Pueraria flower polysaccharide and application thereof in resisting oxidation, preventing and treating non-alcoholic steatohepatitis and regulating intestinal flora disorder

Abstract

The invention belongs to the technical field of medical health food and medicines, and particularly relates to pueraria flower polysaccharide and application thereof in resisting oxidation, preventing and treating non-alcoholic steatohepatitis and regulating intestinal flora disorder. Specifically, the pueraria flower polysaccharide is obtained by taking pueraria flower as a raw material and adopting a water extraction and alcohol precipitation method, an in-vitro anti-oxidation experiment proves that the pueraria flower polysaccharide has in-vitro anti-oxidation capacity, and meanwhile, an animal experiment proves that the pueraria flower polysaccharide has the effects of treating non-alcoholic steatohepatitis, improving intestinal flora disorder and regulating intestinal metabolites; good health care and treatment values are realized. Meanwhile, the extraction and preparation method is simple, can be easily converted into a modern industrial production process, and has great development potential, thereby having good economic and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and health food and drug technology, specifically relating to kudzu flower polysaccharide and its application in anti-oxidation, prevention and treatment of non-alcoholic steatohepatitis and regulation of intestinal flora imbalance. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nonalcoholic steatohepatitis (NASH), a common metabolic liver disease, encompasses the entire process from simple steatosis to liver fibrosis and is considered a major cause of chronic liver disease. Its etiology is complex and closely related to type 2 diabetes, hypertension, hyperlipidemia, metabolic syndrome, and gut microbiota dysbiosis, making it a significant public health issue. However, the specific pathogenesis and progression mechanisms of NASH remain unclear, and relevant treatments are relatively scarce.

[0004] The gut microbiota is linked to diet and host health, and a growing body of research indicates that gut microbiota participate in the pathogenesis of NASH (Neuro-Associated Syndrome). High-fat diets (such as methionine-restricted or choline-deficient diets) can trigger gut microbiota dysbiosis, increasing Firmicutes abundance and decreasing Bacteroidetes abundance, leading to an elevated Firmicutes / Bacteroidetes (F / B) ratio, which in turn exacerbates NASH development. Furthermore, studies have found that germ-free mice lacking gut microbiota exhibit significant resistance to NASH, while mice transplanted with gut microbiota from mice with metabolic syndrome show significant susceptibility to NASH. This demonstrates the close relationship between the development of NASH and gut microbiota.

[0005] Kudzu flower (Pueraria thomsonii Benth.) is the dried flower bud of a plant in the genus Pueraria of the legume family. It has effects such as relieving hangovers, protecting the liver, and clearing heat and reducing inflammation. Modern pharmacological studies have shown that kudzu flower and its active ingredients have antioxidant, liver-protective, lipid-lowering, and gut microbiota-regulating effects. However, the inventors discovered that research on kudzu flower polysaccharides, an important active ingredient in kudzu flower, is relatively scarce. Therefore, conducting systematic research on kudzu flower polysaccharides has significant scientific and application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides kudzu flower polysaccharide and its applications in antioxidation, prevention and treatment of non-alcoholic steatohepatitis (NAH), and regulation of intestinal flora imbalance. Specifically, this invention demonstrates through research that kudzu flower polysaccharide is an acidic polysaccharide with galactose (Gal), glucose (Glc), and galacturonic acid (GalA) as its main monosaccharide components and contains a relatively high amount of acidic sugars (GalA and GlcA). It possesses in vitro antioxidant capacity and pharmacological effects in improving NHA and regulating intestinal flora imbalance. Serological index analysis and stained section microscopy have demonstrated that kudzu flower polysaccharide can reduce serum liver indicators, decrease hepatocyte lipid accumulation, and improve liver inflammation in mice. Simultaneously, 16S rRNA sequencing technology of intestinal flora has clarified its function in improving intestinal flora. Based on the above research results, this invention is thus completed.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides the use of kudzu flower polysaccharide in the preparation of products that have antioxidant properties, prevent and treat non-alcoholic steatohepatitis, and regulate intestinal flora imbalance.

[0009] The products include, but are not limited to, food and pharmaceuticals.

[0010] In this invention, prevention and / or treatment refers to any measure applicable to the treatment of non-alcoholic steatohepatitis and related diseases, or preventive treatment of such diseases or their symptoms, or prevention of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of an already occurring disease, or preemptive intervention to prevent, suppress or reduce the occurrence of such diseases or symptoms.

