A uric acid-lowering compound traditional Chinese medicine extract and preparation and application thereof

Abstract

This invention belongs to the field of traditional Chinese medicine technology, and provides a compound traditional Chinese medicine extract for lowering uric acid, its preparation, and application. Using hawthorn, astragalus, poria cocos, jujube, and tangerine peel as raw materials, this invention obtains ethanol extract S1 and water extract S2 through ethanol extraction and water extraction. Both S1 and S2 have antioxidant activity, targeting DPPH and ABTS. + The free radicals exhibit significant scavenging capabilities and can effectively inhibit xanthine oxidase activity. Furthermore, S1 and S2 can reduce serum and liver XOD activity in hyperuricemic mice, improve liver and kidney function indicators such as AST, ALT, TP, UREA, SCr, and CysC, and decrease serum uric acid levels in a dose-dependent manner; high doses can lower uric acid levels below normal. The extract of this invention possesses uric acid-lowering, antioxidant, and liver and kidney protective effects, showing good therapeutic effects on hyperuricemia and good protective and preventive effects against liver and kidney damage caused by hyperuricemia.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a compound traditional Chinese medicine extract for lowering uric acid and its preparation and application. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease characterized by abnormally elevated serum uric acid levels. It is the core pathological basis for the occurrence and development of gout, and also an independent risk factor for many chronic diseases such as metabolic syndrome, diabetes, cardiovascular disease, and chronic kidney disease. Clinical studies have confirmed that persistently elevated serum uric acid can induce the body to produce a large number of oxygen free radicals and reactive oxygen species, causing oxidative stress damage, which further leads to damage to the structure and function of hepatocytes and renal tubular epithelial cells, seriously affecting liver and kidney homeostasis.

[0003] The production of uric acid in the human body mainly depends on the metabolism of hypoxanthine and xanthine catalyzed by xanthine oxidase (XOD). Therefore, inhibiting xanthine oxidase activity is a key target for clinical intervention in hyperuricemia and reducing endogenous uric acid synthesis. Currently, commonly used uric acid-lowering drugs include allopurinol, febuxostat, and benzbromarone. Although they can rapidly reduce blood uric acid levels, long-term use can easily cause gastrointestinal reactions, liver and kidney damage, allergic reactions, and adverse cardiovascular events, posing significant safety risks and failing to meet the clinical need for long-term safe use.

[0004] Traditional Chinese medicine (TCM) possesses unique advantages in the prevention and treatment of metabolic diseases, including multiple targets, minimal side effects, and holistic regulation, making it an important direction for the research and development of natural drugs for hyperuricemia. Traditional Chinese medicine treatment of hyperuricemia primarily focuses on strengthening the spleen, promoting diuresis, and eliminating dampness; however, it suffers from limitations such as weak targeted formulation, unclear mechanisms of action, and poor synergistic effects in lowering uric acid and protecting organs, resulting in limited clinical efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a compound Chinese herbal extract for lowering uric acid, its preparation and application. Based on the traditional Chinese medicine theory of "promoting digestion and blood circulation, regulating qi and strengthening the spleen, and eliminating dampness and turbidity", a compound Chinese herbal extract composed of hawthorn, astragalus, poria, jujube and tangerine peel is selected. The effective components are obtained by ethanol extraction and water extraction respectively. It has multiple functions such as inhibiting xanthine oxidase, scavenging free radicals, lowering blood uric acid, and protecting liver and kidney function, so as to solve the problems of large side effects of existing Western medicines and single efficacy of traditional Chinese medicine.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a compound Chinese herbal extract for lowering uric acid, wherein the compound Chinese herbal extract is an ethanol extract S1 and a water extract S2, wherein both S1 and S2 are extracted from a compound Chinese herbal composition, and the compound Chinese herbal composition is composed of the following raw materials in parts by weight: 40-60 parts of hawthorn, 15-25 parts of astragalus, 15-25 parts of poria cocos, 15-25 parts of jujube, and 5-15 parts of dried tangerine peel.

