Brassica rapa flavonoid extract with uric acid reducing activity as well as preparation method and application of brassica rapa flavonoid extract

Abstract

The invention discloses a brassica rapa flavonoid extract with uric acid reducing activity as well as a preparation method and application of the brassica rapa flavonoid extract. According to the preparation method disclosed by the invention, process parameters are optimized by combining an ultrasonic-assisted extraction method with response surface analysis, the high-content and high-purity brassica rapa flavonoid extract is prepared, quercetin-3-glucuronide is determined as a main component of the brassica rapa flavonoid extract, and the remarkable treatment effect of the brassica rapa flavonoid extract on hyperuricemia is verified through in-vitro experiments and animal experiments; the brassica rapa flavonoid extract not only can effectively inhibit the activity of xanthine oxidase (XOD) and reduce the level of serum uric acid, but also can relieve inflammatory response of the kidney and improve pathological injury of the kidney, meanwhile, the preparation process is efficient and environmentally friendly, and a new direction is provided for development of safe and effective natural drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant natural active ingredient extraction, and particularly relates to a Chamaeclema flavonoid extract with uric acid lowering activity, a preparation method thereof and application. BACKGROUND

[0002] Hyperuricemia (HUA) is a metabolic disease caused by excessive uric acid production or insufficient excretion. Uric acid is the final product of oxidation of hypoxanthine or xanthine catalyzed by xanthine oxidase. Long-term maintenance of serum uric acid at a high level and an increase in the level may further develop into gout. In China, the standardized overall prevalence of HUA in adults has reached 17.7%, and there is a trend of increasing year by year. At the same time, it is closely related to various cardiovascular diseases, which poses a serious threat to human health. At present, allopurinol (ALL) is a commonly used therapeutic drug in clinical practice, which reduces the generation of uric acid by inhibiting the activity of xanthine oxidase (XOD). However, the use of allopurinol may cause adverse reactions such as skin rash, gastrointestinal discomfort, and renal function damage (Lai S, Comparison of benzbromarone and allopurinol on the risk of chronic kidney disease in people with asymptomatic hyperuricemia, European Journal of Internal Medicine, 2023). Therefore, finding safe and effective natural uric acid-lowering substances has become a current research hotspot.

[0003] In recent years, numerous studies have shown that plant natural active flavonoids have great potential in the treatment of hyperuricemia. Jun Li et al. found that quercetin (IC50=0.03 mg / mL) and kaempferol (IC50=0.11 mg / mL) can reduce the production of uric acid by inhibiting the activity of XOD, and the effect is close to that of allopurinol (IC50=0.06 mg / mL) (Li J, In vitro inhibitory effects of polyphenols from Tartary buckwheat on xanthine oxidase: Identification, inhibitory activity, and action mechanism, Food Chemistry, 2022). Yu Toyoda et al. found that among 7 flavonoids isolated and identified from citrus extracts, naringin (IC50=16.1 μmol / L), hesperetin (IC50=25.7 μmol / L) and nobiletin (IC50=17.6 μmol / L) can significantly inhibit the activity of urate transporter URAT1 and reduce the reabsorption of uric acid (Toyoda Y, Inhibitory effect of Citrus flavonoids on the in vitro transport activity of human urate transporter 1 (URAT1 / SLC22A12), a renal re-absorber of urate, npj Science of Food, 2025). Yuyi Liu et al. found that flavonoid extract of Paederia scandens can improve hyperuricemia in mice by regulating uric acid metabolism and intestinal flora, and reducing liver and kidney damage (Liu Y, Flavonoid-rich extract of Paederia scandens (Lour.) Merrill improves hyperuricemia by regulating uric acid metabolism and gut microbiota, Food Chemistry, 2025).

