Longpimpinella esculenta polysaccharide, preparation method and application thereof in protecting gastric mucosa

Abstract

The invention provides a preparation method of Longpimpinella pedicularis crude polysaccharide, which comprises the following steps: degreasing Longpimpinella pedicularis to obtain degreased Longpimpinella pedicularis; and extracting the degreased longpimpinella pedicularis by using a NaOH solution to obtain the longpimpinella pedicularis crude polysaccharide. The invention also provides a preparation method of the Longpimpinella esculenta polysaccharide, which comprises the following steps: extracting the Longpimpinella esculenta crude polysaccharide by using papain, separating by using a DEAE-52 cellulose column, eluting by using distilled water, and collecting an eluted solution. The invention also provides application of the polysaccharide in gastric mucosa protection. The two polysaccharides have a good gastric mucosa protection effect, especially can protect acute alcoholic gastric mucosal lesion, and can play the gastric mucosa protection effect by resisting oxidation and inflammation and regulating intestinal flora.

Description

Technical Field

[0001] The present invention relates to Longmalu polysaccharide, a preparation method thereof and application thereof in protecting gastric mucosa. Background Art

[0002] Kronopolites svenhedind is the dried whole body of the Myriapoda species Kronopolites svenhedind. According to the Gansu Province Traditional Chinese Medicine Standard (2020 Edition), it has the effects of removing stagnation, soothing the stomach, and reducing swelling and detoxification. Kronopolites svenhedind mainly contains calcium carbonate, polysaccharides, proteins, amino acids, trace elements and other ingredients. Kronopolites polysaccharide ( Kronopolites svenhedind polysaccharides KSP (Kysinus oxysporum) is a member of the animal polysaccharide family, yet its potential biological activity and structure remain largely unexplored. Currently, research on Longmalu primarily focuses on resource distribution. A comprehensive and systematic research framework has yet to be established regarding active ingredient extraction, structural analysis, and mechanism of activity, and remains in its early stages. The challenge currently lies in efficiently identifying KSP's active ingredients, delving deeper into its pharmacological activities, and deepening our understanding of KSP. Summary of the Invention

[0003] To address the problems existing in the prior art, the present invention provides a polysaccharide from Longmalu, a preparation method, and its application in protecting gastric mucosa. The present invention first establishes a preliminary extraction process for KSP and preliminarily verifies its gastric mucosal protective activity. Subsequently, DEAE-52 cellulose column chromatography is used to separate and purify KSP components with potential protective effects, producing a structurally homogeneous, high-purity KSP fraction. Finally, using an in vitro model of ethanol-induced GES-1 cell injury and an in vivo model of acute gastric mucosal injury in rats, the gastric mucosal protective activity of KSP is systematically evaluated, and its mechanism of action is preliminarily explored.

[0004] The first object of the present invention is to provide a method for preparing crude polysaccharide of Longmalu, comprising the following steps: (1) defatting the Longmalu to obtain defatted Longmalu; (2) The defatted Longmalu obtained in step (1) was extracted with NaOH solution to obtain Longmalu crude polysaccharide; the extraction conditions were: NaOH concentration 0.03-0.05 mol / L, extraction temperature 70-90°C, extraction time 2.0-3.0 h, extraction times 2-3 times, and solid-liquid ratio 1:15-1:30.

[0005] Preferably, in step (1), Longmalu is mixed with 95% ethanol in a ratio of 1 g:10 mL, and the mixture is condensed and refluxed for degreasing.

[0006] Preferably, in step (2), the extraction conditions are: NaOH concentration 0.04 mol / L, extraction temperature 78°C, extraction time 2.5 h, extraction times 2 times, and solid-liquid ratio 1:20.

[0007] Preferably, the method further comprises a post-processing step after extraction: collecting the extract, adjusting the pH to neutral, concentrating, and precipitating with alcohol, and freeze-drying to obtain crude polysaccharide from Longmalu.

[0008] The second object of the present invention is to provide crude polysaccharide of Longmalu, which is prepared by the above method.

[0009] The third object of the present invention is to provide a method for preparing Longmalu polysaccharide, comprising the following steps: (1) extracting the crude polysaccharide of the Longmalu truncatum according to claim 5 using papain, removing protein, and obtaining deproteinized crude polysaccharide of the Longmalu truncatum; (2) The deproteinized polysaccharide of Longmalu prepared in step (1) was separated using a DEAE-52 cellulose column. The column was gradient eluted with distilled water, 0.1, 0.2, and 0.5 mol / L NaCl aqueous solutions at a flow rate of 0.7 mL / min. The absorbance at 490 nm was measured using the anthrone-sulfuric acid method. The elution curve was drawn and the same fractions were combined.

[0010] Preferably, in step (1), the specific method of extracting the above-mentioned Longmalu crude polysaccharide using papain is as follows: Longmalu crude polysaccharide is prepared into a solution with distilled water, mixed with papain, and the pH value of the solution is adjusted to 6-7, and enzymatically hydrolyzed at 39-41°C for 3.5-4.5h; Preferably, the weight ratio of papain to Longmalu crude polysaccharide is 1:100.

[0011] The fourth object of the present invention is to provide Longmalu polysaccharide, which is prepared by the above method.

[0012] The fifth object of the present invention is to provide the use of the above-mentioned crude polysaccharide of Longmalu or Longmalu polysaccharide in the preparation of a drug for protecting gastric mucosa; Preferably, the polysaccharide of Longmalu is used in the preparation of a drug for protecting alcoholic gastric mucosal damage; Preferably, the polysaccharide of Longmalu is used in the preparation of a drug for protecting acute alcoholic gastric mucosal damage; Preferably, the concentration of the polysaccharide of Longmalu is 7.8-125 μg / mL; most preferably, the concentration of the polysaccharide of Longmalu is 31.25 μg / mL.

[0013] Preferably, the polysaccharide of Longmalu exerts gastric mucosal protective effects through anti-oxidation, anti-inflammation and regulation of intestinal flora; Preferably, the regulating of intestinal flora includes up-regulating the abundance of beneficial bacteria of Lachnospiraceae and Lactobacillaceae, and down-regulating the abundance of conditionally pathogenic bacteria of Escherichia coli.

[0014] The present invention extracts crude polysaccharides from Longmalu, and further extracts Longmalu polysaccharide KSP-1 with good water solubility. Both polysaccharides have good gastric mucosal protective effects, especially can protect acute alcoholic gastric mucosal damage, and can play a gastric mucosal protective role through anti-oxidation, anti-inflammatory and regulation of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 The results of a single-factor experiment include (A) number of extractions, (B) material-liquid ratio, (C) extraction time, (D) extraction temperature, and (E) NaOH concentration.

[0016] Figure 2 Effects of different concentrations of KSP pretreatment on the survival rate of ethanol-damaged GES-1 cells.

[0017] Figure 3 The effect of different concentrations of KSP pretreatment on ethanol-induced cell damage and oxidative stress.

[0018] Figure 4 The effect of different concentrations of KSP pretreatment on inflammatory factors of cell damage induced by ethanol.

[0019] Figure 5 This is the elution curve of KSP.

[0020] Figure 6 These are purified KSP samples, including A: KSP-1, B: KSP-2, C: KSP-3, D: KSP-4, and E: KSP-5.

[0021] Figure 7 This is the molecular weight spectrum of purified KSP.

[0022] Figure 8 This is the full-wavelength UV scan of purified KSP-1 and KSP-2.

[0023] Figure 9 FTIR spectra of purified KSP-1 and KSP-2.

[0024] Figure 10 This is the gas chromatogram of purified KSP. It contains 1-Rib, 2-Rha, 3-Ara, 4-Xyl, 5-Man, 6:Glc, and 7-Gal.

[0025] Figure 11 KSP-1 1 H NMR spectrum.

[0026] Figure 12 KSP-1 13 C NMR spectrum.

[0027] Figure 13 For KSP-2 1 H NMR spectrum.

[0028] Figure 14 The SEM images of purified KSP are shown in Figure 1. A: KSP-1; B: KSP-2.

[0029] Figure 15 This is the result of the Congo red test.

[0030] Figure 16 The effect of different concentrations of KSP-1 on cell survival rate.

[0031] Figure 17 The effect of different concentrations of KSP-1 pretreatment on the survival rate of ethanol-damaged GES-1 cells.

[0032] Figure 18 Effects of pretreatment with different concentrations of KSP-1 on the morphology of GES-1 cells damaged by ethanol (magnification 20×).

[0033] Figure 19 The effect of different concentrations of KSP-1 pretreatment on ethanol-induced cell damage and oxidative stress.

[0034] Figure 20 The effect of different concentrations of KSP-1 pretreatment on inflammatory factors of ethanol-induced cell damage.

[0035] Figure 21 Figure 5. Body weight changes of rats (n=8).

[0036] Figure 22 Effects of KSP-1 on (A) liver, (B) kidney, and (C) spleen (n=8).

[0037] Figure 23 The gastric tissue morphology of rats. A: blank group; B: blank drug group; C: model group; D: Yang 1 group; E: Yang 2 group; F: KSP-1-L group; G: KSP-1-M group; H: KSP-1-H group.

