Application of pueraria flower isoflavone in resisting oxidation, improving non-alcoholic steatohepatitis and regulating intestinal flora

Abstract

The invention belongs to the technical field of medicines, health foods and medicines, and particularly relates to application of pueraria flower isoflavone in resisting oxidation, improving non-alcoholic steatohepatitis and regulating intestinal flora. Specifically, pueraria flower isoflavone is obtained by taking pueraria flower as a raw material and adopting an ethanol reflux method, and an in-vitro anti-oxidation test proves that the pueraria flower isoflavone has in-vitro anti-oxidation capability and has a synergistic anti-oxidation effect with a cynanchum bungei flower ethyl acetate extract; meanwhile, animal experiments prove that the pueraria flower isoflavone has the effects of treating non-alcoholic steatohepatitis and improving intestinal flora disorder, and has good health care and treatment values. Meanwhile, the extraction and preparation method is simple, can be easily converted into a modern industrial production process, and has great development potential, so that good economic and social benefits can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical health foods and pharmaceuticals, and specifically relates to the application of kudzu flower isoflavones in anti-oxidation, improving non-alcoholic fatty liver disease and regulating intestinal flora. Background Art

[0002] Nonalcoholic steatohepatitis (NASH), a metabolic liver disease that progresses from simple steatosis to liver fibrosis, has become a major cause of chronic liver disease. Its pathogenesis is complex and closely linked to multiple factors, including type 2 diabetes, hypertension, hyperlipidemia, metabolic syndrome, and intestinal dysbiosis. However, its specific mechanisms remain largely unexplained, and effective treatments are lacking.

[0003] Existing research indicates that intestinal microbes, acting as a bridge between diet and host health, are deeply involved in the progression of NASH. A high-fat diet can lead to intestinal flora disorders, manifested by increased abundance of Firmicutes, decreased abundance of Bacteroidetes, and an increased Firmicutes / Bacteroidetes (F / B) ratio, which in turn exacerbates the development of NASH. Experiments on germ-free mice have shown significant resistance to NASH in the absence of intestinal microbes, while transplantation of the microbiome of mice with metabolic syndrome significantly increases susceptibility, confirming that the intestinal microbiome is closely related to the occurrence and development of NASH.

[0004] Flos Puerariae is a plant of the genus Puerariae in the family Leguminosae. Pueraria lobata (Willd.) Ohwi] or bitter kudzu ( Pueraria thomsonii The dried flower buds of the kudzu plant (Pueraria benthamiana) are widely used in traditional Chinese medicine to soothe hangovers, protect the liver, and clear heat and reduce internal heat. Pueraria flowers are often used in teas, decoctions, and medicinal preparations to alleviate alcoholic and non-alcoholic liver damage. Traditional medicine believes they have the effects of soothing alcohol, invigorating the spleen, and clearing heat and dampness. Modern pharmacological research has shown that kudzu flowers and their active ingredients have antioxidant, hepatoprotective, lipid-lowering, and intestinal microbiome-regulating properties. However, research on flavonoids in kudzu flowers remains relatively scarce. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention provides the use of kudzu flower isoflavones in anti-oxidation, improving non-alcoholic steatohepatitis, and regulating intestinal flora. Specifically, the present invention constructs a mouse model of non-alcoholic steatohepatitis by restricting methionine and choline-deficient diet. Experiments have shown that kudzu flower isoflavones have the efficacy of synergistically increasing in vitro antioxidant capacity, improving non-alcoholic steatohepatitis, and regulating intestinal flora. Serological index analysis, stained section microscopy and other techniques have demonstrated that kudzu flower isoflavones can reduce serological liver indicators, reduce hepatocyte lipid accumulation, and improve liver inflammation in mice; at the same time, the intestinal flora 16S rRNA sequencing technology has clarified its function of improving intestinal flora and liver metabolites. Based on the above research results, the present invention has been completed.

[0006] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows: The first aspect of the present invention provides the use of kudzu flower isoflavones in the preparation of products for anti-oxidation, improving non-alcoholic steatohepatitis and / or regulating intestinal flora.

