A kudzu polysaccharide, its preparation method and application

Abstract

This invention discloses a kudzu root polysaccharide, its preparation method, and its applications, relating to the field of biotechnology. The kudzu root polysaccharide is composed of glucose and fructose, and contains six glycosidic bonds: Glcp-(1→, →1)-Fruf-(2→, →3)-Glcp-(1→, →4)-Glcp-(1→, →3,4)-Glcp-(1→ and →4,6)-Glcp-(1→). Functional verification experiments revealed that this kudzu root polysaccharide can effectively prevent acute alcohol poisoning, has a therapeutic effect on oral ulcers, and exhibits antioxidant, anti-inflammatory, antibacterial, and anti-aging activities. This invention, which extracts polysaccharides from kudzu root, not only has significant health benefits but also substantial economic and social benefits. It provides technical support for expanding the application of traditional Chinese medicinal materials and offers a novel, natural solution to various health problems.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a kudzu polysaccharide, its preparation method, and its applications. Background Technology

[0002] Kudzu root is the dried root of Pueraria thomsonii Benth., a plant in the Fabaceae family. It has the effects of relieving muscle tension and reducing fever, promoting body fluid production and quenching thirst, promoting rash eruption, raising yang and stopping diarrhea, clearing the meridians and activating collaterals, and detoxifying alcohol. It can be used for exogenous fever and headache, stiff neck and back pain, thirst, diabetes, measles that fail to erupt, dysentery, diarrhea, dizziness and headache, hemiplegia due to stroke, chest pain and heart pain, and damage to the middle jiao from alcohol poisoning.

[0003] Kudzu root is rich in various active ingredients, such as flavonoids, saponins, and polysaccharides. Among them, kudzu root polysaccharides have attracted widespread attention due to their unique structure and excellent biological activity.

[0004] With changes in modern lifestyles, alcohol abuse and related health problems are becoming increasingly prominent, making the search for natural, safe, and effective hangover remedies a research hotspot. Furthermore, oxidative stress, bacterial infection, and inflammatory responses are common pathological bases for many diseases, making the development of natural drugs with multiple biological activities of great significance. This invention aims to develop a kudzu root polysaccharide to provide technical support for solving the aforementioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a kudzu polysaccharide, its preparation method, and its applications to solve the problems existing in the prior art. This kudzu polysaccharide can effectively prevent acute alcohol poisoning, has a therapeutic effect on oral ulcers, and possesses antioxidant, anti-inflammatory, antibacterial, and anti-aging activities.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a kudzu polysaccharide, which is composed of glucose and fructose and has 6 glycosidic bonds, namely Glcp-(1→,→1)-Fruf-(2→,→3)-Glcp-(1→,→4)-Glcp-(1→,→3,4)-Glcp-(1→ and→4,6)-Glcp-(1→);

[0008] The main chain of the kudzu polysaccharide is linked by glycosidic bonds of →[1)-β-D-Fruf-(2]5→[4)-α-D-Glcp-(1]2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→), while the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain by O-3 of →3,4)-α-D-Glcp-(1→ and O-6 of →4,6)-α-D-Glcp-(1→, respectively.

[0009] The present invention also provides a method for preparing the above-mentioned kudzu polysaccharide, comprising the following steps:

[0010] Pueraria lobata was extracted using a water extraction and alcohol precipitation method to obtain crude polysaccharide.

[0011] After deproteinization, the crude kudzu polysaccharide was purified sequentially using a DEAE-52 cellulose anion exchange chromatography column and a Sephadex G-150 column to obtain the kudzu polysaccharide.

[0012] The present invention also provides the application of the above-mentioned kudzu polysaccharide in any of the following:

[0013] (1) To prepare drugs for the prevention and / or treatment of acute alcohol poisoning;

[0014] (2) To prepare drugs for treating oral ulcers;

[0015] (3) Prepare antioxidant products; the antioxidant products are health products or pharmaceuticals;

[0016] (4) Preparation of anti-inflammatory drugs;

[0017] (5) Preparation of antibacterial drugs;

[0018] (6) Prepare anti-aging drugs.

[0019] Furthermore, the antibacterial drug targets Escherichia coli and / or methicillin-resistant Staphylococcus aureus.

[0020] The present invention also provides a medicament for the prevention and / or treatment of acute alcohol poisoning, the active ingredient of which includes the above-mentioned kudzu polysaccharide.

[0021] The present invention also provides a medicine for treating oral ulcers, the active ingredient of which includes the above-mentioned kudzu polysaccharide.

[0022] The present invention also provides an antioxidant product, the active ingredient of which includes the above-mentioned kudzu polysaccharide;

[0023] The product in question is a health supplement or a medicine.

[0024] The present invention also provides an anti-inflammatory drug, the active ingredient of which includes the above-mentioned kudzu polysaccharide.

[0025] The present invention also provides an antibacterial drug, the active ingredient of which includes the above-mentioned kudzu polysaccharide.

[0026] The present invention also provides an anti-aging drug, the active ingredient of which includes the above-mentioned kudzu polysaccharide.

[0027] The present invention discloses the following technical effects:

[0028] This invention extracts a kudzu polysaccharide from kudzu root. Analysis shows it is composed of glucose and fructose, with six glycosidic bonds: Glcp-(1→、→1)-Fruf-(2→、→3)-Glcp-(1→、→4)-Glcp-(1→、→3,4)-Glcp-(1→ and→4,6)-Glcp-(1→); its main chain linkage is →[1)-β-D-Fruf-(2)5→[ 4)-α-D-Glcp-(1]2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→) glycosidic bonds, while the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain via →3,4)-α-D-Glcp-(1→'s O-3,→4,6)-α-D-Glcp-(1→'s O-6, respectively.

[0029] Functional verification experiments revealed that this kudzu polysaccharide can effectively prevent acute alcohol poisoning, has a therapeutic effect on oral ulcers, and possesses antioxidant, anti-inflammatory, antibacterial, and anti-aging activities.

[0030] The polysaccharide extracted from kudzu root in this invention not only has important health significance, but also has significant economic and social benefits. It provides technical support for expanding the application of traditional Chinese medicinal materials and offers a new and natural solution to various health problems. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The figure shows the results of the single-factor experiment; where A represents the extraction rate of crude kudzu polysaccharide under different material-to-liquid ratios; B represents the extraction rate of crude kudzu polysaccharide under different extraction times; and C represents the extraction rate of crude kudzu polysaccharide under different extraction temperatures.

[0033] Figure 2 The results of the response surface methodology are shown in the figure; where A is the response surface of A2 and A1; D is the profile plot of A2 and A1; B is the response surface of A3 and A1; E is the profile plot of A3 and A1; C is the response surface of A3 and A2; and F is the profile plot of A3 and A2.

[0034] Figure 3 The images show the purification process and physicochemical properties of kudzu polysaccharides. A is the DEAE-52 elution curve; B is the Sephadex G-150 elution curve; C is the HPGPC spectrum of PMPS-A1; D is the UV spectrum of PMPS-A1; E is the FT-IR spectrum of PMPS-A1; and F is the HPLC chromatogram of standard monosaccharides and PMPS-A1. The chromatograms are as follows: 1. Fucose; 2. Galactose hydrochloride; 3. Rhamnose; 4. Arabinose; 5. Glucosamine hydrochloride; 6. Galactose; 7. Glucose; 8. Xylose; 9. Mannose; 10. Fructose; 11. Ribose; 12. Galacturonic acid; 13. Gurouronic acid; 14. Glucuronic acid; 15. Mannuronic acid.