[0038] Figure 24 Macroscopic evaluation score, injury area and injury inhibition rate of rat gastric tissue (n=8).

[0039] Figure 25Pathological sections of rat gastric tissue (HE staining, 100×; a: blank group; b: blank drug group; c: model group; d: Yang 1 group; e: Yang 2 group; f: KSP-1-L group; g: KSP-1-M group; h: KSP-1-H group) (HE staining, 400×; AH: same as above).

[0040] Figure 26 The effect of KSP-1 on gastric injury tissue G-17 (n=4).

[0041] Figure 27 The effect of KSP-1 on oxidative stress in gastric damaged tissue (n=4).

[0042] Figure 28 The effect of KSP-1 on inflammatory factors in gastric injured tissue (n=4).

[0043] Figure 29 The α-diversity analysis results of rats in each group. A: dilution curve; B: Observed_ASV index; C: Shannon index; D: Simpson index; E: Chao1 index; F: ACE index; (n=3).

[0044] Figure 30 The β-diversity analysis results of rats in each group. A: PCA analysis; B: PCoA analysis; C: NMDS analysis; D: UPGMA cluster tree analysis; (Note: In each figure, A: blank group; B: model group; M: KSP-1-M group; G: blank treatment group).

[0045] Figure 31 The bacterial composition of the intestinal contents of rats in each group at the phylum level is shown in Figure 1. A: blank group; B: model group; M: KSP-1-M group; G: blank treatment group.

[0046] Figure 32 The relative abundance of the top ten bacterial phyla in the intestinal contents of rats in each group (n=3).

[0047] Figure 33 The bacterial composition of the intestinal contents of rats in each group at the family level. A: blank group; B: model group; M: KSP-1-M group; G: blank treatment group.

[0048] Figure 34 The relative abundances of the top ten bacterial families in the intestinal contents of rats in each group (n=3).

[0049] Figure 35 The bacterial composition of the intestinal contents of rats in each group at the genus level is shown in Figure 1. A: blank group; B: model group; M: KSP-1-M group; G: blank treatment group.

[0050] Figure 36 The relative abundances of the top ten bacterial genera in the intestinal contents of rats in each group (n=3).

[0051] Figure 37 Figure 2: Analysis of differential bacterial flora in rats. A: Venn diagram based on ASV; B: Linear discriminant analysis histogram based on ASV; C: Evolutionary branch diagram based on ASV. (Note: In each figure, A: blank group; B: model group; M: KSP-1-M group; G: blank treatment group).

[0052] Figure 38 The effect of KSP-1 intervention on the intestinal microbiota of ethanol-induced rats. The heat map in the figure represents the relative abundance of differentially expressed microbiota between the groups; gray circles indicate no significant difference in microbiota between the two groups; light red circles indicate significant difference in microbiota between the two groups; and dark red circles indicate extremely significant difference in microbiota between the two groups. (Note: A: blank group; B: model group; M: KSP-1-M group; G: blank treatment group). DETAILED DESCRIPTION

[0053] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0054] Example 1 Crude Extraction of Longmalu Polysaccharide In the preliminary experiments, by comparing the alkaline extraction method, water extraction method and enzymatic hydrolysis method, the results showed that the alkaline extraction method had a higher KSP yield and a lower protein content. Therefore, the alkaline extraction method was selected for the extraction of KSP.

[0055] Materials: Longmalu was purchased from Lanzhou Foci Pharmaceutical Co., Ltd. Kronopolitus svenhedini The dried whole of (Verhoeff) was authenticated by the chief Chinese medicine pharmacist of the Affiliated Hospital of Gansu University of Chinese Medicine.

[0056] Experimental methods 1.1 Plotting the glucose standard curve Prepare a 0.1 mg / mL glucose (Glc) standard solution by pipetting the standard solution into a test tube in a gradient of 0.00-2.00 mL, and adjust the volume to 2 mL. Add 5% phenol solution (1 mL) and concentrated sulfuric acid (5 mL) sequentially, mix thoroughly, and react in a boiling water bath for 30 min. After cooling, measure the absorbance (A) at 490 nm. Construct a standard curve with Glc concentration as the horizontal axis and A as the vertical axis.

[0057] 1.2 Extraction and determination of polysaccharides from Longmalu Longmalu powder was pulverized and passed through a 40-mesh sieve. The powder was mixed with 95% ethanol at a ratio of 1:10 (g / mL), defatted by condensation and reflux for 1 hour, and then filtered. The defatting process was repeated twice, and the filter residue was air-dried and set aside. The defatted Longmalu powder was dispersed in 0.02 mol / L NaOH solution at a solid-liquid ratio of 1:10 (g / mL). The solution was extracted in a 70°C water bath for 60 minutes. The filtrates were then filtered under reduced pressure and combined. The pH was adjusted to neutral, and the solution was concentrated under reduced pressure to 25% of its original volume. Anhydrous ethanol was added to the concentrate until the ethanol concentration reached 80%. The solution was allowed to stand at 4°C overnight and centrifuged at 4000 rpm for 20 minutes to obtain the precipitate. Crude KSP was obtained by freeze-drying.

[0058] Weigh 10 mg of lyophilized crude KSP and prepare a 0.1 mg / mL test solution with distilled water. Perform the test as described in 1.1, measuring A at 490 nm. Substitute the A value into the regression equation to determine the polysaccharide mass concentration in the test solution. Calculate the crude KSP yield according to Equation 1.

[0059] Formula (1) Where: N is the crude KSP yield (%); C is the mass concentration of polysaccharides in the sample (mg / mL); V is the sample volume (mL); D is the dilution factor; m is the dry weight of crude KSP powder (g).

[0060] 1.3 Single-factor experimental design of Longmalu polysaccharide The extraction was performed according to item 1.2. The crude KSP yield was used as the evaluation index to investigate the effects of extraction times (1-3 times), solid-liquid ratio (1:5-1:30), extraction time (0.5h-3h), extraction temperature (50℃-90℃) and NaOH concentration (0.01mol / L-0.05mol / L) on the crude KSP yield.

[0061] 1.4 Response surface design of polysaccharides from Longmalu Based on a single-factor experiment, the process range for the single factor was determined, with a fixed number of extractions at two and a solid-liquid ratio of 1:20. Subsequently, using Design-Expert 13.0 software and the Box-Behnken design principle within response surface methodology (RSM), a three-factor, three-level experimental design was designed, and a multiple regression model was constructed. NaOH concentration (A), extraction temperature (B), and extraction time (C) were selected as independent variables, and the KSP extraction rate was used as the response value to systematically optimize the alkaline extraction process. The specific independent variables and levels of the response surface experiment are shown in Table 1.

[0062] Table 1 Independent variables and level settings of RSM experimental design

[0063] 1.5 Verification test In order to verify the optimized extraction process conditions, three batches of KSP were prepared in parallel according to this process, taking the crude KSP yield as an indicator.

[0064] 2 Experimental Results and Discussion 2.1 Using Glc as the standard, draw a standard curve regression equation.

[0065] 2.2 Single factor test results of Longmalu polysaccharide In order to obtain a higher yield of KSP from Longmalu, the effects of extraction times, material-liquid ratio, extraction time, extraction temperature and NaOH concentration on the yield of crude KSP were first investigated. Figure 1 The results are from a single-factor experiment. The optimal extraction conditions were determined as follows: 2 extractions, a solid-liquid ratio of 1:20, an extraction time of 2 h, an extraction temperature of 80°C, and a NaOH concentration of 0.04 mol / L.

[0066] 2.3 Response surface analysis results of Longmalu polysaccharide The results of the Box-Behnken response surface experiment are shown in Table 2. The results of the variance analysis are shown in Table 3. The results of the significance test of the model parameters are as follows: the linear effects of the first-order extraction temperature (B) and time (C) are extremely significant ( P <0.0001), which is the key main effect factor affecting the yield; except for the C² term, other quadratic terms (such as A 2 , B²) all reached extremely significant levels ( P <0.001), indicating that the square effect and interaction of the parameters have a strong nonlinear regulation on the yield. The data in Table 3 were subjected to regression analysis using Design-Expert.13 software, and the regression equation was constructed as follows: Y = 12.54-0.1773*A-0.6986*B+0.9149*C+0.0235*AB+0.3159*AC+0.1714*BC-1.91*A 2 -1.97*B 2 -0.7088*C 2 .

[0067] Using Design-Expert 13 software, a systematic analysis was conducted on the interactions among NaOH concentration (A), extraction temperature (B), and extraction time (C). The interaction between NaOH concentration (A) and extraction time (C) was the most critical factor influencing crude KSP yield. Analysis of variance further confirmed this conclusion: among the three-factor interactions, the combined effect of A and C was the strongest, while the interaction between A and B was the weakest, indicating that different parameter combinations have varying sensitivities in regulating yield.