[0007] Such products include, but are not limited to, food and medicine.

[0008] The kudzu flower isoflavones in the present invention are isoflavone components extracted from kudzu flowers. In the present invention, the kudzu flower isoflavones are prepared by ethanol reflux extraction.

[0009] The antioxidant activity is at least manifested as DPPH free radical scavenging ability, ABTS free radical scavenging ability and total antioxidant capacity (T-AOC).

[0010] In particular, the present invention has found through research that the kudzu flower isoflavones and the Cynanchum multiflorum flower ethyl acetate extract have good synergistic antioxidant capacity when used in combination.

[0011] The improvement of non-alcoholic fatty liver disease is at least manifested in the reduction of liver organ index, pathological damage, serological liver index, and reduction of hepatocyte lipid accumulation.

[0012] The regulation of intestinal flora is specifically manifested in regulating intestinal microbial flora imbalance and reducing the abundance of pathogenic bacteria.

[0013] In a specific embodiment of the present invention, the non-alcoholic fatty liver disease and intestinal flora imbalance are mediated by methionine restriction and choline deficiency feeding.

[0014] A second aspect of the present invention provides a method for preparing kudzu flower isoflavones, the method comprising: S1. Soaking the kudzu flower medicinal material: Soak the kudzu flower medicinal material in 90% ethanol overnight to promote the dissolution of the active ingredients; S2. Hot reflux extraction: hot reflux extraction was performed using 90%, 85%, and 70% ethanol solutions in sequence, and the filtrate was filtered to obtain a filtrate; S3. Filtrate treatment: The filtrates obtained by extraction with the ethanol solutions of different concentration gradients are combined, concentrated, and dried to obtain the product.

[0015] Among them, in step S2, the specific conditions of hot reflux extraction are: extraction for each concentration 1-3 times (preferably 2 times), each time for 1-3 hours (preferably 2 hours); the solid-liquid ratio is controlled at 1:6~8, g / mL.

[0016] In the step S3, the concentration is reduced-pressure concentration, and the drying is freeze-drying.

[0017] The UV-visible spectrophotometry method was used, referring to the flavonoid determination method in the "Chinese Pharmacopoeia" (2020 edition), and the total flavonoid content of the prepared dry powder was determined to be 85.06%.

[0018] The inventors have proved through experiments that the kudzu flower isoflavone component prepared by the above method can play an effect in anti-oxidation, improving non-alcoholic fatty liver disease and regulating intestinal flora.

[0019] Therefore, the third aspect of the present invention provides a product for anti-oxidation, improving non-alcoholic fatty liver disease and / or regulating intestinal flora, wherein the product contains at least kudzu flower isoflavones.

[0020] Furthermore, the kudzu flower isoflavones are obtained by the above-mentioned preparation method.

[0021] Furthermore, the product is a combination of kudzu flower isoflavones and Cynanchum multiflorum flower ethyl acetate extract, with the mass ratio of the two being 1-5:5-9.

[0022] The preparation method of the ethyl acetate extract of Cynanchum multiflorum flowers is as follows: The herbal material of Cynanchum multiflorum is crushed and cold-extracted twice with 90% ethanol and once with 80% ethanol; the three medicinal solutions are combined, concentrated, and dried to obtain an extract; the extract is mixed with water, and extracted with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 4 to 8 times, respectively; after each extraction, the organic phase is separated and discarded, and the aqueous phase is retained for the next solvent extraction; the ethyl acetate extracts are combined, concentrated, and dried to obtain the ethyl acetate extract of Cynanchum multiflorum.

[0023] The present invention proves through experiments that the combination of kudzu flower isoflavones and Cynanchum multiflorum flower ethyl acetate extract has a synergistic antioxidant effect, which is specifically manifested in significantly improving the DPPH free radical scavenging ability, ABTS free radical scavenging ability and total antioxidant capacity (T-AOC).

[0024] The products include but are not limited to food and medicine. Obviously, the medicine prepared by the present invention belongs to the category of biological medicine.

[0025] The food can be understood as any edible form. For example, the food in the present invention includes ordinary food and special food. The special food in the present invention includes health food and special medical purpose formula food. Ordinary food is relative to special food and is food suitable for everyone.

