Glycyrrhiza polysaccharide for preventing and treating acute stomach injury as well as preparation method and application of glycyrrhiza polysaccharide

Abstract

The invention discloses glycyrrhiza polysaccharide for preventing and treating acute stomach injury and a preparation method and application thereof. The molecular weight of the glycyrrhiza polysaccharide is 60 kDa, the percentage content of protein is smaller than or equal to 5%, and the mass percentage content is larger than or equal to 60% according to glucose; the preparation method comprises the following steps: carrying out ethanol reflux degreasing on liquorice, carrying out water extraction and alcohol precipitation, removing protein by adopting Sevag, dialyzing by using a 8-13 kDa dialysis bag, carrying out rotary concentration, and freeze-drying to obtain the glycyrrhiza polysaccharide. Animal experiments show that the glycyrrhiza polysaccharide has a protective effect on acute alcoholic stomach injury, the content of MDA and MPO in animal stomach tissues can be reduced, and the stomach injury is relieved by reducing lipid oxidation and neutrophil infiltration level; the content of gastric tissue pepsase and VEGF (vascular endothelial growth factor) can be increased, and the gastric mucosa can be protected in the aspects of maintaining the stomach environment, promoting blood vessel healing and reducing the injury time and degree; and the medicine also has a pathological repair effect on acute stomach injury.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a liquorice polysaccharide for preventing and treating acute gastric injury, and a preparation method and application thereof. Background Art

[0002] At present, commonly used methods for treating gastric ulcers include antacids, gastric mucosal protective agents and antibacterial drugs. These drugs are effective in the short-term treatment of gastric ulcers, but long-term use will produce various side effects and a high recurrence rate.

[0003] As one of the bioactive macromolecules, natural product polysaccharides have been proven to have a variety of biological activities. There are many methods for extracting polysaccharides, and different polysaccharide extraction methods have a significant impact on the polysaccharide structure and bioactive function. The crude polysaccharide was extracted by water extraction and alcohol precipitation, and a relatively uniform licorice polysaccharide was obtained by Sevag method to remove protein, dialysis, concentration, freeze-drying and other operations. The obtained polysaccharide has high purity, which not only controls production costs, but also facilitates subsequent product development.

[0004] Chinese patent CN107361285A discloses a licorice polysaccharide stomach-protecting beverage and a preparation method thereof, wherein the crushed licorice is subjected to ultrasonic defatting treatment with ethanol, the residue is dried until there is no alcohol taste, distilled water is added to the residue for ultrasonic extraction, the extract is combined, concentrated, and centrifuged, the supernatant is mixed with anhydrous ethanol, the mixed solution is precipitated with alcohol, filtered, and the precipitate is dried until there is no alcohol taste to obtain licorice polysaccharide; a flavoring agent with a mass concentration of 0.05% to 0.3%, a stabilizer with a mass concentration of 0.05% to 0.20%, and a preservative with a mass concentration of 0.01% to 0.15% are added to a 0.001 to 0.010 g / mL licorice polysaccharide solution, the pH is adjusted to 4.5 to 7.5, and sterilized to obtain a licorice polysaccharide stomach-protecting beverage. This method uses crude polysaccharide to prepare a stomach-protecting beverage, and the ingredients are relatively complex.

[0005] Chinese patent CN115286721A discloses an active polysaccharide, an active polysaccharide composition and its application in the preparation of products with the effect of preventing or treating damage. The composition obtained by the method is a mixture of codonopsis polysaccharide and licorice polysaccharide in a weight ratio of 3:1. The obtained codonopsis polysaccharide and licorice polysaccharide are both prepared by water extraction and alcohol precipitation, wherein codonopsis polysaccharide is dialyzed in 18~22 kD, 8~12 kD and 2~4 kD dialysis bags in sequence, and the intercepted liquid in the 2~4 kD dialysis bag is retained and concentrated and dried to obtain codonopsis polysaccharide. Licorice polysaccharide is dialyzed in 14~16 kD and 7~9 kD dialysis bags in sequence, and the intercepted liquid in the 7~9 kD dialysis bag is retained and concentrated and dried to obtain licorice polysaccharide. Compared with codonopsis crude polysaccharide, the codonopsis polysaccharide and the active polysaccharide composition prepared by the method of the invention have significantly improved the effect of preventing or treating gastric damage, and have excellent effect of preventing or treating gastric damage. Although licorice polysaccharide is added to the polysaccharide composition of the invention, licorice polysaccharide retained by a 7-9 kDa dialysis bag is used.

