Preparation method of a preparation for treating hyperuricemia and gout and application thereof

Abstract

The application discloses a preparation method of a preparation for treating hyperuricemia and gout and application of the preparation. A star anise is used as raw material to be crushed, and medicinal powder is refluxed and extracted to obtain star anise extract A and star anise extract B after the organic phase and the aqueous phase are separated by standing. A chrysanthemum is used as raw material to be crushed, and medicinal powder is put into water containing a composite enzyme, and chrysanthemum extract A is obtained after extraction liquid is concentrated under reduced pressure and dried. The chrysanthemum extract A is put into a solution containing a hydrolytic enzyme to be enzymolyzed, and chrysanthemum extract B is obtained after the enzymolyzed liquid is concentrated under pressure and dried. The star anise extract A, the star anise extract B and the chrysanthemum extract B are added into a pharmaceutically acceptable carrier to prepare chrysanthemum and star anise preparation for reducing uric acid, such as granules, tablets, capsules, oral liquid and the like. The chrysanthemum and the star anise have a remarkable effect of reducing uric acid under the specific composition, and are further developed into related dosage forms, so that the application of the traditional Chinese medicine compound taking the star anise and the chrysanthemum as cores in the field of treating hyperuricemia and gout is greatly promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to biological medicine, and particularly relates to a preparation method of a preparation for treating hyperuricemia and gout and application thereof. BACKGROUND

[0002] Hyperuricemia (HUA) is a metabolic abnormality syndrome caused by disorder of purine metabolism. Serum uric acid (SUA) level of more than 420 μmol / L twice a day is called hyperuricemia. When the blood uric acid level is above its saturation concentration in blood or tissue fluid, it is easy to produce sodium urate crystals, induce arthritis and tissue damage, i.e. gout. At present, multidisciplinary experts widely believe that HUA and gout are chronic and systemic diseases, which have obvious causal relationship with chronic kidney disease, kidney stones, and increase the risk of hypertension, new-onset diabetes and coronary heart disease.

[0003] Uric acid is mainly produced in the liver, 70% of which is excreted through the kidney, and 30% of which is excreted through the intestinal tract. Both the increase of uric acid production and the decrease of excretion will lead to the accumulation of uric acid in the body, and induce HUA. Among them, xanthine oxidase (XOD) as a key enzyme of uric acid production can catalyze the generation of xanthine from hypoxanthine, and further convert it into uric acid. At the same time, there are uric acid reabsorption proteins including urate-anion transporter 1 (URAT1), uric acid secretion proteins including organic anion transport 1 (OAT1) and organic anion transport 3 (OAT3) on the brush border side (luminal membrane) and the basolateral membrane of renal tubular epithelial cells to realize the regulation of uric acid concentration. And the clinical research results show that 90% of primary HUA belongs to the type of poor uric acid excretion.

[0004] Amomum villosum Lour., Amomum villosum Lour. var. xanthioides TLWu et Senjen, or Amomum longiligulare TLWu are all plants belonging to the ginger family. Its main functions are to resolve dampness and stimulate appetite, warm the spleen and stop diarrhea, regulate qi and calm the fetus. Amomum villosum is rich in various active ingredients such as flavonoids, polysaccharides, polyphenols, volatile oils, and terpenes, among which flavonoids and polyphenols are the main effective components. Liu Xuemei et al. found that the polyphenol extract of Amomum villosum has strong xanthine oxidase inhibitor (XOI) activity and antioxidant activity, making it a potential raw material for developing functional foods with significant uric acid-lowering effects. Zhang et al. studied the interaction between three flavonoids—quercetin, galangin, and myricetin—and XOD, finding that their binding constant was 4.28 × 10⁻⁶. 4 L / mol (quercetin) > 3.60 × 10 4 L / mol (galangin) > 3.24 × 10 4 L / mol (myricetin), the binding process of myricetin and quercetin to XOD is mainly driven by van der Waals forces and hydrogen bonds. Hydrogen bonds and hydrophobic interactions dominate the binding of galangin to XOD, and both quench the intrinsic fluorescence of XOD in a static manner. Du Hongfang et al. measured the IC50 value of protocatechuic acid inhibiting XOD to be 0.38 mmol / L, which is comparable to that of myricetin (IC50 = 0.32 mmol / L). Wang Wenjie et al. used the traditional ethanol reflux method to extract flavonoids from Amomum villosum. Through orthogonal experiments to optimize the process parameters, they found that the highest extraction rate of Amomum villosum flavonoids was achieved under the conditions of 80% ethanol concentration, 60℃, 1:80 material-to-liquid ratio, and extraction for 2.5 h. Zhang Ting et al.'s research showed that the aqueous extract of Amomum villosum can repair mucosal damage in rats to a certain extent and reduce inflammatory response.

[0005] There are reports that confirm that Amomum villosum has antibacterial, antioxidant, anticancer, and hypoglycemic effects, such as the related patents (A compound tablet of traditional Chinese medicine for nourishing the stomach and protecting the liver and a preparation method thereof, CN115252566A; A traditional Chinese medicine for treating colorectal cancer and a preparation method thereof, CN114366800A) disclose the application of Amomum villosum in nourishing the stomach and anticancer. However, there is no related patent or literature that discloses the application of Amomum villosum in the field of HUA and gout. Chrysanthemum is the dry inflorescence of Chrysanthemum morifolium Ramat. of the Compositae family. Its main functions and indications are dispelling wind and clearing heat, calming the liver and improving eyesight, and clearing heat and detoxifying. Beijing Mingyang Huaxia Technology Co., Ltd. has disclosed the application of chrysanthemum in the field of reducing uric acid. This medicine takes chrysanthemum and honeysuckle as the core, and adds dandelion and chicory to achieve inhibition of uric acid reabsorption protein URAT1 (CN113274422A). Peng An analyzed the similarities and differences of the polyphenolic compound composition and XOI activity of 15 representative samples of foodborne chrysanthemum varieties, and screened out specific chrysanthemums with the strongest XOI activity to further explore the multi-target mechanism of specific chrysanthemum extract in the intervention of hyperuricemia and its complications. Ke Zunjun found that chrysanthemum water extract can inhibit xanthine oxidase to a certain extent and has a certain effect on reducing the content of uric acid.

[0006] Although some drugs in the prior art have certain effects in reducing uric acid, the effects are not significant, and it is particularly important to develop specific combination reagents to significantly improve the effects of drugs in the treatment of hyperuricemia and gout. SUMMARY

[0007] The present application provides a preparation method of a preparation for treating hyperuricemia and gout. The preparation prepared by the present application combines Amomum villosum and chrysanthemum extracts to precisely treat multiple targets in HUA and gout. At the same time, a detection method is established to determine the addition amount of medicinal materials from different origins, sources, and years under different extraction, purification, and drying methods to determine the prescription composition. Under the specific formula composition of the present application, the combination of Amomum villosum extract and chrysanthemum extract has a significant effect on reducing uric acid, and is further developed into related dosage forms, which greatly promotes the application of traditional Chinese medicine compounds taking Amomum villosum and chrysanthemum as the core in the field of reducing uric acid and gout.

