Application of pueraria flower extract in preparation of medicine for treating obesity and health care food for helping to control body fat

Abstract

The application belongs to the field of medicine and health food, and discloses application of pueraria lobata flower extract in preparation of medicine for preventing and / or treating obesity; the pueraria lobata flower extract is prepared from pueraria lobata flower as raw material and 70-90% ethanol aqueous solution as extraction solvent through reflux extraction, filtration, concentration under reduced pressure and drying. The application also discloses application of the pueraria lobata flower extract in preparation of health food for helping to control fat in the body and application in preparation of health food for helping to maintain blood lipid health level. The pueraria lobata flower extract has in-vitro lipid-lowering capacity; the pueraria lobata flower extract can significantly slow down weight increase of obese mice, reduce serum blood lipid factor level, reduce lipid accumulation in liver, and can obviously improve small fat hypertrophy and make fat tissue cell morphology tend to normal, and can be used for treating and / or preventing obesity and related symptoms caused by high-fat diet.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and health food, and relates to the application of kudzu flower extract in the preparation of weight loss products. Specifically, it relates to the application of kudzu flower extract in the preparation of drugs for treating obesity, in the preparation of health foods that help control body fat, and in the preparation of health foods that help maintain healthy blood lipid (cholesterol / triglyceride) levels. Background Technology

[0002] Obesity is a multifactorial chronic disease related to energy imbalances caused by unhealthy lifestyles. It has become a significant public health problem facing countries worldwide, posing enormous challenges to families and healthcare systems. According to the latest data, obesity causes severe activity and metabolic disorders, making the body a breeding ground for various chronic diseases, including diabetes, chronic inflammation, cardiovascular disease, and liver disease. These obesity-related diseases are often the direct cause of impaired life and quality of life for obese patients, posing a significant threat to human health.

[0003] Common methods for preventing and treating obesity include dieting and exercise interventions. Research data shows that only 1% of obese patients successfully lose weight, making it a challenging task. Currently approved weight-loss drugs mainly include oral solid dosage forms (tablets and capsules) and injections (subcutaneous long-acting preparations, such as semaglutide, represented by GLP-1 receptor agonists). Oral solutions are also used. Oral tablets and capsules are the most commonly used due to their mature dosage forms and high patient compliance, while injections achieve complete absorption and stable blood drug concentrations, but have limitations such as inconvenient injection. Weight-loss health foods are more diverse, mainly including tablets, capsules, granules, oral liquids, mixtures, syrups, and powders. In recent years, new dosage forms in the form of ordinary food items such as solid beverages, candies, and chocolates have also emerged. Beverage formulations are popular due to their convenience and ease of adherence. However, the current market for weight-loss beverage products is largely untapped, making the search for natural, non-toxic, and low-cost weight-loss products crucial for the prevention and treatment of obesity. Compared with traditional drug treatment, the use of edible Chinese medicine has better safety, a wider range of applications, and good market development prospects.

[0004] Kudzu flower (Puerariae Flos) is the dried flower bud of the legume Pueraria lobata (Willd.) Ohwi. It is rich in flavonoids, polysaccharides, proteins, amino acids, vitamins, etc. Traditional Chinese medicine uses it as a good medicine for relieving hangovers and protecting the liver. Modern pharmacological research shows that kudzu flower and its active ingredients have the potential to regulate lipid metabolism and inhibit adipocyte differentiation. Developing weight-loss products using kudzu flower as a raw material has the following advantages: First, its medicinal and edible properties avoid the toxic side effects of synthetic drugs; second, the ethanol extraction process can selectively enrich fat-soluble active ingredients, and the novel components of kudzu flower are conducive to discovering new targets; third, it turns waste into treasure, improves the utilization rate of kudzu flower, and creates a new comprehensive development model of "one plant, multiple effects".

[0005] Given that existing weight loss methods all have certain difficulties and risks, it is necessary to provide a safe and effective option for treating obesity. Summary of the Invention

[0006] The inventors demonstrated through in vitro experiments that kudzu flower extract (PFE) can significantly inhibit the differentiation and lipid accumulation of preadipocytes and reduce the accumulation and formation of oleic acid-induced lipid droplets in cells, exhibiting in vitro lipid-lowering ability. Further, by constructing a high-fat diet model, they found that kudzu flower extract can reduce the weight of obese mice, significantly slow weight gain, downregulate serum lipid factors (total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C)) levels, reduce lipid accumulation in the liver, and significantly improve fat hypertrophy in mice, normalizing adipose tissue cell morphology and reducing perirenal and epididymal fat wet weight. It can be used to treat and / or prevent obesity and related symptoms caused by a high-fat diet. The purpose of this invention is to provide the application of kudzu flower extract in the preparation of drugs for treating obesity, in the preparation of health foods that help control body fat, and in the preparation of health foods that help maintain healthy blood lipid (cholesterol / triglyceride) levels.

[0007] The purpose of this invention is to provide the use of kudzu flower extract in the preparation of medicaments for the prevention and / or treatment of obesity.

[0008] Preferably, the kudzu flower extract is used in the preparation of a medicament for the prevention and / or treatment of obesity by: reducing body weight, decreasing the accumulation and weight of body fat, and downregulating the levels of lipid factors.

[0009] The lipid factors mentioned are serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).

[0010] Preferably, the drug is a pharmaceutically acceptable formulation prepared with the kudzu flower extract as the main or sole active ingredient and pharmaceutically acceptable excipients.

[0011] Preferably, the preparation is an oral solid dosage form, an oral solution, or an injection.

[0012] Preferably, the oral solid dosage form is a tablet or capsule.

[0013] Another object of the present invention is to provide the application of kudzu flower extract in the preparation of health foods that help control body fat.