[0011] The kudzu flower polysaccharide in this invention is a polysaccharide component extracted from kudzu flowers. In this invention, the kudzu flower polysaccharide is prepared by water extraction and alcohol precipitation.

[0012] The antioxidant properties include at least total antioxidant capacity (T-AOC), DPPH free radical scavenging capacity, and ABTS free radical scavenging capacity.

[0013] The prevention and treatment of non-alcoholic steatohepatitis (NAH) is manifested at least in reducing liver organ indices, pathological damage, serological liver markers, reducing lipid accumulation in hepatocytes, and improving obesity.

[0014] The regulation of intestinal flora imbalance specifically manifests as improving intestinal microbiota dysbiosis and reducing the abundance of pathogenic bacteria.

[0015] In one specific embodiment of the present invention, the non-alcoholic steatohepatitis and intestinal flora imbalance are mediated by a methionine-restricted, choline-deficient feeding method.

[0016] In a second aspect, the present invention provides a method for preparing kudzu flower polysaccharide, the method comprising preparing kudzu flower polysaccharide by water extraction and alcohol precipitation.

[0017] Specifically, the preparation method includes:

[0018] S1. After drying the kudzu flowers, ethanol was added for reflux extraction to remove fat-soluble substances and obtain the residue.

[0019] S2. Add water to the residue and boil it to extract the water extract. Centrifuge to remove insoluble components and collect the supernatant.

[0020] S3. Add ethanol to the supernatant for alcohol precipitation, collect the precipitate to obtain crude polysaccharide, and remove the protein from the crude polysaccharide to obtain the final product.

[0021] In step S1, the drying can be a drying process, such as drying at 40-60℃ (preferably 50℃).

[0022] The specific method for ethanol reflux extraction includes: using high-concentration ethanol (such as 95% ethanol) for reflux extraction 1 to 5 times (preferably 3 times), each time for 1 to 5 hours (preferably 2 hours); the ratio of kudzu flower to ethanol is 1:2-6 (preferably 1:4, kg / L).

[0023] In step S2, the specific conditions for adding water and boiling include: a material-to-liquid ratio of 1:1 to 10 (kg / L), preferably 1:5; a boiling time of 1 to 3 hours, preferably 2 hours; and 1 to 5 times of adding water and boiling (preferably 3 times).

[0024] In step S3, the alcohol precipitation method includes: adding 95% ethanol to the supernatant, allowing it to stand, and then collecting the precipitate; the standing time is 10-40 hours (preferably 24 hours); protein removal can be performed using the Sevage method, specifically, using Sevage reagent (CH3Cl:BuOH = 4:1, v / v) to remove protein 5-8 times.

[0025] Furthermore, the present invention also includes lyophilizing and purifying the polysaccharide obtained in step S3 by column elution.

[0026] The specific method for column elution purification includes: dissolving the lyophilized kudzu polysaccharide in water and eluting it sequentially with 1-4 column volumes (preferably 2 times) of distilled water, 0.2M NaCl, 0.5M NaCl, and 1.0M NaCl solutions using a DEAE agarose gel FF column (3.0×50cm) at a flow rate of 0.5-2 mL / min (preferably 1.0 mL / min); collecting the eluent in 10 mL test tubes and determining the sugar content using an anthrone-sulfuric acid method at 630 nm with a UV-Vis spectrophotometer, and plotting a separation curve; combining test tubes with the same peak (tubes 100-200), concentrating, and lyophilizing to obtain prepared kudzu polysaccharide GHP-E2; then using Chromdex... The prepared kudzu flower polysaccharide GHP-E2 was separated using a 75PG column (1.6cm × 100cm) at a flow rate of 0.5-2mL / min (preferably 1.0mL / min) with 1-4 column volumes (preferably 1 column volume) of distilled water. The eluent was collected in 5mL test tubes, and the sugar content was measured using the anthrone-sulfuric acid method to plot the separation curve. Test tubes with the same peak (tubes 100-200) were combined, concentrated, and lyophilized to obtain the prepared kudzu flower polysaccharide GHPE2-F1. Experimental results showed that the kudzu flower polysaccharide obtained by the above purification method has excellent antioxidant, non-alcoholic fatty liver disease prevention and treatment, and intestinal flora regulation effects.