[0006] Chinese patent CN110343188A discloses a method for extracting and purifying licorice polysaccharides, which comprises crushing licorice through a 40-60 mesh sieve, extracting with 8 times water decoction for 0.5 h, decocting for a second time for 0.5 h, concentrating the decoction, adding 20% ​​diatomaceous earth by volume of the concentrate, and keeping it warm at 80-82°C for 40-50 min, filtering and removing impurities while hot, mixing the filtrate with chloroform: n-butanol = 15: 4: 1, oscillating for 25 min, retaining the water layer, and obtaining water-soluble and fat-soluble polysaccharides by graded alcohol precipitation, and drying all the precipitates. This method has a short extraction time, avoids the precipitation of too many impurities, and also reduces the yield of polysaccharides. The purity of the obtained polysaccharides is relatively low, and the total sugar purity is 50.8-57.4%.

[0007] Therefore, it is necessary to seek and develop safer and more effective natural active substances to achieve effective prevention and treatment of acute gastric injury. Extraction, separation and purification of natural active polysaccharides for the treatment of alcoholic gastric injury is an important method for developing functional foods such as stomach nourishing and anti-gastric ulcer. Therefore, how to optimize the separation and extraction of active ingredients, reduce drug side effects, and improve the protective effect on gastric mucosa is of great significance. Summary of the invention

[0008] The purpose of the present invention is to provide a licorice polysaccharide for preventing and treating acute gastric injury.

[0009] The present invention is achieved through the following technical solutions.

[0010] The licorice polysaccharide for preventing and treating acute gastric injury of the present invention has a molecular weight of 60 kDa, a protein content of less than or equal to 5%, and a mass content of greater than or equal to 60% in terms of glucose.

[0011] The licorice polysaccharide comprises fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galactose acid and gluconic acid.

[0012] The preparation method of liquorice polysaccharide for preventing and treating acute gastric injury of the present invention comprises the following steps.

[0013] (1) Dry the licorice raw material, grind it, and pass it through an 80-mesh sieve to obtain licorice powder.

[0014] (2) Take licorice powder and add 70-90% ethanol solution at a ratio of 1 g of licorice powder to 5-20 mL of ethanol. Reflux in a 70-90°C water bath for 3 hours. Repeat the reflux once for the residue and centrifuge to obtain the first precipitate.

[0015] (3) The first precipitate is collected and dried, and then extracted by water extraction and alcohol precipitation method at a volume ratio of 1 g of the first precipitate after drying to water of 10-40 mL, and protein is removed by Sevag method, and then dialyzed by 8-13 kDa dialysis bag, concentrated by rotation, and freeze-dried to obtain licorice polysaccharide.

[0016] Preferably, in step (2), 80% ethanol solution is added and the reflux temperature is 80°C.

[0017] Preferably, the water extraction and alcohol precipitation method in step (3) is to mix the first precipitate with water, extract with hot water for 2 h at an extraction temperature of 95°C, centrifuge, take the supernatant, extract the residue once again in the same way, combine the two filtrates, concentrate under reduced pressure in a 60°C water bath, mix the concentrated filtrate with a 95% ethanol solution, let it stand, and take the precipitate.

[0018] Preferably, in step (2), the volume ratio of the licorice powder to ethanol is 1 g: 10 mL.

[0019] In the preferred step (3), the volume ratio of the mass of the first precipitate after drying to water is 1 g: 20 mL.