[0004] Brassica rapa L., also known as turnip, is a biennial herbaceous plant of the Brassica genus in the Brassicaceae family, widely distributed in the northwest of China. In particular, in Xinjiang and Tibet, turnip is considered a food or medicinal material with a long history. Its tubers are rich in nutrients such as polysaccharides (0.13-0.21 g / 100 g), flavonoids (0.08-0.14 g / 100 g), and saponins (0.51-0.78 g / 100 g), and have various biological activities such as antioxidant, hypoglycemic, and immunomodulatory activities (Liu Xiaojiao, Nutritional functions and processing status of turnip, Grain and Food Industry, 2020). Currently, there are few studies on the anti-hyperuricemia effects of turnip flavonoid extracts.

[0005] The patent document "Application of extract of fructus amomi in preparation of UA-lowering and anti-gouty arthritis drugs" (CN116211978 B) by Dong Li et al. shows that the 95% ethanol extract (EA) of fructus amomi has significant inhibitory activity on xanthine oxidase (XOD) in vitro, with an IC50 value of 54.65 μg / mL, which is significantly better than the water extract and methanol extract of fructus amomi. In a hyperuricemic mouse model, EA can significantly reduce serum uric acid (UA) levels, with the serum UA level of the high-dose group (390 mg / kg) decreasing to 144.49 μmol / L, close to that of the normal group. In addition, EA also plays a role in anti-gouty arthritis by inhibiting the release of inflammatory factors such as IL-6, TNF-α, and IL-1β. In a gouty arthritis mouse model, the high-dose group (390 mg / kg) of EA can significantly reduce the swelling degree of the paw tissue, and the effect is comparable to that of the positive control drug colchicine. However, the preparation process of the fructus amomi extract in this patent document involves multiple reflux extractions and freeze-drying, which is relatively complicated, and the composition of the extract is complex, so further research is needed to study the specific mechanism of action of the active ingredients.

[0006] The patent document "White Peony Root Extract for Treating Hyperuricemia, Preparation Method and Application" (CN118615349 B) by Li Ning et al. shows that the white peony root extract prepared by a specific extraction process has a significant therapeutic effect on hyperuricemia. The extract mainly reduces uric acid production by inhibiting the activity of xanthine oxidase in serum and liver, while down-regulating the levels of kidney tissue proteins URAT1 and GLUT9, up-regulating the levels of kidney tissue proteins OAT1 and OAT3, and promoting uric acid excretion. In a hyperuricemia rat model, the high-dose group (800 mg / kg) of white peony root extract can significantly reduce blood uric acid levels in a concentration-dependent manner. In addition, when the extract is used in combination with allopurinol, it not only enhances the uric acid-lowering effect, but also improves the gastrointestinal adverse reactions and kidney damage caused by allopurinol. However, the preparation process of the white peony root extract in this patent document involves multiple ethanol extractions, ether impurity removal, and multiple solvent extractions, which is complex and costly, and needs to be further optimized to improve extraction efficiency and reduce costs. SUMMARY

[0007] The present application aims to provide a Chamaeclemais formosana flavonoid extract with uric acid-lowering activity, as well as its preparation method and application. The present application uses ultrasonic-assisted extraction combined with response surface analysis to optimize process parameters, and prepares a Chamaeclemais formosana flavonoid extract with high content and high purity. The therapeutic effect of the extract on hyperuricemia is verified through in vitro experiments and animal experiments.

[0008] The preparation process of the present application is efficient and environmentally friendly. The obtained Chamaeclemais formosana flavonoid extract can be used to prepare drugs and functional foods for preventing and / or treating hyperuricemia. The present application overcomes the shortcomings of existing treatment drugs and provides a new direction for the development of safe and effective natural drugs.

[0009] The technical solution of the present application is as follows:

[0010] A preparation method of a Chamaeclemais formosana flavonoid extract, comprising:

[0011] (1) Raw material processing: Take Chamaeclemais formosana tubers, wash, slice, dry, crush, and sieve to obtain Chamaeclemais formosana dry powder;

[0012] Preferably, after drying at 60°C, crush and sieve through a 60-mesh sieve;

[0013] (2) Ultrasonic extraction: Mix the Chamaeclemais formosana dry powder with an ethanol aqueous solution, perform ultrasonic extraction, then filter, collect the filtrate, and obtain a Chamaeclemais formosana flavonoid crude extract;

[0014] Preferably, the volume fraction of the ethanol aqueous solution is 61%;

[0015] Preferably, the mass-to-volume ratio of the Chamaeclemais formosana dry powder to the ethanol aqueous solution is 1:20, g / mL;

[0016] The power of the ultrasonic extraction is preferably 436 W, and the time is 24 min.