[0027] Therefore, in a third aspect, the present invention provides a product for anti-oxidation, prevention and treatment of non-alcoholic steatohepatitis and / or regulation of intestinal flora imbalance, said product comprising at least kudzu polysaccharide.

[0028] Furthermore, the kudzu polysaccharide is obtained by the above preparation method.

[0029] The products include, but are not limited to, food and pharmaceuticals.

[0030] The term "food" can be understood as any edible form. For example, the food in this invention includes ordinary food and special food. The special food in this invention includes health food and food for special medical purposes. Ordinary food, in contrast to special food, is food suitable for everyone.

[0031] The drug can be administered in unit doses, and the dosage form can be liquid or solid. Liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, emulsions, granules, suppositories, lyophilized powder for injection, inclusion complexes, implants, patches, and liniments. The drug can be used by humans and non-human animals.

[0032] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0033] The above technical solution uses kudzu flower as raw material and obtains kudzu flower polysaccharide through water extraction and alcohol precipitation. In vitro antioxidant experiments have demonstrated that kudzu flower polysaccharide possesses in vitro antioxidant capacity. Animal experiments have also shown that kudzu flower polysaccharide has therapeutic effects on non-alcoholic steatohepatitis and improves intestinal flora imbalance and regulates intestinal metabolites, demonstrating good health and therapeutic value. Furthermore, the extraction and preparation method of this invention is simple and easily adaptable to modern industrial production processes, exhibiting significant development potential and thus possessing good economic and social benefits. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 Elution curves of kudzu polysaccharides; (A) elution curve of DEAE sepharose FF ion exchange column chromatography, (B) elution curve of Chromdex 75PG gel filtration column chromatography.

[0036] Figure 2 HPLC chromatogram and infrared spectrum of GHPE2F1: A: Chromatogram of standard, B: Chromatogram of sample, C: Infrared spectrum.

[0037] Figure 3 The in vitro antioxidant capacity of kudzu polysaccharides: (A) DPPH; (B) ABTS; (C) T-AOC.

[0038] Figure 4 Mouse body weight determination.

[0039] Figure 5 Measurement of the specific gravity of mouse liver and the specific gravity of mouse epididymal fat. A: Specific gravity of mouse liver; B: Specific gravity of mouse epididymal fat. Figure 6 Effects of kudzu polysaccharide on serum lipid disorders and liver function indicators: (A) serum TG content; (B) serum TC content; (C) serum ALT content; (D) serum AST content; (E) serum LDL-C content.

[0040] Figure 7 Pathological damage to the liver of NASH mice: (A) H&E staining: 10X 100μm (top); 20X 50μm (bottom); (B) Oil Red O staining: 10X 100μm (top); 20X 50μm (bottom); (C) NAS score; (D) Quantification of positive area of ​​Oil Red O staining.

[0041] Figure 8 Alpha diversity.

[0042] Figure 9Beta diversity; (A) Venn diagram; (B) Principal coordinate analysis diagram.

[0043] Figure 10 Kudzu flower polysaccharides regulate intestinal flora dysbiosis in mice at the phylum level.

[0044] Figure 11 Kudzu flower polysaccharides regulate intestinal flora dysbiosis in mice at the species level.

[0045] Figure 12 Evolutionary clade diagram of the effects of kudzu polysaccharide on the gut microbiota of mice.

[0046] Figure 13 LEfSe analysis of the effect of kudzu polysaccharide on the regulation of intestinal flora disorder in mice. Detailed Implementation

[0047] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0049] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0050] Example 1: Preparation of kudzu flower polysaccharides

[0051] Kudzu flower polysaccharides were prepared using the following steps:

[0052] The polysaccharide from kudzu flower (kudzu flower) was extracted using a water extraction and alcohol precipitation method.

[0053] First, dry the kudzu flowers in a 50℃ oven and slice them (1.0kg).

[0054] Extract by reflux with 1:4 (kg / L) 95% ethanol for 2 hours, repeat 3 times to remove fat-soluble substances.

[0055] Boil 5L of pure water for 2 hours, and repeat this step 3 times.

[0056] Combine the water extracts and concentrate to 1L. Centrifuge at 4000rpm for 15min to remove insoluble components and collect the supernatant.