[0068] Table 2 Box-Behnken response surface experiment results

[0069] Table 3 Analysis of variance of regression model

[0070] 2.4 Analysis of verification test results Using Design-Expert.13 software for optimization analysis, the crude KSP yield (Y) was used as the response indicator. The optimal KSP extraction process was determined to be: NaOH concentration of 0.041 mol / L, temperature of 78.368°C, and extraction time of 2.545 hours. The theoretical crude KSP yield was 12.87%. Based on actual conditions, these conditions were modified to: NaOH concentration of 0.04 mol / L, extraction temperature of 78°C, extraction time of 2.5 hours, two extractions, and a solid-liquid ratio of 1:20.

[0071] Under these process conditions, the crude KSP yield was 12.02%±0.21%, which was only 0.85% different from the predicted value of 12.87%. This shows that the optimized extraction parameters showed the advantages of high efficiency and stability after three verifications, fully meeting the experimental needs.

[0072] Example 2 Protective effect of Longmalu crude polysaccharide on ethanol-induced GES-1 cell damage Ethanol induction has become a modeling approach in the study of gastric mucosal injury. The GES-1 cell line is an immortalized cell line derived from human gastric epithelium. Its biological properties are highly similar to those of normal gastric epithelial cells, and it can stably express gastric epithelial cell markers under in vitro culture conditions. These characteristics make GES-1 cells an important cell model for in vitro research related to gastric mucosa. Therefore, we established a GES-1 cell injury model using ethanol, and systematically investigated the gastric mucosal protective effects of KSP from the perspectives of oxidative stress and inflammatory factor levels.

[0073] Experimental Materials Experimental material: crude KSP (prepared according to the optimized extraction method in Example 1).

[0074] Cells: Human gastric epithelial cell line GES-1 (Starfish Biotechnology).

[0075] Experimental methods 2.1 Culture and passage of human gastric epithelial cell line GES-1 2.2 Establishment of ethanol-induced cell model GES-1 cells in the exponential growth phase were collected and resuspended in serum-containing RPMI 1640 to the target concentration (1×10 5cells / mL) and then seeded into 96-well plates. The plates were incubated in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. The cells were then cultured in an incomplete medium containing 3% ethanol for 6 hours. At this point, the cell viability was approximately 54.80 ± 0.86%, which met the requirements for model establishment in this experiment.

[0076] (Equation 2) 2.3 Screening of the optimal KSP concentration The experiment was divided into two groups: a control group and a KSP-treated group. The control group received complete medium containing FBS as a normal growth control; the treated groups received complete medium containing varying concentrations of KSP (0.156, 0.315, 0.625, 1.25, 2.5, 5, and 10 mg / mL), with six replicate wells per group. After addition, the 96-well plates were returned to the incubator and cultured for 24 and 48 hours, respectively. Cell viability was assessed using the MTT assay, and cell survival was calculated according to Equation 2. Low, medium, and high concentrations that had no effect on cell viability were selected and used in subsequent experiments.

[0077] 2.4 Effect of KSP on cell survival in the ethanol-induced injury model The experiment was divided into the following five groups: control group, model group, and KSP low (0.625 mg / mL), medium (1.25 mg / mL), and high (2.5 mg / mL) dose groups. First, complete medium containing varying concentrations of KSP was added to GES-1 cells in each experimental group and cultured for 24 hours. After 24 hours, the medium in each well was carefully aspirated. Next, incomplete medium containing 3% ethanol was added to cells in all groups except the blank group to establish an oxidative damage model, and culture was continued for 6 hours. After the incubation period, cell viability was assessed using the MTT assay, and cell survival rate was calculated according to Equation 2 to evaluate the effect of KSP in the ethanol-induced cell damage model.

[0078] 2.5 Determination of biochemical indices of cells in the ethanol-induced injury model by KSP GES-1 cells in the logarithmic growth phase were taken and 4×10 5 cells / mL, accurately pipette 2 mL of cell suspension into each well of a 6-well plate. Follow the experimental grouping and cell treatment procedures in 2.4. After treatment, carefully aspirate the cell supernatant and save it for analysis. Add an appropriate amount of trypsin for digestion, resuspend the cells in PBS, and disrupt them with a cell disruptor to obtain a homogenate.

[0079] SOD and MDA levels in cell homogenates were determined using commercial assay kits strictly according to the manufacturer's instructions. IL-1β, IL-6, and TNF-α concentrations in the supernatant were determined using commercially available Elisa kits according to the manufacturer's instructions.

[0080] 2.6 Data Analysis Data The statistical analysis was performed using GraphPad Prism 8.0 software (ANOVA test for differences between groups), and the graphs were drawn jointly by GraphPad Prism 8.0 and Origin 9.0. Compared with the Control group: # P <0.05 and ## P <0.01; compared with the Model group: * P <0.05 and ** P <0.01.

[0081] Experimental results and discussion 3.1 Results of screening for optimal KSP concentration Compared with the control group, KSP had no inhibitory effect on the survival rate of GES-1 cells after 24 h and 48 h of culture. After 24 h of culture, KSP had a significant proliferation effect in the concentration range of 0.625~5 mg / mL ( P <0.05, 0.01). Therefore, 0.625, 1.25, and 2.5 mg / mL were selected as the low, medium, and high doses of KSP for subsequent experimental concentrations.

[0082] 3.2 Results of the effect of KSP on cell survival in the ethanol-induced injury model like Figure 2 Ethanol significantly inhibited the proliferation of GES-1 cells (the survival rate of the Model group was lower than that of the Control group, P < 0.01), confirming that the injury model was successfully established. KSP pretreatment reversed this effect in a dose-dependent manner: compared with the Model group, the cell survival rate of each concentration group was significantly increased ( P <0.01), and the survival rate increased gradually with the increase of drug concentration, indicating that KSP alleviated ethanol-induced gastric mucosal epithelial cell damage in a concentration-dependent manner.

[0083] 3.3 KSP measurement results of biochemical indices of ethanol-induced damage model cells The results of oxidative stress related indicators are as follows Figure 3 As shown, the SOD activity in the model group was significantly lower than that in the control group ( P<0.01), while with the increase of KSP concentration, SOD activity gradually increased, and the SOD activities of the three treatment groups of 0.625, 1.25, and 2.5 mg / mL were significantly higher than those of the model group ( P <0.01). However, the MDA content showed the opposite trend, and the MDA content in the model group was significantly higher than that in the control group ( P <0.01), while with the increase of KSP concentration, the MDA content gradually decreased, and the SOD activities of the three treatment groups of 0.625, 1.25, and 2.5 mg / mL were significantly lower than those of the model group ( P <0.01). The results showed that KSP could effectively alleviate ethanol-induced oxidative stress damage by enhancing antioxidant enzyme activity and inhibiting lipid peroxidation.

[0084] Excessive release of cytokines and inflammatory mediators can easily cause cell damage. Therefore, inhibiting the secretion of inflammatory factors can reduce cell damage to a certain extent. Figure 4 The levels of IL-1β, IL-6 and TNF-α in the model group were significantly higher than those in the control group ( P <0.01). However, as the concentration of KSP increased, the levels of IL-1β, IL-6, and TNF-α gradually decreased, and the levels of IL-1β, IL-6, and TNF-α in the 0.625, 1.25, and 2.5 mg / mL treatment groups were significantly lower than those in the model group ( P <0.01, 0.05). The results showed that KSP could effectively alleviate ethanol-induced gastric mucosal inflammatory damage by regulating the expression of inflammatory factors.

[0085] Example 3 Isolation, purification and structural analysis of crude polysaccharides from Longmalu Polysaccharides obtained by direct extraction typically contain non-sugar components such as inorganic salts and macromolecular proteins. This is especially true for animal polysaccharides, which are high in protein and impurity content. To obtain highly homogeneous polysaccharides to meet subsequent research requirements, such as structural identification, effective separation and purification methods are essential. This study combines enzymatic and Sevage methods to remove proteins from KSP. KSP is then separated using a DEAE-52 cellulose anion exchange chromatography column. The purified KSP is then characterized using methods such as HPGPC-RID, GC, FTIR, SEM, and NMR.

[0086] Reagents and instruments All reagents used in the experiments were of analytical grade.

[0087] Experimental material: crude KSP (prepared according to the optimized extraction method in Example 1).

[0088] Experimental methods 2.1 Isolation and purification of KSP Deproteinization and column chromatography purification of KSP: (1) Crude KSP (prepared according to the optimized extraction method in Example 1) was prepared into a 1 mg / mL solution with distilled water, and 1 mg of papain was added to 100 mL of crude KSP solution. The pH value of the solution was adjusted to about 6.5. Subsequently, the solution was placed in a water bath at 40°C and stirred for 4 h. After the reaction was completed, the solution was quickly transferred to boiling water and boiled for 10 min to inactivate the papain. Then, the treated solution was transferred to a centrifuge, the speed was set to 4000 r / min, and the centrifugation time was 20 min. After the centrifugation was completed, the upper supernatant was transferred to a new container and the lower precipitate was discarded. Finally, the supernatant was treated by the Sevage method. The specific operation is to mix the supernatant with the Sevage reagent in a certain proportion, shake it thoroughly, and then let it stand to separate the layers to remove the protein layer at the interface. The extraction operation was repeated until the supernatant no longer appeared at the interface after being mixed and shaken with the Sevage reagent. Finally, the protein-removed solution was placed in a dialysis bag (3500 Da) and dialyzed for 48 h, concentrated by rotary evaporation, and freeze-dried to obtain refined KSP.