[0017] Specifically, the method for collecting the filtrate can be high-speed centrifugation followed by filtration; the filtration can be performed by using a Buchner funnel filter paper for negative pressure suction filtration.

[0018] (3) Purification: The crude extract of QMAG flavonoids is rotary evaporated to remove ethanol, and then subjected to static adsorption by AB-8 macroporous resin, followed by elution of impurities with distilled water, and then elution of the target components with an ethanol aqueous solution; the eluate is concentrated by rotary evaporation, and then freeze-dried to obtain the QMAG flavonoids extract.

[0019] Preferably, the volume-to-mass ratio of the crude extract of QMAG flavonoids to AB-8 macroporous resin is 1:1, mL / g.

[0020] Preferably, the volume ratio of the crude extract of QMAG flavonoids to the distilled water and the ethanol aqueous solution is 1:2:2.

[0021] Preferably, the static adsorption time is 4 h.

[0022] Preferably, the volume fraction of the ethanol aqueous solution is 70%.

[0023] The rotary evaporation is performed in a water bath at 60°C.

[0024] The freeze-drying is performed at a temperature of -45°C for 48 h.

[0025] The present application relates to the QMAG flavonoids extract prepared by the above preparation method.

[0026] The QMAG flavonoids extract can be used for preparing a medicament for preventing and / or treating hyperuricemia. The medicament is preferably an oral preparation; and the medicament preferably comprises the QMAG flavonoids extract and a pharmaceutically acceptable excipient, and the type and amount of the excipient are not particularly limited.

[0027] The QMAG flavonoids extract can be used for preparing a functional food for preventing and / or treating hyperuricemia. The functional food uses the QMAG flavonoids extract as an effective component; and the preparation method of the functional food is not particularly limited, and a conventional preparation method of a functional food in the art can be used.

[0028] The innovation points of the present application include:

[0029] The present application determines the extraction parameters for the characteristics of the QMAG matrix by the response surface method, and the optimal extraction conditions are as follows: ultrasonic time 24 min, power 436 W, solid-to-liquid ratio 1:20, and volume fraction of the ethanol aqueous solution 61%. In the obtained QMAG flavonoids extract, the highest content of the substance is quercetin-3-glucuronide, and there is no report on its uric acid-lowering activity at present.

[0030] The present application provides a quercetin-3-glucuronide flavonoid extract, and the significant therapeutic effect of the extract on hyperuricemia is verified by in vitro and animal experiments. The flavonoid extract of Qamagou can effectively inhibit the activity of xanthine oxidase (XOD), reduce the serum uric acid level, alleviate the inflammatory response of the kidney, and improve the pathological damage of the kidney.

[0031] The present application has the following beneficial effects:

[0032] The present application uses ultrasonic-assisted extraction method combined with response surface analysis to optimize the process parameters, so that the content of the flavonoid extract of Qamagou is significantly improved, the average content reaches 5.20 mg / g, and the purity of the freeze-dried sample of the final extract reaches 68.34%. The method has high extraction efficiency, short time, energy saving and environmental protection.

[0033] The significant therapeutic effect of the flavonoid extract of Qamagou on hyperuricemia is verified by in vitro and animal experiments. The in vitro experiment shows that the extract has inhibitory effect on xanthine oxidase (XOD), and the IC50 value is 68.83 μg / mL, and it is competitive inhibition. The animal experiment results show that the flavonoid extract of Qamagou can significantly reduce the serum uric acid level of hyperuricemia mice, and the content of the high-dose group is decreased by 36.89% (P<0.0001) compared with the model group, while the activities of XOD and adenosine deaminase (ADA) in the liver are inhibited, the levels of serum creatinine (Cr) and urea nitrogen (BUN) are reduced, the inflammatory response of the kidney is alleviated, and the pathological damage of the kidney is improved.