[0020] In the preferred step (3), in the water extraction and alcohol precipitation method, the volume ratio of the concentrated filtrate to the 95% ethanol solution is 1:5.33.

[0021] The invention discloses an application of the liquorice polysaccharide in the preparation of medicine or food having a repairing effect on gastric damage.

[0022] The protective properties of the licorice polysaccharide having preventive and therapeutic effects on acute gastric injury of the present invention include but are not limited to the acute gastric injury caused by a one-time intake of a large amount of alcohol.

[0023] Animal experiments in vivo have confirmed that the licorice polysaccharide described in the present invention has good preventive and therapeutic effects on acute gastric damage in mice caused by alcohol, has a protective effect on gastric mucosa, can relieve damage and repair the barrier, has a pathological repair effect on gastric tissue damage, and improves acute gastric damage caused by alcohol.

[0024] The licorice polysaccharide can relieve gastric tissue bleeding, ulcers and the like caused by alcohol and promote the healing of ulcers and the like.

[0025] The licorice polysaccharide can reduce the damages such as epithelial shedding, glandular atrophy, inflammatory infiltration and the like of gastric tissue caused by alcohol, and reduce pathological histological damage.

[0026] The licorice polysaccharide can reduce the lipid oxidation level and the activity of myeloperoxidase in gastric tissue, reduce oxidative stress damage, and alleviate gastric digestive damage.

[0027] The licorice polysaccharide can restore the level of vascular endothelial growth factor and the activity of pepsin, repair the gastric mucosal barrier, and enhance the mucosal defense ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the glucose standard curve of the liquorice polysaccharide prepared by the present invention.

[0029] Figure 2 This is the protein standard curve of the liquorice polysaccharide prepared by the present invention.

[0030] Figure 3 This is the infrared spectrum of the liquorice polysaccharide prepared by the present invention.

[0031] Figure 4 This is a gel chromatogram of various liquorice prepared in the present invention.

[0032] Figure 5 The ion chromatogram of the liquorice polysaccharide prepared in the present invention.

[0033] Figure 6 The licorice polysaccharide prepared by the present invention intervenes in the gastric tissue morphology of mice with acute gastric mucosal injury.

[0034] Figure 7 This is an image of a section stained with hematoxylin-eosin staining (HE) in mice with acute gastric mucosal injury intervened by licorice polysaccharide prepared in the present invention.

[0035] Figure 8 The results are as follows: the licorice polysaccharide prepared by the present invention intervenes in gastric mucosal injury and the inhibition rate of acute gastric mucosal injury in mice.

[0036] Fig. 9The results of intervention of the prepared licorice polysaccharide in the present invention on lipid oxidation (Malondialdehyde, MDA) content in gastric tissue. Compared with the blank control group: # P<0.05, ## P<0.01, ### P<0.001; compared with the model group: * P<0.05, ** P<0.01, *** P<0.001.

[0037] Fig.10 The results of the intervention of the licorice polysaccharide prepared by the present invention on the activity of myeloperoxidase (MPO) in gastric tissue. Compared with the blank control group: # P<0.05, ## P<0.01, ### P<0.001; compared with the model group: * P<0.05, ** P<0.01, *** P<0.001.

[0038] Fig.11 The results of the intervention of the licorice polysaccharide prepared by the present invention on the activity of pepsin in gastric tissue. Compared with the blank control group: # P<0.05, ## P<0.01, ### P<0.001; compared with the model group: * P<0.05, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0039] The present invention will be further described by way of embodiments with reference to the accompanying drawings. Example 1

[0040] Preparation of Licorice Polysaccharide.

[0041] (1) The dried licorice raw material is crushed and passed through an 80-mesh sieve to obtain licorice powder.

[0042] (2) Add 80% ethanol solution in a ratio of 1 g of licorice powder to ethanol by volume of 10 mL, reflux in a water bath at 80°C for 3 hours, repeat the reflux once, and centrifuge to obtain the first precipitate.