[0089] (2) Accurately weigh 500 mg of deproteinized KSP and dissolve it in 20 mL of distilled water. After centrifugation (4000 r / min, 10 min), load the supernatant onto a well-equilibrated DEAE-52 column, pre-wash with distilled water, and gradient elute with distilled water, 0.1, 0.2, and 0.5 mol / L NaCl aqueous solution (flow rate 0.7 mL / min, 10 mL collected in each tube). Measure the absorbance at 490 nm using the anthrone-sulfuric acid method, draw an elution curve, and combine the same fractions. After concentration, dialyzation, and freeze-drying, purified KSP was obtained.

[0090] 2.2 Analysis of basic components of purified KSP Total sugar content was determined using the anthrone-sulfuric acid method with Glc as the standard. Protein content was determined using the Coomassie brilliant blue method with bovine serum albumin as the standard. Uric acid content was determined using the m-hydroxydiphenyl method with galacturonic acid (GalA) as the standard.

[0091] 2.3 Relative molecular weight analysis of purified KSP The relative molecular weight of purified KSP was determined using the HPGPC-RID method. Sample preparation: Dissolve the purified KSP powder in distilled water to a 5 mg / mL solution, filter through a 0.45 μm filter, and use. Liquid chromatography conditions were as follows: columns: TSKPWxl-3000 coupled with TSKPWxl-2500 (7.8 mm × 30 cm, 10 μm); mobile phase: water; flow rate: 1 mL / min; column temperature: 35°C; injection volume: 10 μL.

[0092] 2.4 UV spectral analysis of purified KSP The purified KSP was prepared into a 1 mg / mL solution, and distilled water was used as the blank calibration baseline. The solution was detected by UV full wavelength scanning to detect the presence of impurities such as proteins and nucleic acids.

[0093] 2.5 Infrared spectroscopy analysis of purified KSP A dried, purified KSP sample was ground with anhydrous KBr at a ratio of 1:200. The IR spectrum of the purified KSP was characterized by scanning the spectrum from 4000 to 400 cm⁻¹ at a resolution of 1 cm⁻¹.

[0094] 2.6 Monosaccharide composition analysis of purified KSP The monosaccharide composition of purified KSP was analyzed by nitrile acetate derivatization coupled with GC. Monosaccharide standards included rhamnose (Rha), arabinose (Ara), ribose (Rib), xylose (Xyl), mannose (Man), glucose (Glc), and galactose (Gal). GC conditions were as follows: column: OV-101 (50 m ± 0.25 mm id); detector: FID; flow rate: N₂:H₂:air = 30:30:300 mL / min; injector temperature: 230°C; detector temperature: 260°C; injection volume: 2 μL; temperature program: 175°C (15°C / min → 190°C / 5 min) → 15°C / min → 250°C / 1.5 min.

[0095] 2.7 Methylation analysis of purified KSP After uronic acid reduction, methylation, hydrolysis, reduction, and acetylation, the purified KSP was analyzed for its linkage pattern by GC-MS to determine the polysaccharide linkage order. GC-MS conditions were as follows: column: Rxi-5MS (30 cm × 0.32 mm × 0.25 μm); carrier gas: helium; inlet temperature: 250°C; ion source temperature: 230°C; injection volume: 1 μL; temperature program: 50°C (3 min) → 5°C / min to 100°C → 10°C / min to 250°C (7 min).

[0096] 2.8 NMR analysis of purified KSP 30 mg of purified KSP was weighed, dissolved in 0.5 mL of deuterated water, and freeze-dried for three cycles before being redissolved in 0.5 mL of deuterated water. The supernatant was collected by centrifugation and subjected to 400 MHz NMR analysis.

[0097] 2.9 Ultrastructural analysis of purified KSP In order to determine the ultrastructure of purified KSP, SEM was used for microstructural observation.

[0098] 2.10 Analysis of the physicochemical properties of purified KSP 2.10.1 Iodine-potassium iodide reaction The iodine-potassium iodide (I-KI) reaction was used to determine the presence of starch or starch-like structures in the sample. KSP was dissolved in distilled water to prepare a 1 mg / mL solution. An appropriate amount was placed in a clean test tube. Two control tubes were also prepared: an equal amount of distilled water alone was added to the negative control tube, and an equal amount of starch solution was added to the positive control tube. The I-KI solution was slowly added dropwise to the experimental and positive control tubes. After standing, the color changes of the three solutions were observed and recorded.

[0099] 2.10.2 Ferric chloride reaction The presence of phenolic hydroxyl groups in the sample was detected using the ferric chloride (FeCl3) reaction. KSP was dissolved in distilled water to prepare a 1 mg / mL solution. An appropriate amount was placed in a clean test tube. Two control tubes were also prepared: an equal amount of distilled water alone was added to the negative control tube, and an equal amount of phenol solution was added to the positive control tube. The FeCl3 solution was slowly added dropwise to the experimental and positive control tubes. After standing, the color changes of the three solutions were observed and recorded.

[0100] 2.11 Analysis of the triple helical structure of purified KSP The Congo red reaction was used to determine whether a triple helix structure existed in the sample. The experimental procedure was as follows: Purified KSP (5 mg) was dispensed into three EP tubes. 2 mL of distilled water and 2 mL of Congo red reagent were added to each tube. NaOH solution was added dropwise to a final concentration of 0-0.5 mol / L. After standing for 5 minutes, a UV scan was performed using distilled water as a blank, and the maximum absorption wavelength and the A The presence of a triple helix structure was determined by the linear relationship between NaOH concentration and wavelength.

[0101] Experimental results and discussion 3.1 Isolation and purification results of KSP DEAE-52 cellulose column chromatography was used, and the gradient elution was distilled water, 0.1, 0.2, and 0.5 mol / L NaCl aqueous solution, respectively. Five components were obtained, namely KSP-1 (water wash), KSP-2 (0.1 NaCl wash), KSP-3 (0.2 NaCl wash), KSP-4 (0.5-1 NaCl wash) and KSP-5 (0.5-2 NaCl wash). The elution curve is shown in Figure 2. Figure 5 shown.

[0102] 3.2 Analysis of basic components of purified KSP The purified KSP powder is brown-yellow, among which KSP-1 and KSP-4 are relatively darker in color; KSP-1 has strong viscosity and is flaky; the rest of the powder is fine ( Figure 6 The basic components of different elution samples are shown in Table 4.

[0103] Table 4 Basic components of different elution samples

[0104] 3.3 Relative molecular weight analysis results of purified KSP HPGPC-RID analysis ( Figure 7 ) showed that the purified KSP components (KSP-1 and KSP-2) all showed a single symmetrical chromatographic peak, indicating that KSP-1 and KSP-2 were homogeneous components, while KSP-3~5 did not have a single symmetrical chromatographic peak, indicating that they were not homogeneous components. Since KSP-3~5 were non-homogeneous components, the subsequent structural analysis did not involve the separated components KSP-3~5. Based on the standard curve established with different molecular weight dextran, the regression equation was Y=0.3967X+11.134, R 2 =0.9901, and the relative molecular mass of each component calculated by retention time is shown in Table 5.

[0105] Table 5 Relative molecular weight of different components

[0106] 3.4 UV spectral analysis results of purified KSP UV-Vis analysis showed that ( Figure 8 ). No nucleic acids were detected in KSP-1 and KSP-2, but a weak absorption peak at 280 nm suggested the presence of low-level protein. This suggests that the protein may be bound as a glycoprotein, making separation more difficult.

[0107] 3.5 Infrared spectral analysis results of purified KSP IR spectrum of purified KSP ( Figure 9) showed that its spectral characteristics were consistent with those of polysaccharides, showing multiple typical absorption peaks. Among them, 3400 cm -1 The broad peak at 1200-1000 cm is the vibration and stretching peak of OH in sugars. -1 The characteristic band of polysaccharide, 1060 cm -1 The vibration absorption of polysaccharide COC and COH. The CH contraction vibration peak is at 2940 cm -1 Nearby, the C=O contraction vibration peak is at 1618 cm -1 830 cm -1 There are absorption peaks at both ends, which indicates that the structures of purified KSP-1 and KSP-2 contain furanose rings.

[0108] 3.6 Monosaccharide composition analysis of purified KSP Gas chromatogram of purified KSP ( Figure 10 ) showed that KSP-1 is composed of Rib:Rha:Ara:Xyl:Man:Glc:Gal in a ratio of 0.04:0.02:1.03:0.17:2.12:8.26:0.09; KSP-2 is primarily composed of Man. Their molar ratios are shown in Table 6.