[0035] Figure 4 The TG-DTG curve of PMPS-A1;

[0036] Figure 5 The experimental analysis diagram of PMPS-A1 with Congo red;

[0037] Figure 6 Here are the SEM images of PMPS-A1; where A and D are SEM images with magnifications of 100×, 500×, 1.0K×, and 2.0K×, respectively.

[0038] Figure 7 For PMPS-A1 1 H NMR spectrum;

[0039] Figure 8 For PMPS-A1 13 C NMR spectrum;

[0040] Figure 9 DEPT-135 diagram for PMPS-A1;

[0041] Figure 10 COSY plot for PMPS-A1;

[0042] Figure 11 The HSQC diagram for PMPS-A1;

[0043] Figure 12 HMBC diagram for PMPS-A1;

[0044] Figure 13NOESY plot for PMPS-A1;

[0045] Figure 14 This is a structural schematic diagram of PMPS-A1;

[0046] Figure 15 The following figures illustrate the therapeutic effects of crude polysaccharides from different parts of Pueraria lobata on ethanol-induced acute alcohol poisoning in mice: A represents mouse body weight; B represents the time to intoxication; C represents the GUI score for gastric injury; D represents images of gastric injury in each group; Control group represents the blank group; Model group represents the model group; Positive control group represents the positive control group; Dried head of Pueraria lobata or Dried head represents the crude polysaccharide treatment group; Dried and Powdered Pueraria lobata or Dried and Powdered represents the starch treatment group; Pueraria lobata Root or Root represents the crude polysaccharide treatment group.

[0047] Figure 16 For efficacy verification, here are pathological sections of gastric tissue from each experimental group in Example 1; where Model is the model group, Positive control is the positive control group, Dried head is the group treated with crude polysaccharide from kudzu root, Dried and Powdered is the group treated with kudzu starch, and Root is the group treated with crude polysaccharide from kudzu root.

[0048] Figure 17 For efficacy verification, here are pathological sections of liver tissue from each experimental group in Example 1; where Model is the model group, Positive control is the positive control group, Dried head is the group treated with crude polysaccharide from kudzu root, Dried and Powdered is the group treated with kudzu starch, and Root is the group treated with crude polysaccharide from kudzu root.

[0049] Figure 18 Statistical graphs showing ulcer healing in each experimental group of rats in Example 2 to verify the effectiveness;

[0050] Figure 19 HE staining images of oral skin tissue from rats in each experimental group for efficacy verification;

[0051] Figure 20 The results of the test on the antioxidant activity of PMPS-A1 are shown in the figure; where A is a statistical graph of the scavenging rate of PMPS-A1 on hydroxyl radicals; and B is a statistical graph of the scavenging rate of PMPS-A1 on DPPH radicals.

[0052] Figure 21Figure 1 shows the results of the in vitro anti-inflammatory activity test of PMPS-A1; where A represents the effect of PMPS-A1 on the activity of RAW264.7 macrophages; B represents the effect of PMPS-A1 on the relative expression of IL-6 in macrophages induced by lipopolysaccharide; and C represents the effect of PMPS-A1 on the relative expression of nitric oxide in macrophages induced by lipopolysaccharide. Compared with the control group, ### P<0.001; compared with the lipopolysaccharide group, * P<0.05, *** P<0.001;

[0053] Figure 22 The results show the detection of ROS levels in different experimental groups; where AD represents the fluorescence detection graphs of ROS in the blank control group, 1 mg / mL, 3 mg / mL, and 5 mg / mL of nematodes, respectively; E is a statistical graph of the relative fluorescence intensity of ROS in nematodes; compared with the blank group, *** P<0.001;

[0054] Figure 23 The results show the detection of lipofuscin levels in different experimental groups; AD represents the fluorescence detection graphs of ROS in the blank control group, 1 mg / mL, 3 mg / mL, and 5 mg / mL nematodes (lipofuscin produces blue fluorescence), respectively; E is a statistical graph of the relative fluorescence intensity of lipofuscin in nematodes; compared with the blank group, ** P<0.01, *** P<0.001. Detailed Implementation

[0055] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0056] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0057] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0058] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0059] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0060] Example 1: Extraction, content determination, and process optimization of kudzu root polysaccharides

[0061] Fresh kudzu root was dried at 60℃, ground into powder, and boiled in deionized water at 80℃ for 4 hours at a liquid-to-solid ratio of 1g:30mL. This process was repeated three times. The supernatant was centrifuged at 4000rpm for 10min. The supernatant was then collected and concentrated to one-third of its original volume at 80℃. Precipitation was carried out with four times the volume of anhydrous ethanol at 4℃ for 24 hours, followed by centrifugation (4000r / min, 20min). The precipitate was then freeze-dried to obtain crude kudzu root polysaccharide (PMPS). The contents of total sugar, protein, and uronic acid were determined using the phenol-sulfuric acid method (with glucose as the standard), the Bradford method (with bovine serum albumin as the standard), and the sulfuric acid-carbazole method (with galacturonic acid as the standard), respectively.

[0062] The extraction rate (Y1, %) of kudzu root is Y1 = M1 / M2 × 100, where M1 is the mass of freeze-dried kudzu root crude polysaccharide and M2 is the mass of kudzu root raw powder.

[0063] 1. Univariate analysis

[0064] To explore the preliminary range of extraction variables, a single-factor design was used to study the effects of liquid-to-material ratio (A1: 10, 20, 30, 40 and 50 mL / g), extraction time (A2: 1 h, 2 h, 3 h, 4 h and 5 h), and extraction temperature (A3: 60℃, 70℃, 80℃, 90℃ and 100℃) on polysaccharide yield.

[0065] like Figure 1As shown in Figure A, the polysaccharide yield increases significantly with increasing liquid-to-solid ratio (p<0.05). The larger the solution-to-solid ratio, the more pronounced the concentration difference and the faster the polysaccharide molecules diffuse. On the other hand, the polysaccharide extraction rate reaches its peak at a liquid-to-solid ratio of 30:1, and then begins to decline. In the presence of excess solvent, non-sugar substances are dissolved, hindering the dissolution of polysaccharides and leading to a decrease in the total polysaccharide yield.

[0066] like Figure 1 As shown in Figure B, the yield of PMPS increased significantly between 0.5 and 3 hours, reaching a maximum at 3 hours. Furthermore, prolonged extraction time also led to the destruction and degradation of the polysaccharide structure.

[0067] like Figure 1 As shown in Figure C, the yield increases significantly with increasing temperature, peaking at 90°C. From this temperature onward, the yield declines, likely due to carbohydrate decomposition and inactivation caused by extreme temperatures. Therefore, 90°C is the ideal extraction temperature.

[0068] 2. Experimental Design Using Response Surface Methodology (RSM)

[0069] A 17-step Box-Behnken (BBD) design with three variables and three levels was constructed using Design Expert software to determine the ideal range of the extracted variables (Tables 1 and 2), based on the results of the single-factor experiments. A quadratic polynomial model was used to fit the experimental data. The quadratic polynomial equation is as follows:

[0070]

[0071] Table 1 Box-Benken Experimental Design

[0072]

[0073] Note: A1, A2 and A3 represent the liquid-to-solid ratio (mL / g), extraction time (h) and extraction temperature (°C), respectively.

[0074] 3. Optimization of polysaccharide extraction conditions

[0075] 3.1. Statistical Analysis and Model Fitting

[0076] To optimize extraction parameters and analyze the interaction effects of three factors on the extraction rate, BBD under RSM was used, as shown in Table 1. With a liquid-to-solid ratio of 30 (mL / g), an extraction time of 3 h, and an extraction temperature of 90℃, the highest Y1 was 9.77%. Multiple regression analysis yielded a second-order polynomial equation from the experimental data:

[0077] Y1=9.55-0.08A1-0.12A2+0.093A3+0.047A1A2+0.042A1A3+0.31A2A3- 2.99A1 2- 2.00A2 2- 2.10A3 2 .