[0014] Another object of the present invention is to provide the application of kudzu flower extract in the preparation of health food products that help maintain healthy blood lipid levels; wherein the blood lipids are cholesterol / triglycerides.

[0015] The dosage forms of the health food products mentioned are oral liquids, granules, tablets, capsules, powders, solid beverages, and beverages.

[0016] The total flavonoid content in the kudzu flower extract is approximately 20-21%.

[0017] The kudzu flower extract is obtained by using kudzu flowers as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, reflux extraction, filtration, and concentration and drying of the filtrate under reduced pressure.

[0018] Preferably, the kudzu flower extract is obtained by using kudzu flower as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, reflux extraction at 75 to 85℃ for 2 to 3 times at a material-to-liquid ratio of 1:13 to 1:19 g / mL, combining the extracts, filtering, and then concentrating and drying the filtrate under reduced pressure.

[0019] Preferably, the kudzu flower is crushed, passed through a 40-mesh sieve, mixed with the extraction solvent, stirred evenly, and then subjected to reflux extraction.

[0020] Preferably, the extraction solvent is an 80%–84% aqueous ethanol solution.

[0021] Preferably, the material-to-liquid ratio is 1:15 g / mL.

[0022] Preferably, the reflux extraction temperature is 85°C.

[0023] Preferably, the reflux extraction is performed twice.

[0024] Preferably, the drying method is freeze drying.

[0025] Another object of the present invention is to provide a composition with weight loss effect, the composition comprising a kudzu flower extract solution, a sweetener, an acidulant, an antioxidant, a preservative, a flavoring agent, and water; wherein the sweetener, acidulant, antioxidant, preservative, and flavoring agent are respectively 4-8%, 0.08-0.16%, 0.01%, 0.1%, and 0.01% of the total weight of the kudzu flower extract solution and water; and the concentration of the kudzu flower extract solution is 20 mg / mL.

[0026] Preferably, the total weight of the kudzu flower extract solution and water is 100%, and the kudzu flower extract solution accounts for 40% of the total weight of the kudzu flower extract solution and water; the sweetener, acidulant, antioxidant, preservative and flavoring agent are 8%, 0.12%, 0.01%, 0.1% and 0.01% of the total weight of the kudzu flower extract solution and water, respectively.

[0027] The kudzu flower extract solution is prepared by the following method: adding kudzu flower extract to pure water, dissolving by ultrasonication, centrifuging, and filtering to obtain the kudzu flower extract solution.

[0028] The sweetener is at least one of xylitol, erythritol, sucralose, etc.

[0029] The acidulant mentioned is at least one of citric acid, malic acid, etc.

[0030] The antioxidant mentioned is at least one of D-ascorbic acid and sodium ascorbate;

[0031] The preservative is at least one of sodium benzoate, potassium sorbate, etc.

[0032] The flavoring agent is white peach oolong flavoring, honey peach oolong flavoring, or osmanthus oolong flavoring.

[0033] Preferably, the composition is a beverage.

[0034] Another object of the present invention is to provide a method for preparing a composition with weight loss effect, comprising: stirring and mixing a kudzu flower extract solution, a sweetener, an acidulant, an antioxidant, a flavoring agent and pure water.

[0035] The beneficial effects of this invention are:

[0036] This invention uses kudzu flower as raw material and obtains kudzu flower extract by ethanol reflux method. In vitro lipid differentiation induction experiment proves that kudzu flower extract has in vitro lipid-lowering ability. At the same time, animal experiment proves that kudzu flower extract has the effect of treating hyperlipidemia and obesity. While reducing the weight of mice, it also alleviates liver damage. It has great potential for the development of weight loss and liver protection drugs, and has good health care and treatment value, providing patients with more choices.

[0037] This invention fully leverages the advantages of kudzu flower as both a food and a medicine, developing a safe, natural kudzu flower extract and its beverage products that control body fat. This changes the low utilization rate of kudzu flower resources, significantly enhances its comprehensive value, and has good application prospects and market value. Attached Figure Description

[0038] Figure 1 The results of single-factor investigation of the preparation process of kudzu flower extract are shown below; where A: extraction method, B: number of extractions, C: extraction temperature, D: extraction solvent, E: ethanol concentration, and F: solid-liquid ratio.

[0039] Figure 2 The response surface methodology is used to optimize the extraction process of kudzu flower extract. A represents the interaction between extraction temperature and solid-liquid ratio, B represents the interaction between ethanol volume fraction and solid-liquid ratio, and C represents the interaction between ethanol volume fraction and extraction temperature.

[0040] Figure 3 Results of CCK-8 assay for detecting the cytotoxic activity of kudzu flower extract against 3T3-L1 cells.

[0041] Figure 4 The lipid-lowering effect of kudzu flower extract on 3T3-L1 cells; the left figure is the Oil Red O staining image, and the right figure is the lipid quantification result; *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant difference.

[0042] Figure 5 Results of CCK-8 assay for detecting the cytotoxic activity of kudzu flower extract against HepG2 cells.

[0043] Figure 6 To verify the lipid-lowering and hepatoprotective effects of kudzu flower extract in HepG2 cells; the left image is the Oil Red O staining image, and the right image is the lipid quantification results; *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant difference.

[0044] Figure 7 The effect of administration of kudzu flower extract on mouse body weight; where A: experimental flowchart, B: mouse body weight change graph, C: mouse body weight on the last day, D: mouse body weight gain; *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant difference.

[0045] Figure 8The effects of administration of kudzu flower extract on fat and liver wet weight and organ indices in mice were investigated. A: Wet fat weight in each group of mice; B: Wet liver weight in each group of mice; C: Wet epididymal fat weight in each group of mice; D: Wet perirenal fat weight in each group of mice; E: Liver index in each group of mice; F: Epididymal fat index in each group of mice; G: Perirenal fat index in each group of mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

[0046] Figure 9 HE staining of epididymal adipose tissue in mice after administration of kudzu flower extract.