[0007] Preferably, the compound traditional Chinese medicine composition consists of the following raw materials in parts by weight: 50 parts hawthorn, 20 parts astragalus, 20 parts poria cocos, 20 parts jujube, and 10 parts dried tangerine peel.

[0008] The present invention further provides a method for preparing the above-mentioned compound Chinese herbal extract.

[0009] Preferably, the steps include:

[0010] (1) After the compound Chinese medicine composition is dried at a constant temperature to a constant weight, the extractant is added and soaked for 30 min;

[0011] (2) Boil and reflux for 15 minutes each time, filter by suction, repeat 3 times, and combine the filtrates;

[0012] (3) Concentrate the filtrate obtained in step (2) to a syrup-like state, and freeze-dry it to obtain the compound Chinese medicine extract.

[0013] Preferably, the extractant in step (1) is 80% ethanol or water.

[0014] Preferably, the concentration in step (3) is carried out by using a rotary evaporator at 70 °C.

[0015] The present invention further provides the application of the above-mentioned compound Chinese herbal extract.

[0016] Preferably, the compound Chinese herbal extract is used to prepare a drug for treating hyperuricemia, and the active ingredient of the drug is one of S1 or S2.

[0017] Preferably, the active ingredient of the drug is S1.

[0018] Preferably, the drug reduces uric acid production in the blood and liver by inhibiting xanthine oxidase activity.

[0019] Preferably, the drug also has antioxidant and hepatoprotective and renal-protective effects.

[0020] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0021] The beneficial effects of this invention are:

[0022] 1. Inhibits xanthine oxidase activity, thereby reducing uric acid production.

[0023] The compound extract of this invention can significantly inhibit the in vivo and in vitro activity of xanthine oxidase (XOD), reduce the level of XOD in serum and liver, block the conversion of hypoxanthine and xanthine into uric acid, and thus reduce the concentration of blood uric acid, and has a significant therapeutic effect on hyperuricemia.

[0024] 2. It possesses in vitro antioxidant activity and can alleviate oxidative stress damage.

[0025] Ethanol extract S1 and water extract S2 on DPPH, ABTS + These free radicals have good scavenging ability, which can reduce the level of oxidative stress in the body under hyperuricemia, reduce free radical-mediated cell and tissue damage, and provide important protection for improving pathological damage related to hyperuricemia.

[0026] 3. It also has a protective effect on liver and kidney function, achieving a synergistic effect of lowering uric acid and protecting organs.

[0027] This compound herbal extract can significantly improve liver and kidney function indicators in hyperuricemia model animals, and reduce serum levels of AST, ALT, TP, ALP, UREA, UA, SCr, and CysC. While reducing uric acid, it protects liver and kidney function, effectively improves liver and kidney dysfunction caused by hyperuricemia, and demonstrates the overall regulatory advantage.

[0028] 4. Mild effects, suitable for long-term intervention.

[0029] This compound Chinese medicine extract is composed of Chinese medicinal herbs that are both food and medicine. It has no obvious toxic side effects and can be used safely for a long time. It makes up for the shortcomings of commonly used chemical drugs in clinical practice, which are prone to causing adverse reactions such as liver and kidney damage, gastrointestinal reactions and allergies when used for a long time. It is more suitable for the long-term management of people with hyperuricemia. Attached Figure Description

[0030] Figure 1 This is a diagram showing the scavenging effect of S1 and S2 on DPPH in this invention;

[0031] Figure 2 In this invention, S1 and S2 are for ABTS + The result diagram of the · removal effect;

[0032] Figure 3 This is a graph showing the in vitro inhibitory effect of S1 and S2 on XOD in this invention;

[0033] Figure 4 This is a graph showing the inhibitory effects of S1 and S2 on serum XOD in this invention.

[0034] Figure 5This invention relates to the effect of S1 on liver function in model mice.