[0008] The present application also provides a preparation for treating hyperuricemia and gout and an application.

[0009] Technical scheme: In order to achieve the above-mentioned purpose, the preparation method of a preparation for treating hyperuricemia and gout provided by the present application is characterized in that it comprises the following steps:

[0010] (1) Amomum villosum is used as the raw material, powdered by a powdering machine, sieved, and uniformly mixed to serve as Amomum villosum powder to be extracted;

[0011] (2) The ground long pepper obtained in step (1) is put into an extraction solvent, and reflux extraction is performed;

[0012] (3) The extraction liquid obtained in step (2) is subjected to extraction after the extraction solvent is removed, and is allowed to stand to separate into organic phase and aqueous phase;

[0013] (4) The organic phase obtained in step (3) is dried after being concentrated under reduced pressure to obtain long pepper extract A;

[0014] (5) The aqueous phase obtained in step (3) is subjected to extraction, and is dried after being concentrated under reduced pressure to obtain long pepper extract B;

[0015] (6) Chrysanthemum is ground by a powdering machine, is sieved, and is mixed to obtain ground chrysanthemum as chrysanthemum to be extracted;

[0016] (7) The ground chrysanthemum obtained in step (6) is put into a buffer solution containing a complex enzyme for enzymolysis;

[0017] (8) The enzymolysis system obtained in step (7) is subjected to ultrasonic extraction after pH adjustment;

[0018] (9) The extraction liquid obtained in step (8) is dried after being concentrated under reduced pressure to obtain chrysanthemum extract A;

[0019] (10) The chrysanthemum extract A obtained in step (9) is put into a solution containing a hydrolytic enzyme for enzymolysis;

[0020] (11) The enzymolysis liquid obtained in step (10) is dried after being concentrated under reduced pressure to obtain chrysanthemum extract B;

[0021] (12) Long pepper extract A, long pepper extract B, and chrysanthemum extract B are mixed to prepare a preparation.

[0022] In step (1), the particle size is 60-100 mesh.

[0023] In step (2), the extraction solvent is a 50%-90% ethanol aqueous solution, the ratio of the ground long pepper to the extraction solvent is 1:10-1:30 g / mL, the extraction time is 30-90 min, and the extraction temperature is 60-80°C.

[0024] In step (3), the volume ratio of the extraction liquid to which the extraction solvent is removed to extraction ether is 1:1-1:3 ml / ml, the extraction time is 5-30 min, and the extraction times is 1-3 times.

[0025] In step (5), the volume ratio of the aqueous phase to extraction ethyl acetate is 1:1-1:3 ml / ml, the extraction time is 5-30 min, and the extraction times is 1-3 times.

[0026] The particle size of the screening in step (6) is 60-100 mesh.

[0027] The complex enzyme in step (7) includes any one or more of cellulase, pectinase or papain, the enzyme solution is phosphate buffer, the enzyme dosage is 0.1%-0.3% of the solvent mass, the enzyme solution temperature is 30-50 DEG C, the enzyme solution time is 30 min-1 h, and the ratio of the chrysanthemum powder to the enzyme solution is 1:10-1:20 mg / mL.

[0028] The pH range in step (8) is adjusted to 8-12, the ultrasonic power is 150-300 W, and the extraction time is 0.5-2 h.

[0029] The hydrolytic enzyme in step (9) includes any one or more of beta-glucosidase, naringinase or snail enzyme, the enzyme solution is phosphate buffer, the enzyme dosage is 0.1%-0.3% of the solvent mass, the enzyme solution temperature is 30-50 DEG C, the enzyme solution time is 30 min-1 h, and the ratio of the chrysanthemum extract A to the enzyme solution is 1:10-1:20 mg / mL.

[0030] The mass ratio of the extract A of Amomum villosum, the extract B of Amomum villosum and the extract B of chrysanthemum in step (12) is 1:3:1-3:2:2.

[0031] Preferably, the mass ratio of the extract A of Amomum villosum, the extract B of Amomum villosum and the extract B of chrysanthemum in step (12) is 1:3:1.

[0032] In the chrysanthemum sand extract, the total polyphenol content of the extract A of Amomum villosum is 23.48-50.32 mg / g, the total flavone content of the extract B of Amomum villosum is 12.59-22.35 mg / g, the chrysosplenine content of the extract A of chrysanthemum is 27.64-42.15 mg / g, the quercitrin content of the extract A of chrysanthemum is 19.64-38.47 mg / g, the chrysosplenine conversion rate in the extract B of chrysanthemum is 53.71%-95.13%, and the quercetin conversion rate is 47.35%-78.36%.

[0033] The preparation method of the application is used for preparing a preparation for treating hyperuricemia and gout, and the preparation includes the extract A of Amomum villosum, the extract B of Amomum villosum, the extract B of chrysanthemum and a pharmaceutically acceptable carrier.

[0034] Further, the daily dosage of the preparation contains total polyphenol not less than 10 mg, total flavone not less than 13 mg, chrysosplenine not less than 8 mg and quercetin not less than 6 mg.

[0035] The preparation of the application is used for preparing a medicine for treating hyperuricemia and gout.

[0036] The detection method of the chrysanthemum sand extract for treating hyperuricemia and gout and the preparation thereof according to the present application uses ultraviolet spectrophotometry and high performance liquid chromatography to accurately detect the content of flavonoids, polyphenols, luteolin and quercetin in the chrysanthemum sand extract, and comprises the following steps:

[0037] (1) Preparation of the control solution

[0038] A certain amount of luteolin, luteolin, quercetin and quercetin control samples are precisely weighed, and methanol is added to prepare a control solution stock solution, 1 ml of which contains 0.5 mg of luteolin, 0.5 mg of luteolin, 0.5 mg of quercetin and 0.5 mg of quercetin.

[0039] A certain amount of rutin control sample is precisely weighed, and a certain amount of methanol is added to dissolve after ultrasonic treatment to prepare a rutin control sample stock solution of 0.5 mg / mL.

[0040] A certain amount of gallic acid control sample is precisely weighed, and a certain amount of ethanol is added to dissolve after ultrasonic treatment to prepare a gallic acid control sample stock solution of 1.0 mg / mL.

[0041] (2) Preparation of the test solution

[0042] 5.0 g of the preparation for reducing uric acid prepared in the present application is precisely weighed into a conical flask, and 50 mL of methanol is precisely added for ultrasonic extraction. After cooling, the weight is made up and filtered through a 0.45 μm microporous filter to obtain the test solution.

[0043] (3) Preparation of the standard curve

[0044] The control solution prepared in step (1) is precisely taken and diluted with methanol to obtain a series of control solution solutions with concentration gradients of 1-fold, 2-fold, 4-fold, 8-fold, 16-fold and 32-fold, respectively. 10 μl of each sample is injected, HPLC analysis is performed, the peak area is taken as the vertical coordinate, and the concentration is taken as the horizontal coordinate to draw the standard curve.