[0026] The drug can be administered in a unit dosage form, and the dosage form can be a liquid dosage form or a solid dosage form. The liquid dosage form can be a true solution, a colloid, a microgranular dosage form, an emulsion dosage form, or a suspension dosage form. Other dosage forms include tablets, capsules, dripping pills, aerosols, pills, powders, solutions, emulsions, granules, suppositories, lyophilized powder injections, inclusion compounds, landfills, patches, and liniments. The drug can be for use in humans and non-human animals.

[0027] Beneficial technical effects of one or more of the above technical solutions: In the above technical solution, kudzu flower isoflavones are obtained using ethanol reflux method using kudzu flower as raw material. In vitro antioxidant tests have demonstrated that kudzu flower isoflavones have antioxidant capacity. Animal experiments have also demonstrated that kudzu flower isoflavones are effective in treating non-alcoholic steatohepatitis and improving intestinal flora disorders, demonstrating their excellent health and therapeutic value. Furthermore, the extraction and preparation method of the present invention is simple and can be easily converted to a modern industrial production process, thus possessing great development potential and potentially achieving significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The in vitro antioxidant capacity of the kudzu flower isoflavones of the present invention is shown in Figure 2. (A) DPPH; (B) ABTS; (C) T-AOC.

[0029] Figure 2 The in vitro antioxidant capacity of the Pueraria flower complex of the present invention, (A) T-AOC; (B) DPPH; (C) ABTS.

[0030] Figure 3 This is the body weight measurement of mice of the present invention.

[0031] Figure 4 The liver specific gravity and epididymal fat specific gravity of mice of the present invention are measured. (A) The liver specific gravity of mice; (B) The epididymal fat specific gravity of mice.

[0032] Figure 5 The effects of kudzu flower isoflavones of the present invention 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.

[0033] Figure 6 Figure 2 shows the pathological damage of the liver in NASH mice of the present invention. (A) H&E staining: the scale bar in the upper figure is 100 μm; the scale bar in the lower figure is 50 μm; (B) Oil Red O staining: the scale bar in the upper figure is 100 μm; the scale bar in the lower figure is 50 μm; (C) NAS score; (D) quantification of the Oil Red O-positive area.

[0034] Figure 7 Alpha diversity of the present invention; (A) Chao1 index; (B) Shannon index; (C) Simpson index.

[0035] Figure 8 This is the Beta diversity of the present invention.

[0036] Figure 9 The kudzu flower isoflavones of the present invention regulate the intestinal flora disorder of mice at the phylum level.

[0037] Figure 10 The kudzu flower isoflavones of the present invention regulate the intestinal flora disorder of mice at the species level. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0039] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0040] The present invention will be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the sales company. Materials and reagents used in the examples are commercially available unless otherwise specified.

[0041] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0042] Example 1: Preparation of Pueraria lobata isoflavones and Cynanchum multiflorum flower ethyl acetate extracts Pueraria lobata isoflavones were prepared by the following steps: Pueraria flowers were extracted under reflux using 90%, 85%, and 70% (v / v) ethanol solutions, sequentially at a mass-to-volume ratio of 1:8 (i.e., 100 g of the material to 800 mL of solvent). Each extraction step was repeated twice, lasting 2 hours each. (Note: The Pueraria flowers were soaked in 90% ethanol overnight before the first extraction.) The combined filtrates were concentrated under reduced pressure and freeze-dried to prepare a Pueraria flower crude extract.

[0043] The ethyl acetate extract of Cynanchum multiflorum flowers was prepared by the following steps: 4 kg of Cynanchum multiflorum flowers were ground into a fine powder and extracted twice with 90% ethanol (32 L and 24 L for 15 and 10 days, respectively), followed by a single cold extraction with 80% ethanol (24 L for 8 days). The three extracts were combined, concentrated under reduced pressure, and freeze-dried to yield 1.2 kg of an extract. The extract was then mixed with 1200 mL of water. The extract suspension (1200 mL) was extracted with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol in a gradient fashion, with each solvent extracted 4 to 8 times. After each extraction, the organic phase was separated and discarded, and the aqueous phase was retained for extraction with the next solvent. The ethyl acetate extracts were combined, concentrated under reduced pressure, and freeze-dried to yield 36.5 g of Cynanchum multiflorum flower ethyl acetate extract.