[0035] Figure 6 This invention relates to the effect of S2 on liver function in model mice.

[0036] Figure 7 This invention relates to the effect of S1 on renal function in model mice.

[0037] Figure 8 This invention relates to the effect of S2 on renal function in model mice. Detailed Implementation

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] The reagents used in the following examples are as follows: Ethanol, potassium dihydrogen phosphate, sodium hydroxide, and dimethyl sulfoxide were purchased from Sinopharm Chemical Reagent Co., Ltd., and were of analytical grade; potassium oxonate, uric acid, xanthine, DPPH, and ABTS were purchased from Shanghai Maclean Biotechnology Co., Ltd., and were of analytical grade; xanthine oxidase (Shanghai Maclean Biotechnology Co., Ltd., 50 U / mg), xanthine oxidase assay kit (Xanthine Oxidase (XOD) assay kit (Colorimetric method), Nanjing Jiancheng Bioengineering Institute, catalog number A002-1-1), and cystatin C assay kit (QuicKey Pro Human Cys-C (Cystatin C) ELISA Kit, Elabscience, catalog number E-OSEL-H0021).

[0042] Example 1

[0043] 1. Preparation of compound traditional Chinese medicine extracts

[0044] 1.1 Ethanol Extract S1

[0045] Traditional Chinese medicine composition formula: 50 g hawthorn, 20 g astragalus, 20 g poria cocos, 20 g jujube, 10 g dried tangerine peel.

[0046] The above-mentioned Chinese herbal medicine composition was dried at a constant temperature to constant weight, soaked in 1000 mL of 80% ethanol for 30 min, and then boiled and refluxed for 15 min each time. After filtration, the extraction was repeated 3 times and the filtrates were combined. The filtrates were concentrated to a syrupy state using a rotary evaporator at 70 °C and then freeze-dried using a freeze vacuum dryer to obtain the compound Chinese herbal medicine ethanol extract S1.

[0047] 1.2 Water extract S2

[0048] The formulation of the traditional Chinese medicine composition is the same as that of the ethanol extract S1. The traditional Chinese medicine composition is dried at a constant temperature to a constant weight, soaked in 1000 mL of pure water for 30 min, and then boiled and refluxed for 15 min each time. After filtration, the extraction is repeated 3 times and the filtrates are combined. The mixture is concentrated to a syrup state using a rotary evaporator at 70 °C and then freeze-dried using a freeze vacuum dryer to obtain the compound traditional Chinese medicine water extract S2.

[0049] 2. Performance Testing

[0050] The relevant performance tests were performed on the ethanol extract S1 and the water extract S2.

[0051] 2.1 Inhibitory effect on xanthine oxidase

[0052] Preparation of xanthine solution: Weigh 15 mg xanthine, add 1 mL concentrated ammonia, and heat in a warm water bath until completely dissolved. Adjust the volume to 50 mL with potassium phosphate buffer (pH=7.5) to prepare a 2 mmol / L xanthine stock solution. Dilute 4 times before use to obtain a 0.5 mmol / L xanthine working solution.

[0053] Preparation of xanthine oxidase solution: Weigh 0.001 g of xanthine oxidase and dilute to 50 mL with distilled water, i.e., 1.0 U / mL.

[0054] 2.2 Antioxidant effect

[0055] DPPH removal: A 25 mg / L DPPH solution was prepared using anhydrous ethanol. Sample solutions were prepared using 80% ethanol, with final reaction concentrations of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg / mL. 100 L of each sample solution was added to 3.9 mL of DPPH solution, and the reaction was carried out at room temperature for 30 min. A was then measured. 517 (Absorbance at 517 nm), the control group without sample solution was used. The DPPH scavenging rate was calculated using the following formula:

[0056]

[0057] In the formula A 0，1 A is a solution of deionized water and DPPH ethanol. 517A1 represents the concentrations of aqueous solutions and DPPH ethanol solutions of different samples. 517 A2 represents the concentration of anhydrous ethanol in aqueous solutions of samples at different concentrations. 517 .