[0034] The flavonoid extract of Qamagou is natural and has high safety, and has good development prospect. Moreover, the preparation method is simple and efficient, the extraction conditions are mild, no toxic and harmful organic solvents are needed, it is green and environmentally friendly, the purity of the obtained extract is high, the content of active ingredients is rich, it provides a scientific basis for developing hyperuricemia prevention and treatment drugs based on natural products, and has significant social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The process flow chart of the present application.

[0036] Figure 2 The effects of different conditions on the content of flavonoids; A: the effect of ultrasonic time on the content of extracted flavonoids; B: the effect of ultrasonic power on the content of extracted flavonoids; C: the effect of liquid-to-material ratio on the content of extracted flavonoids; D: the effect of the volume fraction of ethanol aqueous solution on the content of extracted flavonoids.

[0037] Figure 3 The effect of the flavonoid extract of Qamagou on the serum UA content of HUA mice.

[0038] Figure 4 Effect of flavonoids extract from Qamagou on serum Cr and BUN levels in HUA mice.

[0039] Figure 5 Effect of flavonoids extract from Qamagou on XOD and ADA activities in liver of HUA mice.

[0040] Figure 6 Effect of flavonoids extract from Qamagou on inflammation in kidney of HUA mice.

[0041] Figure 7 Effect of flavonoids extract from Qamagou on histopathology of kidney of HUA mice.

[0042] Figure 8 In vitro inhibitory effect of flavonoids extract from Qamagou on XOD.

[0043] Figure 9 Total ion chromatogram of flavonoids extract from Qamagou; A: negative ion mode; B: positive ion mode.

[0044] Figure 10 Visualization result of molecular docking of quercetin-3-glucuronide with XOD. DETAILED DESCRIPTION

[0045] The present application will be further described below by specific examples, but the scope of protection of the present application is not limited to this.

[0046] Example 1: Preparation of flavonoids extract from Qamagou by single factor test

[0047] (1) Raw material treatment: Qamagou tubers provided by Shengquan Industry Co., Ltd. in Keping County, Aksu Region, Xinjiang were cleaned, sliced, dried at 60℃, and then ground through a 60-mesh sieve to obtain Qamagou dry powder.

[0048] (2) Ultrasonic extraction: 3.0 g of Qamagou dry powder was accurately weighed and extracted for 5 min, 10 min, 15 min, 20 min, and 25 min, respectively, with a solid-liquid ratio of 1:15 (g / mL) and a volume fraction of 50% ethanol aqueous solution, and an ultrasonic power of 423 W.

[0049] The ultrasonic extraction was performed using an ultrasonic cell crusher with an adjustable range of 0-100% and corresponding wattage of 0-650 W. The test results are shown in Table A. Figure 2 The crude flavonoids extract was obtained by filtration.

[0050] Example 2: Extraction at ultrasonic power of 293 W, 358 W, 423 W, 488 W, 553 W, respectively, solid-liquid ratio of 1:15, volume fraction of ethanol aqueous solution of 50%, ultrasonic time of 15 min, and other conditions according to Example 1. The test results are as follows: Figure 2 Medium B.

[0051] Example 3: Extraction at solid-liquid ratio of 1:10, 1:15, 1:20, 1:25, 1:30, respectively, volume fraction of ethanol aqueous solution of 50%, ultrasonic power of 358 W, ultrasonic time of 15 min, and other conditions according to Example 1. The test results are as follows: Figure 2 Medium C.

[0052] Example 4: Extraction at volume fraction of ethanol aqueous solution of 10%, 30%, 50%, 70%, 90%, respectively, solid-liquid ratio of 1:15, ultrasonic power of 358 W, ultrasonic time of 15 min, and other conditions according to Example 1. The test results are as follows: Figure 2 Medium D.