[0044] Preparation of a complex of ethyl acetate extract from Cynanchum multiflorum flowers and kudzu flower isoflavones: Dry and pulverize the ethyl acetate extract from Cynanchum multiflorum flowers and kudzu flower isoflavones, respectively, and pass through a 200-mesh sieve (0.0750 mm diameter) for later use. For the complex of the two components, add kudzu flower isoflavones to the ethyl acetate extract from Cynanchum multiflorum flowers according to the mass ratio. The kudzu flower isoflavones are added in a ratio of 10% to 40% and mixed thoroughly to obtain the kudzu flower isoflavone-Cai Shou Wu flower complex, hereinafter referred to as the "kudzu flower complex."

[0045] The total flavonoid content was determined by UV-visible spectrophotometry according to the Chinese Pharmacopoeia (2020 edition). The kudzu flower isoflavones were obtained and used in subsequent Examples 2 to 4.

[0046] Example 2: In vitro antioxidant capacity testing of kudzu flower isoflavones and kudzu flower complexes Determination of the in vitro antioxidant capacity of kudzu flower isoflavones, ethyl acetate extract of Cynanchum multiflorum flower and kudzu flower complex: The antioxidant activity determination method (T-AOC method) includes the following steps: taking the sample to be tested and preparing a test solution with a mass concentration of 0.025, 0.5, 1, 2, 4, 8, and 16 mg / mL with deionized water.

[0047] Calibration curve preparation: 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.

[0048] Prepare standard solution: Take 100 μL of each concentration of Fe 2+ Add 100 μL of FRAP working solution to the standard solution. Mix well at room temperature, react for 10 minutes, and measure the absorbance at 593 nm. Record A 标准 and A1 空白 , calculate ΔA 标准 = A 标准 - A1 空白 .

[0049] Drawing a standard curve: Fe 2+ The final concentration (0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125µmol / mL) is the horizontal axis (x), and ΔA 标准 With the ordinate (y) as the standard curve, we can get the linear regression equation y = kx + b.

[0050] Sample Assay: Add reagents: Add 180 µL of FRAP working solution to each well of a 96-well plate. Add 10 µL of the sample to the assay group and 10 µL of distilled water to the blank group (only one or two assays are required). React: Mix thoroughly and incubate at room temperature for 10 minutes.

[0051] Absorbance measurement: Measure the absorbance at 593 nm and record A 测定 and A2 空白 , calculate ΔA = A 测定 - A2 空白 .

[0052] Calculate sample concentration: Substitute the ΔA value into the standard curve equation y = kx + b to calculate the sample Fe 2+ Concentration x (µmol / mL).

[0053] Calculation of total antioxidant capacity: Total antioxidant capacity (μmol / ml) = x * V 反 / V 样 The antioxidant synergistic ability of the kudzu flower isoflavone complex was calculated based on the Chou-Talalay method, namely the combination index (CI); if CI < 1, the two have a synergistic effect, and the smaller the value, the stronger the synergistic effect; CI = 1, it is an additive effect; CI > 1, it is an antagonistic effect, and the larger the value, the stronger the antagonistic effect.

[0054] Table 1 T-AOC synergistic analysis of five kudzu flower isoflavone complexes

[0055] The Chou-Talalay method was used to analyze the synergistic effect of kudzu flower isoflavones and ethyl acetate extract of Cynanchum multiflorum on total antioxidant capacity (T-AOC). The combination index (CI) was calculated to assess the nature of the interaction. The results showed that at 10%, 20%, 30%, 40%, and 50% ratios of the kudzu flower isoflavone complex, the CI values ​​were 0.3098, 0.3380, 0.2968, 0.2713, and 0.2092, respectively, all less than 1, indicating a significant synergistic effect between kudzu flower isoflavones and ethyl acetate extract of Cynanchum multiflorum in this experimental system. As the ratio of the kudzu flower isoflavone complex increased, the CI value decreased, and the synergistic effect strengthened, reaching the highest synergistic effect at 50% ratio (CI = 0.2092).