[0047] Figure 10 HE staining of perirenal adipose tissue in mice after administration of kudzu flower extract.

[0048] Figure 11 HE staining of mouse liver tissue after administration of kudzu flower extract.

[0049] Figure 12 Results of serum lipid levels in mice after administration of kudzu flower extract; *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant difference.

[0050] Figure 13 The taste evaluation of the beverage product made from kudzu flower extract; where A: amount of extract added, B: amount of xylitol added, C: amount of citric acid added, and D: amount of D-ascorbic acid added.

[0051] Figure 14 Image of a test sample of a composition that has the effect of controlling body fat. Detailed Implementation

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0053] Example 1

[0054] I. Single-factor experiment

[0055] Using the total peak area (representing the total flavonoid content of kudzu flower) as an indicator, the optimal extraction conditions for kudzu flower extract (PFE) were screened.

[0056] Dried kudzu flowers were pulverized and passed through a 40-mesh sieve. A precise weight of the uniformly mixed kudzu flower powder was determined. Single-factor experiments were conducted on extraction method, extraction solvent, solid-liquid ratio, and extraction time. Each group was divided into triplicates, with 5 g of kudzu flower powder per sample, all other conditions kept constant. After extraction, the extract was filtered through filter paper, and the filtrate was centrifuged at 5000 r / min for 20 min. The supernatant was collected and analyzed by HPLC, with the total peak area as the indicator. HPLC analysis conditions: Phenomenex C18 column (4.6 mm × 250 mm, 5.0 μm); mobile phase A was 0.1% formic acid-water, and mobile phase B was acetonitrile, with gradient elution (Table 1); flow rate was 1 mL / min; column temperature was 25°C; detection wavelength was 254 nm; and injection volume was 10 μL.

[0057] Table 1. Gradient elution conditions

[0058]

[0059] 1. Extraction method investigation: Each 5 g portion of kudzu flower powder was added to 95 mL of 80% ethanol and extracted according to the following methods: ultrasonic treatment at room temperature for 30 min (ultrasonic power 100 W), reflux at 80℃ for 1.5 h, and cold soak at room temperature for 12 h. Each extraction method was repeated once.

[0060] from Figure 1 In A, it can be observed that the total peak area of ​​the three extraction methods is in the order of hot reflux > ultrasonication > cold soaking. The total amount of flavonoids obtained by hot reflux and ultrasonic extraction is significantly higher than that obtained by cold soaking at room temperature. Therefore, hot reflux was selected as the extraction method for further investigation.

[0061] 2. Extraction times: Each 5 g portion of kudzu flower powder was added to 95 mL of 80% ethanol and refluxed at 80℃ for 1.5 h, and extracted 1, 2, and 3 times respectively.

[0062] from Figure 1 From B, we can observe that the total peak area is in the order of 3 times of reflux extraction ≈ 2 times > 1 time. It can be seen that 2 extractions are sufficient to achieve full extraction. Therefore, it is recommended to extract 2 to 3 times, with 2 extractions being the preferred method.

[0063] 3. Extraction temperature study: Each 5 g portion of kudzu flower powder was added to 95 mL of 80% ethanol and refluxed for 1.5 h at different extraction temperatures (65, 70, 75, 80, 85℃). Each extraction temperature was repeated once.

[0064] from Figure 1As can be observed from C, the total peak area first increases and then decreases with increasing extraction temperature, reaching its maximum at an extraction temperature of 80℃. Response surface methodology was then performed within the 75–85℃ range.

[0065] 4. Extraction solvent test: Each 5 g portion of kudzu flower powder was added to 95 mL of different extraction solvents (pure water, 50%, 60%, 70%, 80%, and 90% ethanol aqueous solution by volume), refluxed at 80℃ for 1.5 h, and the extraction was repeated once.

[0066] from Figure 1 As can be observed from D, the total peak area of ​​the extract obtained by water extraction is much lower than that of the extract obtained by ethanol aqueous solution extraction. Figure 1 The E values ​​indicate that the total peak area first increases and then decreases with increasing ethanol concentration in the aqueous ethanol solution, with the largest total peak area obtained from 80% ethanol extraction. Response surface methodology was optimized within the 70–90% ethanol range.

[0067] 5. Material-liquid ratio analysis: Each portion of kudzu flower powder (5 g) was added to different volumes (65, 80, 95, 110, 125 mL, corresponding to material-liquid ratios of 1:13, 1:16, 1:19, 1:22, 1:25 g / mL) of 80% ethanol, and refluxed at 80℃ for 1.5 h. The extraction was repeated once.

[0068] from Figure 1 As can be observed from F, the total peak area first increases and then tends to stabilize as the feed-to-liquid ratio increases. The feed-to-liquid ratio reaches its maximum when the total peak area is 1:16 g / mL. Therefore, the range of 1:13 to 1:19 mL / g is selected for response surface optimization.

[0069] II. Response Surface Optimization

[0070] Table 2. Experimental Factor Levels

[0071]

[0072] Table 3. Results of response surface methodology experiments

[0073]

[0074] Table 4. Analysis of Variance

[0075]

[0076] Note: * indicates a significant difference (P<0.05); ** indicates a highly significant difference (P<0.01); *** indicates an extremely significant difference (P<0.001).

[0077] Design-Expert 13 software was used for experimental design and data processing, and a response surface model was constructed based on the Box-Behnken experimental design (BBD) principle. Ethanol volume fraction (A), extraction temperature (B), and solid-liquid ratio (C) were used as experimental variables, with three levels (high, medium, and low) coded as 1, 0, and -1, respectively. The factor level codes are shown in Table 2. A total of 17 experiments were conducted according to the BBD design scheme (including 5 center-point replicates for error estimation).

