A traditional Chinese medicine composition for treating hyperuricemia and its preparation method and application

Abstract

The present invention belongs to the technical field of traditional Chinese medicine, and in particular to a Chinese medicine composition for treating hyperuricemia, and its preparation method and application. The Chinese medicine composition comprises the following components and weight parts: 53-54 parts of Atractylodes lancea, 107-108 parts of Caulis Lonicerae, 53-54 parts of Pheretima, 80-81 parts of Fructus Forsythiae, 53-54 parts of Phellodendron chinense, 107-108 parts of Semen Coicis, 53-54 parts of Saposhnikovia divaricata, 80-81 parts of Cyathulae chinensis, 80-81 parts of Radix Trichosanthis, 107-108 parts of Ramulus Mori, 80-81 parts of Stephania tetrandra, and 64-65 parts of Radix Clematidis. The Chinese medicine composition of the present invention is exquisitely compatible, and has the effects of invigorating the spleen and removing dampness, promoting blood circulation, removing blood stasis and relieving pain, reducing uric acid synthesis, and promoting uric acid excretion, thereby playing a role in treating hyperuricemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating hyperuricemia, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Currently, the clinical incidence of hyperuricemia is increasing annually, and the incidence is increasingly younger. Hyperuricemia can cause multiple damages to the body. For example, it can affect joints and cause gout; damage the kidneys, leading to hyperuricemia nephropathy and uric acid stones; affect blood vessel walls, causing atherosclerosis and cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, and stroke; and damage pancreatic B cells, leading to diabetes. Hyperuricemia is closely associated with damage to multiple target organs, so it must be given due attention.

[0004] Hyperuricemia is rooted in spleen and kidney deficiency, primarily due to dietary indiscriminateness, high stress, prolonged sitting, and staying up late. Dampness, blood stasis, qi stagnation, and phlegm congestion are the pathological consequences. Hyperuricemia affects the spleen, stomach, joints, kidneys, brain, and heart.

[0005] Traditional Chinese Medicine (TCM) treatment for hyperuricemia focuses on strengthening the spleen and kidneys, and removing dampness and turbidity. Treating both the spleen and kidneys addresses the underlying cause, while removing dampness and turbidity clears the source. Based on the specific symptoms, TCM then applies methods such as regulating Qi, activating blood circulation, and resolving phlegm.

[0006] The 2019 Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout in China determine whether hyperuricemia patients require drug treatment based on serum uric acid levels. Based on comorbidity stratification, patients without comorbidities are recommended to maintain a serum uric acid level below 420 μmol / L, while patients with comorbidities are recommended to maintain a serum uric acid level below 360 μmol / L. Reports indicate that approximately 30% of gout patients with serum uric acid levels below 420 μmol / L for approximately 5 years experience a recurrence of gouty arthritis within 4 years after discontinuation of treatment. The 2019 Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout in China state that all patients should be aware of the need to maintain a target serum uric acid level of 240–420 μmol / L throughout their lives, potentially requiring long-term or even lifelong uric acid-lowering medication. Therefore, long-term treatment for hyperuricemia and gout may be necessary to control serum uric acid levels. Oral preparations, taken multiple times daily, have poor patient compliance, and are subject to incomplete absorption and low bioavailability. Injectable formulations, on the other hand, are costly, inconvenient, and may also present with adverse reactions.

[0007] Traditional Chinese medicine preparation methods often involve decoction followed by solvent extraction to extract the active ingredients, ultimately producing TCM preparations. These traditional solvent extraction methods, such as decoction, are complex, time-consuming, and result in significant loss of active ingredients, low extraction yields, and low product purity. This reduces the drug content of the resulting preparations, leading to increased dosages and hindering patient compliance and reducing fluctuations in blood drug concentrations following administration. Summary of the Invention

[0008] In order to solve the problems of traditional solvent extraction methods such as cumbersome processes, long cycles, large loss of active ingredients, low extraction rates, and low product purity, while also reducing the problem of large dosages of the original preparations, the present invention aims to provide a Chinese medicine composition for treating hyperuricemia, and its preparation method and application. The present invention, guided by traditional Chinese medicine theory, uses supercritical fluid extraction to obtain a Chinese medicine composition, and, based on modern pharmaceutical dosage form requirements, combined with clinical medication needs and disease characteristics, utilizes modern pharmaceutical preparation technologies and methods to prepare sustained-release tablets from the composition.