[0057] The supernatant was precipitated overnight at room temperature using 6 L of 95% ethanol to obtain crude polysaccharide. The crude polysaccharide was completely dissolved in pure water.

[0058] Protein contaminants were removed five times using Sevage reagent (CH3Cl:BuOH = 4:1, v / v).

[0059] Finally, the polysaccharide solution was freeze-dried to obtain kudzu flower polysaccharide (21.7 g).

[0060] The sugar content of kudzu flower was determined to be 77.07% by the phenol-sulfuric acid method. 100 mg of deproteinized kudzu flower polysaccharide was dissolved in 30 mL of distilled water and eluted through a DEAE agarose gel FF column (3.0 × 50 cm) at a flow rate of 1.0 mL / min with two column volumes of distilled water, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solutions. The eluent was collected in 10 mL tubes, and the sugar content was determined by UV-Vis spectrophotometry at 630 nm using the anthrone-sulfuric acid method. A separation curve was plotted. Tubes with the same peak from tubes 100-200 were combined, concentrated, and lyophilized to obtain the prepared kudzu flower polysaccharide GHP-E2. Figure 1 A). Dissolve 100 mg of GHP-E2 in 200 mL of distilled water. Separate the kudzu polysaccharide GHP-E2 from a Chromdex 75PG column (1.6 cm × 100 cm) at a flow rate of 1.0 mL / min with one column volume of distilled water. Collect the eluent in a 5 mL tube and measure the sugar content using the anthrone-sulfuric acid method to plot the separation curve. Combine the tubes with the same peak from tubes 100-200, concentrate, and lyophilize to obtain the prepared kudzu polysaccharide GHPE2-F1. Figure 1 B).

[0061] A standard curve for polysaccharide and monosaccharide composition analysis was established. Polysaccharide samples were hydrolyzed with TFA to break glycosidic bonds and release monosaccharides. After washing with methanol to remove impurities, the monosaccharides were dissolved and ready for use. To improve the sensitivity and specificity of monosaccharide detection, PMP was added for derivatization, giving it UV absorption capability for easy detection by liquid chromatography. Excess PMP was removed by chloroform extraction before injection. The chromatographic system used a C18 reversed-phase column with phosphate buffer elution, and detection was performed at 250 nm to separate and quantify each derived monosaccharide. Figure 2 A, B). Another portion of the polysaccharide sample was mixed with KBr and compressed into a tablet. Infrared scanning was used to identify its functional group structure, further confirming it as an acidic polysaccharide. Figure 2 C).

[0062] The prepared kudzu flower polysaccharide GHPE2-F1 (later named PFP) was used in Examples 2-4.

[0063] Example 2: This example is used for the in vitro antioxidant capacity detection of kudzu flower polysaccharides:

[0064] Determination of the in vitro antioxidant capacity of kudzu flower polysaccharides:

[0065] The method for determining antioxidant activity (T-AOC method) includes the following steps: preparing test solutions with mass concentrations of 0.025, 0.5, 1, 2, 4, 8, and 16 mg / mL using crude kudzu flower polysaccharide and deionized water.

[0066] Establishment of calibration curves: Dilute the 40 μmol / mL FeSO4 standard solution with distilled water to the following concentrations: 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 μmol / mL.

[0067] Preparation of standard solutions: Take 100 μL of Fe at various concentrations 2+ Add 100 μL of FRAP working solution to the standard solution. Mix well at room temperature and react for 10 minutes. Measure the absorbance at 593 nm. Record the values ​​for standard A and blank A1, and calculate ΔAstandard = Astandard - Ablank.

[0068] Plotting the standard curve: using Fe 2+ With the final concentrations (0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 μmol / mL) as the abscissa (x) and ΔA standard as the ordinate (y), a standard curve was plotted, yielding the linear regression equation y = kx + b.

[0069] Sample determination: Add reagents: Add 180 μL of FRAP working solution to each well of a 96-well plate. Add 10 μL of the test sample to the assay group, and add 10 μL of distilled water to the blank group 2 (only 1-2 measurements are required). Reaction: Mix well at room temperature and react for 10 minutes.

[0070] Absorbance measurement: Measure the absorbance at 593 nm, record A measurement and A2 blank, and calculate ΔA = A measurement - A2 blank.