[0109] Table 6 Molar ratio of monosaccharide composition of different components

[0110] 3.7 Methylation analysis of purified KSP The purified, fully methylated derivatized KSP was analyzed by GC-MS and compared with the NIST 11 database. The results in Table 7 indicate that KSP-1 possesses four glycosidic linkage types, while KSP-2 contains only 2-, 3-, and 6-linked Glc configurations. The molar ratios of each linkage (calculated based on normalized peak areas) are consistent with the monosaccharide composition.

[0111] Table 7 Methylation analysis of different components

[0112] 3.8 NMR analysis results of purified KSP NMR technology provides key information for the structural analysis of polysaccharide repeating units. 1 In H NMR spectra, anomeric proton signals are commonly found in the δ 4.5–5.5 ppm range. Generally, proton signals above δ 4.9 ppm correspond to the α configuration, while those below δ 4.9 ppm correspond to the β configuration. Due to the D2O solvent, the sample produces a signal near δ 4.8 ppm. Figure 11The chemical shifts of the anomeric protons of KSP-1 are δ5.18, δ4.58, and δ4.57 ppm, indicating the coexistence of α- and β-glycosidic bonds. Typically, δ110-90 is assigned to terminal carbons, while δ90-60 is assigned to non-terminal carbons. Figure 12 middle 13 No signals were detected at δ 82–88 in the C NMR spectrum, indicating that the sugar residues of KSP-1 exist in a pyranose structure. 13 The presence of low-field signals at δ 161–180 ppm in C NMR indicates that KSP-1 contains uronic acid. Three strong signals at δ 111.03, δ 95.83, and δ 92.02 ppm are consistent with the H spectroscopy results, indicating that KSP-1 contains both α- and β-configured glycosidic bonds. The NMR signal assignments for KSP-1 were based on reported monosaccharide composition and chemical shift data (Table 8).

[0113] KSP-2 1 H NMR spectrum ( Figure 13 ) in δ 4.5-5.5 ppm mainly show two peaks, indicating that KSP-2 contains both α-type glycosidic bonds and β-type glycosidic bonds. However, due to the high concentration of uronic acid in KSP-2, its solubility in D2O and deuterated solvents is insufficient, resulting in a decrease in the signal-to-noise ratio of the carbon spectrum, so its 13 C NMR spectrum. According to the methylation 1 The H NMR spectrum results suggested that there might be a 1,4-linked 2,3,6-Me3-Manp structure in the structure of KSP-2.

[0114] Table 8 Assignment of KSP-1 NMR chemical shifts

[0115] 3.9 Ultrastructural analysis of purified KSP Depend on Figure 14 It can be seen that KSP-1: spherical particles; KSP-2: irregular shapes.

[0116] 3.10 Analysis of the physicochemical properties of purified KSP 3.10.1 Iodine-potassium iodide reaction The I-KI reactions of the purified KSP were negative, indicating that the purified KSP-1 and KSP-2 were non-starch structures.

[0117] 3.10.2 Ferric chloride reaction The FeCl3 reaction of the purified KSP was negative, indicating that the purified KSP-1 and KSP-2 did not contain phenolic hydroxyl groups.

[0118] 3.11 Analysis of the triple helical structure of purified KSP The increase in NaOH concentration leads to the disintegration of the triple helix structure of polysaccharides. The maximum absorption wavelength of samples containing this structure shows a trend of first increasing and then decreasing, while the absorption peak change of samples without triple helix structure is consistent with that of blank control ( Figure 15 The experimental results showed that the trends of purified KSP-1 and KSP-2 were completely consistent with those of the blank solution, confirming that they did not have a triple helical structure.

[0119] Example 4 Protective Effect of KSP-1 on Ethanol-Induced GES-1 Cell Damage and Ethanol-Induced Acute Gastric Mucosal Injury in Rats A KSP-1 fraction, characterized by its well-defined monosaccharide composition, highest content, and ease of purification, was selected to systematically investigate its gastric mucosal protective effects and mechanisms, both in vitro and in vivo. First, an ethanol-induced GES-1 cell injury model was established. The cytoprotective effects of KSP-1 were evaluated by measuring cell viability and the expression of oxidative stress and inflammatory factors. Furthermore, an ethanol-induced rat gastric mucosal injury model was constructed, and the interventional effects and mechanisms of KSP-1 on gastric mucosal protection were comprehensively revealed from multiple perspectives, including macroscopic scores, histopathology, serum oxidative stress markers, inflammatory factors, and intestinal flora.

[0120] Reagents and instruments Experimental materials: KSP-1 (prepared according to the method in step 2.1 of Example 3).

[0121] Experimental cells: human gastric epithelial cell line GES-1 (Starfish Biotechnology).

[0122] Experimental Animals: 64 SPF male Sprague-Dawley rats (4 weeks old, 180 ± 20 g) were purchased from the Experimental Animal Center of Lanzhou University (License No. SYXK (Gan)-2023-0004). The experimental protocol was approved by the Lanzhou University Animal Ethics Committee. Animals were housed in a barrier system under environmental conditions of 23 ± 2°C, 50 ± 10% humidity, a 12-h alternating light-dark photoperiod, and free access to food. Acclimation was performed for 5 days.

[0123] Experimental methods 2.1 Screening of the optimal KSP-1 concentration The experiment was divided into two groups: a control group and a KSP-1 treatment group. In the treatment group, cells were treated with KSP-1 at a concentration gradient of 7.8, 15.62, 31.25, 62.5, 125, 500, 1000, 2000, and 10000 μg / mL. The remaining procedures were the same as those in Example 2, Section 2.3.

[0124] 2.2 Effects of KSP-1 on cell survival and morphology in the ethanol-induced injury model The experiment was divided into the following five groups: control group, model group, and KSP-1 low-, medium-, and high-dose groups. The remaining procedures were the same as those in 2.4 of Example 2. Finally, the cell morphology was observed and photographed under an inverted microscope.

[0125] 2.3 Determination of biochemical indices of cells in the ethanol-induced injury model by KSP-1 Same as in Example 2, item 2.5.

[0126] 2.4 Animal Modeling and Grouping and Dosing Each group of SD rats was gavaged daily at 9:00 AM for 14 days to establish an acute alcohol-induced gastric mucosal injury model in SD rats. For 24 hours prior to the final administration, the rats were fasted but not watered. Three hours after the final administration, the rats were gavaged with 95% ethanol at a dose of 6 mL / kg, except for the blank and blank-treated groups.

[0127] Grouping and Dosing: Before the formal experiment, 64 healthy SD rats were randomly assigned to 8 groups, each with 8 rats: blank group, blank treatment group, model group, omeprazole (Yang 1 group), Kangfuxin solution (Yang 2 group), and KSP-1 (high, medium, and low) treatment groups. The blank and model groups received daily oral administration of normal saline (10 mL / kg), the blank treatment group received oral administration of KSP-1 (200 mg / kg), the Yang 1 group received oral administration of omeprazole (10 mg / kg), and the Yang 2 group received oral administration of Kangfuxin solution (5 mL / kg). The different KSP-1 treatment groups received oral administration of KSP-1 solutions at doses of 100 mg / kg, 200 mg / kg, and 400 mg / kg.

[0128] 2.5 Blood sample and gastric tissue collection Blood sampling: After the last dose, the animals were fasted for 24 hours (with free access to water). Blood was collected via the abdominal aorta after anesthesia with an intraperitoneal injection of 3% sodium pentobarbital (1.5 mL / kg). 2 mL of whole blood was aliquoted into anticoagulant tubes (for routine blood tests). The remaining blood was transferred to standard blood collection tubes. After 2 hours of rest, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was aliquoted into EP tubes and stored frozen at -80°C until further use.

[0129] Gastric tissue collection: After blood collection, rats were sacrificed. Gastric tissue was rapidly dissected, surface blood was washed with saline, and then blotted dry. The tissue was weighed and its morphological characteristics recorded. The stomach was cut longitudinally along the greater curvature and divided equally into two portions: one portion was fixed in 4% paraformaldehyde, and the other portion was frozen at -80°C until further analysis.

[0130] 2.6 Determination of organ index The liver, spleen and thymus were removed by dissection, washed with physiological saline, dried and weighed. The organ coefficient of each group was calculated according to formula 3.

[0131] (Formula 3) 2.7 Macroscopic Observation and Evaluation of Gastric Tissue Gastric tissue was unfolded and macroscopic evaluation and scoring was performed according to the Guth criteria. Each SD rat's score was summed to obtain a total lesion score. The mean score for each group was then calculated as the macroscopic evaluation score for that group. Subsequently, the gastric lesion area was calculated using Image J image analysis software, and the gastric lesion inhibition rate was calculated according to Equation 4.

[0132] (Formula 4) 2.8 Pathological Observation and Evaluation of Gastric Tissue Gastric tissue fixed in 4% paraformaldehyde was dehydrated and embedded in paraffin to prepare tissue sections. The sections were stained with HE and the pathological changes of gastric mucosa were evaluated under light microscopy.

[0133] 2.9 Determination of rat serum biochemical parameters Serum SOD, MDA, IL-1β, IL-6, TNF-α, and G-17 levels were determined using commercial kits, and the operating procedures were referred to the instructions.