[0078] The experimental design is shown in Table 3. Regression fitting was performed on the experimental results in Table 3, and the results are shown in Table 4. A quadratic polynomial model was obtained, yielding the equation:

[0079] Y=70620.720+673.013A+652.563B-455.125C-99.400AB+243.075AC+47.525BC-643.435A 2 -170.935B 2 -802.360C 2 ;

[0080] In the formula, Y is the predicted peak area of ​​the extracted components from kudzu flower, A is the volume fraction of ethanol, B is the extraction temperature, and C is the solid-liquid ratio.

[0081] Analysis of variance (ANOVA) was used to evaluate the applicability of the regression model. The significance of each factor in the regression equation was tested using F-values ​​and P-values. A P-value < 0.05 indicates successful modeling. The F-value of the regression model was 30.39, and P < 0.0001, indicating that the model is highly significant and can adequately describe the relationship between the response value and the extraction process parameters. The model correlation R-value was [value missing]. 2 =0.9750, Corrected coefficient of determination adj R 2 =0.9430, the difference between the two is small and close to 1.0, indicating that the model fits the actual value and the predicted value well, and can well reflect the relationship between each factor and the response value. The p-value of the lack-of-fit term is 0.8958>0.05, indicating that the lack-of-fit term is not significantly related to its pure error, indicating that the model can fit the experimental situation well. Therefore, this model can be used to analyze and predict the extraction of components from kudzu flower. Linear terms A, B, interaction term AC, and quadratic term A 2 C 2 The effect on the extraction yield of effective components from kudzu flower was extremely significant (P < 0.001); the linear term C, the interaction term AB, and the quadratic term B 2 The effect of each factor on the yield of effective components from kudzu flower was significant (P < 0.05). The F-values ​​showed that the order of influence of each factor on the yield of effective components from kudzu flower, from largest to smallest, was: ethanol volume fraction > temperature > solid-liquid ratio.

[0082] The results in Table 3 were analyzed using Design-Expert 13 software to obtain a three-dimensional (3D) response surface plot, illustrating the relationship between extraction parameters and kudzu flower extract. While controlling for other experimental variables, the influence of interaction terms on the extraction rate of kudzu flower components was explored, and the pairwise interactions between experimental factors were comprehensively evaluated using response surface analysis. The steepness of the response surface indicates the degree to which the experimental variables affect the extraction rate; the steeper the slope, the greater the interaction effect. Figure 2 It was found that the interaction surfaces between temperature and ethanol volume fraction, and between the solid-liquid ratio and ethanol volume fraction, were steeper, indicating that these two sets of interactions had a stronger impact on the extraction rate, which is consistent with the results of the analysis of variance. To determine the optimal extraction conditions for kudzu flower, response surface methodology (RSM) was performed using Design Expert software. Based on the regression fitting equation, the optimal extraction conditions for kudzu flower extract were: 84% ethanol volume fraction, 85℃ temperature, and a solid-liquid ratio of 1:15 g / mL. To verify the accuracy, the experiment was repeated three times using the above parameters. The average total peak area of ​​the PFE extract was 70693.4, which is close to the theoretical prediction of 71258.6. These conclusions demonstrate that the RSM process optimization parameters are accurate and reliable.

[0083] Example 2

[0084] Preparation of kudzu flower extract: Dried kudzu flowers were pulverized and passed through a 40-mesh sieve to obtain kudzu flower powder. The kudzu flower powder was mixed with 84% ethanol-water at a material-to-liquid ratio of 1:15 g / mL. The mixture was refluxed twice at 85℃ for 1.5 h each time. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to a thick paste. The filtrate was then freeze-dried to obtain a dry solid kudzu flower extract.

[0085] The total flavonoid content in kudzu flower extract was determined by the NaNO2-Al(NO3)3-NaOH colorimetric method: 5 mg of dried kudzu flower extract was accurately weighed, placed in an EP tube, and about 5 mL of pure water was added. The extract was dissolved by sonication for 30 min, centrifuged, and filtered to obtain the kudzu flower extract (1 mg / mL) for the determination of total flavonoid content.

[0086] Take an appropriate amount of kudzu flower filtrate, add 5% NaNO2 solution, mix well, and let stand for 6 min; add 10% Al(NO3)3 solution, mix well, and let stand for 6 min; add 1 mol / L NaOH solution, mix well, and let stand for 10–15 min; measure the absorbance at a wavelength of about 500 nm, and the average absorbance of total flavonoids in kudzu flower is y=0.62; plot a standard curve with rutin as a reference: y=1.2708x + 0.3596 (R²=0.9991), with a linear range of 0.0625–1 mg / mL; calculate that the total flavonoid content in kudzu flower (PFE) extract is 20.8% (±1.9%, n=3).

[0087] Example 3

[0088] In vitro lipid-lowering effect of kudzu flower extract on adipocytes

[0089] Preparation: Take dried kudzu flower extract (Example 2) and put it into a 1.5 mL EP tube. Prepare it to the required concentration with serum-free DMEM medium, filter it and set it aside.

[0090] 1. 3T3-L1 cell culture modeling and drug treatment

[0091] 1) Cell culture: Mouse 3T3-L1 (CL-173, ATCC) cells (mouse preadipocytes) were cultured in DMEM medium containing 10% (v / v) newborn calf serum (NBS), penicillin G (100 IU / mL) and streptomycin (100 μg / mL).