[0053] Example 5: Response surface optimization test

[0054] The optimal process parameters for ultrasonic extraction of Chamaecyparis class flavonoids were determined by response surface analysis using a four-factor three-level rotation combination, with flavonoid content as the response value. The factor level coding table is as follows:

[0055] Table 1 Response surface factors and levels

[0056]

[0057] According to the central composite design test scheme, a four-factor three-level test was conducted, and the results are as follows:

[0058] Table 2 Test results

[0059]

[0060]

[0061] The test data were fitted in Design Expert 13 to obtain a quadratic regression equation with flavonoid content as the objective function: Y = 5.15 + 0.4673*A + 0.3276*B - 0.4335*C + 0.2591*D + 0.2286*AB

[0062] -0.1067*AC + 0.3352*AD - 0.2362*BC + 0.1067*BD - 0.4267*CD - 0.5864*A 2 -0.712*B 2

[0063] -0.8307 * C - 0.7769 * D 2 .

[0064] The analysis of variance of the regression equation is as follows:

[0065] Table 3 Analysis of variance of the regression equation

[0066]

[0067] From the software analysis, it can be seen that the regression equation has a maximum point, i.e. the maximum predicted flavonoid content of Qiamagu is 5.46 mg / g, and the corresponding optimal extraction process parameters are ultrasonic time of 24.19 min, ultrasonic power of 435.96 W, solid-liquid ratio of 1:20.41, and volume fraction of ethanol aqueous solution of 60.92%. For the convenience of actual operation, the parameters are adjusted appropriately, i.e. ultrasonic time of 24 min, ultrasonic power of 436 W, solid-liquid ratio of 1:20, and volume fraction of ethanol aqueous solution of 61%, and the average flavonoid content is 5.20 mg / g in three verification experiments, which is basically consistent with the theoretical value.

[0068] Example 6: Purification of Qiamagu flavonoid extract

[0069] The 500 mL crude flavonoid extract obtained according to the optimized parameters in Example 5 is rotary evaporated in a 60°C water bath, and after removing ethanol, static adsorption is performed for 4 h through 500 g AB-8 macroporous resin (Macklin, reagent grade, 500 g). First, 1000 mL of distilled water is used to elute impurities, and then 1000 mL of ethanol aqueous solution with a volume fraction of 70% is used to elute the target component. The eluate is rotary evaporated in a 60°C water bath, and then freeze-dried to obtain Qiamagu flavonoid extract powder. The flavonoid content is 68.34% as determined.

[0070] Example 7: Therapeutic effect of Qiamagu flavonoid extract on hyperuricemia mice

[0071] (1) Animal grouping and administration: 36 8-week-old male Balb / c mice are randomly divided into 6 groups, 6 mice in each group, namely normal control group (NC), model control group (MC), positive control group (AP), Qiamagu flavonoid extract low-dose group (FB-L), medium-dose group (FB-M), and high-dose group (FB-H). Except for the normal control group, the rest of the mice are given hypoxanthine 300 mg / Kg by gavage, and 1 h later, the positive control group is given allopurinol solution 15 mg / kg by gavage, and the Qiamagu flavonoid extract low-, medium-, and high-dose groups are given 75, 125, and 250 mg / kg by gavage, respectively. The normal control group and the model control group are given the same volume of 0.5% CMC-Na solution by gavage, and the administration is continued for 7 days.

[0072] (2) Modeling and Tissue Collection: Mice were fasted for 2 hours before drug administration on day 7. One hour later, each group of mice was intraperitoneally injected with potassium oxonate 300 mg / kg to establish a hyperuricemia model. One hour after modeling, blood was collected from the eye sockets of the mice, and serum was separated by centrifugation to detect serum uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), and other indicators. Mice were euthanized by cervical dislocation, and liver and kidney tissues were collected to detect XOD and ADA activity in the liver, as well as the levels of inflammatory factors and pathological changes in the kidneys.