[0078] Table 1 shows the ANOVA results for this model. The R-squared value of the fitted model... 2 and R 2 adj The values ​​were 0.9952 and 0.9891, respectively. The model was considered significant (P < 0.05) because its P-value was less than 0.001, and the P-value for no significance was 0.73. The coefficients of other parameters were not significant, but the coefficients of the interaction term (A2A3), the single term (A2), and the quadratic term (A3A3) were significant. 12 A 22 and A 32 The variance coefficient (3.82) of the polynomial model equation is significant. Therefore, the relationship between extraction temperature (A3) and extraction time (A2) has a significant impact on the extracted PMPS and requires careful investigation.

[0079] Table 2. Regression coefficient estimation and significance analysis of the PMPS output quadratic polynomial model.

[0080]

[0081]

[0082] Note: *P<0.05, ***P<0.001.

[0083] 3.2 Response Surface Analysis

[0084] The generated 2D profile plots and 3D response surfaces show the relationships between process variables. The Y1(...) of PMPS... Figure 2 The value first increased with increasing values ​​of two variables (A2 and A3), then decreased. The highest Y1 for PMPS was obtained at an extraction temperature of 90.20℃ and an extraction time of 2.97 h. (Based on Table 2 and...) Figure 2 The interaction between A2A3 is stronger than the interaction between A1A2 and A1A3. Furthermore, the higher the density of the elliptical profile, the greater the influence of the interactions between variables.

[0085] 3.3 Optimization and Validation

[0086] Numerical optimization results show that the optimal conditions for PMPS Y1 are an extraction temperature of 90℃, as shown in the analysis of variance (Table 2) and reaction surface plot (…). Figure 2As shown in Figure AC, the extraction time was 2.96 h, and the liquid-to-solid ratio was 29.86 (mL / g). Under these conditions, the model estimated PMPS Y1 to be 9.55%. Considering experimental feasibility, the extraction temperature was set to 90 °C, the extraction time to 2.96 h, and the liquid-to-solid ratio to 29.86 (mL / g). To demonstrate the reliability of the response surface methodology model, three replicate experiments were conducted under realistic conditions. Using n=3, the experimental PMPS Y1 was 9.78 ± 0.09%. This indicates that the BBD model is effective, as it is close to the expected 9.55%. The study found that the optimized polysaccharide extraction rate was lower than that of some BBD design experiments. However, compared with single-factor experiments, the optimized PMPS extraction rate was higher, indicating the accuracy and effectiveness of the model.

[0087] Example 2: Purification and physicochemical characterization of kudzu polysaccharide

[0088] 1. Preparation of crude kudzu polysaccharide

[0089] Fresh kudzu root was dried at 60℃, ground into powder, and boiled in deionized water at 90℃ for 2.96 h (liquid-to-solid ratio: 1 g: 29.86 mL). This process was repeated three times. The supernatant was centrifuged at 4000 rpm for 10 min. The supernatant was then collected and concentrated to one-third of its original volume at 80℃. Precipitation was carried out with four times the volume of anhydrous ethanol at 4℃ for 24 h, followed by centrifugation (4000 rpm, 20 min). The precipitate was then redissolved in deionized water to obtain a crude kudzu root polysaccharide solution with a concentration of 1 g / mL.

[0090] 2. Purification of kudzu polysaccharides

[0091] Free proteins were separated using Sevage's reagent. Sevage's reagent was mixed with crude kudzu polysaccharide solution at a volume ratio of 1:4, and the mixture was kept in a constant-temperature shaking water bath at 30°C for 30 to 60 min. To remove salts and other small molecules, the supernatant after centrifugation was dialyzed in distilled water for 48 h using a dialysis bag (MWCO 3500 Da). The retentate was then freeze-dried and purified using a DEAE-52 column (5.5 × 40 cm, Beijing Ruida Heng Hui Technology Development Co., Ltd.). Elution was performed using a gradient solution of distilled water and NaCl (0.1, 0.2, 0.3, and 0.5 mol / L) at a flow rate of 4 mL / min. Samples were taken every 3 min, and the total sugar content was calculated using the phenol-sulfuric acid method. Tubes with identical elution peaks were collected and dialyzed for 48 h (MWCO 3500 Da), and the retentate was then freeze-dried to prepare PMPS-A. Then, 20 mL of deionized water was added to 220 mg of PMPS-A. PMPS-A was then purified again using a Sephadex G-150 column (1.0 × 50 cm, Borui Sugar Biotechnology Co., Ltd.). Elution was performed using a gradient solution of distilled water. Tubes with identical elution peaks were collected and dialyzed for 48 h (MWCO 3500 Da). The retentate was then freeze-dried to prepare PMPS-A1.

[0092] The conventional method for extracting crude polysaccharides (PMPS) yielded 3% ± 0.14%. The product was washed with deionized water, 0.1 mol / L NaCl, 0.2 mol / L NaCl, 0.3 mol / L NaCl, 0.5 mol / L NaCl, and a DEAE-52 cellulose anion exchange chromatography column, followed by washing with deionized water. Purification was performed using a Sephadex G-150 column, and the elution curve was determined by the phenol-sulfuric acid method. Figure 3 (AB). Based on the ion column elution curve, the polysaccharide with the highest absorbance was collected in this invention and named PMPS-A. Furthermore, the HPGPC spectrum ( Figure 3 Image B) shows two peaks for PMPS-A, indicating a purity of approximately 80%. The total sugar content of PMPS-A is approximately 95% ± 0.78%. To obtain a homogeneous polysaccharide, it was purified on a Sephadex G-150 column. The peak with the highest polysaccharide content was collected based on its elution curve and named PMPS-A1. The HPGPC spectrum shows that PMPS-A1 is a singlet, indicating a purity > 99%. Using the phenol-sulfuric acid method, the total sugar content in PMPS-A1 is approximately 99% ± 0.48%. The protein content of PMPS-A1 is 0.032% ± 0.0016%, and the uronic acid content is 0.6% ± 0.021%. This also indicates that PMPS-A1 is a neutral sugar.

[0093] 3. Physicochemical properties of PMPS-A1

[0094] 3.1 Analysis of the molecular weight and monosaccharide composition of PMPS-A1

[0095] Accurately weigh 5 mg of PMPS-A1 and dissolve it in 1 mL of mobile phase (0.05 mol / L NaCl solution). After vortexing and centrifugation (12000 rpm, 10 min), the supernatant was collected and filtered through a 0.22 μm water microporous membrane. PMPS-A1 was then placed in a 1.8 mL syringe. The HPGPC system used a BRT105-103-101 tandem gel column (8 × 300 mm) and a RID-20A differential detector (Shimadzu, Japan), with a column temperature of 40 °C and a flow rate of 0.7 mL / min. 5 mg of PMPS-A1 was placed in an ampoule. After adding 2 mL of 3M TFA, PMPS-A1 was hydrolyzed at 60 °C for 3 h. After accurate absorption, the acid-hydrolyzed solution was transferred to a test tube. It was dried under nitrogen and thoroughly mixed with 5 mL of water. Add 950 μL of deionized water to 50 μL of the solution and centrifuge at 12,000 rpm for 5 min. Use the supernatant for IC analysis. Analyze HPIC using an electrochemical detector and a Dionex Carbopac™ PA20 column (3 × 150 mm, Thermal Scientific, USA).