[0134] 2.10 Determination of rat intestinal flora High-throughput sequencing of the 16S rRNA gene of the intestinal flora was performed on the intestinal content samples of rats in the blank group, model group, blank drug group, and KSP-1-M group. The sequencing of the samples was completed by Wuhan Maiwei Biotechnology Co., Ltd.

[0135] The experimental process includes sample processing, DNA amplification, sequencing and bioinformatics analysis: genomic DNA was extracted from rat intestinal contents using the CTAB method. After electrophoresis verification, the 16S rRNA gene was amplified using barcoded primers and high-fidelity PCR. The PCR products were standardized, purified, quantified, and then library construction was performed for high-throughput sequencing. After barcode splitting, quality filtering, and chimera removal, the offline data were clustered into ASVs and annotated to species. The community structure was evaluated using α-diversity indices (Chao1, Shannon) and rarefaction curves. The differences in microbial communities among samples were compared using β-diversity analysis (PCA, NMDS) and LEfSe differential analysis.

[0136] 2.11 Data Analysis Same as Item 2.6 of Example 2. In animal experiments, compared with the blank group: # P <0.05 and ## P <0.01; compared with the model group: * P <0.05 and **P <0.01.

[0137] Experimental results and discussion 3.1 Results of screening for optimal KSP-1 concentration The concentration selection in this part has no direct correlation with Example 2, but is based on the results of MTT assay for cell viability, and the concentration range showing cell proliferation is selected for subsequent experiments. Figure 16 , showing the effect of different concentrations of KSP-1 on cell survival rate. Figure A shows the results of KSP-1 treatment for 24 hours. In the concentration range of 7.8~500 μg / mL, KSP-1 can significantly promote cell proliferation ( P <0.01). However, when the KSP-1 concentration exceeded 500 μg / mL, cell survival began to decrease, presumably due to excessive drug concentration. Figure B shows the results after 48 hours of KSP-1 treatment, showing that cell survival was lower than that of the control group. At a concentration threshold of 500 μg / mL, cell viability decreased, with a gradual downward trend in cell survival. Based on these experimental results, KSP-1 concentrations of 7.8, 31.25, and 125 μg / mL, respectively, after 24 hours of KSP-1 treatment, were selected as low, medium, and high-dose groups for subsequent experiments.

[0138] 3.2 Effects of KSP-1 on cell viability and cell morphology in the ethanol-induced injury model like Figure 17 As shown, ethanol significantly inhibited the proliferation of GES-1 cells (the survival rate of the Model group was lower than that of the Control group, P <0.01), confirming that the injury model was successfully established. KSP-1 pretreatment reversed this effect in a dose-dependent manner: compared with the Model group, the cell survival rate in each concentration group was significantly increased ( P <0.01), and the survival rate increased gradually with the increase of drug concentration, indicating that KSP-1 alleviated ethanol-induced gastric mucosal epithelial cell damage through a concentration-dependent mechanism.

[0139] Inverted microscope showed that the cells in the control group were typical spindle-shaped, closely attached to the wall to form a dense monolayer, and in good growth condition; the cells in the model group were obviously wrinkled and rounded, with enlarged gaps and reduced connections, and a large number of them floated in the culture medium, which was consistent with the reports of other scholars; in each KSP-1 treatment group, as the drug concentration increased, the cell morphology and adhesion state gradually returned to the level of the control group. The specific changes are shown in Figure 18 The above experimental results further verified the improvement effect of KSP-1 on ethanol-induced gastric mucosal epithelial cell damage from the two dimensions of cell survival rate and cell morphology.

[0140] 3.3 Results of KSP-1 assay on biochemical indices of ethanol-induced cell damage model The results of oxidative stress related indicators are as follows Figure 19 As shown, the SOD activity in the model group was significantly lower than that in the control group ( P <0.01), and with the increase of KSP-1 concentration, SOD activity gradually increased, and the SOD activities of the three treatment groups of 7.8, 31.25, and 125 mg / mL were significantly higher than those of the model group ( P <0.01). However, the MDA content showed the opposite trend, and the MDA content in the model group was significantly higher than that in the control group ( P <0.01), while with the increase of KSP-1 concentration, MDA content gradually decreased, and the SOD activities of the three treatment groups of 7.8, 31.25, and 125 mg / mL were significantly lower than those of the model group ( P <0.01). The results showed that KSP-1 could effectively alleviate ethanol-induced oxidative stress damage by enhancing antioxidant enzyme activity and inhibiting lipid peroxidation.

[0141] Inflammatory factor levels Figure 20 The levels of IL-1β, IL-6 and TNF-α in the model group were significantly higher than those in the control group ( P <0.01). However, as the concentration of KSP-1 increased, the levels of IL-1β, IL-6, and TNF-α gradually decreased, and the levels of IL-1β, IL-6, and TNF-α in the 7.8, 31.25, and 125 mg / mL treatment groups were significantly lower than those in the model group ( P <0.01, 0.05). The results showed that KSP-1 could effectively alleviate ethanol-induced gastric mucosal inflammatory damage by regulating the expression of inflammatory factors.

[0142] 3.4 Effects of KSP-1 on the physical signs and body weight of rats The general observation showed that the rats in the eight groups had normal loose hair, normal eating and drinking, and normal mental state. Figure 21 As shown, the body weights of rats in the eight groups increased steadily. After administration of the positive drug and KSP-1, the changes in body weight were greater than those in the blank group and the model group, indicating that the positive drug and KSP-1 had no adverse effects on body weight.

[0143] 3.5 Effects of KSP-1 on blood routine in rats The results of routine blood tests are shown in Table 9. Compared with the blank group, the red blood cell count and hemoglobin content in each experimental group were similar to those of the blank group. The white blood cell, platelet, and lymphocyte counts in the model group were significantly increased (P < 0.05), which may be related to the congestion and acute bleeding in the gastric tissue after oral administration of ethanol. These changes in blood indicators further confirm the damage caused by ethanol to gastric tissue.

[0144] Table 9 Comparison of blood routine indicators in rats (n=3)

[0145] 3.6 Effects of KSP-1 on rat organ indices like Figure 22 As shown in the results, compared with the blank group, all organs in each group showed changes to varying degrees, but there were no significant differences in the liver, kidney, and spleen between the groups. Combined with the results on rat body weight, blood routine, and liver, spleen, and kidney indicators, there were no significant adverse effects, indicating that KSP-1 has no obvious toxic side effects.

[0146] 3.7 Effects of KSP-1 on Macroscopic Observation of Gastric Tissue Anhydrous ethanol, as a classic gastric ulcer-causing substance, can induce erosion, ulceration and punctate bleeding in the gastric mucosa. Its typical pathological features are linear hemorrhagic damage in the mucosal layer accompanied by extensive edema under the mucosa, reflecting the typical pathological mechanism of alcoholic gastric injury. Figure 23 As shown, the gastric mucosa of rats in the blank group (A) and the blank treatment group (B) showed no damage, with intact mucosal folds and no signs of congestion or edema. The gastric mucosa in the model group (C) exhibited typical lesions, including darker color, hemorrhagic spots, and linear hemorrhagic bands. The gastric wall elasticity decreased, with flattened folds, consistent with literature reports. After treatment with the positive drug and KSP-1 (D–H), the gastric mucosa became lighter, congestion and edema were reduced, and gastric wall elasticity and fold morphology improved. The KSP-1-M group (F) showed a superior protective effect on the gastric mucosa compared with the KSP-1-L group (G) and the KSP-1-H group (H), indicating that KSP-1 can effectively alleviate ethanol-induced gastric mucosal damage.

[0147] As shown in Table 10 and Figure 24 As shown in Figure 2, the macroscopic injury score and area in the model group were significantly higher than those in the other groups ( P <0.01), indicating that the gastric mucosal damage was serious. Compared with the model group: there was no significant difference in the macroscopic scores of the positive drug group and the KSP-1 group ( P >0.05, but the lesion area was significantly reduced ( P <0.01); inhibition rate analysis (Figure C) showed that the protective effect of KSP-1-L group (48.32%) and KSP-1-M group (57.68%) was significant ( P <0.01, while no significant difference was found in the KSP-1-H group ( P >0.05). The results showed that KSP-1 has a protective effect on ethanol-induced gastric mucosal damage, and the best effect was achieved by intragastric administration of equal doses.

