Pueraria thomsonii polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses pueraria thomsonii polysaccharide as well as a preparation method and application thereof, and relates to the technical field of biology. The pachyrhizua angulatus polysaccharide consists of glucose and fructose, and has six glucosidic bonds, namely Glcp-(1->,-> 1)-Fruf-(2->,-> 3)-Glcp-(1->,-> 4)-Glcp-(1->,-> 3, 4)-Glcp-(1-> and-> 4, 6)-Glcp-(1-). Functional verification experiments show that the pachyrhizua angulatus polysaccharide can effectively prevent acute alcoholism, has a treatment effect on oral ulcer, and has antioxidant, anti-inflammatory, antibacterial and anti-aging activities. The method for extracting the polysaccharide from the pachyrhizua angulatus not only has important health significance, but also has remarkable economic and social benefits, provides technical support for expanding application of traditional Chinese medicinal materials, and also provides a brand-new and natural solution for solving various health problems.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to kudzu vine polysaccharide and a preparation method and application thereof. Background Art

[0002] Pueraria thomsonii Benth. is the dried root of Pueraria thomsonii Benth., a plant of the Leguminosae family. It has the effects of relieving muscles and reducing fever, promoting fluid production and quenching thirst, clearing rashes, raising yang and stopping diarrhea, promoting blood circulation and activating collaterals, and detoxifying alcohol. It can be used for exogenous fever and headache, stiff neck and back pain, thirst, polydipsia, measles that does not break out, heat dysentery, diarrhea, dizziness and headache, hemiplegia caused by stroke, chest pain and heart pain, and alcohol poisoning.

[0003] Pueraria lobata is rich in various active ingredients, such as flavonoids, saponins and polysaccharides, among which Pueraria lobata polysaccharides have attracted extensive attention due to their unique structure and excellent biological activity.

[0004] With the changes in modern lifestyles, alcohol abuse and related health problems have become increasingly prominent, and the search for natural, safe and efficient alcohol detoxification drugs has become a research hotspot. In addition, oxidative stress, bacterial infection and inflammatory response are also the common pathological basis of many diseases, and the development of natural drugs with multiple biological activities is of great significance. The present invention intends to develop a kudzu polysaccharide in order to provide technical support for solving the above technical problems. Summary of the invention

[0005] The purpose of the present invention is to provide a kudzu vine polysaccharide and its preparation method and application to solve the problems existing in the above-mentioned prior art. The kudzu vine polysaccharide can effectively prevent acute alcohol poisoning, has a therapeutic effect on the ulcer surface of oral ulcers, and has antioxidant, anti-inflammatory, active, antibacterial and anti-aging activities.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a kudzu vine 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 connection mode of the kudzu vine polysaccharide is a glycosidic bond of →[1)-β-D-Fruf-(2]5→[4)-α-D-Glcp-(1]2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→, and the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain through O-3 of →3,4)-α-D-Glcp-(1→, O-6 of →4,6)-α-D-Glcp-(1→, respectively.

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

[0010] The kudzu root was extracted by water extraction and alcohol precipitation to obtain kudzu root crude polysaccharide.

[0011] After the crude kudzu vine polysaccharide is deproteinized, it is purified using a DEAE-52 cellulose anion exchange chromatography column and a Sephadex G-150 column in sequence to obtain the kudzu vine polysaccharide.

[0012] The present invention also provides the use of the above-mentioned Pueraria lobata polysaccharide in any of the following items:

[0013] (1) Preparation of drugs for the prevention and / or treatment of acute alcohol intoxication;

[0014] (2) preparing medicines for treating oral ulcers;

[0015] (3) preparing antioxidant products; the antioxidant products are health products or medicines;

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

[0017] (5) Preparation of antibacterial drugs;

[0018] (6) Preparation of anti-aging drugs.

[0019] Furthermore, the pathogens targeted by the antibacterial drug are Escherichia coli and / or methicillin-resistant Staphylococcus aureus.

[0020] The present invention also provides a medicine for preventing and / or treating acute alcohol poisoning, wherein the active ingredient comprises the above-mentioned Pueraria lobata polysaccharide.

[0021] The present invention also provides a medicine for treating oral ulcers, wherein the active ingredient comprises the above-mentioned Pueraria lobata polysaccharide.

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

[0023] The product is a health product or a medicine.

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

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

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

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

[0028] The invention extracts a kudzu vine polysaccharide from kudzu vine. After detection, the kudzu vine 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 connection mode is →[1)-β-D-Fruf-(2]5→[ 4)-α-D-Glcp-(1]2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→) glycosidic bond, while the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain through O-3 of →3,4)-α-D-Glcp-(1→, O-6 of →4,6)-α-D-Glcp-(1→, respectively.

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

[0030] The invention extracts polysaccharides from Pueraria lobata not only has important health significance, but also has significant economic and social benefits, provides technical support for expanding the application of traditional Chinese medicinal materials, and also provides a new and natural solution for solving various health problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The figure is the result of the single factor experiment; A is the extraction rate of crude kudzu polysaccharide under different solid-liquid ratios; B is the extraction rate of crude kudzu polysaccharide under different extraction times; C is the extraction rate of crude kudzu polysaccharide under different extraction temperatures;

[0033] Figure 2 The results of the response surface experiment analysis are shown in Figure 1. A is the response surface of A2 and A1; D is the contour diagram of A2 and A1; B is the response surface of A3 and A1; E is the contour diagram of A3 and A1; C is the response surface of A3 and A2; F is the contour diagram of A3 and A2;

[0034] Figure 3 Figure 1 is a characterization diagram of the purification process and physicochemical properties of Pueraria lobata polysaccharide; A is the elution curve of DEAE-52; B is the elution curve of Sephadex G-150; 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; F is the HPIC chromatogram of standard monosaccharides and PMPS-A1, where 1 is fucose; 2 is galactosamine hydrochloride; 3 is rhamnose; 4 is arabinose; 5 is glucosamine hydrochloride; 6 is galactose; 7 is glucose; 8 is xylose; 9 is mannose; 10 is fructose; 11 is ribose; 12 is galacturonic acid; 13 is guluronic acid; 14 is glucuronic acid; 15 is mannuronic acid;

[0035] Figure 4 It is the TG-DTG curve of PMPS-A1;

[0036] Figure 5 This is the Congo red assay analysis diagram of PMPS-A1;