[0092] 2) CCK-8 assay for cytotoxic activity: 3T3-L1 cells were cultured at 1×10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured in DMEM complete medium. When the cells reached 70% confluence, the complete medium was discarded, and the plates were divided into a control group (DMEM medium without serum, PFE concentration of 0 μg / mL) and groups with different concentrations of PFE (DMEM medium containing 100, 200, 400, 800, 1000, 1500, 2000, and 2500 μg / mL of kudzu flower extract). Unseeded wells were also included to eliminate background differences in detection. After 24 or 48 hours of culture, 100 μL of 10% CCK-8 solution was added to each well, and the plates were incubated for 30 min. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated and plotted. Results are shown below. Figure 3 The results showed that after 48 hours of administration, the survival rate of 3T3-L1 cells was greater than 80% when PFE was below 1500 μg / mL, and it was non-toxic to 3T3-L1 cells within this range.

[0093] 3) Oil Red O staining to determine the adipogenic content of 3T3-L1 cells: Modeling: 3T3-L1 cells were seeded into 6-well plates. After the cells reached 100% confluence and contact inhibition occurred (recorded as day 0), the medium was changed and cultured for 2 days. On day 2, the medium was changed to DMEM medium containing 1% penicillin antibiotics, 10% fetal bovine serum (FBS), 10 μg / mL insulin, 1 μM dexamethasone, and 0.5 mM IBMX (recorded as induction differentiation medium I). Induction was carried out for 2 days (day 4). The medium was then changed to DMEM medium containing 1% penicillin antibiotics, 10% FBS, and 10 μg / mL insulin (recorded as induction differentiation medium II). Induction was continued for 2 more days (day 6). Induction was then stopped and the medium was changed to DMEM medium containing only 1% penicillin antibiotics, 10% FBS, and DMEM, allowing lipid droplets to continue to accumulate and form an adipogenic model (day 7). On day 7 of adipogenic differentiation, based on the obtained safe concentration of kudzu flower extract, 3T3-L1 cells were treated for 2 days with PFE at concentrations of 200, 400, and 800 μg / mL (in DMEM medium containing kudzu flower extract at concentrations of 200, 400, and 800 μg / mL, respectively). The treatment without PFE was used as the model group. The culture medium was then carefully and gently poured off, the cells were gently washed with PBS, fixed with 10% paraformaldehyde for 30 min, diluted with Oil Red Stock Solution (Oil Red:deionized water volume ratio = 3:2), filtered through filter paper, and incubated at room temperature for 10 min, followed by staining for approximately 20 min. Destaining was performed by washing with 75% isopropanol to remove excess dye, washing with PBS, observing under a microscope, and photographing the staining results. Cells were then lysed with 60% isopropanol, and the OD value at 510 nm was measured using a microplate reader, once every 10 min, for a total of 3 measurements. The results of adipogenic differentiation of 3T3-L1 cells are shown below. Figure 4 The results showed that a large number of red lipid droplets appeared in the 3T3-L1 cells of the model group. After treatment with kudzu flower extract, the number and size of lipid droplets in 3T3-L1 cells decreased in a dose-dependent manner, and the PFE inhibition rate of 800 μg / mL was as high as 30% (P<0.001), indicating that kudzu flower extract can effectively inhibit adipogenic differentiation.

[0094] 2. HepG2 cell culture and drug treatment

[0095] 1) Cell culture: HEPG2 (human liver cancer) cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin G (100 IU / mL) and streptomycin (100 μg / mL).

[0096] 2) CCK-8 assay for cytotoxic activity: HepG2 cells in good condition were used, and cytotoxic activity was determined by CCK-8 assay at a concentration of 1×10⁻⁶. 4Cells were seeded at a density of 10 cells / mL in 96-well plates and cultured in DMEM complete medium. When the cells reached 70% confluence, the complete medium was discarded, and the plates were divided into a control group (DMEM medium without serum, PFE concentration of 0 μM) and groups with different concentrations of PFE (DMEM medium containing 10, 20, 50, 100, 200, 400, 800, and 1600 μg / mL of kudzu flower extract). Unseeded wells were also included to eliminate background differences in detection. Cells were cultured for 24 or 48 h, followed by drug administration for 24 h / 48 h. 100 μL of 10% CCK-8 solution was added to each well, and the plates were incubated for 30 min. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated and plotted. Results are shown below. Figure 5 The results showed that when PFE concentration was no higher than 800 μg / mL, the survival rate of HepG2 cells was higher than 80% at both 24 h and 48 h after administration, indicating that PFE was non-toxic to HepG2 cells within this concentration range.

[0097] 3) Oil Red O staining to determine fat content: Modeling: HepG2 cells in the logarithmic growth phase were seeded at an appropriate density in 6-well plates and cultured at 37℃ in a 5% CO2 incubator for 24 h until the cells adhered well. The cells were then divided into 5 groups: control group (CON group), model group (OA group), and low, medium, and high dose groups of kudzu flower extract (PFE concentrations of 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively). Control group cells were cultured routinely in DMEM complete medium containing 10% FBS; other groups were induced to accumulate lipids by adding DMEM complete medium containing 0.5 mM oleic acid (OA) (using BSA as a carrier). After 24 h of induction, obvious lipid droplet formation was observed under a microscope. After induction, the culture medium was discarded, and cells were treated with PFE at concentrations of 100 μg / mL, 200 μg / mL, and 400 μg / mL (corresponding to low, medium, and high dose groups, respectively). The treatment without PFE was used as the model group. 24 h after PFE treatment, cells were processed according to the "Oil Red O staining for lipid content determination in 3T3-L1 cells" method. Results are shown below. Figure 6 The results showed that in HepG2 cells, the model group showed diffuse orange-red lipid droplets after oleic acid induction. The lipid droplets decreased after treatment with kudzu flower extract in a dose-dependent manner. Both 200 and 400 μg / mL PFE significantly reduced the lipid accumulation rate (P<0.001), and the lipid accumulation rate of cells treated with 400 μg / mL PFE decreased by about 50%.