[0045] The rutin control solution prepared in step (1) is precisely taken and diluted with methanol to obtain a series of control solution solutions with concentration gradients of 1-fold, 2-fold, 4-fold, 8-fold, 16-fold and 32-fold. 1 mL of the series of rutin control solution solutions with concentration gradients is taken, 1 mL of 2% AlCl3 ethanol solution is added, and the mixture is mixed and placed at room temperature for 1 h. Methanol is used as a blank control, and the absorbance is measured at 420 nm. The determination is repeated for 3 times. The absorbance (Y) is taken as the vertical coordinate, and the rutin mass concentration (X) is taken as the horizontal coordinate to draw the standard curve. The content of total flavonoids in the sample is calculated by using the standard curve, and the content of total flavonoids is expressed as the number of milligrams of rutin per gram of dry matter.

[0046] The gallic acid control solution prepared in step (1) was diluted with ethanol to obtain a series of control solutions with different concentrations. 200 μl of the gallic acid control solution was taken, 200 μl of 0.5 mol / L Folin phenol reagent was added, 600 μl of 10% Na2CO3 aqueous solution was added, 1 mL of distilled water was added to make up the volume, and the mixture was reacted at 30°C for 30 min. The absorbance was measured at 750 nm. The standard curve was plotted with the absorbance (Y) as the vertical coordinate and the concentration of gallic acid (X) as the horizontal coordinate. The content of total polyphenols in the sample was calculated by using the standard curve, and the content of total polyphenols was expressed as mg of gallic acid per g of dry matter.

[0047] (4) Content determination

[0048] 1 mL of the test sample solution of the uric acid-lowering preparation prepared in step (2) was taken, 1 mL of 2% AlCl3 ethanol solution was added, and the mixture was mixed and placed at room temperature for 1 h. The absorbance was measured at 420 nm to detect the content of total flavonoids in the test sample.

[0049] 200 μl of the test sample solution of the uric acid-lowering preparation prepared in step (2) was taken, 200 μl of 0.5 mol / L Folin phenol reagent was added, 600 μl of 10% Na2CO3 aqueous solution was added, 1 mL of distilled water was added to make up the volume, and the mixture was reacted at 30°C for 30 min. The absorbance was measured at 750 nm to detect the content of total polyphenols in the test sample.

[0050] 10 μl of the test sample solution of the uric acid-lowering preparation prepared in step (2) was taken and injected into HPLC for detection. The content of each chemical component, luteolin and quercetin, in the test sample was detected by using the regression equation obtained in step (3).

[0051] In step (3) and step (4), the HPLC chromatographic conditions are as follows:

[0052] Column: Agilent zobax C18; mobile phase: acetonitrile as mobile phase A and water as mobile phase B; gradient elution program: 0-10 min, 20% A; 10-15 min, 30% A; 15-30 min, 60% A; flow rate: 1.0 ml / min; column temperature: 30°C; injection volume: 10 μl; UV detection wavelength: 350 nm.

[0053] In step (3), the regression equation is as follows:

[0054]

[0055]

[0056] The present application improves the extraction and post-treatment method of chrysanthemum, fully extracts and processes luteolin and quercetin with obvious XOD inhibitory activity and anti-inflammatory activity, and combines with the extract of litseae fructus, which is beneficial to control the uric acid level of patients with high uric acid and play an anti-inflammatory role in the acute gout attack period.

[0057] The present application uses litseae fructus and chrysanthemum as raw materials, realizes the full extraction of effective components by optimizing the extraction time and frequency, and the time and temperature of compound enzymolysis, determines the efficacy and prescription of the pharmacodynamic components, and further develops the corresponding preparation. At the same time, the detection method is established for evaluation, and the addition amount of medicinal materials of different producing areas, sources and years under different extraction, purification and drying methods is determined, which greatly promotes the application of traditional Chinese medicine compound with litseae fructus and chrysanthemum as the core in the field of reducing uric acid.

[0058] At present, TCM has a long history in the treatment of HUA and gout, which can be divided into damp-heat accumulation, blood stasis, liver and kidney deficiency, spleen deficiency and dampness, etc. However, the components of traditional Chinese medicine compound are more, the mechanism is not clear, and the quality of decoction pieces is uneven, so it is difficult to evaluate the products in multiple batches. It has been reported that litseae fructus has the pharmacological effects of resolving dampness, warming spleen to stop diarrhea, regulating qi and preventing miscarriage, but there is no related patent and literature to disclose the application of litseae fructus in the field of HUA and gout. Chrysanthemum contains various chemical components including volatile oil and terpenes, flavonoids, phenolic acids, polysaccharides, etc., among which flavonoids are the main effective components. The present application combines specific chrysanthemum and litseae fructus extract, accurately treats multiple targets in HUA and gout, and establishes a detection method to determine the addition amount of medicinal materials of different producing areas, sources and years under different extraction, purification and drying methods, so as to determine the prescription composition and further develop related dosage forms, which greatly promotes the application of traditional Chinese medicine compound with litseae fructus and chrysanthemum as the core in the field of reducing uric acid and gout.

[0059] The present application utilizes specific methods to prepare chrysanthemum and litseae fructus extract to synergistically reduce uric acid. The present application optimizes the extraction method, concentrates flavonoids and polyphenol components in litseae fructus through two-step extraction, and fully extracts luteolin and quercetin in chrysanthemum through two-step enzymatic treatment and further converts them into luteolin and quercetin. At the same time, by adding a commonly used carrier in pharmacy, related preparations are prepared, and the contents of polyphenols, flavonoids, luteolin, quercetin, and quercetin are analyzed by high performance liquid chromatography for quality evaluation, and finally the preparation is prepared with a daily dosage of total flavonoids not less than 13 mg, total polyphenols not less than 10 mg, luteolin not less than 8 mg, and quercetin not less than 6 mg. This dose has a significant effect on reducing uric acid, and the preparation has high effectiveness through pharmacodynamic evaluation.

[0060] The present application innovatively develops a new application of chrysanthemum and combined with amomum villosum in the field of gout and hyperuricemia, the chrysanthemum extract is obtained by treating chrysanthemum through two-step enzyme method to fully extract acacetin and quercitrin and further converting acacetin and quercitrin into luteolin and quercetin, which can effectively reduce the uric acid level of hyperuricemia mice and play a role in treating gout and hyperuricemia, and further combined with the amomum villosum extract prepared in the present application, the curative effect of amomum villosum in treating gout and hyperuricemia is greatly improved.

[0061] More importantly, the amomum villosum extract A, the amomum villosum extract B and the chrysanthemum extract B prepared by the specific method of the present application have a certain effect on the treatment of hyperuricemia and gout, and further the specific preparation for treating hyperuricemia and gout prepared in the present application is mixed to prepare a preparation, which significantly improves the single use effect of the amomum villosum extract A and the amomum villosum extract B and the chrysanthemum extract B, and has a significant synergistic effect.

[0062] Beneficial effects: compared with the prior art, the present application has the following advantages:

[0063] The present application innovatively develops a new application of chrysanthemum and combined with amomum villosum in the field of gout and hyperuricemia. The present application uses amomum villosum and chrysanthemum as raw materials, realizes the full extraction of effective components through the optimization of extraction method, accurately treats multiple targets in HUA and gout, and further develops corresponding preparations. At present, there is no related patent and literature to disclose the application of amomum villosum in the field of HUA and gout, and the present application combines amomum villosum with chrysanthemum, which greatly improves the curative effect of chrysanthemum in treating gout and hyperuricemia.