[0043] (3) Take the first precipitate after drying, and extract it in a water bath at 95℃ for 2 hours in a ratio of 1g:20mL of the mass of the first precipitate after drying to the volume of water. Filter and centrifuge. Extract the residue once again in the same way. Combine the two filtrates, concentrate under reduced pressure at 60℃, take it out, cool it, add 5.33 times the volume of 95% ethanol solution, place it in a refrigerator at 4℃ for 12 hours, centrifuge it, collect the precipitate, add water to re-dissolve it, and use the Sevag method to deproteinize. The Sevag reagent is a mixture of 3 volumes of chloroform and 1 volume of n-butanol. The volume ratio of the re-dissolved sample solution to the volume of Sevag is 4:1. After fully shaking for 10 minutes, centrifuge it at 4800 rpm for 10 minutes, take the supernatant, and repeat the deproteinization method three times. The supernatant was collected, and after removing the organic matter, it was dialyzed in a 8-13 kDa dialysis bag with running water for 2 days and distilled water for 1 day. The retentate was concentrated under reduced pressure at 60°C and freeze-dried to obtain licorice polysaccharide. Example 2

[0044] (1) The dried licorice raw material is crushed and passed through an 80-mesh sieve to obtain licorice powder.

[0045] (2) Add 90% ethanol solution in a ratio of 1 g of licorice powder to ethanol by volume of 1 mL, reflux in a 90°C water bath for 3 hours, repeat the reflux once, and centrifuge to obtain the first precipitate.

[0046] (3) Take the first precipitate after drying, and extract it in a water bath at 95℃ for 2 hours in a ratio of 1g:10mL of the mass of the first precipitate after drying to the volume of water. Filter and centrifuge. Extract the residue once again in the same way. Combine the two filtrates, concentrate under reduced pressure at 60℃, take it out, cool it, add 5.33 times the volume of 95% ethanol solution, place it in a refrigerator at 4℃ for 12 hours, centrifuge it, collect the precipitate, add water to re-dissolve it, and use the Sevag method to deproteinize. The Sevag reagent is a mixture of 3 volumes of chloroform and 1 volume of n-butanol. The volume ratio of the re-dissolved sample solution to the volume of Sevag is 4:1. After fully shaking for 10 minutes, centrifuge it at 4800 rpm for 10 minutes, take the supernatant, and repeat the deproteinization method three times. The supernatant was collected, and after removing the organic matter, it was dialyzed in a 8-13 kDa dialysis bag with running water for 2 days and distilled water for 1 day. The retentate was concentrated under reduced pressure at 60°C and freeze-dried to obtain licorice polysaccharide. Example 3

[0047] (1) The dried licorice raw material is crushed and passed through an 80-mesh sieve to obtain licorice powder.

[0048] (2) Add 70% ethanol solution in a ratio of 1 g of licorice powder to ethanol by volume of 1 g: 20 mL, reflux in a 70°C water bath for 3 hours, repeat the reflux once, and centrifuge to obtain the first precipitate.

[0049] (3) Take the first precipitate after drying, and extract it in a water bath at 95℃ for 2 hours in a ratio of 1g:40mL of the mass of the first precipitate after drying to the volume of water. Filter and centrifuge. Extract the residue once again in the same way. Combine the two filtrates, concentrate under reduced pressure at 60℃, take it out, cool it, add 5.33 times the volume of 95% ethanol solution, place it in a refrigerator at 4℃ for 12 hours, centrifuge it, collect the precipitate, add water to reconstitute it, and use the Sevag method to deproteinize it. The Sevag reagent is a mixture of 3 volumes of chloroform and 1 volume of n-butanol. The volume ratio of the reconstituted sample solution to the volume of Sevag is 4:1. After fully shaking for 10 minutes, centrifuge it at 4800 rpm for 10 minutes, take the supernatant, and repeat the deproteinization method three times. The supernatant was collected, and after removing the organic matter, it was dialyzed in a 8-13 kDa dialysis bag with running water for 2 days and distilled water for 1 day. The retentate was concentrated under reduced pressure at 60°C and freeze-dried to obtain licorice polysaccharide. Example 4

[0050] Determination of neutral sugar content of licorice polysaccharide (taking the licorice polysaccharide product of Example 1 as the object, the same below).