[0037] Figure 6 is the SEM image of PMPS-A1; wherein, AD 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] Fig. 9 This is the DEPT-135 diagram of PMPS-A1;

[0041] Fig.10 COSY image of PMPS-A1;

[0042] Fig.11 is the HSQC diagram of PMPS-A1;

[0043] Fig.12 is the HMBC diagram of PMPS-A1;

[0044] Fig.13is the NOESY image of PMPS-A1;

[0045] Fig.14 It is a schematic diagram of the structural formula of PMPS-A1;

[0046] Fig.15 The results of investigating the therapeutic effects of crude polysaccharides from different parts of Pueraria lobata on ethanol-induced acute alcohol intoxication in mice; A is a statistical graph of mouse weight; B is a statistical graph of the time when mice entered drunkenness; C is a statistical graph of gastric injury GUI score; D is a picture of gastric injury in each group; Control is a blank group, Model is a model group, and Positive control is a positive drug group; Dried head of Pueraria lobata or Dried head is a group treated with crude polysaccharides from Pueraria lobata head; Dried and Powdered Pueraria lobata or Dried and Powdered is a group treated with Pueraria starch; Pueraria lobata Root or Root is a group treated with crude polysaccharides from Pueraria lobata root;

[0047] Fig.16 The stomach tissue pathological sections of each experimental group in the effect verification example 1; Model is the model group, Positive control is the positive drug group; Dried head is the group administered with kudzu head crude polysaccharide; Dried and Powdered is the group administered with kudzu starch; Root is the group administered with kudzu root crude polysaccharide;

[0048] Fig.17 The liver tissue pathological sections of each experimental group in the effect verification example 1; Model is the model group, Positive control is the positive drug group; Dried head is the group administered with kudzu head crude polysaccharide; Dried and Powdered is the group administered with kudzu starch; Root is the group administered with kudzu root crude polysaccharide;

[0049] Fig.18 This is a statistical chart of the ulcer healing status of rats in each experimental group in effect verification example 2;

[0050] Fig.19 HE staining of oral skin tissue of rats in each experimental group of effect verification example 2;

[0051] Fig. 20 The results of testing the antioxidant activity of PMPS-A1 are shown in Figure 1; wherein A is a statistical graph showing the scavenging rate of hydroxyl radicals by PMPS-A1; and B is a statistical graph showing the scavenging rate of DPPH radicals by PMPS-A1.

[0052] Fig.21The results of the in vitro anti-inflammatory activity test of PMPS-A1; A is the effect of PMPS-A1 on the activity of RAW264.7 macrophages; B is the effect of PMPS-A1 on the relative expression of IL-6 in macrophages induced by lipopolysaccharide; C is the effect of PMPS-A1 on the relative expression of nitric oxide in macrophages induced by lipopolysaccharide; compared with the blank group, ### P<0.001; compared with the lipopolysaccharide group, * P<0.05, *** P < 0.001;

[0053] Fig. 22 The results of ROS level detection in different experimental groups; AD are the fluorescence detection diagrams of ROS in nematodes of blank control group, 1mg / mL, 3mg / mL and 5mg / mL, respectively; E is the statistical diagram of the relative fluorescence intensity of ROS in nematodes; compared with the blank group, *** P < 0.001;

[0054] Fig.23 The results of the lipofuscin level test in different experimental groups; AD are the fluorescence detection diagrams of ROS in nematodes of blank control group, 1 mg / mL, 3 mg / mL and 5 mg / mL, respectively (lipofuscin produces blue fluorescence); E is the statistical diagram of the relative fluorescence intensity of lipofuscin in nematodes; compared with the blank group, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

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

[0056] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0057] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0058] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

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

[0060] Example 1 Extraction, content determination and process optimization of Pueraria lobata polysaccharide

[0061] Fresh kudzu vine was dried at 60°C, ground into powder, and boiled in deionized water at 80°C for 4h, with a liquid-to-solid ratio of 1g:30mL. The above process was repeated three times in total. The supernatant was centrifuged at 4000rpm for 10min. Then, the supernatant was collected and concentrated to one-third of the solution volume at 80°C. Four times the amount of anhydrous ethanol was used for precipitation at 4°C for 24h, centrifuged (4000r / min, 20min), and then the precipitate was freeze-dried to obtain kudzu vine crude polysaccharide (PMPS). The contents of total sugar, protein and uronic acid were determined by phenol-sulfuric acid method (with glucose as standard), Bradford method (with bovine serum albumin as standard) and sulfuric acid-carbazole method (with galacturonic acid as standard).

[0062] The extraction rate of Pueraria lobata (Y1, %) is Y1=M1 / M2×100, wherein M1 is the mass of freeze-dried Pueraria lobata crude polysaccharide, and M2 is the mass of Pueraria lobata original powder.

[0063] 1. Univariate analysis

[0064] To explore the preliminary range of extraction variables, a single-factor design was used to investigate 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 °C, 70 °C, 80 °C, 90 °C, and 100 °C) on polysaccharide yield.

[0065] like Figure 1As shown in Figure A, as the liquid-to-solid ratio increases, the polysaccharide yield increases significantly (p<0.05). The larger the solution-to-solid ratio, the more obvious the concentration difference and the faster the polysaccharide molecules diffuse. On the other hand, the polysaccharide extraction rate reaches a 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 and the dissolution of polysaccharides is hindered, resulting in a decrease in the total polysaccharide yield.

[0066] like Figure 1 As shown in B, the yield of PMPS increased significantly from 0.5 to 3 h, reaching a maximum at 3 h. In addition, longer extraction time will also lead to the destruction and degradation of polysaccharide structure.

[0067] like Figure 1 As shown in Figure C, the yield increased significantly with increasing temperature and reached a peak at 90°C. From this temperature, the yield began to decrease, probably because of the decomposition and inactivation of carbohydrates caused by extreme temperature. Therefore, 90°C is an ideal extraction temperature.

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

[0069] A three-variable, three-level 17-step Box–Behnken (BBD) design was constructed by Design Expert software to determine the ideal range of the extracted variables (Tables 1 and 2) based on the results of the single-factor experiment. The 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 liquid-to-solid ratio (mL / g), extraction time (h) and extraction temperature (℃), respectively.