[0096] The molecular weight, purity, number-average molecular weight (Mn), and polydispersity index (Mw / Mn) of PMPS-A1 were determined by HPGPC. The molecular weight of PMPS-A1 was 12198 Da. Based on y = -0.1948x + 11.649R (R = 0.9947), the Mn of PMPS-A1 was calculated to be 12168 Da. Figure 3 (B) The Mw / Mn ratio of PMPS-A1 is 1.00, indicating high homogeneity of PMPS-A1. The monosaccharide composition of PMPS-A1 was determined by ion chromatography. Figure 3 (F). The results showed that PMPS-A1 is composed of glucose and fructose.

[0097] 3.2 UV-Vis and IR spectral analysis of PMPS-A1

[0098] PMPS-A1 was scanned between 200 and 500 nm using an ultra-micro UV spectrophotometer (Germany NP80 Touch) at a concentration of 1 mg / mL.

[0099] PMPS-A1 was mixed with KBr powder and thoroughly ground to form tablets. Then, a TENSOR 27FT-IR spectrometer (Brunker, Germany) was used to analyze the spectra at 400-4000 cm⁻¹. -1Measurements are taken within the specified range.

[0100] UV spectrum of PMPS-A1 ( Figure 3 The D-wavelength (D) showed no obvious absorption peak in the 260nm-280nm range, indicating that PMPS-A1 does not contain nucleic acids or proteins. The functional groups in PMPS-A1 were determined using infrared spectroscopy. Figure 3 The FT-IR spectrum shown in E indicates that at 4000-500 cm⁻¹ -1 Within the range, at approximately 3394cm -1 The strong and broad absorption band observed at 2929 cm⁻¹ is attributed to the stretching vibration of OH groups, while the band at 2929 cm⁻¹ is... -1 The small absorption band at 1644 cm⁻¹ corresponds to the stretching vibration of CH₄; these two absorption bands are characteristic absorption bands of polysaccharides. -1 The spectral bands in this region are caused by associated water, and the FT-IR bands are due to the stretching vibrations of the pyranose ring in the 930-1153 cm⁻¹ region. -1 There is a strong spectral band in between. 849cm -1 The absorption at 1025 and 1153 cm⁻¹ indicates the presence of an α-glycosidic bond. -1 The absorption at these sites also indicates that the sugar is a pyranose. At 930, 854, and 708 cm⁻¹... -1 The absorption band at that location indicates that PMPS-A1 is a D-glucopyranose derivative.

[0101] 3.3. Thermal Performance Analysis

[0102] Based on thermogravimetric (TG) and differential thermogravimetric (DTG) methods, a simultaneous thermal analyzer manufactured by Mettler Toledo was used.

[0103] (Mettler TGA / DSC3, Switzerland) The thermal properties of PMPS-A1 were studied. PMPS-A1 (10 mg) was placed on Al2O3 aluminum, using empty aluminum as raw material, and experiments were conducted in an N2 environment at a heating rate of 10 °C / min at temperatures ranging from 30 to 500 °C.

[0104] Thermal stability is an important physicochemical property of polysaccharides in the food industry. TG and DTG are commonly used to evaluate the thermal properties of polysaccharides, and the results are as follows: Figure 4 As shown. Figure 4As shown, the TG and DTG curves of PMPS-A1 mainly exhibit weight loss in the 30-200℃ and 200-500℃ ranges. The decomposition process of PMPS-A1 can be divided into three stages. The first stage occurs at around 30℃, with a weight loss rate of 10.98%. In this stage, PMPS-A1 loses water adsorbed on the polysaccharides due to physical action, indicating that a small amount of water is adsorbed on PMPS-A1. The second stage occurs in the temperature range of 200-400℃, during which the weight loss is most significant, reaching 68.03%. The kudzu polysaccharides undergo rapid decomposition within this temperature range, leading to a significant decrease in the mass of PMPS-A1. The third stage occurs in the temperature range of 400-500℃. In this stage, the weight change of PMPS-A1 tends to be gradual, representing a slow carbonization stage. In this stage, most of the sample decomposes into ash and inorganic components. In conclusion, PMPS-A1 exhibits good thermal stability below 200℃.

[0105] 3.4 PMPS-A1 Congo Red Analysis

[0106] An 80 μmol / L Congo red solution was mixed with PMPS-A1 (3 mg / mL), followed by the addition of a 1 mol / L NaOH solution. The final NaOH concentrations were found to be 0, 0.05, 0.1, 0.15, 0.2, 0.3, and 0.4 mol / L. After standing for 5 min, the maximum absorption wavelength of the solution was measured using an ultraviolet spectrophotometer (Germany NP80 Touch), which was in the range of 400-600 nm.

[0107] Figure 5 The maximum absorption wavelengths of PMPS-A1 and Congo red in NaOH solutions of different concentrations (0-0.4M) are shown. Compared with Congo red, the maximum absorption wavelength of PMPS-A1 did not show a significant red shift with increasing NaOH concentration, indicating that PMPS-A1 does not have a triple helix structure.

[0108] 3.5 SEM Analysis

[0109] PMPS-A1 should be freeze-dried, quenched to expose its cross-section, and then adhered to a copper platform with its surface and cross-section facing upwards. Gold was sputtered on an ion sputterer for 5 min, and the surface morphology of PMPS-A1 was examined using a scanning electron microscope (Sigma-300, Germany).

[0110] SEM images are a powerful analytical tool used to detect the morphological properties of biopolymers, including polysaccharides and proteins. Figure 6 The image shows SEM micrographs of kudzu polysaccharide at magnifications of 200×, 500×, 1.0K×, and 2.0K×. The images show a small number of pores, small spherical shapes, and rod-shaped shapes, dispersed in their distribution.

[0111] 3.6 Methylation analysis of PMPS-A1

[0112] Weigh 2-3 mg of PMPS-A1 and add 1 mL of anhydrous DMSO. Quickly add methylation reagent A.

[0113] The methylation reagent B (iodomethane) solution was added and dissolved. The reaction was carried out at 30 °C for 60 min. Then, 2 mL of ultrapure water was added to the mixture to terminate the methylation reaction. After dialyzing for 24 h, the mixture was freeze-dried using a 1000 Da dialysis bag. After dialyzing, 2 mg and 200 mg of KBr samples were accurately weighed and mixed, compressed into tablets, and scanned and recorded in a Fourier transform infrared spectrometer (FT-IR650). After confirming the completion of methylation, subsequent experiments were performed. The methylated PMPS-A1 was taken and hydrolyzed with 1 mL of 2 mol / L LTFA for 90 min. The mixture was evaporated to dryness using a rotary evaporator. 2 mL of double-distilled water was added to the residue, followed by reduction with 60 mg of NaBH4 for 8 h, neutralization with glacial acetic acid, rotary evaporation, and drying in an oven at 101 °C. Finally, 1 mL of acetic anhydride was added for acetylation at 100 °C for 1 h, followed by cooling. Then, 10 mL of pure water was added to terminate the reaction. The acetylated product was dissolved in 3 mL of CH₂Cl₂ and transferred to a separating funnel. A small amount of distilled water was added and the mixture was shaken thoroughly to remove the aqueous solution from the top. This process was repeated four times. The CH₂Cl₂ layer was dried over an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid chromatography vial. The partially methylated ardiol acetates (PMAAs) were then determined by gas chromatography-mass spectrometry (GC-MS) on an HP-INNOVAX column (30 m × 0.32 mm × 0.25 μm). The programmed heating conditions were: initial temperature 140 °C, temperature ramp to 230 °C at a rate of 1 °C / min; inlet temperature 250 °C, detector temperature 250 °C, and helium as the carrier gas.

[0114] The methylation results of PMPS-A1 are shown in Table 3. PMPS-A1 has 6 glycosidic bonds, namely Glcp-(1→,

[0115] →1)-Fruf-(2→, →3)-Glcp-(1→, →4)-Glcp-(1→, →3,4)-Glcp-(1→ and →4,6)-Glcp-(1→).

