KI solution, slowly heat to 20 ° C, ultrasonic for 30 minutes, then add 1 mL of 4 mmol / L Na2SO3 aqueous solution, mix well, add 500 μL chloroform, vortex, stand and take the chloroform layer, repeat extraction three times, combine the solution, add anhydrous Na2SO4 to remove water, centrifuge and filter, blow dry with nitrogen, use infrared to analyze its methylation degree, if the hydroxyl group in the polysaccharide is not completely replaced, repeat the above process until the hydroxyl absorption peak disappears in the infrared spectrum. The methylated product is dissolved in 2 mol / L TFA, hydrolyzed at 120 ° C for 3 h and reduced with deuterated NaBH4, and then acetylated to convert its monosaccharide into partially methylated sugar alcohol acetate. The acetylated samples were analyzed by GC-MS. The peaks of methylated sugars were identified by mass spectrometry, and their relative molecular masses were estimated by combining the peak areas and corresponding responses of GC. The response factors of partially methylated sugar alcohol acetates were calculated by effective carbon reactions.

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

[0134] 2. Experimental results

[0135] Table 4 Methylation analysis results of wax gourd peel homogeneous polysaccharide BEP-1a

[0136]

[0137] The methylation results showed that (see Figure 5and Table 4), the connection 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%), among which the content of branched sugar residues and terminal sugar residues is basically the same, indicating that the polysaccharide is completely methylated. It can be seen that the polysaccharide is composed of about 90% 1,4-Gal galactan and about 10% RG-Ⅰ type pectin.

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

[0139] 1. Experimental methods

[0140] 20 mg of wax gourd peel polysaccharide sample was weighed and dissolved in 2 mL of deuterated water. After freeze-drying, it was redissolved in 600 μL of deuterated water. The ginkgo leaf polysaccharide was characterized by using a Bruker AM 500 NMR spectrometer at room temperature. 1 H spectrum, 13 C spectrum, 1 H- 1 HCosy spectrum, 1 H- 13 C HMBC spectrum and 1 H- 13 C HSQC spectrum scanning analysis.

[0141] 2. Experimental results

[0142] like Figure 6 As shown in A and 6B, the chemical shifts of the terminal carbon and terminal hydrogen of the galactose residue of BEP-1a are δ107.45 and δ5.03, respectively, indicating that galactose is mainly in the α configuration. Figure 6 C) and HSQC spectra ( Figure 6 D) It can be inferred that the carbon and 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). Figure 6 In E), galactose C1 / H4 and C4 / H1 cross peaks can be observed, further proving that the galactose residues of the polysaccharide are linked to each other through 1,4 bonds.

[0143] In summary, the present invention speculates that the homogeneous wax gourd peel polysaccharide BEP-1a has the following Figure 7 The repeating unit structure is shown.

[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 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 the form of irregular stones. 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 damage 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 pure water and heat to dissolve. After complete dissolution, cool the solution to room temperature, add pure water to make up the weight, and place it in a 4°C refrigerator for later use.

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

[0154] Preparation of omeprazole solution: Weigh an appropriate amount of omeprazole powder and dissolve it in 0.5% CMC-Na solution. The intragastric volume of 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, Kunming mice were randomly divided into normal group (Control), model group (Model), omeprazole positive drug group (OME, 20 mg / kg), wax gourd peel polysaccharide low-dose group (BEP-1a-L, 25 mg / kg), wax gourd peel polysaccharide high-dose group (BEP-1a-H, 50 mg / kg), 12 mice in each group. The normal group and model group were given 0.5% CMC-Na solution by intragastric administration once a day for 3 consecutive days, and once 1 hour before modeling on the 4th day.

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

[0158] After the administration on the third day, all mice were fasted for 24 hours and deprived of water for 2 hours. After the administration 1 hour before modeling on the fourth day, all groups of mice were gavaged with 0.2mL / 10g of anhydrous ethanol except the normal group, which was given an equal volume of 0.5% CMC-Na solution. The modeling was completed 1 hour later, and all mice were anesthetized with isoflurane and then blood was taken from the heart and killed.