[0051] The phenol-sulfuric acid method was used to determine the percentage of neutral sugar in glycyrrhiza polysaccharide. The specific operation was as follows: glucose was used as the standard, 25 mg of glucose standard was weighed into a 25 mL volumetric flask, and 5 mL of the solution was transferred to a 50 mL volumetric flask after dissolving and set to volume, and the solution was diluted 10 times as the standard solution; 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of the standard solution were added to each stoppered glass test tube, and the part less than 2 mL was supplemented to 2 mL with ultrapure water. Three groups of parallel measurements were performed for each concentration, 2 mL of 3% phenol solution was added to each tube, and then 4 mL of 95%-98% concentrated sulfuric acid was slowly added, and the solution was shaken while being slowly added, and the solution was allowed to stand for 30 min. After the solution was cooled to room temperature, the absorbance value was measured at 490 nm. The standard curve was drawn with the glucose concentration (mg / mL) as the abscissa and the absorbance value at 490 nm as the ordinate.

[0052] Take 2 mL of 0.1 mg / mL sample solution in a stoppered glass test tube, measure 3 groups in parallel, add 2 mL of 3% phenol solution to each tube, then slowly add 4 mL of 95%~98% concentrated sulfuric acid, shake while slowly adding, let stand for 30 minutes, and measure the absorbance value at 490 nm after the solution cools to room temperature. After the determination is completed, calculate the neutral sugar content of the sample according to the standard curve. The obtained glucose standard curve is as follows: Figure 1 As shown, the neutral sugar content of the sample was calculated to be 64.06% based on the standard curve. Example 4

[0053] Determination of protein content of licorice polysaccharide.

[0054] The protein was determined by the Coomassie brilliant blue method using bovine serum albumin as the standard.

[0055] Preparation of Coomassie Brilliant Blue Solution (G-250): Accurately weigh 50 mg of G-250 in a beaker, dissolve in 25 mL of 95% ethanol, add 50 mL of 85% phosphoric acid H3PO4, and then pour into a 500 mL brown volumetric flask to make up to volume. Protein standard solution: Weigh 10 mg of bovine serum albumin in a 50 mL volumetric flask, dissolve and make up to volume with pure water to obtain a protein standard solution. Pipette 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution into test tubes, respectively, and make up to 1 mL with pure water. Then quickly add 5 mL of Coomassie Brilliant Blue to all test tubes in a dark environment and shake, then measure the absorbance at 595 nm in a cuvette to draw a standard curve. Take 1 mL of 1 mg / mL sample solution in a test tube, then quickly add 5 mL of Coomassie Brilliant Blue to all test tubes for shaking, then measure the absorbance at 595 nm in a cuvette and compare it with the standard curve to calculate its protein content. The obtained protein content standard curve is as follows: Figure 2 As shown, the protein content of the sample was calculated to be 1.11% based on the standard curve. Example 5

[0056] Fourier transform infrared analysis of liquorice polysaccharides.

[0057] Take an appropriate amount of licorice polysaccharide sample and mix it with dry KBr, grind and press it into tablets. -1 Scan within the range to obtain an infrared spectrum, such as Figure 3 shown.

[0058] Depend on Figure 3 It can be seen that licorice polysaccharide is at 3436 cm -1 There is a strong broad absorption peak at 2940 cm, which is the OH stretching vibration and is the characteristic absorption peak of sugars. -1 There is an absorption peak at 1737 cm, which is the CH stretching vibration peak of polysaccharide. -1 The absorption peak attributed to the stretching vibration of C=O is 1639 cm -1 There is an absorption peak at 1432 cm -1 The peak at 1100 - 1000 cm -1 A strong absorption peak was observed at , indicating that the component has a pyranose ring structure. Therefore, the above characteristic absorption peaks indicate that the substance is a polysaccharide. Example 6

[0059] Molecular weight analysis of licorice polysaccharides.