[0073] (3) Results determination: Serum and liver / kidney biochemical index detection: Serum UA, Cr, and BUN levels, and liver XOD and ADA levels were measured using kits, following the manufacturer's instructions. ELISA kits were used to detect IL-1β, IL-6, and TNF-α levels in kidney tissue. Kidney pathological examination: Kidney tissue was fixed with 4% paraformaldehyde solution. After 24 hours, it was dehydrated with ethanol, sectioned, clarified with xylene, embedded in paraffin, stained with hematoxylin and eosin (H&E), and observed under a light microscope for histopathological changes.

[0074] (4) Test results: such as Figure 3 The serum UA level in the model group mice was significantly higher than that in the normal control group (P<0.0001), indicating that the hyperuricemia model was successfully established. After intervention with Chaga mushroom flavonoid extract, the serum UA level in mice in each dose group decreased to varying degrees, with the highest dose group showing the most significant decrease. Compared with the model group, the UA level decreased by 36.89% (P<0.0001), indicating that Chaga mushroom flavonoid extract has a significant uric acid-lowering effect.

[0075] like Figure 4 The serum Cr and BUN levels in the model group mice were significantly higher than those in the normal control group (P<0.01), indicating that hyperuricemia caused severe damage to the kidney function of mice. However, after intervention with Chaga mushroom flavonoid extract, the serum Cr and BUN levels in the medium and high dose groups were significantly reduced (P<0.05), with the medium dose group showing the most significant reduction. There was no significant difference compared to the normal control group (P>0.05), indicating that Chaga mushroom flavonoid extract can effectively improve kidney function damage caused by hyperuricemia and has a certain protective effect on the kidneys.

[0076] like Figure 5The activities of XOD and ADA in the liver of mice in the model group were significantly higher than those in the normal control group (P<0.01), which is consistent with the pathological characteristics of increased uric acid production in hyperuricemia. However, after intervention with Chaga mushroom flavonoid extract, the activities of XOD and ADA in the liver of mice in all dose groups were significantly reduced (P<0.05), with the most significant reduction in the high-dose group, where XOD activity decreased by 41.1% and ADA activity decreased by 46.2%. This indicates that Chaga mushroom flavonoid extract can effectively inhibit the activities of XOD and ADA, thereby reducing uric acid production and exerting a uric acid-lowering effect.

[0077] like Figure 6 The levels of IL-1β, IL-6, and TNF-α in the kidneys of model group mice were significantly higher than those in the normal control group (P<0.01), indicating that hyperuricemia induced an inflammatory response in the kidneys. However, after intervention with Chaga mushroom flavonoid extract, the levels of IL-1β, IL-6, and TNF-α in the kidneys of mice in the medium and high dose groups were significantly lower (P<0.05), indicating that Chaga mushroom flavonoid extract has a significant anti-inflammatory effect and can effectively alleviate the inflammatory response in the kidneys caused by hyperuricemia.

[0078] like Figure 7 In the NC group, the nephrons of mice showed intact structure, with normal morphology of the Bowman's capsule wall, proximal convoluted tubules, and distal convoluted tubules, and no obvious pathological changes such as glomerular atrophy or inflammatory cell infiltration. Compared with the normal group, the MC group showed significant dilation of the proximal and distal convoluted tubules, thickening of the Bowman's capsule wall, and mild glomerular atrophy, indicating that hyperuricemia caused pathological damage to the kidneys. The AP group showed mild glomerular atrophy and mild dilation of the distal convoluted tubules. After intervention with Chaga mushroom flavonoid extract, the FB-L group showed normal glomeruli and Bowman's capsule walls, but nuclear rupture and dissolution of the distal convoluted tubules; the FB-M group showed normal glomeruli and Bowman's capsule walls, and significant relief of distal convoluted tubule nuclear rupture; the FB-H group showed intact nephron structure, no glomerular atrophy, and normal morphology of the Bowman's capsule wall, proximal convoluted tubules, and distal convoluted tubules. It is evident that the FB-H group showed the most significant improvement, with kidney tissue structure similar to the NC group. This indicates that the flavonoid extract of Chamaegu can effectively improve kidney pathological damage caused by hyperuricemia and has a certain protective effect on the kidneys.