[0056] The antioxidant activity determination method (DPPH method) comprises the following steps: Pueraria lobata isoflavones, Cynanchum multiflorum flower ethyl acetate extract and Pueraria lobata flower complex are prepared into test solutions with mass concentrations of 0.00125, 0.0025, 0.01, 0.02, 0.04, 0.08, 0.16 and 0.32 mg / mL with deionized water. Set up the photometer: Preheat the spectrophotometer or microplate reader for more than 30 minutes, adjust the wavelength to 515 nm, and zero the reagent.

[0057] Preparation of Trolox Positive Control (TR): If Trolox needs to be set as a positive control, it is recommended to use a 20 mmol / L Trolox solution.

[0058] Prepare Trolox dilutions of different concentrations (1.6, 1.2, 0.8, 0.4, 0.2, and 0.1 mmol / L) according to the desired final concentration. If the extract concentration is higher, use Reagent 3 with the extract to prepare a Trolox dilution greater than 1.6 mmol / L.

[0059] 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.

[0060] Mixing reaction: After thorough mixing, react in the dark at room temperature for 30 minutes.

[0061] Absorbance determination: The absorbance of each group was measured at 515 nm and recorded as A 测定 、A 对照 , ATR and A 空白 Each dilution TR group and blank group only need to be measured 1-2 times. A control group should be set up for each sample.

[0062] The calculation formula of the antioxidant activity determination method (DPPH method) is: DPPH free radical scavenging rate of the sample (%) = (A 空白 -A 测定 +A 对照 ) / A 空白 ×100% DPPH free radical scavenging rate of Trolox positive control group (%) = (A 空白 -ATR) / A 空白 ×100% Table 2 DPPH synergistic analysis of five kudzu flower isoflavone complexes

[0063] The analysis results showed that the CI values ​​for all five blend ratios were less than 1, indicating a synergistic antioxidant effect between kudzu flower isoflavones and Cynanchum multiflorum flower ethyl acetate extract in DPPH radical scavenging activity. Among them, the CI value at a blend ratio of 30% was the lowest (0.518), indicating the most significant synergistic effect, indicating that this ratio has a superior combination effect in enhancing antioxidant activity.

[0064] The antioxidant activity determination method (ABTS method) comprises the following steps: Pueraria lobata isoflavones, Cynanchum multiflorum flower ethyl acetate extract and Pueraria lobata flower complex are prepared into test solutions with mass concentrations of 0.0025, 0.005, 0.01, 0.02, 0.04, 0.08 and 0.16 mg / mL with deionized water.

[0065] Instrument preheating: Preheat the spectrophotometer or microplate reader for at least 30 minutes, adjust the wavelength to 405 nm, and zero it with distilled water.

[0066] Trolox positive control (TR) configuration: It is recommended to use 20 mmol / L Trolox solution, which can be diluted to 1.6, 1.2, 0.8, 0.4, 0.2, or 0.1 mmol / L as needed to obtain Trolox dilutions of different concentrations.

[0067] Sample and Reagent Preparation: Add the following reagents sequentially to microplate tubes in a 96-well plate: Assay Group: 10 µL of sample, 10 µL of distilled water, 20 µL of Reagent 1, 20 µL of Reagent IV Application Solution, and 170 µL of ABTS Working Solution. Control Group: 10 µL of sample, 190 µL of distilled water, and 170 µL of ABTS Working Solution. TR Group: 10 µL of Trolox Diluent, 10 µL of distilled water, 20 µL of Reagent 1, 20 µL of Reagent IV Application Solution, and 170 µL of ABTS Working Solution. Blank Group: 190 µL of distilled water and 170 µL of ABTS Working Solution. Reaction: After thorough mixing, incubate at room temperature in the dark for 6 minutes.

[0068] Absorbance determination: The absorbance was measured at a wavelength of 405 nm and recorded as A 测定 、A 对照 , ATR and A 空白 Note: Each concentration of TR group and blank group only needs to be measured 1-2 times, and a control tube must be set up for each sample.