[0060] High performance gel permeation chromatography (HPGPC) was used to determine the molecular weight of the polysaccharide. A series of glucose standards (Mw of 10, 40, 70, 180 and 500 kDa) were prepared to establish a standard curve. Licorice polysaccharide was prepared into a 1.0 mg / mL polysaccharide solution using a 0.1 M NaNO3 solution, passed through a double-layer 0.22 μm water filter membrane, and analyzed using a high performance gel chromatography. As a result, the molecular weight of the licorice polysaccharide was 60.098 kDa. Example 7

[0061] Analysis of monosaccharide composition of licorice polysaccharides.

[0062] High performance anion exchange chromatography-pulse integrated amperometry (HPAEC-PAD) was used to determine the monosaccharide composition of polysaccharides. 5 mg of 11 monosaccharides, including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, ribose, fructose, galacturonic acid and glucuronic acid, were accurately weighed to prepare a mixed standard stock solution and diluted to 10, 8, 6, 4, 2, 1, 0.5, and 0.25 μg / mL mass concentration gradients. After injection, the standard curves of each monosaccharide were obtained according to the monosaccharide peak areas under different concentration gradients for qualitative and quantitative analysis. Licorice polysaccharide was weighed in a test tube, 0.5 mL of 12 mol / L concentrated sulfuric acid was added in an ice bath, and stirred with a magnetic stirrer for 30 min. Then, 2.5 mL of ultrapure water was added and stirred in an oil bath at 100°C for hydrolysis for 70 min. After the hydrolyzate was diluted 100 times, it was filtered through a 0.22 μm water filter and analyzed by ion chromatography. As a result, the licorice polysaccharide consisted of rhamnose, arabinose, galactose, glucose, xylose, mannose, galactonic acid and gluconic acid. Example 8

[0063] Evaluation of the activity of licorice polysaccharide against acute alcoholic gastric injury.

[0064] 9.1 Establishment of acute alcoholic gastric injury mouse model + drug administration.

[0065] 8-week-old SPF male Kunming mice, weighing 20 ± 2 g, were housed in a room at 22 ± 1°C, with a 12h / 12h light / dark cycle, free access to food and water, and were allowed to acclimate to the environment for seven days. They were randomly divided into five groups: normal group (Control, NC), model group (MC), low-dose licorice polysaccharide group (Glycyrrhiza polysaccharide Low, GCP-L, 100 mg / kg bw), medium-dose licorice polysaccharide group (Glycyrrhiza polysaccharide Medium, GPC-M, 200 mg / kg bw), and high-dose licorice polysaccharide group (Glycyrrhiza polysaccharide High, GCP-H, 400 mg / kg bw). Each group of mice was gavaged with 10 mL / kg of the test substance, and the normal group and model group were gavaged with normal saline. The drug was administered once a day. After 14 days of gavage, the mice were weighed. All animals were strictly fasted for 24 h (water was not prohibited), and the test substance was also prohibited during this period. Each mouse in the model group and polysaccharide group was gavaged with 0.1 mL / 10 g 70% ethanol. After 1 hour, the mice were anesthetized and blood was collected by eye removal, and the mice were killed by cervical dislocation. The abdominal cavity was opened to remove the stomach, liver and other organs, washed with normal saline, dried with filter paper, weighed and recorded the tissue weight. The stomach tissue was fixed with 4% paraformaldehyde, and the remaining tissue was stored at -80℃.

[0066] 9.2 Determination of gastric mucosal injury and inhibition rate.

[0067] The mouse stomach was cut open along the greater curvature of the stomach, and the residual in the stomach was flushed away with normal saline. The gastric mucosa was unfolded and photographed. The length and width of the bleeding point or bleeding strip were measured with a vernier caliper. The scoring criteria are shown in Table 1.

[0068] Table 1 Scoring criteria for gastric mucosal injury index

[0069] Evaluation index: Gastric mucosal injury index = bleeding point score + erosion length score + erosion width score × 2.