[0116] Table 3 PMPS-A1 methylation results

[0117]

[0118] 3.7 PMPS-A1 NMR Analysis

[0119] Weigh 50 mg of PMPS-A1, dissolve it in 0.5 mL of D2O, and then freeze-dry. Repeat this process, freezing the powder, dissolving it in 0.5 mL of D2O, and then freeze-drying it again to ensure sufficient exchange of active hydrogen.

[0120] mLD₂O was collected and stored at room temperature (25°C) for measurement using a 600 MHz nuclear magnetic resonance instrument. 1 H NMR spectrum, 13 C

[0121] NMR spectra, DEPT-135 one-dimensional and two-dimensional spectra, results are shown in Figures 7-13 .

[0122] The 1D and 2D NMR spectra of PMPS-A1 are as follows: Figures 7-8 As shown, the proton spectrum signal is mainly concentrated between 3.0 and 5.5 ppm. The δ 3.2-4.0 ppm range represents the proton signal of the sugar ring, while the main-terminal matrix proton peaks δ 4.87, 5.18, 5.25, 5.26, and 5.30 are concentrated in the 4.3-5.5 ppm range. (DEPT-135 spectral analysis) Figure 9 The results show that the peaks at 61.27, 64.81, 69.04, 61.48, 61.89, and 61.46 ppm are inverted peaks, indicating a chemical shift of glucose C6 or fructose C1.

[0123] HSQC spectroscopy revealed an anomeric carbon signal of δ 100.92 ppm and a corresponding anomeric hydrogen signal of δ 5.35 ppm, suggesting a possible α-configuration glucose residue. HH-COSY ( Figure 10 The signals for H1-2 are 5.25 / 3.59, corresponding to H2 at δ 3.59 ppm; the signals for H2-3 are 3.59 / 3.69, corresponding to H3 at δ 3.69 ppm; the signals for H3-4 are 3.69 / 3.94, corresponding to H4 at δ 3.94 ppm; the signals for H4-5 are 3.94 / 3.96, corresponding to H5 at δ 3.96 ppm; and the signals for H5-6 are 3.96 / 3.59, corresponding to H6 at δ 359 ppm. Using HSQC spectroscopy, H1 corresponds to δ 5. 35 ppm, C1 corresponds to δ 100.92 ppm; H2 at δ 3.59 ppm corresponds to C2 at δ 100.92 ppm, H3 at δ 3.69 ppm corresponds to C3 at δ 73.62 ppm, H4 at δ 3.94 ppm corresponds to C4 at δ 70.2 ppm, H5 at δ 3.96 ppm corresponds to C5 at δ 71.27 ppm, and H6a at δ 3.59 ppm corresponds to C6 at δ 61.89 ppm. Therefore, this signal should be attributed to the glycosidic bond α-D-Glcp-1→.

[0124] The HSQC spectrum revealed a carbon anomaly signal of δ100.86 ppm, with a corresponding hydrogen anomaly signal of δ5.18 ppm. Using HH-COSY, the signals for H1-2 were 5.18 / 3.44, corresponding to H2 at δ3.44 ppm; H2-3 was 3.44 / 3.52, corresponding to H3 at δ3.52 ppm; H3-4 was 3.52 / 3.27, corresponding to H4 at δ3.27 ppm; H4-5 was 3.27 / 3.61, corresponding to H5 at δ3.61 ppm; and H5-6a was 3.61 / 3.66, corresponding to H6a at δ... 3.66 ppm; further attribution was performed using HSQC spectroscopy, where H1 corresponds to 5.18 ppm and C1 corresponds to 100.86 ppm; H2 at 3.44 ppm corresponds to C2 at 72.33 ppm, H3 at 3.52 ppm corresponds to C3 at 78.02 ppm, H4 at 3.27 ppm corresponds to C4 at 70.26 ppm, H5 at 3.61 ppm corresponds to C5 at 73.52 ppm, and H6a at 3.66 ppm corresponds to C6 at 61.48 ppm. Therefore, this signal should be attributed to the glycosidic bond →3)-α-D-Glcp-(1→).

[0125] The anomeric carbon signal observed by HSQC spectroscopy is δ 100.48, and the corresponding anomeric hydrogen signal is δ 5.30. By HH-COSY, the signals for H1-2 are 5.30 / 3.54; for H2-3, 3.54 / 3.89; and for H3-4, 3.89 / 3.57. This invention deduces that the δ values ​​for H1, H2, H3, and H4 are 100.48, 72.50, 74.15, and 77.91, respectively. However, NOESY spectroscopy observed peaks associated with δ 5.30 and 3.57, and 3.77 and 3.89, with H5 at 3.77 ppm. The corresponding C5 is 71.12; the chemical shift for C6 is δ 61.48, and the corresponding H6a is δ 3.77. Therefore, the signal should be attributed to the glycosidic bond →4)-α-Glcp-(1→).

[0126] HSQC spectroscopy revealed an anomeric carbon signal of δ 99.48 ppm and a corresponding anomeric hydrogen signal of δ 4.87 ppm, indicating a possible α-configuration glucose residue. HH-COSY analysis showed H1-2 signals of 4.87 / 3.48, corresponding to H2 at δ 3.48 ppm; H2-3 signals of 3.48 / 3.66, corresponding to H3 at δ 3.66 ppm; H3-4 signals of 3.66 / 3.4, corresponding to H4 at δ 3.4 ppm; H4-5 signals of 3.4 / 3.56, corresponding to H5 at δ 3.56 ppm; and H5-6 signals of 3.56 / 3.84, corresponding to H6 at δ 3.84 ppm. HSQC spectroscopy assignment showed H1 at δ 4.87 ppm, corresponding to C1 at... δ99.48ppm; H2 is δ3.48ppm, corresponding to C2 δ72.45ppm; H3 is δ3.66ppm, corresponding to C3 δ73.61ppm; H4 is δ3.4ppm, corresponding to C4 δ76.69ppm; H5 is δ3.56ppm, corresponding to C5 δ70.28ppm; H6 is δ3.84ppm, corresponding to C6 δ69.04ppm. The chemical shifts of C1, C4, and C6 to the lower field indicate that, combined with methylation analysis, the D sugar residues are speculated to be →4,6)-α-D-Glcp-(1→).

[0127] HSQC spectroscopy revealed an anomeric carbon signal of δ 100.95 ppm and a corresponding anomeric hydrogen signal of δ 5.26 ppm, indicating a possible α-configuration glucose residue. HH-COSY analysis showed that H1 residue 2 had a signal of 5.26 / 3.49, corresponding to H2 at δ 3.49 ppm; H2-3 had a signal of 3.49 / 3.74, corresponding to H3 at δ 3.74 ppm; H3-4 had a signal of 3.74 / 3.73, corresponding to H4 at δ 3.73 ppm; H4-5 had a signal of 3.73 / 3.84, corresponding to H5 at δ 3.84 ppm; and H5-6 had a signal of 3.84 / 3.65, corresponding to H6 at δ 3.65 ppm. HSQC spectroscopy assignment showed H1 at δ 5.26 ppm and C1 at δ 100.95 ppm. ppm; H2 is δ 3.49ppm, corresponding to δ 72.45ppm for C2, H3 is δ 3.74ppm, corresponding to δ 78ppm for C3, H4 is δ 3.73ppm, corresponding to δ 78.16ppm for C4, H5 is δ 3.84ppm, corresponding to δ 70.76ppm for C5, H6 is δ 3.65ppm, corresponding to δ 61.46ppm for C6. The chemical shifts of C3 and δ C4 to the lower field indicate that the residues have undergone substitution and methylation analysis at the O-1 and O-3 positions. It is speculated that the E sugar residue may be →3,4)-α-D-Glcp-(1→).