[0074] 3. Optimization of polysaccharide extraction conditions

[0075] Statistical analysis and model fitting

[0076] In order to optimize the extraction parameters and analyze the interactive effects of the three factors on the extraction rate, BBD under RSM was used, as shown in Table 1. The liquid-to-solid ratio was 30 (mL / g), the extraction time was 3 h, the extraction temperature was 90 °C, and the maximum Y1 was 9.77%. Multiple regression analysis obtained 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 the model. The R 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 non-significant P value was 0.73. The coefficients of other parameters were not significant, but the coefficients of the interaction term (A2A3), the single term coefficient (A2), and the quadratic term coefficient (A 12 , A 22 and A 32 ) is significant. The low coefficient of variance (3.82) of the polynomial model equation indicates that it has high accuracy and reliability. Therefore, the relationship between extraction temperature (A3) and extraction time (A2) has a great influence on the extracted PMPS and needs to be carefully studied.

[0079] Table 2 Estimation of regression coefficients and significance analysis of the quadratic polynomial model for PMPS production

[0080]

[0081]

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

[0083] 3.2 Response surface analysis

[0084] The generated 2D contour plots and 3D response surfaces show the relationship between process variables. Figure 2 ) first increases when the two variables (A2 and A3) increase, and then decreases. The highest Y1 of PMPS was obtained at an extraction temperature of 90.20 °C and an extraction time of 2.97 h. According to Table 2 and Figure 2 , the interaction between A2A3 is stronger than the interaction between A1A2 and A1A3. In addition, the greater the density of the ellipse contour plot, the greater the interaction effect between the variables.

[0085] 3.3 Optimization and Verification

[0086] The numerical optimization results show that the PMPS Y1 condition is an extraction temperature of 90 °C, as shown in the variance analysis (Table 2) and the reaction surface diagram ( Figure 2AC), the extraction time was 2.96 h and the liquid-solid ratio was 29.86 (mL / g). Under these conditions, the PMPS Y1 estimated by the model was 9.55%. Considering the feasibility of the experiment, the extraction temperature was set to 90 ° C, the extraction time was set to 2.96 h, and the liquid-solid ratio was set to 29.86 (mL / g). In order to demonstrate the reliability of the response surface technology model, three repeated experiments were carried out under realistic conditions. Using n = 3, the experimental PMPS Y1 was 9.78 ± 0.09%. This shows that the BBD model is valid because 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 the single factor experiment, the optimized extraction rate of PMPS was higher, which shows the accuracy and effectiveness of the model.

[0087] Example 2 Purification and characterization of physicochemical properties of Pueraria lobata polysaccharide

[0088] 1. Preparation of Pueraria lobata crude polysaccharide

[0089] Fresh kudzu root was dried at 60°C, ground into powder, and boiled in deionized water at 90°C for 2.96h, with a liquid-to-solid ratio of 1g:29.86mL. The above process was repeated three times in total. The supernatant was centrifuged at 4000rpm for 10min. Then, the supernatant was collected and concentrated to one-third of the solution volume at 80°C. Four times the amount of anhydrous ethanol was used for precipitation at 4°C for 24h, centrifuged (4000r / min, 20min), and then the precipitate was redissolved in deionized water to obtain a kudzu root crude polysaccharide solution with a concentration of 1g / mL.

[0090] 2. Purification of Pueraria lobata polysaccharide

[0091] Sevage reagent was used to separate free proteins. Sevage reagent and kudzu vine crude polysaccharide solution were mixed evenly in 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. In order to remove salt and other small molecules, the supernatant after centrifugation was dialyzed in distilled water for 48 h using a dialysis bag (MWCO 3500Da). The retentate was then freeze-dried and purified using a DEAE-52 column (5.5×40cm, Beijing Ruida Henghui Technology Development Co., Ltd.). A gradient solution of distilled water and NaCl (0.1, 0.2, 0.3 and 0.5 mol / L) was used for elution 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 the same elution peak were collected and dialyzed for 48 h (MWCO3500Da), 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. Then, PMPS-A was purified again using a Sephadex G-150 (1.0 × 50 cm, Borui Sugar Biotechnology Co., Ltd.) column. The elution process was performed using a gradient solution of distilled water. Tubes with the same elution peak were collected and dialyzed for 48 h (MWCO 3500 Da), and the retentate was then freeze-dried to prepare PMPS-A1.

[0092] The yield of the traditional method for extracting crude polysaccharide (PMPS) is 3% ± 0.14%. Deionized water, 0.1 mol / L NaCl, 0.2 mol / L NaCl, 0.3 mol / L NaCl, 0.5 mol / L NaCl, DEAE-52 cellulose anion exchange chromatography column, deionized water washing, Sephadex G-150 column purification, phenol sulfuric acid method determination, plotting elution curve ( Figure 3 According to the ion column elution curve, the present invention collects the polysaccharide with the highest absorbance and names it PMPS-A. In addition, the HPGPC spectrum ( Figure 3 Middle B) shows two peaks of PMPS-A, indicating that the purity of PMPS-A is about 80%. The total sugar content of PMPS-A is about 95% ± 0.78%. In order to obtain a uniform polysaccharide, it was purified on a Sephadex G-150 column. The peak with the highest polysaccharide content was collected according to its elution curve and named PMPS-A1. The HPGPC spectrum shows that PMPS-A1 is a single peak, indicating that the purity of PMPS-A1 is >99%. Using the phenol-sulfuric acid method, the total sugar content in PMPS-A1 is about 99% ± 0.48%. The protein content of PMPS-A1 is: 0.032% ± 0.0016%, and the content of uronic acid is: 0.6% ± 0.021%. This also shows that PMPS-A1 is a neutral sugar.

[0093] 3.Physical and chemical properties of PMPS-A1

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

[0095] PMPS-A15 mg was accurately weighed and dissolved in 1 mL of mobile phase (0.05 mol / L NaCl solution). After vortexing and centrifugation (12000 rpm, 10 min), the supernatant was aspirated and filtered through a 0.22 μm water microporous filter membrane. PMPS-A1 was placed in a 1.8 mL injection bottle. 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 hydrolysis solution was transferred to a test tube. Blow dry with nitrogen and mix thoroughly with 5 mL of water. 950 μL of deionized water was added to 50 μL, and centrifuged at 12,000 rpm for 5 min. For IC analysis, the supernatant was used. HPIC was analyzed using an electrochemical detector and a Dionex Carbopac™ PA20 column (3×150 mm, Thermal Scientific, USA).