[0071] Sample concentration calculation: Substitute the measured value of ΔA into the standard curve equation y=kx+b to calculate the sample Fe concentration. 2+ Concentration x (μmol / mL).

[0072] Total antioxidant capacity calculation: Total antioxidant capacity (umol / ml) = x * V 反 / V 样

[0073] The method for determining the antioxidant activity (DPPH method) includes the following steps: preparing test solutions with mass concentrations of 0.025, 0.5, 1, 2, 4, 8, and 16 mg / mL using raw crude kudzu flower polysaccharide and deionized water.

[0074] Setting up the spectrophotometer: Preheat the spectrophotometer or microplate reader for at least 30 minutes, adjust the wavelength to 515nm, and zero the reagent set.

[0075] Preparation of Trolox positive control (TR): If Trolox is to be used as a positive control, it is recommended to use a 20 mmol / L Trolox solution. Prepare Trolox dilutions of different concentrations (1.6, 1.2, 0.8, 0.4, 0.2, 0.1 mmol / L) according to the required final concentration. If the extract concentration is high, it is recommended to prepare a Trolox dilution greater than 1.6 mmol / L using the extract.

[0076] Reaction system preparation: Add the following reagents to a 96-well plate or centrifuge tube: Assay group: 10 μL sample + 190 μL DPPH working solution. Control group: 10 μL sample + 190 μL DPPH working solution. TR group: 10 μL Trolox diluent + 190 μL DPPH working solution. Blank group: 200 μL DPPH working solution.

[0077] Mixing reaction: After thorough mixing, react at room temperature in the dark for 30 minutes.

[0078] Absorbance measurement: The absorbance of each group was measured at 515 nm and recorded as A measurement, A control, ATR, and A blank, respectively. Each dilution TR group and blank group only needs to be measured 1-2 times. A control group should be set up for each sample.

[0079] The formula for calculating the antioxidant activity (DPPH method) is as follows:

[0080] Sample DPPH radical scavenging rate (%) = (A 空白 -A 测定 +A 对照 ) / A 空白 ×100%

[0081] Trolox positive control group DPPH free radical scavenging rate (%) = (A 空白 -ATR) / A 空白 ×100%

[0082] The method for determining antioxidant activity (ABTS method) includes the following steps: preparing test solutions of kudzu polysaccharide with deionized water at mass concentrations of 0.025, 0.5, 1, 2, 4, 8, and 16 mg / mL.

[0083] Instrument warm-up: Preheat the spectrophotometer or microplate reader for at least 30 minutes, adjust the wavelength to 405nm, and zero the instrument using distilled water.

[0084] Trolox positive control (TR) preparation: If a Trolox positive control curve relationship is required, it is recommended to dilute the 20 mmol / L Trolox solution to 1.6, 1.2, 0.8, 0.4, 0.2, and 0.1 mmol / L, respectively, which are Trolox dilutions of different concentrations.

[0085] Sample and reagent preparation: Add the following reagents sequentially to the microplate tubes of a 96-well plate: Assay group: 10 μL sample, 10 μL distilled water, 20 μL reagent 1, 20 μL reagent 4 working solution, 170 μL ABTS working solution. Control group: 10 μL sample, 190 μL distilled water, 170 μL ABTS working solution. TR group: 10 μL Trolox diluent, 10 μL distilled water, 20 μL reagent 1, 20 μL reagent 4 working solution, 170 μL ABTS working solution. Blank group: 190 μL distilled water, 170 μL ABTS working solution. Reaction: After thorough mixing, incubate at room temperature in the dark for 6 minutes.

[0086] Absorbance measurement:

[0087] The absorbance was measured at a wavelength of 405 nm and recorded as A measurement, A control, ATR, and A blank. Note: Each concentration of TR group and blank group only needs to be measured 1-2 times, and a control tube should be set up for each sample.

[0088] Calculation of ABTS free radical scavenging ability:

[0089] ABTS radical scavenging rate of sample (D) S %)=(A 空白 -A 测定 +A 对照 ) / A 空白 ×100%

[0090] Trolox positive control group free radical scavenging rate (D) TR %)=(A 空白 -ATR) / A 空白 ×100%

[0091] Results analysis: As PFP concentration increases, DPPH ( Figure 3 A) and ABTS Figure 3 B) The free radical scavenging rate increased significantly and plateaued at around 5 mg / mL, indicating that it has a strong free radical scavenging ability. Figure 3The C-value shows that the total antioxidant capacity increases linearly with increasing PFP concentration, indicating that PFP has a concentration-dependent antioxidant effect. In summary, PFP exhibits good antioxidant activity within a certain concentration range.