[0128] The HSQC spectrum showed an anomeric carbon signal of C3 at δ 77.74 ppm and a corresponding anomeric hydrogen signal of H3 at δ 4.1 ppm, indicating that it may be a β-configured fructose residue. HH-COSY analysis showed that the signals for H3-4 were 4.1 / 4.01, corresponding to a δ of 4.01 ppm for H4; the signals for H4-H5 were 4.01 / 3.86, corresponding to a δ of 3.86 ppm for H5; and the signals for H5-6 were 3.86 / 3.46, corresponding to a δ of 3.46 ppm for H6. HSQC spectroscopy assignments showed that H3 was δ 4.1 ppm, corresponding to a δ of 77.74 ppm for C3; H4 was δ 4.01 ppm, corresponding to a δ of 76.59 ppm for C4; H5 was δ 3.86 ppm, corresponding to a δ of 81.73 ppm for C5; and H6 was δ 3.46 ppm, corresponding to a δ of 64.81 ppm for C6. The lower-field chemical shifts of C1 and C2 indicate substitution at the O-1 and O-2 positions of the sugar ring. Combined with methylation analysis, the F sugar residue is speculated to be →1)-β-D-Fruf-(2→).

[0129] The structural diagram of PMPS-A1 is shown below. Figure 14 As shown in Table 4, the C and H chemical shifts of PMPS-A1 are assigned.

[0130] The present invention can infer that the main glycosidic bond structure of PMPS-A1 is as follows: the main chain is connected by glycosidic bonds of →[1)-β-D-Fruf-(2]5→[4)-α-D-Glcp-(1]2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→), while the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain by O-3 of →3,4)-α-D-Glcp-(1→ and O-6 of →4,6)-α-D-Glcp-(1→, respectively.

[0131] Table 4. C and H chemical shifts of PMPS-A1

[0132]

[0133] Example 1 of effect verification

[0134] I. Experimental Materials

[0135] Experimental animals: male Balb / c mice, age: 6-8 weeks; weight: approximately 18-22g.

[0136] Experimental instruments: heating stirrer, paraffin slicer, microscope, syringe, enzyme-linked immunosorbent assay reader, balance, oven, refrigerated centrifuge, etc.

[0137] Experimental reagents: HE staining reagents, PBS solution, etc.

[0138] Preparation method of crude polysaccharide from kudzu root: Fresh kudzu root heads (the part connecting the tuberous root and stem of kudzu) are chopped, added with 10 times the amount of water, boiled over high heat for 0.5 hours, then simmered over low heat for 1 hour. The filtrate is collected by filtering through gauze and concentrated under reduced pressure at 80℃.

[0139] Preparation method of crude kudzu root polysaccharide: Fresh kudzu root (the underground tuberous part of kudzu) is chopped, 10 times the amount of water is added, and it is first boiled over high heat for 0.5 hours, then simmered over low heat for 1 hour. The filtrate is collected by filtering with gauze and concentrated under reduced pressure at 80℃ to obtain crude kudzu root polysaccharide.

[0140] Preparation method of kudzu starch: First, clean the kudzu root, cut it into pieces, and soak it at 30℃ for 2 hours (first soaking), with a material-to-liquid mass ratio of 1:9. After soaking, crush (pulverize) the slurry and filter it through gauze. Let the slurry settle for 24 hours, then soak the residue again (second soaking), with a material-to-liquid mass ratio of 1:9, for 2 hours. Filter it again through gauze and wash the residue with a small amount of water (the residue contains a small amount of starch). Set four groups of slurry to settle for 6, 12, 18, and 24 hours, then wash the slurry (with the same liquid volume as during soaking) and stir for three to ten minutes. After stirring, let the four groups settle for 6, 12, 18, and 24 hours respectively, discard the supernatant, and repeat the washing twice. Finally, discard the supernatant, dry the precipitate, and obtain kudzu starch.

[0141] II. Animal Experiments

[0142] 1. Male Balb / c mice were randomly divided into 6 groups of 7 mice each: a control group (orally administered saline), a model group (orally administered saline), and positive control groups: Haiwang Jinzun (390 mg / kg); Pueraria lobata crude polysaccharide group (400 mg / kg), Pueraria lobata starch group (400 mg / kg), and Pueraria lobata root crude polysaccharide group (400 mg / kg). All groups received the drug solution via gavage for 7 days according to body weight. Thirty minutes after the last administration, all mice except the control group were orally administered 53% ABV liquor (0.25 mL / 20 g) via tube feeding. Prior to the experiment, all mice underwent a 12-hour fast with unlimited water. The onset time of LORR (loss of righting reflex) was calculated from the completion of alcohol administration to the onset of LORR. LORR ended when the animal was able to fall down three times within 30 seconds. The duration of LORR was measured as the time from the onset of LORR to the animal's return to normal movement.

[0143] Mice exhibiting the LORR phenomenon were considered intoxicated, and the number of intoxicated mice was recorded. The mice were weighed daily, and weight changes were observed. Weighing was repeated for 7 consecutive days.

[0144] 2. Mouse euthanasia and tissue sampling

[0145] Two hours after inducing an acute alcohol poisoning model with ethanol, blood was collected from the eyeballs. Mice were euthanized, and stomach and liver tissues were fixed in 4% paraformaldehyde. After graded alcohol dehydration, the tissues were fixed in paraffin blocks onto a Leica microtome. The microtome blade was sharp and free of nicks, with even and gentle pressure applied. Sections were 5 μm thick and continuous. Sections were spread in 40°C water using toothless forceps, retrieved with a glass slide, baked at 60°C for 2 hours, and then stored at room temperature in a slide box for later use. The slides were removed from xylene, placed on tissue paper, and a drop of neutral resin was applied to the tissue. A coverslip was gently placed on top, and the slides were allowed to air dry. Pathological changes were observed under a microscope, photographed, and analyzed.

[0146] 3. Assess the degree of gastric mucosal damage.

[0147] Stomach tissue was dissected and removed along the major curvature of the stomach. The stomach was rinsed with physiological saline to remove blood clots and gastric juice. The tissue was then smeared onto filter paper according to the Guth method, and the gastric ulcer score (GUI) was evaluated. The GUI of mice was measured using the Guth method to assess the degree of gastric mucosal damage. Based on the Guth scoring criteria, abnormalities in the integrity, smoothness, color, and bleeding of the gastric mucosa in each group of mice were observed and assessed. The severity was determined by the size and number of bleeding points in the gastric mucosa. The criteria for gastric ulcers were as follows: 0 points – no pathology; 1 point – small ulcer (1-2 mm); 2 points – moderate ulcer (3-4 mm); 4 points – large ulcer (5-6 mm); 8 points – relatively large ulcer (>6 mm).

[0148] III. Experimental Results

[0149] The results of investigating the therapeutic effects of crude polysaccharides from different parts of kudzu root on ethanol-induced acute alcohol poisoning in mice are shown in [the table below]. Figure 15 .

[0150] like Figure 15 As shown in Figures AB, there was no significant change in the body weight of mice in each group after one week of continuous administration, indicating that the drug had no obvious toxicity. Furthermore, compared to the model group, crude polysaccharide from kudzu root and kudzu starch had the effect of delaying the time it took for mice to become intoxicated.