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

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

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

[0099] PMPS-A1 was mixed with KBr powder and ground thoroughly to form pellets, which were then analyzed using a TENSOR 27 FT-IR spectrometer (Brunker, Germany) at 400–4000 cm -1Measure within the range of

[0100] UV spectrum of PMPS-A1 ( Figure 3 Middle D) shows no obvious absorption peak at 260nm-280nm, indicating that PMPS-A1 does not contain nucleic acid or protein. The functional groups in PMPS-A1 were determined by infrared spectroscopy. Figure 3 The FT-IR spectrum shown in Figure E shows that the -1 In the range of about 3394cm -1 The strong and broad absorption band observed at 2929 cm -1 The small absorption band at 1644cm corresponds to the stretching vibration of CH. These two absorption bands are characteristic absorption bands of polysaccharides. -1 The bands in the region are caused by associated water, and the FT-IR bands are due to the stretching vibration of the pyranose ring at 930-1153 cm -1 There is a strong band between 849cm -1 The absorption at 1025 and 1153 cm indicates the presence of an α-glycosidic bond. -1 The absorption at 930, 854 and 708 cm -1 The absorption band at indicated that PMPS-A1 is a D-glucose pyranose derivative.

[0101] Thermal performance analysis

[0102] Based on thermogravimetric (TG) and differential thermogravimetric (DTG) methods, using a simultaneous thermal analyzer produced by Mettler Toledo

[0103] The thermal properties of PMPS-A1 were investigated (Mettler TGA / DSC3, Switzerland). PMPS-A1 (10 mg) was placed on Al2O3 aluminum using bare aluminum as the raw material and the experiments were performed at 30-500°C at a heating rate of 10°C / min in N2 environment.

[0104] Thermal stability is an important physical and chemical property of polysaccharides in the food industry. TG and DTG are often used to evaluate the thermal properties of polysaccharides. Figure 4 As shown. Figure 4As shown in the figure, the TG and DTG curves of PMPS-A1 mainly show weight loss in the 30-200℃ and 200-500℃ stages. The decomposition process of PMPS-A1 is divided into three stages. The first stage is around 30℃, with a weight loss rate of 10.98%. In this stage, PMPS-A1 loses the moisture adsorbed on the polysaccharide due to physical action, indicating that a small amount of moisture will be adsorbed on PMPS-A1. The temperature range of the second stage is 200-400℃, during which the weight loss is most significant, reaching 68.03%. Pueraria polysaccharide decomposes violently in this temperature range, resulting in a significant decrease in the mass of PMPS-A1. In the third stage, the temperature range is 400-500℃. In this stage, the weight change of PMPS-A1 tends to be flat, which is a slow carbonization stage. In this stage, most samples decompose into ash and inorganic components. In summary, PMPS-A1 exhibits good thermal stability within 200℃.

[0105] 3.4 Congo red analysis of PMPS-A1

[0106] 80 μmol / L Congo red solution was mixed with PMPS-A1 (3 mg / mL), and then 1 mol / L NaOH solution was added. The concentrations of NaOH were finally 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 ultra-micro UV spectrophotometer (NP80 Touch, Germany), which was in the range of 400-600 nm.

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

[0108] 3.5SEM analysis

[0109] PMPS-A1 should be freeze-dried, quenched to expose its cross section, and adhered to a copper platform with its surface and cross section facing up. Gold was sputtered on an ion sputtering instrument 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 for examining the morphological properties of biopolymers, including polysaccharides, proteins, etc. Figure 6 SEM micrographs of Pueraria lobata polysaccharide at 200×, 500×, 1.0K×, and 2.0K× magnifications are shown in FIG. This image has a small number of pores, small spherical shapes, and rod-like shapes, and the shape distribution is dispersed.

[0111] 3.6 Methylation analysis of PMPS-A1

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

[0113] (anhydrous alkali) solution and dissolved, and then the methylated reagent B (iodomethane) solution was added. The reaction was carried out at 30°C for 60min. Then, 2mL of ultrapure water was added to the mixture to terminate the methylation reaction. After dialysis for 24h, the mixture was freeze-dried with a 1000Da dialysis bag. After dialysis, 2mg and 200mg KBr samples were accurately weighed and mixed, pressed into tablets, and scanned and recorded in a Fourier transform infrared spectrometer (FT-IR650). After confirming that the methylation was completed, subsequent experiments were performed. Take the methylated PMPS-A1 and add 1mL of 2mol / LTFA to hydrolyze for 90min. The mixture was evaporated to dryness using a rotary evaporator. 2mL of double distilled water was added to the residue, which was then reduced with 60mg of NaBH4 for 8h, neutralized with glacial acetic acid, rotary evaporated, and dried in an oven at 101°C. Finally, 1mL of acetic anhydride was added for acetylation at 100°C for 1h, and then cooled. Then, 10mL of pure water was added to terminate the reaction. The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separation funnel. A small amount of distilled water was added and shaken thoroughly to remove the upper aqueous solution. This process was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid phase vial. The partially methylated alditol acetate (PMAAs) was then determined by gas chromatography-mass spectrometry GC-MS on an HP-INNOVAX column (30 m × 0.32 mm × 0.25 μm); the program heating conditions were: starting temperature of 140 ° C, heating to 230 ° C, heating rate of 1 ° C / min; inlet temperature of 250 ° C, detector temperature of 250 ° C, and carrier gas of helium.

[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 NMR analysis of PMPS-A1

[0119] Weigh 50 mg of PMPS-A1, dissolve in 0.5 mL of D2O, and then freeze-dry. The freeze-dried powder is then dissolved in 0.5 mL of D2O and freeze-dried again, and this process is repeated to fully exchange the active hydrogen. The sample is dissolved in 0.5

[0120] mLD2O, and placed at room temperature 25℃, using a 600MHz nuclear magnetic resonance instrument to measure 1 H NMR spectrum, 13 C

[0121] NMR spectrum, DEPT-135 one-dimensional spectrum and two-dimensional spectrum, the results are shown in Figure 7-Figure 13 .

[0122] The 1D and 2D NMR spectra of PMPS-A1 are shown in Figure 7-Figure 8 As shown, the hydrogen spectrum signal is mainly concentrated between 3.0 and 5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks δ4.87, 5.18, 5.25, 5.26, and 5.30 are concentrated in the range of 4.3-5.5 ppm. DEPT-135 spectral analysis ( Fig. 9 ) showed that the peaks at 61.2764.81, 69.04, 61.48, 61.89 and 61.46 ppm were inverted peaks, indicating the chemical shift of glucose C6 or fructose C1.