[0092] Example 3: Treatment of non-alcoholic steatohepatitis in mice induced by a high-fat diet (methionine-restricted, choline-deficient diet) with kudzu polysaccharides

[0093] Experimental methods:

[0094] Seventy-two four-week-old male C57BL / 6J mice (SPF grade) were purchased and acclimatized in a clean environment for one week. They were then weighed and randomly divided into six groups of 12 mice each: control group, model group, silymarin positive control group, low-dose pueraria polysaccharide group (PFPL), medium-dose group (PFPM), and high-dose group (PFPH).

[0095] Control group: fed standard feed for 24 weeks, and gavaged with an equal volume of distilled water daily starting from week 16.

[0096] Model group: fed a methionine-restricted, choline-deficient (MCD) diet for 24 weeks, and gavaged with an equal volume of distilled water daily starting from week 16.

[0097] Silymarin group: fed with MCD diet for 24 weeks, and from week 16 onwards, silymarin solution (100 mg / kg / d) was administered by gavage daily.

[0098] PFPL group: fed with MCD diet for 24 weeks, and from week 16 onwards, administered a low dose of kudzu polysaccharide solution (50 mg / kg / d) by gavage daily.

[0099] PFPM group: fed with MCD diet for 24 weeks, and from week 16 onwards, administered a medium dose of kudzu polysaccharide solution by gavage daily (100 mg / kg / d).

[0100] PFPH group: fed with MCD diet for 24 weeks, and from week 16 onwards, administered high dose solution of kudzu polysaccharide by gavage daily (200 mg / kg / d).

[0101] Throughout the experiment, the mice were weighed weekly to record changes in body weight.

[0102] At the end of week 24 of the experiment, mice were fasted for 24 hours but allowed free access to water. They were then euthanized by cervical dislocation after anesthesia. Blood samples were collected for serological marker detection. Liver samples were divided into two parts: one part was stored at -80℃ for Oil Red O staining to detect lipid deposition in the liver; the other part was fixed in 4% paraformaldehyde for paraffin embedding, sectioning, and H&E staining to observe pathological changes in liver tissue.

[0103] Serological tests: After blood collection, the blood was incubated at 4°C for 2 hours, then centrifuged (3500 rpm, 15 min), and the supernatant was collected. The ALT, AST, TG, TC, and LDL-C assays were then performed according to the kit instructions.

[0104] Pathological examination: After the isolated liver tissue has been fixed, embedded, sectioned, stained, etc., the morphological changes of the liver tissue are observed under a microscope.

[0105] Results Analysis: This experiment induced non-alcoholic steatohepatitis (NASH) in mice using a methionine-restricted, choline-deficient diet to evaluate the effect of kudzu flower on NASH. Sixteen weeks after modeling, kudzu flower polysaccharides were injected intragastricly. The gavage dose of kudzu flower polysaccharide group L was 50 mg / kg / day, and the gavage dose of kudzu flower polysaccharide group H was 200 mg / kg / day. During the first 16 weeks, the body weight of mice fed the methionine-restricted, choline-deficient diet was significantly higher than that of the NC group. However, after gavage administration of kudzu flower polysaccharides, the weight gain of obese mice in the kudzu flower polysaccharide group decreased, while the body weight of obese mice in the kudzu flower polysaccharide group decreased to some extent. The accumulation of epididymal fat in mice can reflect the degree of obesity.

[0106] After 24 weeks of methionine-restricted and choline-deficient diets, compared with NC diet mice, liver weight (LW) and liver weight to body weight ratio (LW / BW) were significantly increased, while LW and LW / BW were significantly decreased in mice treated with kudzu polysaccharide. Epididymal fat weight after mouse sacrifice indicated that kudzu polysaccharide reduced epididymal fat accumulation in a dose-dependent manner. Figure 5 Compared with methionine-restricted and choline-deficient diets, the serum lipid levels (TG, TC, AST, ALT, LDL-C) in the kudzu polysaccharide group were significantly lower. Figure 6 A, B, C, D, E).