[0098] In summary, kudzu flower extract can dose-dependently inhibit adipogenic differentiation of 3T3-L1 cells and lipid accumulation in the liver of HepG2 cells.

[0099] Example 4

[0100] All animal experiments were approved by the Ethics Committee of China Pharmaceutical University [YSL-202511049].

[0101] In vivo lipid-lowering effect of kudzu flower extract

[0102] 1. Establishment and experimental design of a mouse hyperlipidemia model

[0103] Thirty-six healthy adult male KM mice, weighing 18–21 g, were used in the experiment. They were acclimatized for one week in an environment with a temperature of 24±2℃, relative humidity of 45%–55%, and no noise. The mice were divided into four groups: a blank control group (CON), a high-fat model group (HFD), a positive drug control group (HFD+OR1), a low-dose PFE group (HFD+PFE-L), a medium-dose PFE group (HFD+PFE-M), and a high-dose PFE group (HFD+PFE-H). Except for the CON group, the mice in the other groups were fed a high-fat diet to establish a high-fat model. Starting from the second week, the high-fat diet was mixed with the normal diet. On the first day, the diet consisted of 20% high-fat diet. The proportion of high-fat diet was gradually increased each day (20%, 40%, 60%, 80%, 100%) until the fifth day when it was replaced with 100% high-fat diet. The high-fat diet was then used to induce a high-fat model for another 8 weeks. Starting in week 5, mice in the three PFE intervention groups (low, medium, and high dose PFE groups) were administered 45, 90, and 180 mg / kg of kudzu flower extract (prepared with purified water) daily by gavage, respectively. Mice in the positive control group were administered 20 mg / kg of orlistat (prepared with purified water) daily by gavage. Mice in the blank control group and the high-fat model group were administered an equal volume of physiological saline by gavage. This intervention continued for 4 weeks. Gavage was performed using a syringe to prevent injury to the mice, and they were fasted for one day before sacrifice. After the experiment, mice were sacrificed by cervical dislocation, and the liver, epididymal fat, and perirenal fat were dissected and completely removed, weighed, and recorded.

[0104] 2. Indicator Measurement and Processing

[0105] 1) Measurement of body weight, liver weight, and wet weight of adipose tissue: The body weight, liver weight, and wet weight of adipose tissue in different parts of the mice in each treatment group were measured.

[0106] 2) Determination of indicators in serum and liver tissue: After anesthetizing mice, blood was collected by enucleation and allowed to stand for 2-4 hours. The blood was then centrifuged at 3000 r / min for 10 min at 4℃. The supernatant was collected, and the levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum were determined according to the kit instructions.

[0107] 3) Pathological observation of liver and adipose tissue: Liver tissue, perirenal fat, and epididymal fat tissue from the same location in the liver of mice in each treatment group were taken, fixed with 4% paraformaldehyde and fat fixative, rinsed with running water, dehydrated with graded ethanol and xylene, permeated with paraffin, embedded, dewaxed and sectioned, and the pathological changes of liver and adipose tissue were observed under a light microscope using the hematoxylin-eosin (HE) staining method.

[0108] Data processing and statistical analysis: GraphPad Prism 8.0 software was used for statistical analysis and graphing. Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no statistical significance.

[0109] 3. Analysis of Experimental Results

[0110] 1) Trends and increases in mouse body weight

[0111] The total duration of the study was 8 weeks, from the modeling period to the gavage period. The body weight of mice in each group was recorded and analyzed. Figure 7 B), the weight change trend of the CON group was relatively stable, the weight of the mice in the HFD group continued to increase, and the weight gain of the other groups showed a decreasing trend in the 6th week.

[0112] At the end of the experiment, the final body weight of the mice in each group was as follows: Figure 7 As shown in C; statistical analysis was performed on the weight gain (final weight - initial weight) of mice in each group, and the results are as follows. Figure 7 As shown in Figure D, compared with the CON group, the HFD group showed a significantly increased weight gain (P<0.001), indicating the successful establishment of a high-fat diet-induced obesity model. Compared with the HFD group, the high-dose PFE group and the positive control group showed significantly decreased weight gain (P<0.001). This indicates that kudzu flower extract can effectively inhibit high-fat diet-induced weight gain in mice, and its effect is dose-dependent.

[0113] 2) To further evaluate the effects of kudzu flower extract on fat accumulation and liver function, epididymal fat and perirenal adipose tissue were isolated and weighed, and the fat coefficient (fat weight / body weight × 100%) was calculated; simultaneously, the liver was isolated and weighed, and the liver coefficient (liver weight / body weight × 100%) was calculated. The results are as follows: Figure 8As shown in the figure, compared with the CON group, the HFD group mice showed significantly increased liver wet weight, liver index, epididymal fat, and perirenal fat, indicating that the high-fat diet successfully induced liver enlargement and visceral fat accumulation. After PFE intervention, the above indicators of mice in the three PFE intervention groups were improved to varying degrees. Among them, the high-dose PFE group and the positive drug control group significantly reduced liver wet weight and liver index, approaching the level of the CON group; compared with the HFD group, the epididymal fat weight of mice in the medium and high-dose PFE groups decreased significantly in a dose-dependent manner; the perirenal fat weight of mice in the high-dose PFE group also decreased to a level similar to that of the CON group. Compared with the HFD group, all three PFE intervention groups showed a trend of fat reduction, with the high-dose PFE group showing the most significant effect. The above results indicate that kudzu flower extract can effectively alleviate liver lipid accumulation and abdominal fat deposition induced by a high-fat diet, and has a clear in vivo lipid-lowering activity.

[0114] 3) Observation of mouse adipocyte morphology

[0115] In the CON group, the adipose tissue cells in both locations showed regular morphology and were neatly and tightly arranged. In the HFD group, the adipocytes were arranged randomly, with cells showing fat accumulation and increased volume, exhibiting varying degrees of differentiation. Gavage administration of PFE improved these conditions to varying degrees. The medium-dose PFE group showed morphology close to that of the CON group, while the high-dose PFE group showed more tightly arranged cells and smaller cell volume than the CK group. Figure 9 , Figure 10 ).