[0069] ABTS free radical scavenging ability calculation: ABTS free radical scavenging rate of samples (D S %) = (A 空白 -A 测定 +A 对照 ) / A 空白 ×100% The free radical scavenging rate of Trolox positive control group (D TR %) = (A 空白 -ATR) / A 空白 ×100% Table 3 ABTS synergistic analysis of five kudzu flower isoflavone complexes

[0070] The results showed that as the proportion of kudzu flower isoflavones increased, the CI value gradually decreased, indicating an enhanced synergistic antioxidant effect. Although the CI value of the 10% compound ratio was close to 1, indicating an approximately additive effect, the CI values ​​of the remaining groups were all less than 1, with the 50% compound ratio having the lowest CI value (0.6657), indicating the most significant synergistic effect.

[0071] Result analysis: With the increase of PFI concentration, DPPH ( Figure 1 A) and ABTS ( Figure 1 Middle B) The free radical scavenging rate increased rapidly and approached saturation at lower concentrations (<0.1 mg / mL), indicating that kudzu flower isoflavones have strong and efficient free radical scavenging ability. Figure 1Figure C shows that the total antioxidant capacity increases rapidly between 0–5 mg / mL and then stabilizes, indicating that the antioxidant capacity of kudzu flower isoflavones is concentration-dependent. Overall, kudzu flower isoflavones exhibit significant antioxidant activity even at low doses. Pueraria flower isoflavones and Cynanchum multiflorum flower ethyl acetate extract all exhibited synergistic effects with CI values ​​less than 1 in the three antioxidant systems of T-AOC, DPPH, and ABTS, indicating that the combination of the two has a good synergistic antioxidant effect in vitro ( Figure 2 In the T-AOC assay, as the proportion of kudzu flower isoflavones increased, the CI value gradually decreased, and the synergistic effect was strongest at a 50% compound ratio (Table 1). In the DPPH system, a 30% compound ratio had the best effect (Table 2). In the ABTS system, the synergistic effect also increased with increasing ratio (Table 3).

[0072] In summary, the combination of the two exhibits stable and significant synergistic effects in different antioxidant mechanisms and has good application potential.

[0073] Example 3: Treatment of non-alcoholic steatohepatitis in mice induced by high-fat diet (methionine restriction, choline-deficient diet) with kudzu flower isoflavones Experimental Methods: Seventy-two 4-week-old male C57BL / 6J mice (SPF grade) were purchased and acclimated in a clean environment for one week. They were then weighed and randomly divided into six groups, each consisting of 12 mice: control group, model group, positive drug silymarin group, low-dose kudzu flower isoflavones group (PFIL), medium-dose kudzu flower isoflavones group (PFIM), and high-dose kudzu flower isoflavones group (PFIH).

[0074] Control group: The mice were fed with a standard diet for 24 weeks and were gavaged with an equal volume of distilled water daily starting from the 16th week.

[0075] The Model group was fed a methionine-restricted, choline-deficient (MCD) diet for 24 weeks and received an equal volume of distilled water by gavage daily starting from week 16. The Silymarin group was fed an MCD diet for 24 weeks and received a silymarin solution (100 mg / kg / d) by gavage daily starting from week 16. The PFIL group was fed an MCD diet for 24 weeks and received a low-dose kudzu flower isoflavone solution (50 mg / kg / d) by gavage daily starting from week 16. The PFIM group was fed an MCD diet for 24 weeks and received a medium-dose kudzu flower isoflavone solution (100 mg / kg / d) by gavage daily starting from week 16. The PFIH group was fed an MCD diet for 24 weeks and received a high-dose kudzu flower isoflavone solution (200 mg / kg / d) by gavage daily starting from week 16.

[0076] During the entire experimental period, the mice were weighed weekly and their body weight changes were recorded.

[0077] At the end of the 24th week of the experiment, mice were fasted for 24 hours but not water and then sacrificed by cervical dislocation after anesthesia. Blood samples were collected for serological analysis, and liver samples were divided into two parts: one part was stored at -80°C and used for Oil Red O staining to detect hepatic lipid deposition; the other part was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E to observe liver tissue pathological changes.