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

[0160] After the mice were killed by cardiac blood sampling, the gastric tissue was removed and cut open along the greater curvature of the stomach. The contents of the gastric tissue were cleaned with ice saline. The cleaned gastric tissue was spread on filter paper and photographed with a camera for archiving to evaluate the macroscopic morphology of the gastric tissue of mice in different treatment groups. Subsequently, Image J software was used to quantify the gastric ulcer area, and the ulcer index and ulcer inhibition rate were calculated.

[0161] The ulcer index calculation formula is: ulcer index = gastric ulcer area / total stomach area × 100

[0162] The ulcer inhibition rate was calculated as follows: (ulcer index of the model group - ulcer index of the drug group) / ulcer index of the model group × 100%

[0163] (5) Histopathological examination of mouse gastric tissue

[0164] ① H&E staining of mouse gastric tissue

[0165] The removed gastric tissue was fixed in 4% paraformaldehyde fixative for 24 hours and then prepared into paraffin sections. The paraffin sections were dewaxed to water and then stained with hematoxylin and eosin in sequence. After staining, the sections were dehydrated and sealed, and the sections were placed in a scanner imaging system to collect images, and then the collected images were scored for H&E histology.

[0166] ②PAS staining of mouse gastric tissue

[0167] The removed gastric tissue was fixed in 4% paraformaldehyde fixative for 24 hours and then prepared into paraffin sections. The paraffin sections were dewaxed to water and then stained with periodic acid, Shevchenko and hematoxylin in sequence. After staining, the sections were dehydrated and sealed, and the sections were placed in a scanner imaging system to collect images, and then the PAS positive staining rate was counted using Image J software.

[0168] 2. Experimental results

[0169] The present invention performs macroscopic examination on mouse gastric tissue to evaluate the protective effect of BEP-1a on ethanol-induced acute gastric ulcer injury. Fig. 9 A is a macroscopic image of the gastric tissue of mice in different treatment groups. As can be seen from the figure, the macroscopic morphology of the gastric tissue of mice in the normal group was normal, without ulceration and bleeding. The gastric tissue of mice in the ethanol group showed large-area bleeding lesions, which were distributed in sheets. Compared with the ethanol group, the gastric tissue bleeding of mice in the positive drug OME group and BEP-1a group was significantly reduced, and the bleeding was distributed in dots and small sheets.

[0170] In order 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 quantify the gastric ulcer area of ​​different treatment groups and calculate the ulcer index and ulcer inhibition rate. The results showed that the gastric ulcer area of ​​the model group was 13442±2284mm 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. The gastric ulcer area of ​​the BEP-1a-H group was 3814±1399mm 2 The gastric ulcer areas in the BEP-1a-L group and the OME group were 5410±2005mm 2 and 8260±3892mm 2 ( Fig. 9 B). Further analysis of the ulcer index revealed that the ulcer index of the model group was 75.48±9.28%, which was significantly higher than that of the normal group. The administration of OME and BEP-1a groups significantly reduced the ulcer index. 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% ( Fig. 9 C). In terms of ulcer inhibition rate, both the OME group and the BEP-1a group increased significantly after administration. 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% ( Fig. 9 D).

[0171] In order 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. H&E staining results ( Fig.10 A) showed that the gastric mucosal glands of the normal group were neatly arranged, and the mucosal layer, submucosal layer and muscular layer were intact. In contrast, the gastric tissue of the mice in the model group was severely damaged, manifested as gastric mucosal erosion and bleeding, inflammatory cell infiltration and edema of the gastric submucosal layer. However, after treatment with OME and BEP-1a, the gastric tissue of the mice was significantly protected, the area of ​​gastric mucosal erosion and bleeding was reduced, the edema of the gastric submucosal layer was alleviated, and there was no obvious inflammatory cell infiltration. H&E staining histological score showed ( Fig.10 B), the score of the model group was 8.78±0.69, and the degree of gastric mucosal damage was the most serious. 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, among which the BEP-1a-H group had a better effect.