[0116] Liver tissue sections from each group of mice were stained with hematoxylin and eosin (HE). Figure 11 In the CON group, hepatocytes were observed to be uniform in size and neatly arranged, with orderly sinusoidal arrangement and no lipid droplets. In the HFD group, a large number of lipid droplets accumulated in the hepatocytes, the sinusoids were disordered, vacuoles appeared, and the hepatocyte structure was incomplete. After gavage administration of PFE, the fatty degeneration in all three PFE intervention groups was reduced compared to the model group. In the low-dose PFE group, lipid droplet aggregation and vacuolation in the liver tissue were reduced compared to the HFD group, but a certain number of lipid droplets were present. The liver tissue sections in the medium-dose PFE group showed significant improvement, with a small number of small lipid droplets in the cells and a clear and orderly arrangement of sinusoids. In the high-dose PFE group, no obvious lipid droplets were observed, and the hepatocytes were uniformly arranged. PFE intervention can alleviate the excessive accumulation of lipids in the liver caused by a high-fat diet.

[0117] The results above indicate that PFE can alleviate excessive fat accumulation and degeneration in the liver, and improve liver inflammation that may be caused by obesity.

[0118] 4) Blood lipid levels in mice

[0119] The inventors studied the effect of PFE on blood lipid levels in HFD mice, and measured the serum TG, TC, LDL-C and HDL-C levels in mice in different treatment groups. Figure 12 Compared with the HFD group, all three doses of PFE (low, medium, and high) significantly reduced serum TG and LDL-C levels (P<0.05). Among them, the high-dose PFE was more effective than the positive control drug orlistat (0.37±0.04 mmol / L) in reducing LDL-C. In conclusion, kudzu flower extract shows promise as a candidate drug for regulating dyslipidemia.

[0120] Example 5

[0121] 1. Preparation of kudzu flower extract

[0122] Accurately weigh 1 g of dried kudzu flower extract (Example 2), place it in an EP tube, add about 50 mL of pure water, sonicate for 30 min to dissolve, centrifuge at 3000 rpm for 20 min, take the supernatant and filter it with qualitative filter paper to obtain kudzu flower extract solution (20 mg / mL), which is used for subsequent beverage preparation.

[0123] 2. Sensory test scoring indicators

[0124] A sensory evaluation team of 15 food workers with professional training and experience in beverage sensory evaluation was selected. Based on other sensory evaluation standards, the evaluation standards suitable for this study were determined, and the beverages were evaluated in terms of color, aroma, taste and texture (Table 5).

[0125] Table 5. Sensory rating indicators

[0126]

[0127] 3. Beverage preparation

[0128] Kudzu flower beverages exhibit a characteristic amber color and a distinctive kudzu flower aroma. Furthermore, as the concentration of kudzu flower extract solution in the beverage increases, the solution gradually thickens and the color darkens. This demonstrates that the amount of kudzu flower extract solution added affects the beverage's color, flavor, and texture.

[0129] In EP tubes, different amounts of kudzu flower extract solution and pure water were mixed, with the addition amounts of kudzu flower extract solution set at 20, 40, 60, 80, and 100 wt% (when the kudzu flower extract solution was 100%, no additional pure water was needed). Then, 8 wt% xylitol (sweetener), 0.12 wt% citric acid (acidifier), 0.008 wt% sodium benzoate, 0.01 wt% D-ascorbic acid (antioxidant), and 0.01 wt% white peach oolong flavoring (flavor enhancer) were added according to the total weight of the kudzu flower extract solution and pure water, and the mixture was stirred until dissolved and homogeneous. The optimal addition amount of kudzu flower extract solution was determined using sensory evaluation (color, aroma, taste, and texture) as the main evaluation indicators. Figure 13 As shown in Figure A, when the addition amount increased from 20% to 40%, the sensory score rose from 70.0 to 79.5, indicating that appropriately increasing the proportion of extract solution is beneficial to improving flavor and texture. When the addition amount exceeded 40%, the score gradually decreased, indicating that excessively high amounts of extract solution may lead to undesirable flavor or texture changes. Therefore, the optimal addition amount of extract solution is 40%.

[0130] In an EP tube, a kudzu flower extract solution and purified water were mixed, with the kudzu flower extract solution added at a rate of 40 wt%. Then, 2, 4, 6, 8, or 10 wt% xylitol, 0.12 wt% citric acid, 0.008 wt% sodium benzoate, 0.01 wt% D-ascorbic acid, and 0.01 wt% white peach oolong flavoring were added, stirred until dissolved and mixed thoroughly. Sensory evaluation (primarily focusing on sweetness palatability and flavor harmony) was used to investigate the effect of xylitol addition on the product flavor and determine its suitable addition range. Figure 13 As shown in Figure B, the sensory score first increases and then decreases with increasing xylitol content. Adding xylitol at 4%-8% can improve the taste of beverage products, reaching a maximum score of 78 points at 8%. Excessive addition of xylitol can lead to sweetness receptor saturation, easily causing a sweetness-masking effect, thereby inhibiting the flavor characteristics of the product's unique flavor compounds and resulting in a taste imbalance. Therefore, the optimal addition amount of xylitol is 8%.