[0107] H&E staining and Oil Red O staining results showed that the methionine-restricted and choline-deficient diet group exhibited extensive hepatocyte steatosis and a small number of ballooning degenerations, while the kudzu polysaccharide treatment group showed significant improvement. Figure 7 A, B).

[0108] In summary, our research indicates that kudzu polysaccharides can alleviate non-alcoholic steatohepatitis in mice induced by methionine restriction and choline deficiency.

[0109] Example 4: Kudzu polysaccharide improves intestinal flora disorder induced by methionine restriction and choline deficiency in mice.

[0110] Experimental methods and animal models: Same as in Example 3.

[0111] Results Analysis: This experiment induced intestinal flora dysbiosis in mice using a methionine-restricted and choline-deficient diet to evaluate the regulatory effect of kudzu flower on intestinal flora dysbiosis. 16S rRNA sequencing of mouse colonic feces and α-diversity analysis showed that kudzu flower polysaccharide supplementation improved the decline in microbiota induced by methionine-restricted and choline-deficient diets. It increased microbial community diversity to a certain extent, and kudzu flower polysaccharide increased the CHAO1 index, Shannon index, and Simpson index (…). Figure 8 ).like Figure 9 As shown in Figure A, the results indicate that the PFPL group had the most endemic species (274), suggesting that this treatment may have caused the most significant changes in the gut microbiota. The model group had 215 endemic species, followed by the PFPM group (208), the control group (Con) (189), the silymarin group (186), and the PFPH group (141). We analyzed β-diversity using the PCoA measured by Bray-Curtis and hierarchical clustering of the samples. The results showed that the model group samples were tightly clustered and significantly distinguished from the other groups, indicating that their microbial community structure had undergone significant changes. The control group (Con), the silymarin group, and the three treatment groups (PFPL, PFPM, and PFPH) were relatively dispersed in the PCoA space, with some overlap between samples, suggesting that these groups had varying degrees of intervention or restoration effects on the gut microbiota structure. In particular, the PFPL group was far from the model group, indicating that PFPL may have a significant effect on regulating the gut microbiota. Figure 9 B). Changes in the relative abundance of gut microbiota at the phylum level. The control group (Con) showed a relatively balanced microbiota structure, containing multiple phyla. In the model group (Model), the relative abundance of Bacteroidota was significantly increased, exceeding half of the overall abundance, while other phyla such as Firmicutes decreased significantly, indicating that modeling caused severe disruption of the gut microbiota structure. After intervention with PFPL, PFPM, PFPH, and Silymarin, the abundance of Firmicutes gradually recovered, while Bacteroidota decreased, and the microbiota tended towards diversification and homeostasis. The PFPM group showed the most significant regulatory effect, with its microbiota composition closer to the normal control level. The results indicate that each intervention improved the model-induced microbiota imbalance to varying degrees, demonstrating a certain potential for gut microbiota regulation. Figure 10-11The figure shows changes in the relative abundance of gut microbiota at the species level. As can be seen from the figure, the control group (Con) had a higher proportion of Bacteroidia and Bacilli, and a relatively balanced microbiota structure; while in the model group (Model), the abundance of Clostridia_258463 was significantly increased, and the proportion of Bacteroidia was significantly decreased, indicating that modeling led to gut microbiota dysbiosis and promoted the enrichment of potential pathogens. After intervention with PFPL, PFPM, PFPH, and Silymarin, the abundance of Bacteroidia and Bacilli gradually recovered, the abundance of Clostridia_258463 decreased, and the microbiota structure tended to normalize. Among them, the PFPM group showed a more obvious recovery trend, and its microbiota composition was closer to that of the normal control group (…). Figure 12 Meanwhile, we used LEfSe analysis to examine the clade map based on OTUs, and the species with LDA values ​​greater than 4 were statistically analyzed as follows: Figure 13 As shown.

[0112] These results indicate that kudzu polysaccharide can improve gut microbiota dysbiosis in mice fed methionine-restricted and choline-deficient diets and reduce the abundance of pathogenic bacteria.

[0113] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of kudzu flower polysaccharides in the preparation of products for anti-oxidation, prevention and treatment of non-alcoholic steatohepatitis, and regulation of intestinal flora imbalance.