[0009] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0010] In the first aspect, the present invention provides a traditional Chinese medicine composition for treating hyperuricemia, comprising the following components and their composition in parts by weight: 53-54 parts of Atractylodes lancea, 107-108 parts of Lonicera japonica, 53-54 parts of Earthworm, 80-81 parts of Forsythia suspensa, 53-54 parts of Phellodendron chinense, 107-108 parts of Coix lachryma-jobi, 53-54 parts of Saposhnikovia divaricata, 80-81 parts of Cyathula capitata, 80-81 parts of Trichosanthes kirilowii, 107-108 parts of Morus alba, 80-81 parts of Stephania tetrandra and 64-65 parts of Clematis chinensis.

[0011] Atractylodes lancea and coix seed have the effects of strengthening the spleen and removing dampness; honeysuckle vine, forsythia suspensa and phellodendron can clear away heat, dry dampness and purge fire; Stephania tetrandra and Radix Trichosanthis can remove dampness and turbidity; Achyranthes bidentata can promote blood circulation, remove blood stasis, remove dampness and promote diuresis; earthworm, saposhnikovia divaricata, clematis root and morulone are commonly used traditional Chinese medicines for removing wind, dampness and relieving pain. The whole formula is exquisitely matched, and together they have the effects of strengthening the spleen and removing dampness, promoting blood circulation, removing blood stasis and relieving pain, which can reduce uric acid synthesis and promote uric acid excretion, thereby playing a role in treating hyperuricemia.

[0012] The chemical components of Atractylodes lancea mainly include enynes, sesquiterpenes, triterpenes, glycosides, and sterols; the chemical components of Lonicera japonica include organic acids, triterpenes, iridoids, flavonoids, and volatile oils; the chemical components of Coix lachryma-jobi include fatty acids, polysaccharides, flavonoids, triterpenes, and sterols; and the chemical components of Clematidis include triterpenes, volatile oils, and flavonoids. Among them, triterpenes are the main active components; Morus alba mainly contains flavonoids, alkaloids, polysaccharides, and coumarins, and also contains amino acids, organic acids, volatile oils, and various vitamins. The most important active ingredients in Saposhnikovia divaricata are chromone compounds; the main active ingredients in Stephania tetrandra are alkaloids, and it contains a variety of alkaloids, mainly tetrandrine (hantelandin), demethyl tetrandrine (hantelandin B), and cyclopentane quaternium; the main chemical components of Phellodendron chinense are alkaloids, sterols, limonin, etc.; the main chemical components of Cyathula capitata are alkaloids, ecdysterone, cyperasterone, polysaccharides, etc.; the chemical components of Forsythia suspensa include phenylethanol and its glycosides, volatile Hair oil, lignans, flavonoids, natural alcohols and their glycosides, triterpenes, alkaloids, etc., among which phenylethanoid glycosides, volatile oils and lignans are the main active ingredients; Dioscorea ternata mainly contains steroidal saponins and aglycones, such as diosgenin, diosgenin A, diosgenin, protodiosgenin, etc., as well as chemical components such as diarylheptanes, lignans, organic acids and lipids; the chemical components of earthworms include proteins, polypeptides, amino acids, enzymes, nucleotides, lipids, trace elements and other ingredients.

[0013] In a second aspect, the present invention provides a method for preparing the traditional Chinese medicine composition for treating hyperuricemia as described in the first aspect, comprising the following steps:

[0014] Crush the Chinese medicine raw materials, pass them through a No. 4 sieve, and put them into the extraction kettle. Set the extraction temperature. When the set temperature is reached and the refrigeration temperature is below 5°C, open the plug and load the material. Adjust the valve to make the extraction pressure equal to the tank pressure.

[0015] Turn on the CO2 pump, and the CO2 is compressed by the compressor and enters the extraction kettle. After the extraction pressure reaches the set value and stabilizes, add the entrainer for supercritical extraction. Collect the extracted liquid from the outlet every 30 minutes until the extracted liquid is completely collected.

[0016] The extracted stock solution was concentrated under reduced pressure using a rotary evaporator until the volume no longer changed, and then freeze-dried to obtain a traditional Chinese medicine composition.