[0131] In an EP tube, a kudzu flower extract solution and purified water were mixed, with the kudzu flower extract solution added at a concentration of 40 wt%. Then, 8 wt% xylitol, 0.04, 0.08, 0.12, 0.16, or 0.2 wt% citric acid, 0.008 wt% sodium benzoate, 0.01 wt% D-ascorbic acid, and 0.01 wt% white peach oolong flavoring were added, and the mixture was stirred until dissolved and homogeneous. Sensory evaluation (primarily based on palatability of acidity and balance of sweet and sour flavors) was used to investigate the effect of citric acid addition on the product's acidity and overall flavor, determining its optimal range. Figure 13As shown in C, when the amount of citric acid added reaches 0.12%, the sensory score of the product reaches a peak of 76, indicating that this level of addition can effectively coordinate the sweet and sour balance of the beverage and present the best sensory quality. Therefore, the optimal amount of citric acid added is 0.12%.

[0132] In an EP tube, a kudzu flower extract solution and purified water were mixed, with the kudzu flower extract solution added at a concentration of 40 wt%. Then, 8 wt% xylitol, 0.12 wt% citric acid, 0.008 wt% sodium benzoate, 0.005, 0.01, 0.015, 0.02, or 0.02 wt% D-ascorbic acid, and 0.01 wt% white peach oolong flavoring were added and stirred until dissolved and mixed. The effect of D-ascorbic acid addition on product stability and overall flavor was investigated using sensory evaluation and beverage oxidation and color change time as evaluation indicators to determine its suitable addition range. Figure 13 As shown in D, when the amount of D-ascorbic acid added is 0.005%, the product shows a darkening of color and precipitation on the 5th day. The sensory scores are not significantly different when the amount added is between 0.01% and 0.02%. Therefore, the optimal amount of D-ascorbic acid added is 0.01%.

[0133] Based on the investigation results, the optimal ratio of the beverage was determined to be 100% kudzu flower extract solution and 40 wt% pure water. The amounts of sweetener, acidulant, antioxidant, and flavoring agent were determined to be 8 wt%, 0.12 wt%, 0.01 wt%, and 0.01 wt% of the total weight of the kudzu flower extract solution and pure water, respectively. The kudzu flower beverage prepared according to the optimal ratio was a uniform yellowish-brown color, with a homogeneous liquid texture, and no visible foreign matter was detected. Figure 14 The kudzu flower beverage is uniform and has good stability. After shaking, it has good fluidity and no layering is observed. It has a slight medicinal aroma, is light and elegant with a slightly sweet aftertaste, and has no abnormal odor.

Claims

1. The application of kudzu flower extract in the preparation of drugs for the prevention and / or treatment of obesity; characterized in that: The kudzu flower extract is obtained by using kudzu flowers as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, reflux extraction, filtration, and concentration and drying of the filtrate under reduced pressure.

2. The application according to claim 1, characterized in that: The application of the kudzu flower extract in the preparation of a medicine for preventing and / or treating obesity by: reducing body weight, reducing the accumulation and weight of body fat, and downregulating the level of lipid factors.

3. The application according to claim 1, characterized in that: The drug is a pharmaceutically acceptable formulation prepared with the kudzu flower extract as the main or sole active ingredient and pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: The formulations mentioned are oral solid dosage forms, oral solutions, and injections.

5. The application of kudzu flower extract in the preparation of health foods that help control body fat; characterized in that: The kudzu flower extract is obtained by using kudzu flowers as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, reflux extraction, filtration, and concentration and drying of the filtrate under reduced pressure.

6. The application of kudzu flower extract in the preparation of health food products that help maintain healthy blood lipid levels; wherein the blood lipids are cholesterol / triglycerides; characterized in that: The kudzu flower extract is obtained by using kudzu flowers as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, reflux extraction, filtration, and concentration and drying of the filtrate under reduced pressure.

7. The application according to claim 1, 5, or 6, characterized in that: The kudzu flower extract is obtained by using kudzu flowers as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, reflux extraction at 75 to 85℃ for 2 to 3 times at a material-to-liquid ratio of 1:13 to 1:19 g / mL, combining the extracts, filtering, and then concentrating and drying the filtrate under reduced pressure.

8. The application according to claim 7, characterized in that: The extraction solvent is an 80%–84% aqueous ethanol solution; the material-to-liquid ratio is 1:15 g / mL; the reflux extraction temperature is 85°C; and the reflux extraction is performed twice.

9. A composition having weight-loss effects, characterized in that: The composition includes a kudzu flower extract solution, a sweetener, an acidulant, an antioxidant, a preservative, a flavoring agent, and water; The total weight of the kudzu flower extract solution and water is 100%, and the kudzu flower extract solution accounts for 40-60% of the total weight of the kudzu flower extract solution and water. The sweetener, acidulant, antioxidant, preservative, and flavoring agent are respectively 4-8%, 0.08-0.16%, 0.01%, 0.1%, and 0.01% of the total weight of the kudzu flower extract solution and water. The concentration of the kudzu flower extract solution is 20 mg / mL; the kudzu flower extract is obtained by using kudzu flower as raw material, 70% to 90% ethanol aqueous solution as extraction solvent, at a material-to-liquid ratio of 1:13 to 1:19 g / mL, through reflux extraction, filtration, and concentration and drying of the filtrate under reduced pressure. The sweetener is at least one of xylitol, erythritol, and sucralose; The acidulant is at least one of citric acid and malic acid; The antioxidant mentioned is at least one of D-ascorbic acid and sodium ascorbate; The preservative is at least one of sodium benzoate and potassium sorbate; The flavoring agent is white peach oolong flavoring, honey peach oolong flavoring, or osmanthus oolong flavoring.

10. The composition with weight-loss effect according to claim 9, characterized in that: The total weight of the kudzu flower extract solution and water is 100%, and the kudzu flower extract solution accounts for 40% of the total weight of the kudzu flower extract solution and water; the sweetener, acidulant, antioxidant, preservative and flavoring agent are 8%, 0.12%, 0.01%, 0.1% and 0.01% of the total weight of the kudzu flower extract solution and water, respectively.

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