[0058] ABTS + • Removal: Prepare 7.4 mmol / L ABTS and 2.6 mmol / L K₂S₂O₈ solutions with deionized water, mix equal volumes, and incubate at room temperature in the dark for 12 h as stock solutions. Dilute the working solution 50-fold with phosphate buffer (pH 7.4). Prepare sample solutions with deionized water, and take ABTS… + • Add 0.4 mL of sample solution to 1.6 mL of the solution. The final sample concentrations are 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg / mL. Mix thoroughly and let stand at room temperature for 10 min. Measure the absorbance A. 734 With the same mass concentration of V C As a positive control, ABTS + The free radical scavenging rate (R2, %) is calculated using the following formula:

[0059]

[0060] In the formula A 0，2 For ABTS + ·A of solution and deionized water 734 A3 is ABTS + • A of the mixture of solution and sample solution 734 A4 represents the A4 concentration of the sample solution mixed with deionized water. 734 .

[0061] 2.3 In vitro inhibition experiment of XOD

[0062] Inhibitor-free reaction system: 0.2 mL of solutions with different sample concentrations (2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 mg / mL) were placed in different test tubes. 1.0 mL of xanthine solution was added to each test tube, followed by 0.05 mL of xanthine oxidase solution (1.0 U / mL). The total volume was brought to 5 mL with potassium phosphate buffer (pH=7.5). The final concentration of xanthine was 0.1 mmol / L, and the final concentration of xanthine oxidase was 0.01 U / mL. The final concentrations of the samples were 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, and 0.24 mg / mL, respectively. After incubating the test tubes in a constant temperature water bath at 37 ℃ for 15 min, A was measured. 290, taking 1 mL of xanthine solution, adding 0.2 mL of sample solutions with different concentrations and 4.8 mL of phosphate buffer as the reference solution, calculate the influence of samples with different concentrations on XOD activity. The calculation formula is:

[0063]

[0064] A 1，290 is the absorbance value at 290 nm without adding the sample solution; A 2，290 is the absorbance value at 290 nm when adding the sample solution.

[0065] 2.4 In vivo experiments

[0066] 2.4.1 Establishment of mouse model

[0067] SPF-grade Kunming mice, weighing 25 - 35 g, were provided by Wuhan Youdu Biotechnology Co., Ltd. (Experimental Animal Use License Number: SYXK(E)2026 - 0115).

[0068] The hyperuricemia mouse model was established by feeding high-fat mouse diet. The mouse diet contained 45% fat (for energy supply), 20% yeast powder, 0.75% adenine. Male KM white mice at 60 days old were fed for 3 weeks, and then the serum uric acid content of the mice was detected to determine whether the model was successfully established. The mice were randomly divided into groups of 10 each. The disease model group (DG) continued to be fed high-fat mouse diet and pure water, and the treatment groups continued to be fed high-fat mouse diet, and the decoction (compound traditional Chinese medicine extract) was used instead of drinking pure water for treatment. The concentrations of S1 and S2 were divided into low-dose group (LG) at 2.0 g / L, mid-dose group (MG) at 4.0 g / L, high-dose group (HG) at 8.0 g / L, and the control group (CG) was fed according to normal diet.

[0069] 2.4.2 Function detection

[0070] Detection of XOD in vivo activity: After continuous feeding for 3 weeks, the mice were fasted for 12 h before sampling, and the activity of XOD in the liver and serum of the mice was detected using a kit (Xanthine Oxidase (XOD) assay kit (Colorimetric method), Nanjing Jiancheng Bioengineering Institute, product number A002 - 1 - 1)). ​Liver function tests: After 3 weeks of continuous feeding, fasting for 12 hours was performed, and blood was collected from the orbital vein. The serum was separated by centrifugation, and the levels of aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), and total protein (TP) in the serum were tested using a fully automated biochemical analyzer.