[0017] Supercritical fluid extraction (SCFE) is a novel technology developed in recent years. It uses supercritical fluid as an extractant to extract certain high-boiling-point components from liquids and solids for the purpose of separation or purification. Supercritical fluids are fluids above their critical temperature and pressure. Compared to conventional liquid solvents, they enable faster mass transfer and reach equilibrium in a shorter time, resulting in efficient separation. This is particularly advantageous for extracting certain components from solid materials, as the solvent's high diffusion coefficient, low viscosity, and excellent permeability simplify pre-processing for solid pulverization. This technology offers excellent selectivity. By controlling pressure and temperature to alter the density of the supercritical fluid CO2, and thus its solubility, it can be used to selectively extract specific components from traditional Chinese herbal medicines, completing the entire extraction and separation process in a single step. This method offers advantages such as no residual solvent, strong extraction capacity, high extraction efficiency, easy control of operating parameters, resistance to decomposition and destruction of active and thermally labile components, low energy consumption, safety, and affordability. It can extract substances that are inaccessible to traditional methods. By adjusting the extraction parameters such as extraction temperature, pressure, and addition of entrainers, significant advantages are shown in the extraction of active ingredients such as volatile oils, terpenes, flavonoids, and alkaloids.

[0018] Preferably, the extraction temperature is 30-70°C.

[0019] Preferably, the extraction pressure is 15-55 MPa.

[0020] Preferably, the entrainer is 10%-90% ethanol.

[0021] Preferably, the extraction time is 30-150 min.

[0022] In a third aspect, the present invention provides use of the traditional Chinese medicine composition for treating hyperuricemia as described in the first aspect in the preparation of a drug for treating hyperuricemia.

[0023] In a fourth aspect, the present invention provides a sustained-release tablet, comprising the following components and content in weight ratio: 29%-63% of the traditional Chinese medicine composition for treating hyperuricemia as described in the first aspect, 20%-40% HPMC, 5%-15% dextrin and 12%-16% microcrystalline cellulose.

[0024] Preferably, the HPMC is HPMCK15M or HPMCK100M.

[0025] In a fifth aspect, a method for preparing the sustained-release tablet according to the fourth aspect comprises the following steps:

[0026] The traditional Chinese medicine composition for treating hyperuricemia as described in the first aspect, HPMC, dextrin and microcrystalline cellulose are weighed in proportion, and a soft material is prepared using 95% ethanol as a wetting agent until it forms a ball when lightly gripped and breaks when lightly pressed. The material is granulated using a 20-mesh sieve, dried at 35-45° C. for 20-40 minutes, sieved through an 18-mesh sieve, and 0.5% talc powder is added as a lubricant. After mixing evenly, the material is pressed into sustained-release tablets weighing 0.24-0.26 g.

[0027] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0028] The Chinese medicine composition of the present invention has an exquisite compatibility and can strengthen the spleen and eliminate dampness, activate blood circulation, remove blood stasis and relieve pain. It can reduce uric acid synthesis and promote uric acid excretion, thus playing a role in treating hyperuricemia.

[0029] The present invention uses supercritical fluid extraction to prepare important compositions, and by adjusting parameters such as extraction temperature, pressure, and addition of entrainers, it exhibits significant advantages in the extraction of active ingredients such as volatile oils, terpenes, flavonoids, and alkaloids.

[0030] The invention prepares the traditional Chinese medicine composition into a sustained-release tablet, which can realize slow release, and the release of the medicine can reach about 80% 8 hours after taking it.

[0031] The present invention uses potassium oxonate and adenine to construct a mouse hyperuricemia model to investigate the efficacy of a Chinese medicine composition in lowering uric acid. Through experimental studies, it was found that compared with the hyperuricemia model group, after treatment with the composition, the serum UA, Cr, BUN levels, inflammatory factors TNF-α, IL-1β, and liver XOD activity in the model group mice were significantly increased compared with the normal group (P < 0.05, P < 0.01); compared with the model group, the serum UA, BUN, Cr, TNF-α, IL-1β levels and liver XOD activity in the Chinese medicine composition group and high-dose group mice were reduced to varying degrees (P < 0.05, P < 0.01). The Chinese medicine composition of the present invention achieved a therapeutic effect similar to that of allopurinol and febuxostat with a smaller dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 The effects of different extraction temperatures on the yields of total flavonoids, total alkaloids, total triterpenes and volatile oil in Example 1 are shown;