[0172] PAS staining was used to measure the glycoproteins on the surface of the gastric mucosa. After PAS staining, the glycoproteins on the surface of the gastric mucosa produced a positive reaction and appeared purple-red. The gastric mucosa of the mice in the normal group 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. The model group showed large-area epithelial cell shedding, and the PAS staining rate was 0.21±0.01%, which was significantly reduced compared with the normal group. 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, which were significantly higher than those of the model group, indicating that after treatment with BEP-1a, the gastric tissue of the mice was significantly protected, reducing the loss of glycoproteins on the surface of the gastric mucosa. The above results show that BEP-1a-H has the best effect in protecting mice from gastric mucosal damage ( Fig.10 CD).

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

[0174] 1. Experimental methods

[0175] (1) GES-1 cell culture

[0176] ①Cell recovery

[0177] Take out the cryovial containing GES-1 cells from the liquid nitrogen tank, quickly put it into a 37℃ water bath, gently rotate the cryovial until only a small piece of ice core is observed to remain in the cryovial, immediately transfer it to the clean bench, wipe the seal with an alcohol cotton ball, transfer the cryovial into an EP tube containing 2mL of complete culture medium, centrifuge at 1000r / min for 4min, discard the supernatant, add 2mL of complete culture medium, blow and mix, inoculate into a culture bottle containing 2mL of complete culture medium, mix, and place in a carbon dioxide cell culture incubator for culture. After 12h, the cells adhere to the wall, replace with new complete culture medium, and continue to culture.

[0178] ② GES-1 cell passaging

[0179] When the growth density of GES-1 cells is 80%-90% under a microscope, the cells can be subcultured. The original culture medium in the cell culture bottle is aspirated, and 2 mL of PBS buffer is added to wash twice, and then 500 μL of 0.25% trypsin solution is added for digestion. When the cells are observed under a microscope to be digested and shrink into a round shape, the digestion is complete (about 3 minutes). The trypsin in the culture bottle is aspirated, and 3 mL of complete culture medium is added to terminate the digestion. The cells at the bottom of the cell culture bottle are blown off the wall, and the cell suspension is centrifuged at 1000 r / min for 4 minutes, the supernatant is discarded, and 3 mL of new complete culture medium is added to mix by blowing, and the cells are transferred to a new cell culture bottle at a ratio of 1:3 to continue culturing.

[0180] ③ Cryopreservation of GES-1 cells

[0181] GES-1 cells are cultured in a cell culture bottle. When the cell growth density reaches 80-90% under a microscope, the cells can be cryopreserved. After the cells are digested according to the method "② GES-1 cell passage", the cell suspension is centrifuged at 1000r / min for 4min, the supernatant is discarded, and 1mL of cell freezing solution is added. After resuspending, it is added to the cell freezing tube, placed in a freezing box, placed in a -80℃ refrigerator, and transferred to a liquid nitrogen tank for long-term storage after 24h.

[0182] (2) MTT assay for cell viability

[0183] The survival rate of GES-1 cells was determined by the MTT method. The cells in the 96-well plate were incubated with 10 μl of MTT solution at 37°C for 4 h until blue-purple formazan crystals were formed, the liquid in the wells was discarded, 100 μL of DMSO solution was added to each well, and the wells were gently shaken and incubated in an incubator for 15 min. After the blue-purple formazan crystals were completely dissolved, the absorbance (OD value) of each well was measured at a wavelength of 490 nm using an enzyme marker to calculate the cell survival rate.