[0064] The present application has a clear mechanism of action and high safety, and has less side effects compared with allopurinol and benzbromarone, and has a clear mechanism of action compared with traditional Chinese medicine preparations. In addition, the present application has a clear dosage and high effectiveness. Compared with the prior art, the present application introduces a Chinese medicine component containing flavonoids, and further converts acacetin and quercitrin in chrysanthemum into luteolin and quercetin, and combines with amomum villosum extract, significantly enhances the inhibition ability of XOD and serum uric acid concentration (SUA), and effectively improves the curative effect of treating gout and hyperuricemia. The present application adds a commonly used carrier in pharmacy to prepare related preparations, and uses high performance liquid chromatography and ultraviolet spectrophotometry to analyze the content of polyphenols, flavonoids, luteolin and quercetin for quality evaluation, and the results show that the present application has a significant uric acid lowering effect, and in addition, the preparation has high effectiveness through pharmacodynamic evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1The image shows the ultraviolet spectrum of Amomum villosum extract A prepared in Example 1 of this invention.

[0066] Figure 2 The image shows the ultraviolet spectrum of Amomum villosum extract B prepared in Example 1 of this invention.

[0067] Figure 3 This is the HPLC chromatogram of chrysanthemum extract A obtained in Example 4 of the present invention;

[0068] Figure 4 This is the HPLC chromatogram of chrysanthemum extract B obtained in Example 4 of the present invention;

[0069] Figure 5 This is an ultraviolet image of the cypermethrin-based uric acid-lowering preparation obtained in Example 10 of the present invention;

[0070] Figure 6 This is an HPLC chromatogram of the cypermethrin-based uric acid-lowering preparation obtained in Example 10 of the present invention.

[0071] Figure 7 The effect of uric acid-lowering agents on serum uric acid concentration (SUA) in hyperuricemic mice (compared with the hyperuricemia model group, ###p<0.001, ns represents no significant difference; compared with groups d and f, ***p<0.001)

[0072] Figure 8 The effect of uric acid-lowering agents on XOD activity in liver tissue homogenate of hyperuricemic mice (compared with the hyperuricemia model group, ###p<0.001, ns represents no significant difference; compared with groups d and f, ***p<0.001);

[0073] Figure 9 The effect of uric acid-lowering agents on urinary uric acid concentration (UUA) in hyperuricemic mice (compared with the hyperuricemia model group, ###p<0.001, ns represents no significant difference; compared with groups d and f, ***p<0.001). Detailed Implementation

[0074] To make the present invention easier to understand, the present invention will be further described below with reference to specific embodiments. These embodiments are not intended to limit the present invention in any way. They are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modifications or changes to the present invention that are easily implemented by those skilled in the art without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

[0075] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0076] The cellulase, pectinase, papain, snailase, β-glucosidase, naringinase in the hydrolytic enzyme are respectively purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai Yuan Ye Biotechnology Co., Ltd., Shanghai Yuan Ye Biotechnology Co., Ltd., Shanghai Yuan Ye Biotechnology Co., Ltd., Shanghai McLean Biochemical Technology Co., Ltd. Among them, the cellulase is 10000U / g (item number C 140864), the pectinase is 30000U / g (item number P 116864), the papain is 800U / mg (item number S10011), the snailase is 90% of the broken wall rate (the dissolution activity of the yeast cell wall) (item number S10083), the β-glucosidase is 100U / g (item number S24786), and the naringinase is 100U / g (item number N787363).

[0077] The standard samples of quercitrin, quercetin, luteoloside, luteolin, rutin and gallic acid are all purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0078] Example 1

[0079] The sand ginger extract A and the sand ginger extract B are prepared according to the following process with sand ginger as the raw material

[0080] (1) The sand ginger is used as the raw material, is ground by a powder machine, is passed through a 100-mesh sieve, is mixed, and is used as sand ginger to be extracted;

[0081] (2) The sand ginger to be extracted is put into a 70% ethanol aqueous solution according to a solid-liquid ratio of 1:20 g / mL, and is extracted at 60°C for 1 h;

[0082] (3) The ethanol in the extract obtained in step (2) is removed, and the remaining water phase is extracted with diethyl ether at room temperature for 15 min according to a water phase to diethyl ether volume ratio of 1:2, and is extracted for a total of two times. After standing, the diethyl ether phase and the water phase are separated;

[0083] (4) The diethyl ether phase obtained in step (3) is dried after being concentrated under reduced pressure, and the sand ginger extract A is obtained. The total polyphenol content is 50.32 mg / g detected by ultraviolet detection. The ultraviolet spectrum is shown in Figure 1 .

[0084] (5) The water phase obtained in step (3) is extracted with ethyl acetate, and the water phase to ethyl acetate phase volume ratio is 1:2. The extraction is carried out at room temperature for 15 min, and the extraction is carried out for a total of two times. After standing, the ethyl acetate phase is taken, is concentrated under reduced pressure, and is dried to obtain the sand ginger extract B. The total flavonoid content is 22.35 mg / g detected by ultraviolet detection. The ultraviolet spectrum is shown in Figure 2 .

[0085] Example 2

[0086] The extract A and the extract B of the litse cubeba are prepared according to the following process with the litse cubeba as the raw material

[0087] (1) The litse cubeba is used as the raw material, is ground by a powdering machine, is mixed uniformly after being passed through a 80-mesh sieve, and is used as the powder to be extracted;

[0088] (2) The powder to be extracted is put into a 90% ethanol aqueous solution according to a material-liquid ratio of 1:10 g / mL, and is extracted at 70°C for 1.5 hours;

[0089] (3) The ethanol in the extract obtained in the step (2) is removed, the remaining water phase is extracted with ether at room temperature according to a water phase-ether volume ratio of 1:3 for 30 minutes, and the ether phase and the water phase are separated after one extraction.

[0090] (4) The ether phase obtained in the step (3) is dried after being concentrated under reduced pressure, and the extract A of the litse cubeba is obtained, and the total polyphenol content is 33.97 mg / g according to ultraviolet detection;

[0091] (5) The water phase obtained in the step (3) is extracted with ethyl acetate according to a water phase-ethyl acetate volume ratio of 1:3 at room temperature for 30 minutes, and the ethyl acetate phase is separated after three extractions, and the extract B of the litse cubeba is obtained after being dried after being concentrated under reduced pressure, and the total flavonoid content is 16.48 mg / g according to ultraviolet detection.

[0092] Example 3

[0093] The extract A and the extract B of the litse cubeba are prepared according to the following process with the litse cubeba as the raw material

[0094] (1) The litse cubeba is used as the raw material, is ground by a powdering machine, is mixed uniformly after being passed through a 60-mesh sieve, and is used as the powder to be extracted;

[0095] (2) The powder to be extracted is put into a 50% ethanol aqueous solution according to a material-liquid ratio of 1:30 g / mL, and is extracted at 80°C for 30 minutes;

[0096] (3) The ethanol in the extract obtained in the step (2) is removed, the remaining water phase is extracted with ether at room temperature according to a water phase-ether volume ratio of 1:1 for 5 minutes, and the ether phase and the water phase are separated after three extractions.