[0151] like Figure 15 Chinese CD and Figure 16 As shown in the images and Guth scores, kudzu root polysaccharides and kudzu stalk polysaccharides significantly alleviate gastric mucosal damage and gastric bleeding. Pathological results showed that the gastric mucosal tissue structure of mice in the blank control group was neatly arranged, with no inflammatory cell infiltration; while in the model group, gastric mucosal epithelial cells were sloughed off, and inflammatory cells were visible in the lamina propria. Compared with the model group, the degree of gastric mucosal damage was reduced in each treatment group, with less sloughing of mucosal epithelial cells and milder inflammatory cell infiltration; indicating that alcohol treatment caused varying degrees of damage to the gastric mucosal cells of mice, and that kudzu root polysaccharides and kudzu stalk polysaccharides alleviated gastric damage.

[0152] In addition, such as Figure 17 As shown, the liver tissue cells of mice in the blank control group had intact structures; in the model group, the intercellular spaces of hepatocytes were loose, and cell vacuolation was observed. The liver lobule structures of the groups treated with kudzu root polysaccharide and kudzu starch were basically intact, with no obvious inflammatory cell infiltration, and hepatocyte damage could be alleviated.

[0153] Example 2: Anti-inflammatory effect of PMPS-A1 on oral ulcers in rats.

[0154] I. Experimental Materials

[0155] Experimental animals: 24 male SD rats; weight: approximately 180-220g, purchased from Zhuhai Beston Biotechnology Co., Ltd.

[0156] Experimental instruments: heating stirrer, paraffin slicer, microscope, syringe, balance, oven, refrigerated centrifuge, etc.

[0157] Experimental reagents: HE staining reagents, PBS solution, etc.

[0158] II. Animal Experiments

[0159] 1. Establishment of a rat model of oral ulcers:

[0160] ① Modeling drug: Phenol (aladdin, catalog number: 108-95-2); Experimental drug: PMPS-A1 prepared in Example 2;

[0161] ② Modeling method: An oral ulcer model was established using SD rats. 95% phenol was placed inside the left side of the rat's oral cavity and left for 120 seconds. Oral application was continued for 7 consecutive days starting the next day.

[0162] 2. Animal grouping: Control group, Model group, Low-dose group (PMPS-A1 dosage of 200 mg / kg) and High-dose group (PMPS-A1 dosage of 400 mg / kg), with 6 animals in each group.

[0163] 3. Animal administration: The low-dose group and the high-dose group were given PMPS-A1 by topical application according to different dosage groups, once a day for 7 consecutive days, and then euthanized by intraperitoneal injection of an excessive amount of 3% sodium pentobarbital.

[0164] 4. Animal sampling: After the rats were sacrificed, oral skin tissue was collected.

[0165] 5. Observation of indicators: ulcer healing rate and pathology in rats.

[0166] (1) Measurement of ulcer healing: The area of ​​oral ulcers in rats was observed and measured daily, and the daily ulcer healing status was calculated. The results are as follows: Figure 18 As shown, compared with the Model group, the High group rats showed better healing of oral ulcers.

[0167] (2) After fixing, dehydrating and embedding the oral skin tissue, HE staining was performed, and finally the pathological changes were observed under a microscope, photographed and analyzed.

[0168] Microscopic observation shows (see) Figure 19 In the Control group, rats had a small number of inflammatory cells in their oral cavity; in the Model group, the oral epithelial layer of rats was thickened and a large number of inflammatory cells infiltrated the lamina propria; compared with the Model group, the oral epithelial layer of rats in each drug administration group was thinner and the number of inflammatory cells in the lamina propria was reduced.

[0169] Example 3: Antioxidant activity of PMPS-A1

[0170] The antioxidant activity of PMPS-A1 prepared in Example 2 was tested:

[0171] 1. DPPH free radical scavenging assay

[0172] Vitamin C (Vc) was used as a positive control. Different concentrations of PMPS-A1, from 1-5 mg / L, were set as sample groups. The experiment consisted of a measurement group (80 μL sample + 120 μL reagent), a control group (80 μL sample + 120 μL extract), and a blank group (80 μL extract + 120 μL reagent), each added to a 96-well plate. The absorbance at 515 nm was then measured using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan M2001, Tecan, Mannedorf, Switzerland) after 30 min of exposure in the dark at room temperature (De Backer & Dorman, 2017). The calculation formula was based on the method used in the kit provided by Shanghai Enzyme-Link Biotechnology Co., Ltd., as follows:

[0173] DPPH free radical scavenging rate (%) = (A b -A m / A b )×100%;

[0174] Among them, A b A represents the absorbance value of the blank control in the working fluid. m This indicates the absorbance value of the crude polysaccharide sample in the working solution.

[0175] The ability of PMPS-A1 to scavenge DPPH free radicals is expressed as a scavenging rate. The higher the scavenging rate, the stronger the antioxidant activity of the extract. Figure 20As shown in Figure B, within a certain concentration range, the scavenging efficiency of PMPS-A1 against DPPH free radicals is dose-dependent with respect to polysaccharide concentration. The scavenging rate of polysaccharides against DPPH free radicals gradually increases with increasing polysaccharide concentration.

[0176] 2. Hydroxyl radical scavenging test

[0177] According to the instructions provided by Shanghai Enzyme-Link Biotechnology Co., Ltd., the working solution was diluted at a ratio of 1:9 (stock solution: distilled water) to prepare reagent four. Reagents one, two, and three were prepared at a ratio of 2:1:2. The sample was set as a gradient concentration of 1-5 mg / mL PMPS-A1. A measurement group (50 μL working solution + 50 μL sample + 50 μL reagent four), a control group (50 μL working solution + 50 μL distilled water + 50 μL reagent four), and a blank group (50 μL working solution + 100 μL distilled water) were set up. After mixing, the mixture was incubated at 37°C for 60 min, then centrifuged at 25°C with a centrifugal force of 8000 g for 5 min. 200 μL was then absorbed into a 96-well plate, and the absorbance was measured at 536 nm using a Tecan M2001 microplate reader (Tecan, Mannodorf, Switzerland). The calculation formula is as follows:

[0178] Hydroxyl radical scavenging rate (%) = [(A m -A c ) / A b -A c ]×100%;

[0179] Among them, A m A c and A b These represent the absorbance values ​​of the measurement group, control group, and blank group, respectively.

[0180] like Figure 20 As shown in Figure A, PMPS-A1 exhibits a certain scavenging effect on hydroxyl radicals in vitro, and this scavenging effect gradually increases with increasing polysaccharide concentration. When the PMPS-A1 concentration is 6 mg / mL, its ability to scavenge hydroxyl radicals is relatively strong, with a scavenging rate of 40%.

[0181] Example 4: In vitro anti-inflammatory activity of PMPS-A1

[0182] The in vitro anti-inflammatory activity of PMPS-A1 prepared in Example 2 was tested:

[0183] 1. Cell viability assay

[0184] RAW 264.7 cells were cultured in 96-well plates and treated with different concentrations of PMPS-A1 (1.25, 2.5, and 5 mg / mL) for 24 h. Fresh medium containing 10% CCK-8 was added to each well, and the plates were incubated at 37°C for 1 h. Absorbance was measured at 450 nm using a microplate reader (Tecan M2001, Thermo Fisher, USA). Cell viability was determined using the following formula:

[0185] Cell viability = [(A s -A b ) / (A c -A b )]×100%;

[0186] Among them, A s A represents the absorbance of the sample. b A represents the absorbance of the blank. c This indicates the absorbance of the control.

[0187] 2. Measurement of cytokines

[0188] RAW 264.7 cells were grown at 3 × 10⁻⁶ cells per well. 5 Cells were seeded at a density in 12-well plates and cultured overnight at 37°C with 5% CO2. Experimental groups included: a blank control group (Control, without lipopolysaccharide (LPS) stimulation); a positive control group (dexamethasone (DEX), 1 μM); an LPS stimulation group (1 μg / mL); and three treatment groups (PMPS-A1 doses of 1.25, 2.5, and 5 mg / mL, respectively). Cells were pretreated with PMPS-A1 for 2 h. Except for the blank control group, all experimental groups were induced with 1 μg / mL LPS for 4 h.