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

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

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

[0187] Human gastric epithelial cells GES-1 in logarithmic growth phase were cultured at 5×10 3 / well were inoculated in a 96-well cell culture plate and cultured in a cell culture incubator. After 24 hours, they were divided into a blank group and a treatment group (the concentrations of wax gourd polysaccharide BEP-1a were: 1000, 500, 100, 50, 10, 5, 1, 0.5, 0.1 μg / mL). After corresponding treatment, different groups were cultured in a 37°C, 5% CO2 cell culture incubator. After 24 hours, the MTT method was used to detect the toxic effect of wax gourd polysaccharide BEP-1a on GES-1 cells.

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

[0189] Human gastric epithelial cells GES-1 in logarithmic growth phase were cultured at 5×10 3 / well were inoculated in a 96-well cell culture plate and cultured in a cell culture incubator. After 12 hours, they were divided into a blank group, a model group and a drug group (the concentrations of wax gourd polysaccharides BEP-1a and BEP-1a were: 200, 100, 50, 25, 12.5 μg / mL). After different groups were pretreated for 12 hours, modeling was carried out under the optimal modeling conditions, and 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 determined by the MTT method.

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

[0191] Human gastric epithelial cells GES-1 in the logarithmic growth phase were cultured at 25×10 4 / well were inoculated in a 96-well cell culture plate and cultured in a cell culture incubator. After 24 hours, they were divided 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). The administration and modeling were carried out under the same time conditions as in step "(3) Establishment of ethanol-induced GES-1 cell injury model". After the modeling, the cells were digested and collected with 0.25% trypsin, and stained according to the instructions of the Annexin V-FITC / PI apoptosis detection kit. After the staining, the cells were detected by ultra-high-speed flow cytometry.

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

[0193] After drug administration and modeling as in method (5), staining was performed according to the instructions of the Calcein-AM / PI cell activity detection kit, and fluorescent images were collected using an intelligent cell imaging system.

[0194] 2. Experimental results

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

[0196] In order to determine the optimal concentration of ethanol for modeling, normal GES-1 cells were treated with different concentrations of ethanol for 2 hours to detect the effect of ethanol on the survival rate of GES-1 cells. The results showed that as the concentration of ethanol increased, the survival rate of GES-1 cells decreased. When the ethanol concentration was 4%, the survival rate of GES-1 cells was 59.36±6.66%, which met the model requirements required by the experiment ( Fig.11 A). Therefore, in subsequent experiments, 4% ethanol was used to co-incubate GES-1 cells for 2 hours as the optimal modeling condition.

[0197] The toxicity of the drug needs to be investigated before the biological activity. Therefore, the present invention co-incubated different concentrations of BEP-1a and GES-1 cells to determine the safe concentration range of BEP-1a. Fig.11B It can be seen that as the concentration of BEP-1a increases, it produces certain cytotoxicity to GES-1 cells. When the BEP-1a administration concentration is 1000μg / mL and 500μg / mL, it shows significant cytotoxicity compared with the normal group. Therefore, in the ethanol-induced GES-1 cell injury model, the present invention selects a concentration below 500μg / 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 ( Fig.11 C), compared with the normal group, the cell survival rate of the model group was 33.74±0.71%, and different concentrations of BEP-1a showed different degrees of protection. When the concentration of BEP-1a was 200μg / mL and 100μg / mL, the protection rates were 45.35±2.73% and 42.43±3.35%, respectively, showing a significant protective effect. Therefore, 200μg / mL and 100μg / mL were used as high and low doses of BEP-1a in subsequent in vitro experiments.

[0198] The MTT test results of BEP-1b showed that ( Fig.12 ), BEP-1b could not enhance the cell survival rate of GES-1 cells damaged by ethanol.