[0078] Serological testing: After blood collection, incubate at 4°C for 2 hours, centrifuge (3500 rpm, 15 minutes), and collect the supernatant. ALT, AST, TG, TC, and LDL-C levels will then be tested according to the kit instructions.

[0079] Pathological examination: After the ex vivo liver tissue is fixed, embedded, sectioned, and stained, the morphological changes of the liver tissue are observed under a microscope.

[0080] Results: This study used a methionine-restricted, choline-deficient diet to induce nonalcoholic fatty liver disease in mice to evaluate the effect of Pueraria lobata on NASH. Sixteen weeks after modeling, Pueraria lobata isoflavones were injected intragastrically. The low-dose Pueraria lobata isoflavones group received an oral dose of 50 mg / kg / d, while the high-dose Pueraria lobata isoflavones group received an oral dose of 200 mg / kg / d. Figure 3 As shown, during the first 16 weeks, mice fed a methionine-restricted, choline-deficient diet weighed significantly more than the control group. However, the obese mice in the high-dose kudzu flower isoflavone group experienced a modest weight loss. Epididymal fat accumulation in mice can reflect the degree of obesity.

[0081] After 24 weeks of feeding with a methionine-restricted, choline-deficient diet, liver weight (LW) and liver weight to body weight ratio (LW / BW) increased significantly compared with mice fed a control diet, while LW and LW / BW of mice treated with kudzu flower isoflavones decreased significantly. Epididymal fat weight after mice were sacrificed indicated that kudzu flower isoflavones reduced epididymal fat accumulation in a dose-dependent manner ( Figure 4 A to B). Compared with the methionine-restricted and choline-deficient diets, the serum lipid levels (TG, TC, AST, ALT, and LDL-C) of the kudzu flower isoflavone group were significantly reduced ( Figure 5 A to E).

[0082] The results of H&E staining and Oil Red O staining showed that the methionine-restricted and choline-deficient diet groups showed extensive hepatocyte fatty degeneration and a small amount of ballooning, while the kudzu flower isoflavones-treated group showed significant improvement ( Figure 6 A to B).

[0083] In summary, the research in this example shows that kudzu flower isoflavones can alleviate non-alcoholic fatty liver disease in mice induced by methionine restriction and choline deficiency diet.

[0084] Example 4: Pueraria lobata isoflavones improve intestinal flora disturbance in mice induced by methionine restriction and choline deficiency diet Experimental methods and animal models: same as in Example 3.

[0085] Results: This study used a methionine-restricted, choline-deficient diet to induce intestinal microbial disturbances in mice to evaluate the regulatory effects of kudzu flower on intestinal microbial disturbances. 16S rRNA sequencing and α-diversity analysis of mouse colonic feces demonstrated that kudzu flower isoflavone supplementation ameliorated the decrease in microbial diversity induced by the methionine-restricted, choline-deficient diet. Supplementation with kudzu flower isoflavones increased microbial diversity to a certain extent, and kudzu flower isoflavones increased the Chao1 index ( Figure 7 A), Shannon Index ( Figure 7 B) and Simpson index ( Figure 7 (C). Figure 8 As shown, each group has 77 core genes in common, among which the low-dose kudzu flower isoflavone group has the largest number of specific genes (253); the model group, the medium-dose kudzu flower isoflavone group and the positive drug silymarin group have 215, 194 and 186 specific genes, respectively; the high-dose kudzu flower isoflavone group has the least, at 169. PCoA1 and PCoA2 explained a total of 70.35% of the total variation (55.75% for PCoA1 and 14.6% for PCoA2). The samples in the control group were obviously clustered, and were clearly distinguished from the model group and the low-dose kudzu flower isoflavone group, suggesting that there were structural differences in the microbial composition between the different treatment groups. Among them, the low-dose kudzu flower isoflavone group was relatively far away from the model group, indicating that its intervention had a more significant effect on the structure of the microbial community. In the phylum-level difference analysis ( Figure 9 ), the results showed that some bacterial genera in the model group (such as CAG-485, Dubosiella ) abundance increased significantly, while Lactobacillus The abundance of beneficial bacteria decreased, suggesting that the modeling led to an imbalance in the intestinal flora. After different treatments, the low-dose group of kudzu flower isoflavones, the medium-dose group of kudzu flower isoflavones, the high-dose group of kudzu flower isoflavones, and the positive drug silymarin group all regulated the flora structure to a certain extent, as shown by Lactobacillus 、 Muribaculum The abundance of probiotics such as kudzu flower isoflavones rebounded, and the effect was most obvious in the low-dose group, and the bacterial composition was closer to the normal control group, suggesting that it has good potential in regulating intestinal flora. In the species level difference analysis ( Figure 10 ), as the treatment groups progressed from the control group to the model group, then to the low-dose group of kudzu flower isoflavones, the medium-dose group of kudzu flower isoflavones, the high-dose group of kudzu flower isoflavones, and finally to the positive drug silymarin group, Muribaculum gordoncarteri The relative abundance of Dubosiella sp900403415The relative abundance of gradually increased, indicating that kudzu flower isoflavones play an important role in regulating the composition of the microbial community. These results indicate that kudzu flower isoflavones can improve the intestinal microbiota imbalance in mice fed a methionine-restricted or choline-deficient diet and reduce the abundance of pathogenic bacteria.