[0079] Example 8: In vitro uric acid-lowering activity of Chamaegu flavonoid extract

[0080] (1) Determination of xanthine oxidase inhibition rate: The in vitro inhibitory effect of Chaga mushroom flavonoid extract on xanthine oxidase activity was determined by ultraviolet spectrophotometry. Extract solutions of 25, 50, 100, 200, and 400 μg / mL were prepared, with allopurinol used as a positive control (0.2, 0.5, 1, 2, and 5 μg / mL). 100 μL of each concentration of extract, allopurinol solution, and blank solution (PBS) and 50 μL of 0.08 U / mL enzyme solution were added sequentially to a 96-well plate, with three replicates for each concentration. The plate was heated at 37℃ for 10 min, and then 50 μL of pre-warmed xanthine solution (400 μmol / L) was added to initiate the enzymatic reaction. After reacting at 37℃ for 30 min, the absorbance was measured at 293 nm. The formula for calculating the xanthine oxidase inhibition rate is as follows:

[0081] Inhibition rate / % = [1 - (AB) / (CD)] x 100%

[0082] In the formula:

[0083] (A) Absorbance of sample tube with or without enzyme addition;

[0084] (B) Absorbance of sample tube without enzyme or sample addition;

[0085] (C) - Add enzyme and sample tube absorbance;

[0086] (D) - Absorbance of sample tube without enzyme addition.

[0087] (2) Inhibition type of xanthine oxidase: The xanthine oxidase concentration was fixed (0.10 U / mL), and different concentrations (0.00, 1.00 and 3.00 mg / mL) of Chamaegu flavonoid extract solution were added respectively. The initial rate was measured when the xanthine solution concentration was 0.25, 0.50, 1.00, 1.50, 2.00 and 2.50 mmol / L. The inhibitory type of Chamaegu flavonoid extract on xanthine oxidase was determined by plotting the reciprocal of the initial rate (V) against the reciprocal of the substrate concentration [S] using the Lineweaver-Burks double reciprocal plot method.

[0088] (3) The test results are as follows Figure 8 In Figure A, as the concentration of the Chaga mushroom flavonoid extract increased, the XOD activity gradually decreased, indicating that its inhibitory effect on XOD was dose-dependent. The IC50 values ​​of the Chaga mushroom flavonoid extract and allopurinol were 68.83 μg / mL and 6.33 μg / mL, respectively. Figure 8In Figure B, plotting the Lineweaver-Burk double reciprocal equation, the Lineweaver-Burk double reciprocal curves of different concentrations of Chaga mushroom flavonoid extract almost intersect at the same point on the Y-axis, indicating that VMAX remains unchanged, meaning that the presence of Chaga mushroom flavonoid extract does not cause a change in the maximum reaction rate VMAX of xanthine oxidase. As the concentration of Chaga mushroom flavonoid extract increases, the slope of the curve increases, indicating that the Michaelis constant KM increases, showing that the inhibition type of xanthine oxidase by Chaga mushroom flavonoid extract is competitive inhibition.

[0089] Example 9: Component Analysis of Chamaegu Flavonoid Extract

[0090] (1) The components of the Chamaegu flavonoid extract were identified using UPLC-Q-TOF-MS / MS.

[0091] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (1.8 μm 2.1 × 100 mm), mobile phase: 0.1% formic acid water (A), 0.1% formic acid acetonitrile (B), gradient elution (0-1.5 min, 95% A; 1.5-2.5 min, 95%-90% A; 2.5-14 min, 90%-60% A; 14-24 min, 60%-5% A; 24-27 min, 5% A; 27-27.1 min, 5%-95% A; 27.1-30 min, 95% A), flow rate 0.4 mL / min, sample chamber temperature 10℃, column temperature 40℃, injection volume 4 μL.