[0034] Figure 2 The effects of different extraction pressures on the yields of total flavonoids, total alkaloids, total triterpenes and volatile oil in Example 1 are shown;

[0035] Figure 3 The effects of different extraction times on the yields of total flavonoids, total alkaloids, total triterpenes and volatile oil in Example 1 are shown;

[0036] Figure 4 The effect of different extraction entrainer concentrations on the yields of total flavonoids, total alkaloids, total triterpenes and volatile oil in Example 1 is shown;

[0037] Figure 5 is the cumulative release percentage of total flavonoids under different HPMC specifications in Example 2;

[0038] Figure 6 is the cumulative release percentage of total flavonoids under different fillers in Example 2;

[0039] Figure 7 This is the cumulative release percentage curve of total flavonoids from three batches of sustained-release tablets in Example 2. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0041] Example 1

[0042] 53.9g of Atractylodes lancea, 107.8g of Honeysuckle Caulis, 53.9g of Earthworm, 80.9g of Forsythia, 53.9g of Phellodendron chinense, 107.8g of Coix Seed, 53.9g of Saposhnikovia divaricata, 80.9g of Cyathula, 80.9g of Radix Trichosanthis, 107.8g of Morus alba, 80.9g of Stephania tetrandra, and 64.7g of Radix Clematidis were crushed, passed through a No. 4 sieve, and loaded into an extraction kettle. The extraction temperature was set. When the set temperature was reached and the refrigeration temperature was below 5°C, the plug was opened and loaded. The valve was adjusted to equalize the extraction pressure and the tank pressure. The CO2 pump was turned on, and CO2 was compressed by the compressor into the extraction kettle. After the extraction pressure reached the set value and stabilized, an entrainer was added for supercritical extraction. The extracted stock solution was collected from the outlet every 30 minutes until the extracted stock solution was completely collected. The above-mentioned extracted stock solution was concentrated under reduced pressure with a rotary evaporator until the volume no longer changed, and then freeze-dried to obtain a Chinese medicine composition.

[0043] In this example, the effects of extraction temperature (30°C, 40°C, 50°C, 60°C and 70°C), extraction pressure (15MPa, 25MPa, 35MPa, 45MPa and 55MPa), extraction time (30min, 60min, 90min, 120min and 150min), and entrainer concentration (10%, 30%, 50%, 70% and 90%) on the yield of total flavonoids, total alkaloids, total triterpenes and volatile oil were investigated respectively.

[0044] Determination of total flavonoid yield

[0045] (1) Drawing of rutin standard curve

[0046] Accurately weigh the rutin standard solution and place it into a 25mL volumetric flask. Dose to volume with 70% ethanol, shake well, and refrigerate to cool. Use a pipette to accurately pipette 0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0mL of the rutin standard solution into 25mL colorimetric tubes. Add 70% ethanol to 10.0mL. Add 1mL of 5% NaNO₂, shake well, and let stand for 6 minutes. Then, add 1mL of 10% Al(NO₃)₃·9H₂O, shake well, and let stand for 6 minutes. Then, take 10mL of 4% NaOH, dilute to volume with purified water, shake well, and let stand for 15 minutes. Add these solutions sequentially to a cuvette, place in a UV spectrometer, and measure the absorbance (A) at 510nm.

[0047] (2) Sample measurement

[0048] Take the extract, add an appropriate amount of 70% ethanol, ultrasonically extract for 30 minutes, centrifuge, take the supernatant as the extract solution to be tested, transfer 1 mL into a 25 mL colorimetric tube, prepare according to the method described in (1), measure the absorbance, calculate the sample concentration, and calculate the flavonoid extraction rate in the extract sample according to the following formula:

[0049] Total flavonoid extraction rate % = C × V × N / M × 10 3 ×100%,

[0050] Where, C: total flavonoids content, mg / mL;

[0051] V: volume of the extract, mL;

[0052] N: dilution multiple;

[0053] M: mass of extract, g.

[0054] Determination of total alkaloids

[0055] Take an appropriate amount of the extract, add it to a 50ml conical flask, add an appropriate amount of water to dissolve, make up to volume, and use it as the extract solution to be tested. Titrate it with 0.1mol / L hydrochloric acid solution to a pH of about 3, add a few drops of phenolphthalein indicator, and titrate it with 0.1mol / L sodium hydroxide solution to neutrality. Record the required titrant. Calculate the total alkaloid content of the sample based on the amount of alkali consumed in the titration in mg / g. Perform a blank experiment with water as the blank solvent.