[0148] Table 10 Macroscopic evaluation results of gastric mucosal injury in rats (n=8)

[0149] 3.8 Effect of KSP-1 on gastric tissue pathology HE staining results Figure 25 The gastric mucosal structure was intact in the blank and blank-dose groups, with densely arranged glands and normal morphology. Inflammatory cell infiltration was absent, and the lamina propria showed no congestion or edema. The cells were monolayered and columnar with clear cytoplasm. The model group exhibited typical pathological features of alcohol-induced toxicity: blurred mucosal boundaries, epithelial necrosis and exfoliation, lymphocytic infiltration of the lamina propria, dilated and congested mucosal capillaries, and disorganized glandular arrangement, consistent with reported results. Compared with the model group, the positive drug groups (Yang 1 and Yang 2) and the KSP-1-M group showed the best mucosal repair effect, with reduced superficial epithelial necrosis, improved congestion, and intact glandular structure. The KSP-1-L group showed some repair effect, but the effect was weaker than that of the medium-dose group. The KSP-1-H group showed the worst repair effect, which may be related to the exacerbation of mucosal damage caused by residual organic reagents during the purification process of the high-concentration polysaccharide. This study confirmed that KSP-1 ameliorates ethanol-induced gastric mucosal pathological damage in a dose-dependent manner, with the KSP-1-M group showing the best effect after oral administration.

[0150] 3.9 Analysis of KSP-1 on serum biochemical indicators 3.9.1 Changes in Rat Serum G-17 G-17 is a gastrointestinal hormone secreted by gastric G cells. It stimulates the secretion of gastric acid and pepsinogen, promotes gastric mucosal cell proliferation, and enhances gastrointestinal motility. High concentrations can overactivate gastric acid and pepsinogen secretion, leading to autodigestive damage to the gastric mucosa (such as erosion or ulceration).

[0151] like Figure 26 As shown in Figure 2, the expression level of serum G-17 in the model group was significantly higher than that in the blank group ( P <0.01). After oral administration of positive drugs and KSP, the level of G-17 was significantly lower than that of the model control group ( P <0.01), among which the KSP-1-M group had the most significant inhibitory effect ( P <0.01), indicating that oral administration of KSP-1 can reduce the level of G-17, alleviate the excessive secretion of gastric acid and pepsin, and play a protective role in gastric mucosa.

[0152] 3.9.2 Changes in Rat Serum SOD and MDA SOD and MDA are key markers of oxidative stress, and their contents can be used to assess the degree of lipid peroxidation and the level of oxidative stress in the body. 2−It destroys free radicals and protects oxygen-metabolizing cells from the harmful effects of superoxide radicals. MDA, as the end product of lipid peroxidation, can indirectly reflect the degree of cellular damage. Excessive levels of MDA and other oxidative products in the body can directly cause gastric mucosal damage. Therefore, SOD and MDA are selected as indicators of oxidative stress to reflect the level of oxidative damage in the gastric mucosa.

[0153] The results of oxidative stress related indicators are as follows Figure 27 Compared with the blank group, the SOD level in the model group was significantly downregulated ( P <0.01), indicating that the antioxidant capacity was impaired. However, the intervention of KSP-1 and positive drugs restored the SOD level ( P <0.01), and the KSP-1 concentration was dependent. Compared with the blank group, the MDA level in the model group was significantly increased ( P <0.01), reflecting the aggravation of lipid peroxidation. However, MDA was significantly reduced after oral administration of KSP-1 and positive drugs ( P <0.01). The results showed that KSP-1 can protect the gastric mucosa by resisting oxidative stress.

[0154] 3.9.3 Changes in Rat Serum IL-1β, IL-6, and TNF-α Excessive alcohol consumption can directly damage the gastric mucosal surface, weaken the protective effects of gastric mucosal defense factors, and promote the infiltration of inflammatory cells into the gastric mucosal tissue. The inflammatory response to gastric mucosal injury is primarily manifested by increased expression of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β. Among inflammatory cytokines, TNF-α is a core factor mediating gastric mucosal injury, promoting the expression of inflammatory mediators by inhibiting local microcirculation and activating the NF-κB pathway. Therefore, IL-1β, IL-6, and TNF-α are used to reflect the level of inflammation and the extent of injury.

[0155] Inflammatory factor levels Figure 28 Compared with the blank group, the levels of IL-1β, IL-6 and TNF-α in the model group were significantly increased ( P <0.05, P <0.01), indicating a severe inflammatory response. After administration of positive drugs and KSP-1, the levels of IL-1β, IL-6 and TNF-α were significantly reduced ( P <0.05, P <0.01). The results showed that KSP-1 can protect the gastric mucosa by inhibiting inflammatory response, and the best effect is achieved at a moderate dose.

[0156] 3.10 Analysis of KSP-1 on rat intestinal flora 3.10.1 Alpha Diversity Figure 29The effect of ethanol on the structure of intestinal microbial communities was revealed. The dilution coverage curves of the blank group, model group, KSP-1-M group, and blank administration group all reached a stable platform with the increase of sequencing depth, indicating that the current amount of data is sufficient to cover the microbial diversity of the sample and the sequencing depth is reasonable. Observed_ASV, Chao1, and ACE can be used to calculate colony abundance; Shannon index and Simpson index are often used to evaluate microbial diversity in samples. Shannon index is positively correlated with diversity, while Simpson index is negatively correlated. Figure 29 As shown in Figure 2, the Observed_ASV, Chao1, and ACE index of the model group were lower than those of the blank group, indicating that the diversity of the bacterial community was reduced ( P <0.05), while the Observed_ASV, Chao1 and ACE index of the drug group increased compared with the model group, but there was no significant difference. In addition, the Shannon index increased slightly and the Simpson index decreased slightly compared with the model group, but there was no significant difference. In summary, oral administration of ethanol led to a significant decrease in the α diversity of intestinal microorganisms in rats ( P <0.05), which was manifested as a decrease in microbial richness and total species count; although KSP-1-M failed to significantly improve the Shannon and Simpson indices, its upward trend in abundance indicators suggested that it might partially reverse ethanol-induced diversity damage by regulating microbial community structure.

[0157] 3.10.2 Beta Diversity Figure 30 The effects of the samples on the β-diversity of the intestinal microbiota were revealed. The samples of the blank group, blank treatment group, model group, and KSP-1-M group were clearly separated in PCA, PcoA, and NMDS, indicating differences in species composition. UPGMA analysis clustered the blank group and blank treatment group samples into one category, indicating that the microbial species composition of the intestinal content samples of the two groups of rats was similar. In addition, there was some overlap between the model group and the KSP-1-M group, but they were still roughly divided into two categories. This indicates that the composition of the intestinal microbial community in rats after KSP-1-M administration was similar to that of the blank group, but ethanol modeling could change the community composition of the rat intestinal microbial community.

[0158] 3.10.3 Species composition analysis The abundance of each bacterial group in the intestinal contents of rats can reflect the differences in the species composition of the intestinal flora of rats in different groups. The composition characteristics of the intestinal flora of rats in each group were systematically compared at the three taxonomic levels of phylum, family, and genus. Figure 31 for door horizontal distribution).

[0159] The results showed that Firmicutes, Bacteroidota, Desulfobactcrota, Verrucomicrobiota, Actinobacteriota, Actinobacteria, Proteobacteria, unidentified_Bacteria, Campylobacterota and Cyanobacteria. Among them, Firmicutes and Bacteroidota accounted for a relatively high proportion, and their total abundance generally exceeded 80% in each group. Figure 32 It can be seen that there was no significant difference in the relative abundance of bacterial flora in the intestinal contents of rats in each group; compared with the model group, the relative abundance of Cyanobacteria in the blank administration group was significantly reduced ( P Compared with the blank group, the relative abundance of Firmicutes, Verrucomicrobiota, and Actinobacteriota in the intestinal contents of rats in the model group decreased, while the relative abundance of Bacteroidota and Proteobacteria increased. This is associated with intestinal dysfunction, disrupting host-microbe homeostasis and increasing the risk of pathogenic bacterial infection. At the phylum level, administration of KSP-1 to rats with ethanol-induced gastric mucosal damage inhibited the increase in the relative abundance of Bacteroidota and Proteobacteria in the intestinal contents to a certain extent, while preventing the decrease in the relative abundance of Firmicutes, Verrucomicrobiota, and Actinobacteriota.

[0160] Figure 33The results at the family level showed that the Lachnospiraceae, Muribaculaceae, Lactobacillaceae, Oscillospiraceae, Erysipelotrichaceae, Desulfovibrionaceae, Peptostreptococcaceae, Akkermansiaceae, Ruminococcaceae and Christensenellaceae were the most abundant. Among them, Lachnospiraceae, Muribaculaceae and Lactobacillaceae accounted for a relatively high proportion, and their total abundance generally exceeded 60% in each group. Figure 34 It can be seen that there was no significant difference in the relative abundance of bacteria in the intestinal contents of rats in each group; the relative abundance of Peptostreptococcaceae bacteria in the blank group was significantly increased compared with the model group ( P Compared with the blank group, the intestinal contents of the model rats showed decreased abundance of Lachnospiraceae, Lactobacillaceae, Akkermansiaceae, and Ruminococcaceae, while increased abundance of Erysipelotrichaceae and Peptostreptococcaceae. This may be related to intestinal inflammatory disease, leading to reduced production of short-chain fatty acids and weakened intestinal barrier function, making the intestine more susceptible to pathogen invasion and inflammatory stimulation. At a scientific level, KSP-1 administration can, to a certain extent, correct the imbalance in the intestinal microbial community in rats with ethanol-induced gastric mucosal injury. Specifically, it inhibited the increase in the relative abundance of Erysipelotrichaceae and Peptostreptococcaceae, while alleviating the decrease in the relative abundance of Lachnospiraceae, Lactobacillaceae, Akkermansiaceae, and Ruminococcaceae.