[0092] Mass spectrometry conditions: The AB 5600 Triple TOF mass spectrometer can perform primary and secondary mass spectrometry data acquisition based on IDA function under the control software (Analyst TF 1.7, ABSciex). In each data acquisition cycle, the molecular ions with the strongest intensity and greater than 100 are selected for the corresponding secondary mass spectrometry data acquisition. The primary acquisition range is 50-1200, bombardment energy: 30 eV, and 15 secondary spectra are acquired per cycle. The ESI ion source parameters are set as follows: nebulizer pressure (GS1): 60 Psi, auxiliary gas pressure: 60 Psi, curtain gas pressure: 35 Psi, temperature: 550℃, spray voltage: 5500V (positive ion mode) or -4500V (negative ion mode).

[0093] (2) Experimental Results: UPLC-Q-TOF-MS / MS analysis, compared with databases such as Metlin, MassBank, and MoNA, revealed that the Chamaegu flavonoid extract contained 187 flavonoid compounds, mainly flavones, flavonols, dihydroflavones, flavanols, biflavones, and chalcones, with quercetin-3-glucuronide as the main component. The total ion chromatogram is shown below.Figure 9 .

[0094] Example 10: Molecular docking simulation analysis of the interaction between quercetin-3-glucuronide and XOD

[0095] The 3D crystal structure of guanine-XOD (PDB ID: 3NVW) was downloaded from the PDB database (https: / / www.rcsb.org / ). Small molecule ligands such as H2O and guanine were removed, and redundant peptide chains were deleted using PyMoL v2.4.0 software. The SDF file of quercetin-3-glucuronide, the flavonoid compound with the highest proportion in the LC-MS identification results, was downloaded from the NCBI website (https: / / pubchem.ncbi.nlm.nih.gov / ) and converted to PDB format using OpenBabel v3.1.1. The prepared quercetin-3-glucuronide ligand and XOD receptor were docked using AutoDock Vina software. Based on the principle that lower binding energies indicate more stable binding, all docking results were selected using the lowest binding energy. The main conformations of quercetin-3-glucuronide and XOD binding were screened and imported into PyMoL v2.4.0 software for analysis and plotting.

[0096] The molecular docking results are shown in Table 4, and the schematic diagram is shown in [the diagram]. Figure 10 .

[0097] Table 4 Results of XOD docking of flavonoids

[0098]

Claims

1. A method for preparing a flavonoid extract from Chamaegu, characterized in that, include: (1) Raw material processing: Take Chamaegu tuber, wash, slice, dry, crush and sieve to obtain Chamaegu dry powder; (2) Ultrasonic extraction: Mix dried Chamaegu powder with ethanol aqueous solution, perform ultrasonic extraction, filter, collect the filtrate, and obtain crude extract of Chamaegu flavonoids. (3) Purification: The crude extract of Chamaegu flavonoids was rotary evaporated to remove ethanol, statically adsorbed through AB-8 macroporous resin, then impurities were washed off with distilled water, and the target components were washed off with an ethanol aqueous solution. The eluent was collected, concentrated by rotary evaporation, and freeze-dried to obtain the Chamaegu flavonoid extract.

2. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (2), the volume fraction of the ethanol-water solution is 61%.

3. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (2), the mass-to-volume ratio of dried Chamaegu powder to ethanol-water solution is 1:20, g / mL.

4. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (2), the ultrasonic extraction power is 436W and the time is 24min.

5. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (3), the volume-to-mass ratio of the crude extract of Chamaegu flavonoids to AB-8 macroporous resin is 1:1, mL / g.

6. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (3), the volume ratio of the crude extract of Chamaegu flavonoids to distilled water and ethanol aqueous solution is 1:2:

2.

7. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (3), the static adsorption time is 4 hours.

8. The method for preparing the Chamaegu flavonoid extract as described in claim 1, characterized in that, In step (3), the volume fraction of the ethanol-water solution is 70%.

9. The flavonoid extract of Chamaegu prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the Chamaegu flavonoid extract as described in claim 9 in the preparation of drugs and functional foods for the prevention and / or treatment of hyperuricemia.

Images