2. The application as described in claim 1, characterized in that, The products include food and pharmaceuticals.

3. The application as described in claim 1, characterized in that, The kudzu flower polysaccharide was prepared by water extraction and alcohol precipitation.

4. The application as described in claim 1, characterized in that, The antioxidant properties are at least characterized by total antioxidant capacity, DPPH free radical scavenging capacity and ABTS free radical scavenging capacity; The prevention and treatment of non-alcoholic steatohepatitis (NAH) is at least manifested in reducing liver organ indices, pathological damage, serological liver indicators, reducing lipid accumulation in hepatocytes, and improving obesity. The regulation of intestinal flora imbalance specifically manifests as improving intestinal microbiota dysbiosis and reducing the abundance of pathogenic bacteria.

5. A method for preparing kudzu flower polysaccharide, characterized in that, The preparation method includes preparing kudzu flower polysaccharides using a water extraction and alcohol precipitation method. Preferably, the preparation method includes: S1. After drying the kudzu flowers, ethanol was added for reflux extraction to remove fat-soluble substances and obtain the residue. S2. Add water to the residue and boil it to extract the water extract. Centrifuge to remove insoluble components and collect the supernatant. S3. Add ethanol to the supernatant for alcohol precipitation, collect the precipitate to obtain crude polysaccharide, and remove the protein from the crude polysaccharide to obtain the final product.

6. The preparation method according to claim 5, characterized in that, In step S1, the drying is a drying process, specifically a drying process performed at 40-60℃ (preferably 50℃). The specific method for ethanol reflux extraction includes: using high-concentration ethanol (such as 95% ethanol) for reflux extraction 1 to 5 times (preferably 3 times), each time for 1 to 5 hours (preferably 2 hours); the ratio of kudzu flower to ethanol is 1:2-6 (preferably 1:4, kg / L).

7. The preparation method according to claim 5, characterized in that, In step S2, the specific conditions for adding water and boiling include: a material-to-liquid ratio of 1:1 to 10 (kg / L), preferably 1:5; a boiling time of 1 to 3 hours, preferably 2 hours; and 1 to 5 times of adding water and boiling (preferably 3 times).

8. The preparation method according to claim 5, characterized in that, In step S3, the alcohol precipitation method includes: adding 95% ethanol to the supernatant, allowing it to stand, and collecting the precipitate; the standing time is 10-40 hours (preferably 24 hours); protein removal is performed using the Sevage method, specifically, using Sevage reagent (CH3Cl:BuOH = 4:1, v / v) to remove protein 5-8 times. Furthermore, the polysaccharide obtained in step S3 is subjected to lyophilization and column elution purification. The specific method for column elution purification includes: dissolving the lyophilized kudzu polysaccharide in water and eluting it sequentially with 1-4 column volumes (preferably 2 times) of distilled water, 0.2M NaCl, 0.5M NaCl, and 1.0M NaCl solutions using a DEAE agarose gel FF column (3.0×50cm) at a flow rate of 0.5-2 mL / min (preferably 1.0 mL / min); collecting the eluent in 10 mL test tubes and determining the sugar content using an anthrone-sulfuric acid method at 630 nm with a UV-Vis spectrophotometer, and plotting the separation curve; combining test tubes with the same peak, concentrating, and lyophilizing to obtain prepared kudzu polysaccharide GHP-E2; and then using Chromdex... The prepared kudzu flower polysaccharide GHP-E2 was separated using a 75PG column (1.6cm×100cm) at a flow rate of 0.5-2mL / min (preferably 1.0mL / min) with 1-4 times (preferably 1 times) column volume of distilled water. The eluent was collected in a 5mL test tube, and the sugar content was measured using the anthrone-sulfuric acid method. The separation curve was plotted. Test tubes with the same peak were combined, concentrated, and lyophilized to obtain the prepared kudzu flower polysaccharide GHPE2-F1.

9. A product for antioxidation, prevention and treatment of non-alcoholic steatohepatitis and / or regulation of intestinal flora imbalance, characterized in that, The product contains at least kudzu flower polysaccharides.

10. The product as described in claim 9, characterized in that, The kudzu flower polysaccharide is obtained by the preparation method according to any one of claims 5-8; The products include food and pharmaceuticals.

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