[0189] To investigate the anti-inflammatory activity of PMPS-A1, a cytotoxicity experiment was conducted. Compared with the control group, PMPS-A1 had no effect on cell viability (P>0.05). Figure 21 (A) Compared with the LPS group, both dexamethasone and PMPS-A1 significantly (P<0.05 or 0.001) downregulated 1L-6 expression. Figure 21 (B) Compared with the LPS group, both dexamethasone and PMPS-A1 significantly (P<0.05 or 0.001) downregulated NO expression. Figure 21 (C)

[0190] Example 5: In vitro antibacterial activity of PMPS-A1

[0191] The in vitro antibacterial activity of PMPS-A1 prepared in Example 2 was tested:

[0192] Lipid diffusion assay was used to measure the antibacterial activity of PMPS-A1 at two different concentrations (20, 25, 30, and 35 mg / mL). *Escherichia coli* and methicillin-resistant Staphylococcus aureus (MRSA) were inoculated onto agar plates. 7 mm diameter wells were drilled into the agar medium. PMPS-A1 solutions of different concentrations were added to each well. 100 μg / mL ampicillin was used as a positive control to confirm the results. The plates were incubated at 4°C for 4 h to allow PMPS-A1 to diffuse in the agar, and then incubated at 37°C for 24 h. Antibacterial activity was assessed by measuring the diameter of the inhibition zone (including the 7 mm well diameter). All tests were performed triplicate, and the values ​​presented are the average of the three replicates.

[0193] As shown in Table 5, the antibacterial activity gradually increased with increasing PMPS-A1 concentration. Furthermore, studies have shown that polysaccharides may impair the permeability of the cell walls and membranes of *E. coli* and MRSA, such as through a sharp increase in water-soluble intracellular proteins or by inhibiting bacterial growth by preventing the input of nutrients.

[0194] Table 5 Antibacterial activity of PMPS-A1

[0195]

[0196] Example 6: The anti-aging effect of PMPS-A1 on *C. elegans*

[0197] The anti-aging effect of PMPS-A1 prepared in Example 2 was tested using *C. elegans*.

[0198] 1. Culture of Escherichia coli

[0199] All *C. elegans* were infected with *E. coli* OP50 at 20°C when grown on *NGM* agar plates. *E. coli* was incubated at 37°C for 12 h. Eggs were obtained using bleaching solution and then washed with M9 buffer. After a 48-h synchronization period, L4 nematodes were ready for further testing.

[0200] 2. Determine the level of reactive oxygen species (ROS).

[0201] Synchronized L4 nematodes were divided into four groups: a control group, three PMPS-A1-treated groups (1 mg / mL, 3 mg / mL, and 5 mg / mL). Forty-eight hours post-treatment, the nematodes were incubated for 15 minutes at a concentration of 10 mmol / mL H2O2. Each nematode was collected and washed three times with M9 buffer. The nematodes were then incubated for 30 minutes at 37°C in 10 mM DCFH-DA fluorescent probe dye solution (Beyotime, Shanghai, China) to measure ROS levels. The relative intensity of ROS fluorescence was measured and examined using fluorescence microscopy (EVOS, Thermo Fisher Scientific, Massachusetts, USA) and ImageJ software (NIH, Bethesda, Maryland, USA).

[0202] Reactive oxygen species (ROS) are closely related to aging in organisms. Appropriate amounts of ROS can act as signaling molecules, participating in various intracellular physiological processes such as cell growth, differentiation, and apoptosis, while excessive ROS can lead to cell damage and aging. Figure 22 As shown, compared with the control group, the fluorescence accumulation level of ROS gradually decreased with increasing PMPS-A1 concentration. The evidence clearly indicates that kudzu polysaccharide can reduce ROS accumulation in nematodes, thereby delaying the senescence of nematodes.

[0203] 3. PMPS-A1 reduces lipofuscin accumulation in *C. elegans*.

[0204] Synchronized L4 nematodes were selected and placed on NGM medium plates containing PMPS-A1 and OP50. After two days of treatment, the nematodes were treated with 10 mmol / mL H2O2 for 15 min and then transferred to a centrifuge tube containing M9 buffer. To precipitate the nematodes, the centrifuge tubes containing them were centrifuged at 3000 rpm for 2 min. The precipitated nematodes were placed on a 2% agarose plate, and the supernatant was discarded. They were studied using a fluorescence microscope. After observing their shape in bright field and dark field, images were acquired and fluorescence counts were performed using ImageJ. Statistical evaluation of light intensity was performed using Graphpad Pism 8.

[0205] As we age, lipofuscin typically accumulates in neurons, heart muscle, liver, and other tissue cells, and it also contributes to age spots on the skin. Reducing lipofuscin accumulation may help slow aging because it is closely related to the aging process. The autofluorescence of lipofuscin in nematodes can be observed using a fluorescence microscope. Figure 23 As shown, compared with the blank control group, the fluorescence accumulation level of lipofuscin gradually decreased with the gradual increase of polysaccharide concentration.

[0206] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A kudzu root polysaccharide, characterized in that, The kudzu polysaccharide is composed of glucose and fructose and has 6 glycosidic bonds, namely Glcp-(1→,→1)-Fruf-(2→,→3)-Glcp-(1→,→4)-Glcp-(1→,→3,4)-Glcp-(1→ and→4,6)-Glcp-(1→). The main chain of the kudzu polysaccharide is linked by glycosidic bonds of →[1)-β-D-Fruf-(2)5→[4)-α-D-Glcp-(1)2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→), while the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain by O-3 of →3,4)-α-D-Glcp-(1→, and O-6 of →4,6)-α-D-Glcp-(1→); The molecular weight of the kudzu polysaccharide is 12198 Da.

2. A method for preparing kudzu polysaccharide as described in claim 1, characterized in that, Includes the following steps: Pueraria lobata was extracted using a water extraction and alcohol precipitation method to obtain crude polysaccharide. After deproteinization, the crude kudzu polysaccharide was purified sequentially using a DEAE-52 cellulose anion exchange chromatography column and a Sephadex G-150 column to obtain the kudzu polysaccharide.

3. The use of the kudzu polysaccharide as described in claim 1 in any of the following: (1) To prepare drugs for the prevention and / or treatment of acute alcohol poisoning; (2) Preparation of drugs for treating oral ulcers; (3) Preparation of antioxidant products; the antioxidant products are pharmaceuticals; (4) Preparation of anti-inflammatory drugs; (5) Preparation of antibacterial drugs; (6) Prepare anti-aging drugs.

4. The application according to claim 3, characterized in that, The antibacterial drug targets Escherichia coli and / or methicillin-resistant Staphylococcus aureus.

5. A drug for the prevention and / or treatment of acute alcohol poisoning, characterized in that, The active ingredient includes the kudzu polysaccharide as described in claim 1.

6. A medicine for treating oral ulcers, characterized in that, The active ingredient includes the kudzu polysaccharide as described in claim 1.

7. An antioxidant product, characterized in that, The active ingredient includes the kudzu polysaccharide as described in claim 1; The product in question is a pharmaceutical product.

8. An anti-inflammatory drug, characterized in that, The active ingredient includes the kudzu polysaccharide as described in claim 1.

9. An antibacterial drug, characterized in that, The active ingredient includes the kudzu polysaccharide as described in claim 1.

10. An anti-aging drug, characterized in that, The active ingredient includes the kudzu polysaccharide as described in claim 1.

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