[0123] The anomeric carbon signal can be observed by HSQC spectrum: δ100.92ppm, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ5.35ppm, indicating that it may be an α-configuration glucose residue. Fig.10 ), the signal of H1-2 is 5.25 / 3.59, corresponding to H2 at δ3.59ppm; the signal of H2-3 is 3.59 / 3.69, corresponding to H3 at δ3.69ppm; the signal of H3-4 is 3.69 / 3.94, corresponding to H4 at δ3.94ppm; the signal of H4-5 is 3.94 / 3.96, corresponding to H5 at δ3.96ppm; the signal of H5-6 is 3.96 / 3.59, corresponding to H6 at δ359ppm; the HSQC spectrum is used for assignment, H1 corresponds to δ5. 35ppm, C1 corresponds to δ100.92ppm; H2 at δ3.59ppm corresponds to C2 at δ100.92ppm, H3 at δ3.69ppm corresponds to C3 at δ73.62ppm, H4 at δ3.94ppm corresponds to C4 at δ70.2ppm, H5 at δ3.96ppm corresponds to C5 at δ71.27ppm, and H6a at δ3.59ppm corresponds to C6 at δ61.89ppm. Therefore, the signal should be attributed to the glycosidic bond α-D-Glcp-1→.

[0124] Through the HSQC spectrum, it can be observed that the carbon abnormal signal is δ100.86ppm, and the corresponding hydrogen abnormal signal in the HSQC spectrum is δ5.18ppm. Through HH-COSY, the signal of H1-2 is 5.18 / 3.44, and the corresponding H2 is δ3.44ppm; the signal of H2-3 is 3.44 / 3.52, and the corresponding H3 is δ3.52ppm; the signal of H3-4 is 3.52 / 3.27, and the corresponding H4 is δ3.27ppm; the signal of H4-5 is 3.27 / 3.61, and the corresponding H5 is δ3.61ppm; the signal of H5-6a is 3.61 / 3.66, and the corresponding H6a is δ 3.66ppm; further attribution was performed using the HSQC spectrum, where H1 corresponded to δ5.18ppm, C1 corresponded to δ100.86ppm; H2 at δ3.44ppm corresponded to C2 at δ72.33ppm, H3 at δ3.52ppm corresponded to C3 at δ78.02ppm, H4 at δ3.27ppm corresponded to C4 at δ70.26ppm, H5 at δ3.61ppm corresponded to C5 at δ73.52ppm, and H6a at δ3.66ppm corresponded to C6 at δ61.48ppm. Therefore, the signal should be attributed to the glycosidic bond →3)-α-D-Glcp-(1→.

[0125] Through the HSQC spectrum, it can be observed that the anomeric carbon signal is δ100.48, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ5.30. Through HH-COSY, the signal of H1-2 is 5.30 / 3.54; the signal of H2-3 is 3.54 / 3.89; the signal of H3-4 is 3.89 / 3.57; the present invention can infer that the δ of H1, H2, H3 and H4 are 100.48, 72.50, 74.15 and 77.91, respectively. However, the NOESY spectrum observed peaks associated with δ5.30 and 3.57, 3.77 and 3.89, among which H5 is 3.77ppm. The corresponding C5 is 71.12; the chemical shift of C6 is δ61.48, and the corresponding H6a is δ3.77. Therefore, the signal should be attributed to the glycosidic bond →4)-α-Glcp-(1→.

[0126] Through the HSQC spectrum, it can be observed that the anomeric carbon signal is δ99.48ppm, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.87ppm, indicating that it may be an α-configuration glucose residue. Through HH-COSY, the signal of H1-2 is 4.87 / 3.48, and the corresponding H2 is δ3.48ppm; the signal of H2-3 is 3.48 / 3.66, and the corresponding H3 is δ3.66ppm; the signal of H3-4 is 3.66 / 3.4, and the corresponding H4 is at δ3.4ppm; the signal of H4-5 is 3.4 / 3.56, and the corresponding H5 is δ3.56ppm; the signal of H5-6 is 3.56 / 3.84, and the corresponding H6 is δ3.84ppm; using the HSQC spectrum for assignment, H1 is δ4.87ppm, and the corresponding C1 is δ99.48ppm; H2 is δ3.48ppm, corresponding to C2 is δ72.45ppm, H3 is δ3.66ppm, corresponding to C3 is δ73.61ppm, H4 is δ3.4ppm, corresponding to C4 is δ76.69ppm, H5 is δ3.56ppm, corresponding to C5 is δ70.28ppm, H6 is δ3.84ppm, corresponding to C6 is δ69.04ppm, the chemical shifts of C1, C4, and C6 to the downfield indicate that combined with the methylation analysis, it is speculated that the D sugar residue may be →4,6)-α-D-Glcp-(1→

[0127] Through HSQC spectrum, it can be observed that the anomeric carbon signal is δ100.95ppm, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ5.26ppm, indicating that it may be an α-configuration glucose residue. Through HH-COSY, the signal of H1 No. 2 is 5.26 / 3.49, and the corresponding H2 is δ3.49ppm; the signal of H2-3 is 3.49 / 3.74, and the corresponding H3 is δ3.74ppm; the signal of H3-4 is 3.74 / 3.73, and the corresponding H4 is at δ3.73ppm; the signal of H4-5 is 3.73 / 3.84, and the corresponding H5 is δ3.84ppm; the signal of H5-6 is 3.84 / 3.65, and the corresponding H6 is δ3.65ppm; using HSQC spectrum for assignment, H1 is δ5.26ppm, and the corresponding C1 is δ100.95 ppm; H2 is δ3.49ppm, corresponding to δ72.45ppm of C2, H3 is δ3.74ppm, corresponding to δ78ppm of C3, H4 is δ3.73ppm, corresponding to δ78.16ppm of C4, H5 is δ3.84ppm, corresponding to δ70.76ppm of C5, H6 is δ3.65ppm, corresponding to δ61.46ppm of C6. The chemical shifts of C3 and δC4 to the downfield indicate that the residues are substituted at the O-1 and O-3 positions. Combined with the methylation analysis, it is speculated that the E sugar residue may be →3,4)-α-D-Glcp-(1→