[0086] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of kudzu flower isoflavones in the preparation of products for antioxidant, improvement of non-alcoholic fatty liver disease and / or regulation of intestinal flora.

2. The use according to claim 1, characterized in that The products include food and medicine.

3. The use according to claim 1, characterized in that The kudzu flower isoflavones are prepared by ethanol reflux extraction.

4. The use according to claim 1, characterized in that The antioxidant capacity is at least DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and total antioxidant capacity; The improvement of non-alcoholic fatty liver disease is at least manifested in a reduction in liver organ index, pathological damage, serological liver indicators, and a reduction in hepatocyte lipid accumulation; The regulation of intestinal flora is specifically manifested in regulating intestinal microbial flora imbalance and reducing the abundance of pathogenic bacteria.

5. A method for preparing kudzu flower isoflavones, characterized in that: The preparation method comprises: S1. Soaking the kudzu flower medicinal material: Soak the kudzu flower medicinal material in 90% ethanol overnight; S2. Hot reflux extraction: hot reflux extraction was performed using 90%, 85%, and 70% ethanol solutions in sequence, and the filtrate was filtered to obtain a filtrate; S3. Filtrate treatment: The filtrates obtained by extraction with the ethanol solutions of different concentration gradients are combined, concentrated, and dried to obtain the product.

6. The preparation method according to claim 5, wherein In step S2, the specific conditions of the hot reflux extraction are: 1-3 extractions for each concentration, each for 1-3 hours; the solid-liquid ratio is 1:6-8, g / mL; In the step S3, the concentration is reduced-pressure concentration, and the drying is freeze-drying.

7. A product for anti-oxidation, improving non-alcoholic steatohepatitis and / or regulating intestinal flora, characterized in that: The product contains at least kudzu flower isoflavones.

8. The product according to claim 7, characterized in that The kudzu flower isoflavones are obtained by the preparation method according to any one of claims 5-6.

9. The product according to claim 8, characterized in that The product is a combination of kudzu flower isoflavones and Cynanchum multiflorum flower ethyl acetate extract, with the mass ratio of the two being 1-5:5-9; The preparation method of the ethyl acetate extract of Cynanchum multiflorum flowers is as follows: The herbal material of Cynanchum multiflorum is crushed and cold-extracted twice with 90% ethanol and once with 80% ethanol; the three medicinal solutions are combined, concentrated, and dried to obtain an extract; the extract is mixed with water, and extracted with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol for 4 to 8 times, respectively; after each extraction, the organic phase is separated and discarded, and the aqueous phase is retained for the next solvent extraction; the ethyl acetate extracts are combined, concentrated, and dried to obtain the ethyl acetate extract of Cynanchum multiflorum.

10. The product according to claim 7, characterized in that The products include food and medicine.

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