[0097] (4) The ether phase obtained in the step (3) is dried after being concentrated under reduced pressure, and the extract A of the litse cubeba is obtained, and the total polyphenol content is 23.48 mg / g according to ultraviolet detection;

[0098] (5) The water phase obtained in the step (3) is extracted with ethyl acetate according to a water phase-ethyl acetate volume ratio of 1:1 at room temperature for 5 minutes, and the ethyl acetate phase is separated after one extraction, and the extract B of the litse cubeba is obtained after being dried after being concentrated under reduced pressure, and the total flavonoid content is 12.59 mg / g according to ultraviolet detection.

[0099] Example 4

[0100] Chrysanthemum as raw material, according to the following process to prepare chrysanthemum extract A

[0101] (1) chrysanthemum as raw material, with a powder machine, 100 mesh sieve, mix, as chrysanthemum powder;

[0102] (2) chrysanthemum powder into the phosphate buffer solution pH = 7, buffer containing 0.2% cellulase, 50 ℃ enzymolysis 30 min;

[0103] (3) the resulting enzyme solution of step (2) with sodium hydroxide to adjust the pH to 10, ultrasonic power 300 W extraction 2h;

[0104] (4) the resulting extract of step (3) after drying, get chrysanthemum extract A, HPLC detection of syringa glycoside 42.15 mg / g, quercitrin 38.47 mg / g, HPLC spectrum see Figure 3 .

[0105] Example 5

[0106] Chrysanthemum as raw material, according to the following process to prepare chrysanthemum extract A

[0107] (1) chrysanthemum as raw material, with a powder machine, 80 mesh sieve, mix, as powder;

[0108] (2) chrysanthemum powder into the phosphate buffer solution pH = 7, buffer containing 0.1% cellulase, 30 ℃ enzymolysis 1h;

[0109] (3) the resulting enzyme solution of step (2) with sodium hydroxide to adjust the pH to 8, ultrasonic power 200 W extraction 0.5h;

[0110] (4) the resulting extract of step (3) after drying, get chrysanthemum extract A, HPLC detection of syringa glycoside 31.27 mg / g, quercitrin 24.36 mg / g.

[0111] Example 6

[0112] Chrysanthemum as raw material, according to the following process to prepare chrysanthemum extract A

[0113] (1) chrysanthemum as raw material, with a powder machine, 60 mesh sieve, mix, as powder;

[0114] (2) The chrysanthemum to be extracted is put into a phosphate buffer with pH = 7 at a material-liquid ratio of 1:15 g / mL, the buffer contains 0.3% cellulase by mass of solvent, and enzymolysis is carried out at 40°C for 45 min;

[0115] (3) The enzymolysis solution obtained in step (2) is adjusted to pH 12 by sodium hydroxide, and extraction is carried out under ultrasonic power of 150 W for 1 h.

[0116] (4) The extraction solution obtained in step (3) is concentrated under reduced pressure and dried to obtain chrysanthemum extract A, which contains 27.64 mg / g of syringa extract and 19.64 mg / g of quercitrin as detected by HPLC.

[0117] Test Example 1

[0118] Chrysanthemum is used as the raw material to prepare chrysanthemum extract A according to the following process

[0119] (1) Chrysanthemum is used as the raw material, and powdering is carried out by a powdering machine, and the powder is mixed and passed through a 100-mesh sieve to obtain chrysanthemum to be extracted;

[0120] (2) The chrysanthemum to be extracted is put into a phosphate buffer with pH = 7 at a material-liquid ratio of 1:20 g / mL, the buffer contains 0.2% cellulase, pectinase, or papain by mass of solvent, or no enzyme solution, and enzymolysis is carried out at 50°C for 30 min;

[0121] (3) The enzymolysis solution obtained in step (2) is adjusted to pH 10 by dilute hydrochloric acid, and extraction is carried out under ultrasonic power of 300 W for 2 h.

[0122] (4) The extraction solution obtained in step (3) is concentrated under reduced pressure and dried to obtain chrysanthemum extract A, which contains the contents of syringa extract and quercitrin as shown in Table 1.

[0123] Table 1. Effect of pretreatment of different types of hydrolytic enzymes on the extraction contents of syringa extract and quercitrin

[0124]

[0125] As shown in Table 1, when no enzyme is added for pretreatment of the chrysanthemum to be extracted, the cell wall structure is relatively compact, which is not conducive to the extraction of the effective components syringa extract and quercitrin, and further affects the subsequent efficacy. The test example shows that cellulase has more advantages as a hydrolytic enzyme, and the effect is obviously better than that of pectinase and papain.

[0126] Test Example 2

[0127] Chrysanthemum is used as the raw material to prepare chrysanthemum extract A according to the following process

[0128] (1) Chrysanthemum is used as the raw material, and powdering is carried out by a powdering machine, and the powder is mixed and passed through a 100-mesh sieve to obtain chrysanthemum to be extracted;

[0129] (2) The chrysanthemum to be powdered is put into a phosphate buffer with pH = 7 at a material-liquid ratio of 1:20 g / mL, and the buffer contains 0.2% cellulase by mass of the solvent, and enzymolysis is carried out at 50°C for 30 min;

[0130] (3) The enzymolysis liquid obtained in step (2) is adjusted to an acidic / neutral / alkaline environment, and extraction is carried out under ultrasonic power of 300 W for 2 h;

[0131] (4) The extraction liquid obtained in step (3) is concentrated under reduced pressure and dried to obtain chrysanthemum extract A, and the contents of syringidin and quercitrin are shown in Table 2.

[0132] Table 2. Influence of hydrolytic enzyme pretreatment under different pH conditions on the extraction contents of syringidin and quercitrin

[0133]

[0134] The test example 2 of the present application shows that the contents of syringidin and quercitrin in chrysanthemum extracted in an alkaline environment are higher, and the optimal extraction pH is 10, and the extraction efficiency of syringidin and quercitrin obviously decreases when the pH is neutral or acidic. Therefore, the pH of the extraction solvent needs to be adjusted to 10 when extracting syringidin and quercitrin.

[0135] Example 7

[0136] Chrysanthemum extract B is prepared using chrysanthemum extract A obtained in Example 4 as a raw material

[0137] (1) The chrysanthemum extract A is put into a phosphate buffer with pH = 7 at a material-liquid ratio of 1:10 g / mL, and the buffer contains 0.2% snailase by mass of the solvent, and enzymolysis is carried out at 50°C for 45 min;

[0138] (2) The extraction liquid obtained in step (1) is concentrated under reduced pressure and dried to obtain chrysanthemum extract B, and the conversion rate of syringidin to syringin is 95.13% and the conversion rate of quercitrin to quercetin is 78.36% by HPLC detection, and the HPLC spectrum is shown in Figure 4 .