[0070] Damage inhibition rate (%) = (damage index of model group - damage index of polysaccharide group) / damage index of model group.

[0071] 9.3 Pathological observation of gastric tissue by HE staining.

[0072] Hematoxylin and stained (HE) was used to observe the morphology of gastric tissue. Approximately 10 mm × 2 mm tissue was cut from the same part of the gastric mucosal injury of the mouse, fixed in 10% neutral formalin solution, dehydrated, and embedded in paraffin. The tissue wax block was cut into sections with a thickness of 4 μm, and the pathological changes of gastric tissue were observed under an optical microscope (200 times) after HE staining.

[0073] 9.4 Determination of lipid oxidation, vascular endothelial growth factor content, and myeloperoxidase and pepsin activities in gastric tissue.

[0074] Accurately weigh the tissue, add 10 times the volume of 0.01 MPBS buffer at a ratio of weight (g): volume (mL) = 1: 10, homogenize at low temperature (0~4℃) to make a tissue homogenate, and measure the indicators according to the instructions of the kit. The lipid oxidation (MDA) detection kit was purchased from Biyuntian Biotechnology Co., Ltd. (S0101M); the myeloperoxidase (MPO) determination kit was purchased from Nanjing Jiancheng Bioengineering Institute (A044-1-1); the pepsin test kit was purchased from Nanjing Jiancheng Bioengineering Institute (A080-1-1); the vascular endothelial growth factor test kit was purchased from Wuhan Boster Bioengineering Co., Ltd. (EK0541).

[0075] 9.5 Experimental results.

[0076] 9.5.1 Licorice polysaccharide on gastric mucosal damage and inhibition rate.

[0077] The results are as follows Figure 6 , 8 As shown in the figure, a large number of bleeding strips appeared in the gastric mucosa of mice after 70% ethanol modeling. Compared with the model group, the gastric mucosal injury index of each drug-treated group was reduced, among which the GCPH group had the smallest gastric injury index and the highest injury inhibition rate, reaching 47.9%. This shows that licorice polysaccharide has a protective function on gastric mucosal injury and has a certain dose dependence.

[0078] 9.5.2 Pathological observation of gastric tissue by HE staining.

[0079] The results are as follows Figure 7 As shown in the figure, the gastric tissue structure of the normal group was intact, flat and smooth, the mucosal epithelial cells were normal in morphology, arranged regularly and tightly, and no shedding, necrosis, inflammatory infiltration and other phenomena were observed. The gastric mucosa of the model group was severely damaged, with a large number of epithelial cells shedding, inflammatory infiltration in the upper layer of the mucosa, and disordered glandular structure. Compared with the model group, the epithelial shedding phenomenon in the GCPL group was significantly improved, the glandular disorder was alleviated, and the degree of damage in the GCPM group was further reduced. The glandular structure of the GCPH group was basically completed, and a small amount of inflammatory infiltration was observed.

[0080] 9.5.3 Determination of lipid oxidation, vascular endothelial growth factor content, and myeloperoxidase and pepsin activities in gastric tissue.

[0081] The content of lipid oxidation (MDA) is an important parameter reflecting the body's antioxidant potential. It can reflect the rate and intensity of lipid peroxidation in the body, and can also indirectly reflect the degree of tissue peroxidation damage. Myeloperoxidase (MPO) is a member of the heme peroxidase superfamily and is a functional marker and activation marker of neutrophils. Pepsin is secreted by chief cells, activated by gastric acid and provides an acidic environment to play a role in decomposing proteins. Its absence will cause digestive damage. Vascular endothelial growth factor (VEGF) is a powerful specific endothelial cell mitogen that mediates the proliferation, budding, migration and lumen formation of endothelial cells, and is involved in many vascular-related diseases and certain inflammatory reactions.