[0161] Figure 35 The results show that Lactobacillus ( Lactobacillus ), Desulfovibrio ( Desulfovibrio ), Dubois spp. Dubosiella ), Rombutsiella ( Romboutsia ), Lactobacillus mucilaginosus ( Limosilactobacillus ), Lactobacillus spp. ( Ligilactobacillus ), Akkermansia muciniphila ( Akkermansia ), Allobacterium ( Allobaculum ), unidentified Lachnospira Lachnospiraceae ) and Escherichia coli ( Colidextribacter ).in Lactobacillus The proportion was high, and the abundance in each group generally exceeded 15%. Lactobacillus The relative abundance can reach more than 30%, which is significantly different from the model group ( P <0.01). Figure 36 It can be seen that compared with the blank group, the model group Desulfovibrio 、 Allobaculum The relative abundance of bacteria increased, with significant differences ( P <0.01, 0.05), Limosilactobacillus The relative abundance of bacteria decreased significantly ( P <0.05); compared with the model group, after administration of KSP-1 Lactobacillus 、 Dubosiella and Romboutsia The relative abundance of bacteria increased significantly ( P <0.01, 0.05), Allobaculum The relative abundance of bacteria decreased significantly ( P <0.01, 0.05). At the genus level, KSP-1 administration can regulate the relative abundance of intestinal flora in rats with ethanol-induced gastric mucosal damage to a certain extent. Desulfovibrio 、 Limosilactobacillus 、 Colidextribacter The relative abundance increased, while mitigating Lactobacillus 、 Romboutsia and unidentified_ Lachnospiraceae A decrease in the relative abundance of bacteria.

[0162] 3.10.4 Differential Microbial Community Analysis In order to screen out species with significant differences in abundance between groups, we first analyzed the common and unique ASVs between different groups, such as Figure 37 As shown in A. The number of unique ASVs in the blank group, model group, KSP-1-M group and blank treatment group were 26, 11, 12 and 33 respectively, and the total number of ASVs was 299. The linear discriminant analysis LefSe (LDA Effect Size) was further used to find the specific bacterial groups with significant differences between the groups. The results are shown in Figure 37 As shown in B. There were 8, 3, 9 and 5 specific bacterial groups with significant differences between the blank group, model group, KSP-1-M group and blank treatment group, respectively. Among them, the highest abundance of specific bacterial group in the blank group was s_Akkermansia_murinus ; The highest abundance of specific bacterial groups in the model group is g_Desutovibrio, an intestinal pathogen; the highest abundance of specific bacterial groups in the KSP-1-M group was f_Desuioyorionaceae The highest abundance of specific bacterial groups in the blank treatment group was g_Dubosiela . These specific bacterial groups are further distributed in the evolutionary branch diagram, such as Figure 37 C, the relationship between microorganisms at different levels can be seen.

[0163] Finally, combined with the above species composition and differential microbial community analysis, a heat map analysis of the specific changes in the differential microbial community was performed. The results are as follows: Figure 38 Compared with the blank group, the blank group Anaerostipes hadrus There is a significant difference ( P <0.05), and there was no significant difference in other bacterial groups. Anaerostipes hadrus It belongs to the genus Lachnospiraceae and is generally considered to be beneficial to human health. The short-chain fatty acids it produces can provide energy for intestinal epithelial cells, maintain the integrity of the intestinal mucosa, and may also participate in regulating the intestinal immune system and enhancing immune defense. This shows that pretreatment with KSP-1 can promote the growth and reproduction of various bacterial communities, enrich the composition of intestinal microbial communities, strengthen intestinal barrier function, participate in immune regulation and other processes, and play a comprehensive beneficial role in the health of the body. Model group Akkermansia muciniphila 、 Adlercreutzia caecicola 、 Faecalibaculum rodentium 、 Parabacteroides goldsteinii There is a significant difference ( P <0.05, 0.01). When the body's immunity is low, the above-mentioned flora will turn into opportunistic pathogens, causing some intestinal or systemic problems.

[0164] Studies of the intestinal flora have shown that KSP-1 administration significantly increased the abundance of Lachnospiraceae, Lactobacillaceae, and Ruminococcaceae, indicating that KSP-1 promotes these beneficial bacterial families. However, the use of ethanol in modeling disrupts the normal composition of the intestinal flora, disrupting the positive changes in the abundance of these families induced by KSP-1 and exacerbating intestinal microecological imbalances. Romboutsia It shows higher relative abundance in healthy mucosa and lower abundance in patients with gastric disease, and is an indicator of mucosal changes. Colidextribacter Some strains in the gut are opportunistic pathogens. Excessive numbers can cause intestinal infections, which are more harmful when the immune system is weakened or the intestinal barrier function is damaged. Intestinal flora studies have shown that the blank group and the drug-treated group have a higher abundance of Romboutsia ) and lower abundance ColidextribacterThe model group showed the opposite trend. In summary, the results showed that KSP-1 may upregulate the abundance of beneficial bacteria such as Lachnospiraceae and Lactobacillaceae, and downregulate Colidextribacter The abundance of conditional pathogens such as β-lactamase and β-lactamase can change the diversity and structure of the intestinal flora of rats, thereby exerting a protective effect on the gastric mucosa.

[0165] 4 Summary Through the above in vitro and in vivo experiments, the results showed that KSP-1 can play a protective role in gastric mucosa by resisting oxidative stress, inhibiting inflammatory response and regulating intestinal flora.

[0166] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing crude polysaccharide from Longmalu, characterized by: The following steps are involved: (1) defatting the Longmalu to obtain defatted Longmalu; (2) The defatted Longmalu obtained in step (1) was extracted with NaOH solution to obtain Longmalu crude polysaccharide; the extraction conditions were: NaOH concentration 0.03-0.05 mol / L, extraction temperature 70-90°C, extraction time 2.0-3.0 h, extraction times 2-3 times, and solid-liquid ratio 1:15-1:

30.

2. The method according to claim 1, wherein: In step (1), Longmalu was mixed with 95% ethanol in a ratio of 1 g:10 mL, and the mixture was condensed and refluxed for degreasing.

3. The method according to claim 1, wherein: In step (2), the extraction conditions are: NaOH concentration 0.04 mol / L, extraction temperature 78°C, extraction time 2.5 h, extraction times 2 times, and solid-liquid ratio 1:

20.

4. The method according to any one of claims 1 to 3, characterized in that: The invention also includes the following post-processing steps after extraction: collecting the extract, adjusting the pH to neutral, concentrating, precipitating with alcohol, and freeze-drying to obtain the crude polysaccharide of Longmalu.

5. Longmalu crude polysaccharide is prepared by the method according to any one of claims 1 to 4.

6. A method for preparing Longmalu polysaccharide, characterized in that: The following steps are involved: (1) extracting the crude polysaccharide of the Longmalu truncatum according to claim 5 using papain, removing protein, and obtaining deproteinized crude polysaccharide of the Longmalu truncatum; (2) The deproteinized polysaccharide prepared in step (1) was separated using a DEAE-52 cellulose column. The elution was performed with distilled water, 0.1, 0.2, and 0.5 mol / L NaCl aqueous solutions in a gradient manner at a flow rate of 0.7 mL / min. The absorbance at 490 nm was measured using the anthrone-sulfuric acid method. The elution curve was drawn and the same fractions were combined.

7. The method according to claim 6, characterized in that: In step (1), the specific method of using papain to extract the crude polysaccharide of Longmalu according to claim 5 is as follows: the crude polysaccharide of Longmalu is prepared into a solution with distilled water, mixed with papain, and the pH value of the solution is adjusted to 6-7, and enzymatically hydrolyzed at 39-41°C for 3.5-4.5h; Preferably, the weight ratio of papain to Longmalu crude polysaccharide is 1:

100.

8. Longmalu polysaccharide is prepared by the method according to claim 6 or 7.

9. Use of the crude polysaccharide of Longmalu according to claim 5 or the polysaccharide of Longmalu according to claim 8 in the preparation of a medicament for protecting gastric mucosa; Preferably, the polysaccharide of Longmalu is used in the preparation of a drug for protecting alcoholic gastric mucosal damage; Preferably, the polysaccharide of Longmalu is used in the preparation of a drug for protecting acute alcoholic gastric mucosal damage; Preferably, the concentration of the polysaccharide of Longmalu is 7.8-125 μg / mL; most preferably, the concentration of the polysaccharide of Longmalu is 31.25 μg / mL.

10. The use according to claim 9, characterized in that: The polysaccharide of Longmalu exerts gastric mucosal protective effects through anti-oxidation, anti-inflammation and regulation of intestinal flora; Preferably, the regulating of intestinal flora includes up-regulating the abundance of beneficial bacteria of Lachnospiraceae and Lactobacillaceae, and down-regulating the abundance of conditionally pathogenic bacteria of Escherichia coli.

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