[0139] Example 8

[0140] Chrysanthemum extract B is prepared using chrysanthemum extract A obtained in Example 4 as a raw material

[0141] (1) The chrysanthemum extract A is put into a phosphate buffer with pH = 7 at a material-liquid ratio of 1:20 g / mL, and the buffer contains 0.3% β-glucosidase by mass of the solvent, and enzymolysis is carried out at 30°C for 1 h;

[0142] (2) The extract solution obtained in step (1) is dried after being concentrated under reduced pressure to obtain the chrysanthemum extract B. The conversion rate of jujuboside to luteolin is 67.13% and the conversion rate of quercitrin to quercetin is 58.49% as detected by HPLC.

[0143] Example 9

[0144] Example 9

[0145] (1) The chrysanthemum extract A is put into a phosphate buffer with pH = 7 at a material-to-liquid ratio of 1:15 g / mL. The buffer contains naringinase at a solvent mass of 0.1%. Enzymolysis is carried out at 40°C for 30 min.

[0146] (2) The extract solution obtained in step (1) is dried after being concentrated under reduced pressure to obtain the chrysanthemum extract B. The conversion rate of jujuboside to luteolin is 67.13% and the conversion rate of quercitrin to quercetin is 58.49% as detected by HPLC.

[0147] Example 10

[0148] The chrysanthemum extract B obtained in Example 9 is mixed with the extract A obtained in Example 1 and the extract B obtained in Example 7 at a weight ratio of 1:3:1. Lactose and mannitol are added as excipients at a mass ratio of 1:2 (lactose:mannitol = 1:2, total extract:excipient = 2:8). The mixture is crushed and sieved, mixed with 90% ethanol, and granulated through a 20-mesh sieve. The granules are dried for 1 h and sieved through a 16-mesh sieve to obtain granules a. Each 5 g of the granules contains 10 mg of total polyphenols, 13 mg of total flavonoids, 8 mg of luteolin, and 6 mg of quercetin. The UV spectrum is shown in Figure 5 , and the HPLC spectrum is shown in Figure 6 .

[0149] Test Example 3

[0150] The extract A obtained in Example 1 is mixed with lactose and mannitol as excipients at a mass ratio of 1:2 (lactose:mannitol = 1:2, extract A:excipient = 2:8). The mixture is crushed and sieved, mixed with 90% ethanol, and granulated through a 20-mesh sieve. The granules are dried for 1 h and sieved through a 16-mesh sieve to obtain granules b.

[0151] Test Example 4

[0152] The extract B obtained in Example 1 is mixed with lactose and mannitol as excipients at a mass ratio of 1:2 (lactose:mannitol = 1:2, extract B:excipient = 2:8). The mixture is crushed and sieved, mixed with 90% ethanol, and granulated through a 20-mesh sieve. The granules are dried for 1 h and sieved through a 16-mesh sieve to obtain granules c.

[0153] Test Example 5

[0154] The chrysanthemum extract B obtained in Example 7 (the amount is the same as that of the chrysanthemum extract B in Example 10) was added with the auxiliary materials lactose and mannitol (mass ratio, lactose : mannitol = 1 : 2, chrysanthemum extract B : auxiliary materials = 2 : 8), and then ground and sieved. The mixture was mixed with an appropriate amount of 90% ethanol, passed through a 20-mesh sieve, granulated, dried for 1 h, and sieved through a 16-mesh sieve to obtain granules d.

[0155] Test Example 6

[0156] The chrysanthemum extract A obtained in Example 1 (the amount is the same as that of the chrysanthemum extract B in Test Example 5) was added with the auxiliary materials lactose and mannitol (mass ratio, lactose : mannitol = 1 : 2, chrysanthemum extract A : auxiliary materials = 2 : 8), and then ground and sieved. The mixture was mixed with an appropriate amount of 90% ethanol, passed through a 20-mesh sieve, granulated, dried for 1 h, and sieved through a 16-mesh sieve to obtain granules e.

[0157] Test Example 7

[0158] The extract A and the extract B of Amomum villosum Franch. obtained in Example 1 (the amount is the same as that of the extract A and the extract B of Amomum villosum Franch. in Example 10) were mixed in a weight ratio of 1 : 3. The mixture was added with the auxiliary materials lactose and mannitol (mass ratio, lactose : mannitol = 1 : 2, mixed extract : auxiliary materials = 2 : 8), and then ground and sieved. The mixture was mixed with 90% ethanol, passed through a 20-mesh sieve, granulated, dried for 1 h, and sieved through a 16-mesh sieve to obtain granules f.

[0159] Example 11

[0160] The extract A of Amomum villosum Franch., the extract B of Amomum villosum Franch., and the chrysanthemum extract B obtained in Example 7 (the amount is the same as that of the extract A, the extract B of Amomum villosum Franch., and the chrysanthemum extract B in Example 10) were mixed in a weight ratio of 1 : 3 : 1. The mixture was added with an appropriate amount of dextrin, ground, and then encapsulated to obtain chrysanthemum and Amomum villosum Franch. capsules.

[0161] Example 12

[0162] A detection method of a preparation for treating hyperuricemia and gout, comprising the following steps:

[0163] (1) Preparation of the control solution

[0164] An appropriate amount of the control substances of chrysosplenin, luteolin, quercetin, and quercitrin was precisely weighed, and dissolved in methanol to prepare a control solution stock solution containing 0.5 mg of chrysosplenin, 0.5 mg of luteolin, 0.5 mg of quercitrin, and 0.5 mg of quercetin per 1 ml.

[0165] An appropriate amount of the control substance of rutin was precisely weighed, dissolved after ultrasonic treatment with an appropriate amount of methanol, and prepared into a rutin control substance stock solution of 0.5 mg / mL.

[0166] Accurately weigh a certain amount of gallic acid reference substance, dissolve it in distilled water after ultrasonic treatment, and prepare a 1.0 mg / mL gallic acid reference substance stock solution.

[0167] (2) Preparation of test solution

[0168] Accurately weigh 5.0 g of the prepared hypouricemic preparation of the application into a conical flask, accurately add 50 mL of methanol, ultrasonically extract, cool, make up the weight, and pass through a 0.45 μm microporous filter membrane to obtain the solution.

[0169] (3) Preparation of standard curve

[0170] Accurately take the mixed reference substance solution prepared in step (1), dilute it with methanol by 1-fold, 2-fold, 4-fold, 8-fold, 16-fold, and 32-fold respectively to obtain a series of concentration gradient reference substance solutions, take 10 μl of each sample, and use HPLC analysis to prepare a standard curve with peak area as the vertical coordinate and concentration as the horizontal coordinate.

[0171] Accurately take the rutin reference substance solution prepared in step (1), dilute it with methanol by 1-fold, 2-fold, 4-fold, 8-fold, 16-fold, and 32-fold respectively to obtain a series of concentration gradient reference substance solutions, take 1 mL of each sample, add 1 mL of 2% AlCl3 ethanol solution, mix well, stand at room temperature for 1 h, use methanol as a blank control, and measure the absorbance at 420 nm. Repeat the measurement 3 times, and prepare a standard curve with absorbance (Y) as the vertical coordinate and rutin mass concentration (X) as the horizontal coordinate. Calculate the content of total flavonoids in the sample using the standard curve, and express the content of total flavonoids as mg of rutin per g of dry matter.