[0199] The effect of BEP-1a on the cell morphology of GES-1 cells induced by ethanol was observed under an inverted microscope. The cells in the normal group were flat and spindle-shaped, adherent to the wall, and arranged tightly. In the model group, a large number of cells shrank and rounded, the cell adhesion ability weakened, the cell gap increased, and the number of floating cells increased significantly. With the increase of drug concentration, the growth state and morphology of cells in the high and low concentration groups of BEP-1a gradually approached the normal group ( Fig.11 E).

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

[0201] In order to more intuitively examine the protective effect of BEP-1a on GES-1 cells, the present invention uses a Calcein / PI cell activity kit for fluorescence staining. Calcein-AM stains live cells with green fluorescence, and PI stains dead cells with red fluorescence. Fig.11As shown in G, the model group showed a decrease in Calcein-AM positive cells and an increase in PI positive cells, which was manifested by a decrease in the green fluorescence area and an increase in the red fluorescence area, indicating ethanol-induced cell damage. Compared with the model group, the BEP-1a group reversed these trends. As the concentration of BEP-1a increased, the protective effect on ethanol-induced GES-1 cell damage became more significant ( Fig.11 HM).

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

[0203] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A wax gourd peel polysaccharide BEP-1a, characterized in that: The preparation method comprising the following steps is obtained: S1. Extraction of crude polysaccharides from wax gourd peel: crude polysaccharides from wax gourd peel were extracted by water extraction and alcohol precipitation method; S2. Deproteinization and decolorization: The crude wax gourd peel polysaccharide obtained in step S1 was deproteinized and decolorized to obtain wax gourd peel polysaccharide BEP; S3. After preparing the wax gourd peel polysaccharide BEP obtained in step S2 into an aqueous solution, eluting it using a DEAE-Cellulose52 anion chromatography column, using water, 0.1M NaCl, 0.5M NaCl, and 0.5M NaOH solution as eluents for stepwise elution, collecting the 0.1M NaCl eluted fraction, dialyzing it, and then freeze-drying it to obtain wax gourd peel polysaccharide BEP-1; S4. After the wax gourd peel polysaccharide BEP-1 obtained in step S3 is prepared into an aqueous solution, it is purified using a polysaccharide G-25 chromatography column, using 0.1M NaCl as an eluent, and the eluent of the first single symmetrical peak is collected according to the elution curve, and post-processed to obtain wax gourd peel polysaccharide BEP-1a.

2. The wax gourd peel polysaccharide BEP-1a according to claim 1, characterized in that The specific preparation method of the wax gourd peel crude polysaccharide comprises the following steps: soaking the wax gourd peel in water, decocting it, filtering it, concentrating the filtrate to obtain a crude extract, adding an alcohol solvent to the obtained crude extract, fully precipitating it, and drying the precipitate to obtain the wax gourd peel crude polysaccharide.

3. The wax gourd peel polysaccharide BEP-1a according to claim 2, characterized in that: The alcohol solvent includes ethanol or methanol.

4. A wax gourd peel polysaccharide, characterized in that: The relative molecular mass of wax gourd peel polysaccharide is 4.68×10 4 Da, the repeating unit structural formula of the wax gourd peel polysaccharide is 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, and p represents the configuration of pyranose.

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

6. The wax gourd peel polysaccharide according to claim 4, characterized in that: 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.

7. Use of the wax gourd peel polysaccharide BEP-1a according to any one of claims 1 to 3 or the wax gourd peel polysaccharide according to any one of claims 4 to 6 in the preparation of a medicament for preventing and treating gastric ulcer.

8. The use according to claim 7, characterized in that: The gastric ulcer includes alcoholic gastric ulcer.

9. Use of the wax gourd peel polysaccharide BEP-1a according to any one of claims 1 to 3 or the wax gourd peel polysaccharide according to any one of claims 4 to 6 in the preparation of health products or feeds with auxiliary gastric mucosal protection function.

10. A drug, characterized in that The drug comprises the wax gourd peel polysaccharide BEP-1a described in any one of claims 1 to 3 or the wax gourd peel polysaccharide described in any one of claims 4 to 6.

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