[0082] The results are as follows Fig. 9 , 10 As shown in Figures 11, compared with the normal group, the levels of MDA and MPO in the gastric tissue homogenate of the model group mice were significantly increased (P<0.001), that is, ethanol stimulation would enhance the level of lipid oxidation, stimulate neutrophil infiltration and induce inflammation; the level of pepsin was significantly reduced (P<0.01). Combined with pathological analysis, it can be seen that ethanol stimulation increases the pH in the stomach, affects the digestive function of the stomach, and causes an increase in the amount and area of ​​bleeding. Compared with the model group, the level of pepsin in the groups administered with each dose of licorice polysaccharide increased, among which the GCPL group significantly restored its level (P<0.01), and the levels of MDA and MPO were significantly reduced (P<0.01). Comprehensive observation showed that the effect of the GCPL group was better than that of the GCPM group and the GCPH group. The results showed that licorice polysaccharide alleviated the acute gastric injury caused by ethanol by reducing lipid oxidation and neutrophil infiltration, while maintaining the synthesis and activation environment of pepsin, alleviating gastric peptic injury, promoting the healing of ulcers, and protecting the gastric mucosa.

[0083] Studies have shown that ethanol stimulation significantly reduces VEGF content (P<0.001), affecting the proliferation and migration of vascular endothelial cells and causing damage; various doses of licorice polysaccharides can appropriately restore its level, and medium doses of licorice polysaccharides have the best effect in restoring VEGF.

Claims

1. A licorice polysaccharide for preventing and treating acute gastric injury, characterized in that The molecular weight of the licorice polysaccharide is 60kDa, the percentage of protein is ≤5%, and the mass percentage of glucose is ≥60%; The licorice polysaccharide comprises fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galactose acid and gluconic acid.

2. The method for preparing a licorice polysaccharide for preventing and treating acute gastric injury according to claim 1, characterized in that The following steps are involved: (1) drying the licorice raw material, crushing it, and passing it through an 80-mesh sieve to obtain licorice powder; (2) Take licorice powder and add it to an ethanol solution with a concentration of 70-90% at a ratio of 1 g of licorice powder to 5-20 mL of ethanol. Reflux in a water bath at 70-90°C for 3 hours. Repeat the reflux once for the residue and centrifuge to obtain a first precipitate. (3) The first precipitate is collected and dried, and then extracted by water extraction and alcohol precipitation method at a volume ratio of 1 g of the first precipitate after drying to water of 10-40 mL, and protein is removed by Sevag method, and then dialyzed by 8-13 kDa dialysis bag, concentrated by rotation, and freeze-dried to obtain licorice polysaccharide.

3. The method for preparing a licorice polysaccharide for preventing and treating acute gastric injury according to claim 2, characterized in that In step (2), an ethanol solution with a concentration of 80% is added and the reflux temperature is 80°C.

4. The method for preparing a licorice polysaccharide for preventing and treating acute gastric injury according to claim 2, characterized in that The water extraction and alcohol precipitation method in step (3) is to mix the first precipitate with water, extract with hot water for 2 h, extract at 95°C, centrifuge, take the supernatant, extract the residue once again in the same way, combine the two filtrates, concentrate under reduced pressure in a 60°C water bath, mix the concentrated filtrate with a 95% ethanol solution, let it stand, and take the precipitate.

5. The method for preparing a licorice polysaccharide for preventing and treating acute gastric injury according to claim 2, characterized in that In step (2), the volume ratio of the licorice powder to ethanol is 1 g: 10 mL.

6. The method for preparing a licorice polysaccharide for preventing and treating acute gastric injury according to claim 2, characterized in that In step (3), the ratio of the mass of the first precipitate after drying to the volume of water is 1 g: 20 mL.

7. The method for preparing a licorice polysaccharide for preventing and treating acute gastric injury according to claim 4, characterized in that In the water extraction and alcohol precipitation method, the volume ratio of the concentrated filtrate to the 95% ethanol solution is 1:5.

33.

8. Use of the licorice polysaccharide according to claim 1 in the preparation of medicines or foods having a repairing effect on gastric damage.

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