[0172] Accurately take the gallic acid reference substance solution prepared in step (1), dilute it with distilled water by 1-fold, 2-fold, 4-fold, 8-fold, 16-fold, and 32-fold respectively to obtain a series of concentration gradient reference substance solutions, take 200 μl of each sample, add 200 μl of 0.5 mol / L Folin phenol reagent, then add 600 μl of 10% Na2CO3 aqueous solution, add 1 mL of distilled water to make up the volume, react in a 30°C water bath for 30 min, measure the absorbance at 750 nm, and prepare a standard curve with absorbance (Y) as the vertical coordinate and gallic acid mass concentration (X) as the horizontal coordinate. Calculate the content of total polyphenols in the sample using the standard curve, and express the content of total polyphenols as mg of gallic acid per g of dry matter.

[0173] (4) Content determination

[0174] Take 1 mL of the test solution of the hypouricemic preparation prepared in step (2), add 1 mL of 2% AlCl3 ethanol solution, mix well, stand at room temperature for 1 h, measure the absorbance at 420 nm, and detect the content of total flavonoids in the test solution.

[0175] Take 200 μl of the prepared hypouricemic agent test sample solution of step (2), add 200 μl of 0.5 mol / L Folin phenol reagent, then add 600 μl of 10% Na2CO3 aqueous solution, add 1 mL of distilled water to make up the volume, react in a 30°C water bath for 30 min, and measure the absorbance at 750 nm to detect the content of total polyphenols in the test sample.

[0176] Take 10 μl of the prepared hypouricemic agent test sample solution of step (2), inject into HPLC for detection, and detect the content of each chemical component in the test sample by the regression equation obtained in step (3).

[0177] The HPLC chromatographic conditions described in steps (3) and (4) are as follows:

[0178] Chromatographic column: Agilent zobax C18; mobile phase: acetonitrile as mobile phase A and water as mobile phase B, gradient elution program: 0-10 min, 20% A; 10-15 min, 30% A; 15-30 min, 40% A; flow rate: 1.0 ml / min; column temperature: 30°C; injection volume: 10 μl; ultraviolet detection wavelength: 350 nm.

[0179] The regression equation of step (3) is shown in Table 3:

[0180] Table 3 Regression equation

[0181]

[0182] Example 13

[0183] Methodological investigation of a detection method for a preparation for treating hyperuricemia and gout

[0184] 1. Precision test

[0185] The control solution was precisely pipetted and continuously determined for 6 times according to the method of Example 12. According to the peak area and absorbance, the RSD of total flavonol, total polyphenol, luteolin and quercetin was 2.76%, 1.72%, 2.88% and 2.23% respectively, indicating that the instrument precision was good.

[0186] 2. Reproducibility test

[0187] Six portions of the hypouricemic agent prepared according to the method of Example 10 were taken, the test sample solution was prepared according to the method of Example 12 and determined, and according to the peak area and absorbance, the RSD of the content of total flavonol, total polyphenol, luteolin and quercetin in the test sample was 1.25%, 2.53%, 1.98% and 2.69% respectively, indicating that the reproducibility of the method was good.

[0188] 3. Sample addition recovery rate test

[0189] Take 0.5 g of the uric acid-lowering preparation prepared according to the method of Example 10 in six portions, respectively, and accurately add an appropriate amount of gallic acid and rutin standard product, and prepare the sample solution according to the method of Example 12, and measure and calculate. The average recovery rate of the six sample additions is shown in Table 4, and the results show that the accuracy of the method is good.

[0190] Table 4 Results of sample addition recovery rate test of three components in chrysanthemum sand uric acid-lowering preparation

[0191]

[0192]

[0193] The above experimental results show that the detection method provided by the present application can accurately detect the four main effective components in the uric acid-lowering preparation at the same time, and the quality control method provided by the present application has high precision, high accuracy and high sensitivity, and can comprehensively and objectively evaluate the quality of the uric acid-lowering preparation prepared by the present application.

[0194] Test Example 8

[0195] Pharmacodynamic experiment

[0196] The evaluation scheme for the uric acid-lowering effect is as follows:

[0197] a. Animal grouping and feeding

[0198] Take 81 male Kunming mice weighing 18-22 g for one week of adaptive feeding, and after one week, the mice are randomly divided into 9 groups, each group containing 9 mice. The mice in each group are respectively a blank control group, a positive control group, a drug a group (Example 10), a drug b group (Test Example 3), a drug c group (Test Example 4), a drug d group (Test Example 5), a drug e group (Test Example 6), a drug f group (Test Example 7), and a model group.

[0199] Take an appropriate amount of granules of Example 10, Test Examples 3-7, respectively, and prepare a certain concentration of drug solution with phosphate buffer as the drug a group, the drug b group, the drug c group, the drug d group, the drug e group, and the drug f group. The phosphate buffer is used as the blank group. Each group is given intragastrically, and the drug dose is 1 g / kg of mouse body weight. Take a certain amount of allopurinol powder, and prepare a certain concentration of drug solution with phosphate buffer as the positive control group, and the drug dose is 40 mg / kg of mouse body weight. At the same time, take a certain amount of hypoxanthine, ethambutol, and potassium oxonate, and prepare a suspension of 200 mg / kg, 300 mg / kg, and 300 mg / kg, respectively, with 0.5% carboxymethylcellulose sodium aqueous solution.

[0200] b. Establishment of high uric acid mouse model and drug administration

[0201] The mice in each group were orally administered with 300 mg / kg ethambutol, 200 mg / kg hypoxanthine, and 300 mg / kg potassium oxonate at 9 o'clock in the morning, and the blank control group was orally administered with normal saline. The mice in each group were orally administered with drugs at 4 o'clock in the afternoon, and the blank control group was orally administered with normal saline, for 20 days. After the administration at 9 o'clock in the morning on the 20th day, the mice in each group were placed in a metabolic cage to collect urine for 5 hours. The mice in each group were administered with drugs at 9 o'clock in the morning on the 21st day, and were intraperitoneally injected with 300 mg / kg potassium oxonate after 1 hour.

[0202] c. Evaluation of the uric acid-lowering effect

[0203] ① Determination of the serum UA concentration of mice

[0204] After the last administration, the mice were intraperitoneally injected with 300 mg / kg potassium oxonate, and were enucleated to collect blood after 1 hour. The mice were decapitated, and the whole blood was placed at room temperature for 2 hours, and was centrifuged at 3000 rpm at 4℃ for 15 minutes. The supernatant was used to detect the UA content in the serum of mice by using a uric acid (UA) kit.

[0205] ② Determination of the XOD activity in the liver homogenate of mice

[0206] The liver samples of the mice in each group were added with 4℃ pre-cooled normal saline at a mass ratio of 1:9 to prepare 10% liver tissue homogenate, and were centrifuged at 3000 rpm for 10 minutes. The supernatant was used to detect the XOD activity in the liver tissue homogenate of mice by using a xanthine oxidase (XOD) kit.