[0072] Kidney function tests: After 3 weeks of continuous feeding, fasting for 12 hours was performed. The main indicators measured were serum creatinine (SCr), serum blood urea (UREA), serum uric acid (UA), and cystatin C (CysC). CysC was detected using an ELISA kit (QuicKey Pro Human Cys-C (Cystatin C) ELISA Kit, Elabscience, catalog number E-OSEL-H0021). Other indicators were detected using a fully automated biochemical analyzer.

[0073] All experimental results were statistically analyzed using Origin Pro 2021 and GraphPad Prism 11 software. One-way ANOVA was used for comparisons between multiple groups and between groups, with a significance level of α=0.05.

[0074] 3. Results Analysis

[0075] 3.1 Antioxidant effect

[0076] DPPH scavenging: The scavenging rate of DPPH hydroxyl radicals increases with increasing sample concentration. According to the fitted linear equation, the IC50 of S1... 50 =2.645 mg / mL, IC50 of S2 50 =3.830 mg / mL ( Figure 1 (In the figure, sample 1 is S1, sample 2 is S2, and so on in the following figures).

[0077] ABTS + • Removal: ABTS decreases with increasing sample concentration. + The scavenging rate of free radicals also increased accordingly, and there was a good linear relationship between concentration and scavenging rate. The IC50 of S1 was... 50 =2.930 mg / mL, IC50 of S2 50 =4.326 mg / mL ( Figure 2 Both S1 and S2 are related to DPPH and ABTS. +The fact that S1 and S2 have a scavenging effect on free radicals indicates that both have good in vitro antioxidant activity.

[0078] 3.2 In vitro and in vivo inhibitory effects of XOD

[0079] The results showed that the in vitro inhibition rate of XOD gradually increased with increasing sample concentration. At the same concentration, S1 had a significantly higher inhibitory effect on XOD than S2, indicating that the ethanol extract and the water extract have significant differences in chemical composition. Figure 3 In the inhibition of XOD, the IC50 of S1 is... 50 =0.179 mg / mL, IC50 of S2 50 =0.239 mg / mL.

[0080] Furthermore, due to liver dysfunction, the model group mice experienced a large release of XOD into the bloodstream, along with increased liver XOD activity. After treatment with high doses of S1 and S2, serum and liver XOD activity decreased significantly, with levels lower than the control group (P < 0.01). The ethanol extract (S1) was more significantly effective than the water extract (S2) in reducing XOD activity (P < 0.01). Figure 4 ).

[0081] 3.3 Liver function test analysis

[0082] The serum levels of AST, ALT, ALP, and TP in the model group were significantly higher than those in the control group (P < 0.01). Figure 5 In the figure, CG represents the control group, DG the model group, LG the low-dose group, MG the medium-dose group, and HG the high-dose group. This indicates that the liver function of mice was severely impaired after being fed a high-fat diet for 3 weeks. After treatment with the compound Chinese medicine ethanol extract, all four indicators decreased, with significant decreases in AST, ALT, and TP levels (P < 0.01). At the end of the high-dose treatment, there was no significant difference in AST, ALT, and TP levels compared to the control group (P > 0.05), indicating that the therapeutic effect of high S1 was dose-dependent and significantly different from the low-dose and medium-dose groups (P < 0.01). S2 showed the same trend as S1. Figure 6 ).

[0083] In summary, both S1 and S2 at the same dose significantly reduced the concentrations of AST, ALT, ALP, and TP in serum, with a relatively smaller effect on ALP compared to the other three indicators. The water extract was slightly less effective than the ethanol extract to some extent, but both were effective in maintaining stable liver function in the model mice.