[0128] The anomeric carbon signal C3 can be observed at δ77.74 ppm through the HSQC spectrum, and the corresponding anomeric hydrogen signal H3 in the HSQC spectrum is at δ4.1 ppm, indicating that it may be a fructose residue in the β configuration. By HH-COSY, the signals of H3-4 are 4.1 / 4.01, corresponding to H4 at δ4.01ppm; the signals of H4-H5 are 4.01 / 3.86, corresponding to H5 at δ3.86ppm; the signals of H5-6 are 3.86 / 3.46, corresponding to H6 at δ3.46ppm; the HSQC spectrum is used for assignment, H3 is δ4.1ppm, corresponding to C3 at δ77.74ppm, H4 at δ4.01ppm, corresponding to C4 at δ76.59ppm, H5 at δ3.86ppm, corresponding to C5 at δ81.73ppm, H6 at δ3.46ppm, corresponding to C6 at δ64.81ppm, and the chemical shifts of C1 and C2 to the downfield indicate that the residues are substituted at the O-1 and O-2 positions of the sugar ring. Combined with the analysis of the methylation results, it is speculated that the F sugar residue may be →1)-β-D-Fruf-(2→

[0129] The structural diagram of PMPS-A1 is shown in Fig.14 As shown in Table 4, the C and H chemical shift assignments of PMPS-A1 are shown in Table 4.

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

[0131] Table 4 C and H chemical shift assignments of PMPS-A1

[0132]

[0133] Effect verification example 1

[0134] 1. Experimental Materials

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

[0136] Experimental instruments: heating stirrer, paraffin slicer, microscope, syringe, microplate reader, balance, oven, refrigerated centrifuge, etc.

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

[0138] Preparation method of kudzu root crude polysaccharide: chop fresh kudzu root (the part connecting the tuber and stem of kudzu), add 10 times water, boil for 0.5h with high heat and then boil for 1h with low heat, collect the filtrate through gauze, and concentrate under reduced pressure at 80℃

[0139] Preparation method of Pueraria lobata crude polysaccharide: chop fresh Pueraria lobata (the underground tuberous root part of Pueraria lobata), add 10 times water, boil it with high heat for 0.5h, then boil it with low heat for 1h, collect the filtrate through gauze, and concentrate it under reduced pressure at 80℃ to obtain Pueraria lobata crude polysaccharide.

[0140] Preparation method of kudzu starch: firstly, clean the kudzu root, cut it into pieces, soak it at 30℃ for 2h (first soaking), and the material-liquid mass ratio is 1:9; after soaking, crush (beat) and filter the filter pulp with gauze, let it settle for 24h, and then soak the residue (second soaking), the soaking material-liquid mass ratio is 1:9, soaking for 2h; filter it with gauze again, and wash the filter residue of gauze with a small amount of water (the filter residue contains a small amount of starch); set four groups of filter pulp to settle for 6, 12, 18, 24h, then wash the pulp (consistent with the liquid volume during soaking), and stir for three to ten minutes, and then settle the four groups for 6, 12, 18, 24h respectively after stirring, pour out the supernatant, and repeat washing twice. Finally, pour out the supernatant, dry the precipitate, and obtain kudzu starch.

[0141] 2. Animal Experiments

[0142] 1. Male Balb / c mice were randomly divided into 6 groups, 7 mice in each group, and divided into blank group (oral saline), model group (oral saline), positive drug group: Neptune Jinzun (390 mg / kg); Pueraria lobata crude polysaccharide administration group (400 mg / kg), Pueraria lobata starch administration group (400 mg / kg) and Pueraria lobata crude polysaccharide administration group (400 mg / kg). The drug solution was given to the drug groups by oral gavage for 7 days according to body weight. 30 minutes after the last administration, all mice except the blank group were orally gavaged with 53-degree liquor (0.25 mL / 20 g). Before the experiment, all groups of mice were fasted for 12 hours but not water. The start time of LORR (loss of righting reflex) was calculated as the time from the completion of alcohol administration to the start of LORR. LORR ended when the animal was able to lie down three times within 30 seconds. The duration of LORR was measured as the time from the start of LORR to the ability of the animal to resume normal movement.

[0143] Mice with LORR phenomenon were judged as drunk, and the number of drunk mice was recorded. The mice were weighed every day to observe the weight changes of mice. The weight was weighed continuously for 7 days.

[0144] 2. Mouse Sacrifice and Sample Collection

[0145] 2 hours after the ethanol-induced acute alcohol intoxication model, the eyeballs were removed and blood was collected. The mice were killed and the stomach and liver tissues were fixed in 4% paraformaldehyde. After the tissues were dehydrated by alcohol gradient, the tissue paraffin blocks were fixed on the Leica slicer. The slice edge should be sharp and without notches, and the force should be gentle and uniform. The slice thickness should be 5μm and continuous slices should be made. Use toothless tweezers to place the slices in 40℃ water for spreading. After scooping the slices with a glass slide, bake the slices at 60℃ for 2 hours, and then put them in a slice box for storage at room temperature for later use. Take out the glass slide from xylene, place it on toilet paper, add neutral gum to the tissue, gently cover it with a coverslip, and let it dry naturally. Observe the pathological changes under a microscope, take pictures, and analyze.

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

[0147] Gastric tissue was obtained by dissection, and the stomach was cut along the greater curvature, rinsed with saline to remove blood clots and gastric juice, and then spread on filter paper according to the Guth method to evaluate the gastric ulcer score GUI, and then photographed. The Guth method was used to measure the GUI of mice and evaluate the degree of gastric mucosal damage. According to the Guth scoring standard, the abnormalities of gastric mucosal integrity, surface smoothness, color, bleeding, etc. of each group of mice were observed and evaluated. Its severity is determined by the size and number of gastric mucosal bleeding spots. The gastric ulcer criteria are as follows: 0 points - no pathology; 1 point - small ulcer (1-2mm); 2 points - moderate ulcer (3-4mm); 4 points - large ulcer (5-6mm); 8 points - large ulcer (>6mm).

[0148] 3. Experimental Results

[0149] The results of investigating the therapeutic effects of crude polysaccharides from different parts of Pueraria lobata on ethanol-induced acute alcoholism in mice are shown in Fig.15 .

[0150] like Fig.15 As shown in Figures AB, there was no significant change in the body weight of mice in each group after continuous administration for one week, indicating that the drug had no obvious toxicity. In addition, compared with the model group, the kudzu head crude polysaccharide and kudzu starch had the effect of delaying the time when mice entered drunkenness.