[0207] ③ Determination of the UA concentration in the urine of mice

[0208] After the administration at 9 o'clock in the morning on the 20th day, the mice in each group were placed in a metabolic cage to collect urine for 5 hours. Fresh urine samples were centrifuged at 3000 rpm for 10 minutes, and the supernatant was used to detect the UA content in the urine of mice by using a uric acid (UA) kit.

[0209] Experimental results:

[0210] The uric acid-lowering preparation of the present application can reduce the serum uric acid concentration (SUA) of hyperuricemic mice, the XOD activity in the liver tissue homogenate, and the UA concentration in the urine (UUA) as shown in Table 1. Figures 7-9

[0211] ​By comparing the changes in serum uric acid (SUA) levels in mice before and after drug administration, the results showed that administration to groups b, c, and d could reduce serum uric acid levels in hyperuricemic mice to some extent, while group e had no uric acid-lowering effect. Comparing the uric acid-lowering effects of different combinations of drug formulations, the results showed that the combination of polyphenols and flavonoid extracts containing Amomum villosum (group f) enhanced the uric acid-lowering effect. The group further incorporating chrysanthemum extract containing luteolin and quercetin (group a) exhibited the best uric acid-lowering effect, significantly surpassing other groups.

[0212] Meanwhile, the above experimental results showed that prolonged gavage administration of purine substances significantly increased serum uric acid levels in mice, indicating the successful establishment of a hyperuricemia mouse model. Comparison of XOD activity changes revealed that uric acid-lowering preparations containing Amomum villosum extract A, Amomum villosum extract B, and their combinations (groups b, c, and f) could all reduce XOD activity in mouse liver to some extent. A uric acid-lowering preparation containing Chrysanthemum extract B (group d) also reduced XOD activity in mouse liver to some extent, while a uric acid-lowering preparation containing Chrysanthemum extract A (group e) had no significant effect on XOD activity in mouse liver. Observation of UUA concentration showed that only in groups a, c, and f did uric acid excretion increase in the hyperuricemia model mice, with group a showing a significant increase.

[0213] Furthermore, from Figures 7-9 It can be seen that the combination of Amomum villosum extract A and B extracted by the specific method of this invention (drug administration group f) or Chrysanthemum extract B (drug administration group d) can inhibit serum uric acid concentration (SUA) and XOD activity in liver tissue homogenate of hyperuricemic mice to a certain extent, while increasing urine uric acid concentration (UUA), but the effect is not significant. However, the specific combination of Amomum villosum extract A and B with Chrysanthemum extract B (mass ratio 1:3:1, drug administration group a) can significantly enhance the inhibition of serum uric acid concentration (SUA) and XOD activity in liver tissue homogenate of hyperuricemic mice, while significantly increasing urine uric acid concentration (UUA), which is significantly different from the single drug administration groups (d and f), indicating a strong synergistic effect. This further illustrates the synergistic effect of the combination of Amomum villosum extract A and B with Chrysanthemum extract B in the treatment of hyperuricemia and gout.

Claims

1. A method for preparing a preparation for treating hyperuricemia and gout, characterized in that, Includes the following steps: (1) Using Amomum villosum as raw material, grind it into powder using a grinder, sieve it, mix it evenly, and use it as Amomum villosum powder to be extracted; (2) Add the Amomum villosum powder obtained in step (1) into the extraction solvent and reflux for extraction; (3) Evaporate the extraction solvent from the extract obtained in step (2) and extract, let stand to separate the layers, and separate the organic phase and the aqueous phase; (4) The organic phase obtained in step (3) is concentrated under reduced pressure and then dried to obtain Amomum villosum extract A; (5) Extract the aqueous phase obtained in step (3), concentrate and dry under reduced pressure after extraction to obtain Amomum villosum extract B; (6) Using chrysanthemum as raw material, grind it into powder using a grinder, sieve it, mix it evenly, and use it as chrysanthemum powder to be extracted; (7) Add the chrysanthemum powder to be extracted in step (6) into an enzyme-containing buffer solution for enzymatic hydrolysis; (8) Adjust the pH of the enzymatic hydrolysis system obtained in step (7) and extract it by ultrasonication; (9) The extract obtained in step (8) was concentrated under reduced pressure and then dried to obtain chrysanthemum extract A; (10) Add the chrysanthemum extract A obtained in step (9) into a solution containing hydrolytic enzyme for enzymatic hydrolysis; (11) The enzymatic hydrolysate obtained in step (10) was concentrated under reduced pressure and then dried to obtain chrysanthemum extract B; (12) Mix Amomum villosum extract A, Amomum villosum extract B, and Chrysanthemum extract B to prepare a preparation; The extraction solvent in step (2) is an aqueous solution of 50%-90% ethanol, the ratio of the Amomum villosum powder to the extraction solvent is 1:10-1:30 g / mL, the extraction time is 30-90 min, and the extraction temperature is 60-80℃. In step (3), the volume ratio of the extract to the organic solvent after evaporation of the extraction solvent is 1:1-1:3, the extraction time is 5-30 min, and the number of extractions is 1-3. In step (5), the volume ratio of the aqueous phase to the extractant ethyl acetate is 1:1-1:3, the extraction time is 5-30 min, and the number of extractions is 1-3. The enzyme in step (7) includes any one or more of cellulase, pectinase or papain, the amount of enzyme used is 0.1%-0.3% of the solvent mass, the enzymatic hydrolysis temperature is 30℃-50℃, the enzymatic hydrolysis time is 30 min-1 h, and the ratio of chrysanthemum powder to enzymatic hydrolysis solvent is 1:10-1:20 mg / mL. The hydrolytic enzyme in step (10) includes any one or more of β-glucosidase, naringinase or snailase, the amount of enzyme used is 0.1%-0.3% of the solvent mass, the hydrolysis temperature is 30℃-50℃, the hydrolysis time is 30 min-1h, and the ratio of chrysanthemum extract A to hydrolysis solvent is 1:10-1:20 mg / mL; In step (12), the mass ratio of Amomum villosum extract A, Amomum villosum extract B, and Chrysanthemum extract B is 1:3:1-3:2:

2.

2. The method for preparing the preparation for treating hyperuricemia and gout according to claim 1, characterized in that, The enzyme buffer in step (7) is phosphate buffer.

3. The method for preparing the preparation for treating hyperuricemia and gout according to claim 1, characterized in that, In step (8), the pH range is adjusted to 8-12, the ultrasonic power is 150-300W, and the extraction time is 0.5 h-2 h.

4. The method for preparing the preparation for treating hyperuricemia and gout according to claim 1, characterized in that, The solution of the hydrolytic enzyme in step (10) is phosphate buffer.

5. A preparation for treating hyperuricemia and gout prepared by the method of claim 1, characterized in that, The formulation includes Amomum villosum extract A, Amomum villosum extract B, Chrysanthemum extract B, and a pharmaceutically acceptable carrier. The dosage form of the formulation includes granules, tablets, capsules, or oral liquid.

6. The use of the formulation of claim 5 in the preparation of a drug for treating hyperuricemia and gout.

Images