[0084] 3.4 Kidney function test analysis

[0085] After 3 weeks of S1 treatment, the renal function indicators UREA, UA, SCr, and CysC in the treatment group mice were significantly lower than those in the model group (P < 0.01). In the high-dose treatment group, UREA, SCr, and CysC recovered to the control group levels (P > 0.05). The medium-dose treatment group restored UA to normal levels, and the high-dose treatment group significantly reduced serum UA to below normal levels (P < 0.01). Figure 7 S2 and S1 show the same trend (). Figure 8 ).

[0086] In summary, both the water extract and the ethanol extract at the same concentration significantly reduced the serum concentrations of four substances: UREA, UA, SCr, and CysC. This demonstrates that both S1 and S2 can maintain stable renal function in the model mice, although the ethanol extract had a slightly stronger effect.

[0087] Based on the above results, the following conclusions can be drawn:

[0088] (1) via DPPH and ABTS + • Free radical scavenging test showed that both the ethanol extract and the water extract of the compound Chinese medicine had good antioxidant effects. The ethanol extract had a higher free radical scavenging efficiency than the water extract. This antioxidant effect can be used to resist the large number of free radicals caused by hyperuricemia and reduce the risk of free radical oxidation of molecules, cells and organs in the body.

[0089] (2) The determination of the inhibitory effect of the extract on xanthine oxidase in vitro and in vivo showed that both the ethanol extract and the water extract of the compound Chinese medicine had the effect of inhibiting xanthine oxidase activity, which could effectively reduce the production of uric acid in the blood and liver.

[0090] (3) By treating model mice with different doses of the extract, sensitive indicators related to liver and kidney function after treatment were detected. The results showed that both the ethanol extract and the water extract of the compound Chinese medicine could significantly reduce the levels of AST, ALT, TP, ALP, UREA, UA, SCr and CysC in the serum of hyperuricemic mice. Among them, the reduction of UA (uric acid) was the most significant. The ethanol extract was more effective than the water extract, indicating that the compound Chinese medicine extract has the dual effects of reducing uric acid and protecting the liver and kidneys.

[0091] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A compound traditional Chinese medicine extract for lowering uric acid, characterized in that, The compound Chinese medicine extract is an ethanol extract S1 and a water extract S2. Both S1 and S2 are extracted from the compound Chinese medicine composition. The compound Chinese medicine composition consists of the following raw materials in parts by weight: 40-60 parts of hawthorn, 15-25 parts of astragalus, 15-25 parts of poria cocos, 15-25 parts of jujube, and 5-15 parts of dried tangerine peel.

2. The compound traditional Chinese medicine extract according to claim 1, characterized in that, The compound traditional Chinese medicine composition consists of the following raw materials in parts by weight: 50 parts hawthorn, 20 parts astragalus, 20 parts poria cocos, 20 parts jujube, and 10 parts dried tangerine peel.

3. The method for preparing the compound traditional Chinese medicine extract according to claim 1, characterized in that, Includes the following steps: (1) After the compound Chinese medicine composition is dried at a constant temperature to a constant weight, the extractant is added and soaked for 30 min; (2) Boil and reflux for 15 minutes each time, filter by suction, repeat 3 times, and combine the filtrates; (3) Concentrate the filtrate obtained in step (2) to a syrup-like state, and freeze-dry it to obtain the compound Chinese medicine extract.

4. The method according to claim 3, characterized in that, The extractant in step (1) is 80% ethanol or water.

5. The method according to claim 3, characterized in that, The concentration described in step (3) is achieved by using a rotary evaporator at 70 °C.

6. The application of the compound traditional Chinese medicine extract according to claim 1, characterized in that, The drug extract is used to prepare a drug for treating hyperuricemia, and the active ingredient of the drug is one of S1 or S2.

7. The application according to claim 6, characterized in that, The active ingredient of the drug is S1.

8. The application according to claim 6, characterized in that, The drug reduces uric acid production in the blood and liver by inhibiting xanthine oxidase activity.

9. The application according to claim 6, characterized in that, The drug also has antioxidant and liver and kidney protective effects.

10. The application according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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