[0151] like Fig.15 CD and Fig.16 As shown in the figure, according to the stomach pictures and Guth scores, Pueraria lobata and Pueraria lobata root polysaccharides have obvious effects on alleviating gastric mucosal damage and gastric bleeding. Pathological results showed that the gastric mucosal tissue structure of mice in the blank group was neatly arranged, and no inflammatory cell infiltration was observed; the gastric mucosal epithelial cells of mice in the model group fell off, and inflammatory cells were visible in the lamina propria. Compared with the model group, the degree of gastric mucosal damage in each drug-treated group was reduced, the mucosal epithelial cells fell less, and the degree of inflammatory cell infiltration was lighter; this showed that alcohol treatment caused different degrees of damage to the gastric mucosal cells of mice, and Pueraria lobata root polysaccharides and Pueraria lobata head polysaccharides had the effect of alleviating gastric damage.

[0152] In addition, if Fig.17 As shown in the figure, the liver tissue cell structure of the blank group mice was intact; the liver cell gaps in the model group were loose, and cell vacuolation occurred. The liver lobule structure of the kudzu root polysaccharide and kudzu starch administration groups was basically intact, with no obvious inflammatory cell infiltration, which could alleviate liver cell damage.

[0153] Effect verification example 2: Anti-inflammatory effect of PMPS-A1 on the ulcer surface of rats with oral ulcer

[0154] 1. Experimental Materials

[0155] Experimental animals: 24 SD male rats; weight: about 180-220 g, purchased from Zhuhai Baishitong Biological Co., Ltd.

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

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

[0158] 2. Animal Experiments

[0159] 1. Establishment of oral ulcer rat model:

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

[0161] ② Modeling method: SD rats were used to establish the oral ulcer model. 95% phenol was placed inside the left oral cavity of the rat for 120 seconds. The drug was applied orally starting from the second day for 7 consecutive days.

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

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

[0164] 4. Animal sampling: Rats were killed and oral skin tissue was obtained.

[0165] 5. Observation indicators: rat ulcer healing rate, pathology, etc.

[0166] (1) Determination of ulcer surface healing: The area of ​​oral ulcer surface of rats was observed and measured daily, and the daily ulcer healing status was calculated. Fig.18 As shown, compared with the Model group, the oral ulcer healing effect of rats in the High group was better.

[0167] (2) After fixation, dehydration and embedding, the oral skin tissue was stained with HE, and finally the pathological changes were observed under a microscope, photographed and analyzed.

[0168] Microscopic observation showed (see Fig.19 ), there were a small number of inflammatory cells in the oral cavity of rats in the Control group; the oral epithelial layer of rats in the Model group 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-treated group was thinned, and the number of inflammatory cells in the lamina propria decreased.

[0169] Effect Verification 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. PMPS-A1 at different concentrations, from 1-5 mg / L, was set as the sample group. The experiment was divided into a measurement group (80 μL sample group + 120 μL reagent group), a control group (80 μL sample + 120 μL extract) and a blank group (80 μL extract + 120 μL reagent group), which were dropped into a 96-well plate. Then, an enzyme-linked immunosorbent assay (ELISA) reader (Tecan M2001, Tecan, Mannedorf, Switzerland) was used to detect the absorbance at 515 nm after 30 min of exposure in the dark at room temperature (De Backer & Dorman, 2017). According to the method used in the kit provided by Shanghai ELISA Biotechnology Co., Ltd., the calculation formula is as follows:

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

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

[0175] The ability of PMPS-A1 to scavenge DPPH free radicals is expressed by the scavenging rate. The higher the scavenging rate, the stronger the antioxidant effect of the extract. Fig. 20As can be seen from Figure B, within a certain concentration range, the scavenging efficiency of PMPS-A1 on DPPH free radicals is dose-dependent with the polysaccharide concentration. As the polysaccharide concentration increases, the scavenging rate of polysaccharides on DPPH free radicals gradually increases.

[0176] 2. Hydroxyl radical scavenging test

[0177] According to the instructions for use of the kit provided by Shanghai ELISA Biotechnology Co., Ltd., the working solution was diluted at a ratio of 1:9 (mother solution: distilled water) to prepare reagent 4. Reagent 1, reagent 2, and reagent 3 were prepared at a ratio of 2:1:2. The sample was set to a gradient concentration of 1-5 mg / mL PMPS-A1. A measurement group (50 μL working solution + 50 μL sample + 50 μL reagent 4), a control group (50 μL working solution + 50 μL distilled water + 50 μL reagent 4), and a blank group (50 μL working solution + 100 μL distilled water) were set. After mixing, incubate at 37°C for 60 min, then centrifuge at 8000 g for 5 min at 25°C, and then absorb 200 μL into a 96-well plate, and measure the absorbance at 536 nm using a Tecan M2001 microplate reader (Tecan, Manndorf, 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 Represent the absorbance values ​​of the measurement group, control group and blank group, respectively.

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

[0181] Effect Verification 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 plated in 96-well plates and treated with different concentrations of PMPS-A1 (1.25, 2.5, 5 mg / mL) for 24 h. Fresh medium containing 10% CCK-8 was added to each well and then incubated at 37 °C for 1 h. The 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 survival rate = [(A s -A b ) / (A c -A b )]×100%;

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

[0187] 2. Determination of Cytokines

[0188] RAW 264.7 cells were plated at 3 × 10 per well. 5 The cells were seeded in 12-well plates at different densities and cultured overnight in an incubator at 37°C with 5% CO2. The 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). The cells were pretreated with PMPS-A1 for 2 hours. All experimental groups, except the blank control group, were induced with 1 μg / mL LPS for 4 hours.

[0189] In order to study the anti-inflammatory activity of PMPS-A1, the present invention conducted a cytotoxicity experiment. Compared with the control group, PMPS-A1 had no effect on cell viability (P>0.05) ( Fig.21 Compared with the LPS group, dexamethasone and PMPS-A1 significantly (P<0.05 or 0.001) down-regulated the expression of 1L-6 ( Fig.21 Compared with the LPS group, dexamethasone and PMPS-A1 significantly (P<0.05 or 0.001) down-regulated the expression of NO ( Fig.21 Middle C).

[0190] Effect verification 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] The lipid diffusion method was used to measure the antimicrobial activity of PMPS-A1 at two different concentrations (20, 25, 30 and 35 mg / mL). Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) were inoculated on the surface of agar plates. A 7 mm diameter hole was drilled in the agar medium. PMPS-A1 solutions of different concentrations were added to each well. 100 μg / mL of ampicillin was used as a positive control to determine the results. The plates were placed at 4°C for 4 h to allow PMPS-A1 to diffuse in the agar and then incubated at 37°C for 24 h. The antimicrobial activity was evaluated by measuring the inhibition zone diameter (including the 7 mm hole diameter). All tests were performed three times, and the values ​​presented are the average of the three replicates.

[0193] As shown in Table 5, the antibacterial activity gradually increased with the increase of PMPS-A1 concentration. In addition, studies have shown that polysaccharides may damage the cell wall and membrane permeability of E. coli and MRSA, such as a sharp increase in water-soluble intracellular proteins or inhibit bacterial growth by preventing the input of nutrients.

[0194] Table 5 Antibacterial activity of PMPS-A1

[0195]

[0196] Effect Verification Example 6: Anti-aging Effect of PMPS-A1 on Caenorhabditis elegans

[0197] The anti-aging effect of PMPS-A1 prepared in Example 2 was tested using Caenorhabditis elegans:

[0198] 1. Cultivation of Escherichia coli

[0199] All C. elegans were infected with E. coli OP50 at 20°C while growing on root-knot nematode growth medium (NGM) plates. E. coli was grown for 12 h at 37°C. Eggs were obtained using a bleach solution and then washed with M9 buffer. After a 48 h synchronization period, L4 nematodes were ready for subsequent testing.

[0200] 2. Determine the Levels of Reactive Oxygen Species (ROS)

[0201] Synchronized L4 nematodes were divided into four groups: a blank group, three PMPS-A1-administered groups (1 mg / mL, 3 mg / mL, and 5 mg / mL). 48 h after administration, the nematodes were incubated in a concentration of 10 mmol / mL H2O2 for 15 min. After each nematode was collected, it was washed three times with M9 buffer. The nematodes were incubated in a 10 mM DCFH-DA fluorescent probe dye solution (Shanghai Biyuntian, China) for 30 min at 37 °C to measure the ROS level. The relative intensity of ROS fluorescence intensity was measured and examined by fluorescence microscopy (EVOS, Thermo Fisher Scientific, Massachusetts, USA) and ImageJ software (NIH, Bethesda, Maryland, USA).

[0202] ROS is closely related to the aging of organisms. Appropriate reactive oxygen species can act as signal molecules and participate in various physiological processes in cells, such as cell growth, differentiation and apoptosis, while excessive reactive oxygen species can cause cell damage and aging. Fig. 22 As shown, compared with the control group, with the increase of PMPS-A1 concentration, the fluorescence accumulation level of ROS gradually decreased. The evidence clearly shows that Pueraria lobata polysaccharide can reduce the accumulation of ROS in nematodes, thereby delaying the degree of nematode aging.

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

[0204] Synchronized L4 nematodes were selected and placed on NGM medium plates with PMPS-A1 and OP50. After two days of treatment, the nematodes were transferred to a centrifuge tube filled with M9 buffer after treatment with 10mmol / mLH2O2 for 15min. To precipitate the nematodes, the centrifuge tube containing them was centrifuged at 3000rpm for 2min. 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 shapes under bright field and dark field, image acquisition and fluorescence counting were performed using ImageJ. Statistical evaluation of light intensity was performed using Graphpad Pism 8.

[0205] As we age, lipofuscin usually accumulates in neurons, myocardium, liver, and other tissue cells. It also causes age spots on the skin surface. Reducing the accumulation of lipofuscin may help delay aging because it is closely related to the aging process. Using a fluorescence microscope, the spontaneous blue fluorescence of lipofuscin in nematodes can be observed. Fig.23 As shown, compared with the blank control group, with the gradual increase of polysaccharide concentration, the fluorescence accumulation level of lipofuscin gradually decreased.

[0206] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A kudzu vine polysaccharide, characterized in that: The kudzu vine polysaccharide is composed of glucose and fructose and has six 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 connection mode of the kudzu vine polysaccharide is a glycosidic bond of →[1)-β-D-Fruf-(2]5→[4)-α-D-Glcp-(1]2→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→, and the side chains α-D-Glcp-(1→3)-α-D-Glcp-(1→, α-D-Glcp-(1→) are connected to the main chain through O-3 of →3,4)-α-D-Glcp-(1→, O-6 of →4,6)-α-D-Glcp-(1→, respectively.

2. A method for preparing kudzu vine polysaccharide according to claim 1, characterized in that: The following steps are involved: The kudzu root was extracted by water extraction and alcohol precipitation to obtain kudzu root crude polysaccharide. After the crude kudzu vine polysaccharide is deproteinized, it is purified using a DEAE-52 cellulose anion exchange chromatography column and a Sephadex G-150 column in sequence to obtain the kudzu vine polysaccharide.

3. Use of the kudzu vine polysaccharide according to claim 1 in any of the following: (1) Preparation of drugs for the prevention and / or treatment of acute alcohol intoxication; (2) preparing medicines for treating oral ulcers; (3) preparing antioxidant products; the antioxidant products are health products or medicines; (4) Preparation of anti-inflammatory drugs; (5) Preparation of antibacterial drugs; (6) Preparation of anti-aging drugs.

4. The use according to claim 3, characterized in that: The pathogens targeted by the antibacterial drug are Escherichia coli and / or methicillin-resistant Staphylococcus aureus.

5. A drug for preventing and / or treating acute alcohol poisoning, characterized in that: The active ingredient comprises the kudzu vine polysaccharide according to claim 1.

6. A drug for treating oral ulcers, characterized in that: The active ingredient comprises the kudzu vine polysaccharide according to claim 1.

7. An antioxidant product, characterized in that: The active ingredient comprises the kudzu vine polysaccharide according to claim 1; The product is a health product or a medicine.

8. An anti-inflammatory drug, characterized in that: The active ingredient comprises the kudzu vine polysaccharide according to claim 1.

9. An antibacterial drug, characterized in that: The active ingredient comprises the kudzu vine polysaccharide according to claim 1.

10. An anti-aging drug, characterized in that: The active ingredient comprises the kudzu vine polysaccharide according to claim 1.

Images