[0056] Total alkaloid content % = (V-V0) × N × 0.1 × 40 × F × 10 -3 / m×100%

[0057] Where, V: volume of sodium hydroxide solution consumed by the sample to be tested, mL;

[0058] V0: Volume of sodium hydroxide solution consumed by blank sample, mL;

[0059] N: sample dilution multiple;

[0060] F: titrant concentration correction factor

[0061] m: mass of extract, g.

[0062] Determination of total triterpenes

[0063] (1) Drawing of standard curve

[0064] Accurately weigh the oleanolic acid standard and dilute to 50 ml with chloroform as the standard solution. Accurately pipette 0.0, 0.10, 0.20, 0.50, 1.00, and 1.50 ml of the standard solution at different concentrations, heat and evaporate the solvent. Add 0.5 ml of vanillin and 5 ml of sulfuric acid solution, mix thoroughly, and incubate in a 60°C water bath for 30 minutes. Remove from the heat and place in ice water for 15 minutes. In a 1 cm cuvette, adjust the zero point using the 0.0 ml tube of standard solution as a reference. Measure the absorbance at a wavelength of 550 nm and calculate the regression equation.

[0065] (2) Sample measurement

[0066] Take the extract, accurately weigh it, add approximately 20 ml of chloroform, and sonicate for 1 hour. After cooling, dilute to 25 ml with chloroform, filter, and use the filtrate as the test solution. Accurately pipette 0.1-1.0 ml of the test solution into a glass test tube, heat to evaporate and dissolve, add 0.5 ml of vanillin and 5 ml of sulfuric acid solution, mix well, and incubate in a 60°C water bath for 30 minutes. After removing, place in ice water for 15 minutes. In a 1 cm cuvette, adjust the zero point using the standard solution 0.0 tube as a reference. Measure the absorbance at a wavelength of 550 nm to calculate the total triterpene content.

[0067] Total triterpenoid content % = M2 × V1 / M1 / V2 × 10 -3 ×100%

[0068] Where, M1: mass of the sample, g;

[0069] M2: The mass of triterpenoids in the sample solution calculated by the linear regression equation, mg

[0070] V1: volume of the sample to be tested, mL;

[0071] V2: Volume of sample solution to be tested, mL.

[0072] Determination of volatile oil yield

[0073] Take an appropriate amount of the extract and determine the content (%) of volatile oil in the extract according to the volatile oil determination method 2204 of the Chinese Pharmacopoeia.

[0074] The effects of different extraction temperatures (30°C, 40°C, 50°C, 60°C and 70°C) on the yields of total flavonoids, total alkaloids, total triterpenes and volatile oil were investigated at an extraction pressure of 25 MPa, an extraction time of 120 min and an entrainer of 70% ethanol. Figure 1 As shown, below 50°C, the extraction rate of each component increases with increasing temperature. Above 50°C, however, the rate of extraction decreases. This is because increasing temperature accelerates molecular motion, increasing the dissolution rate and facilitating extraction. However, above a certain temperature, the density of the supercritical fluid decreases, reducing its dissolving power and even destroying certain chemical components, leading to a decrease in extraction rate. Therefore, 50°C is an appropriate extraction temperature.

[0075] When the extraction temperature was 50°C, the extraction time was 120 min, and the entrainer was 70% ethanol, the effects of different extraction pressures (15MPa, 25MPa, 35MPa, 45MPa and 55MPa) on the yield of total flavonoids, total alkaloids, total triterpenes and volatile oils were investigated. It is generally believed that increasing the pressure will increase the density of CO2 while also reducing the mass transfer distance, which is beneficial to increasing the mass transfer efficiency. As the pressure increases, the convection and diffusivity of the fluid decrease, and the mass transfer rate decreases. The increase in pressure will also promote the swelling of the matrix, thereby accelerating the rate of outward diffusion of the effective ingredients and promoting mass transfer during the extraction process. At the same time, when the extraction pressure is high, the tissue will deform and become loose, the cell wall will be damaged, the permeability will increase, and the solubility of each component will be enhanced. For example Figure 2 As shown in the figure, when the pressure reaches 35-45MPa, the extraction rate reaches the maximum, and then the extraction rate decreases with the increase of pressure. This may be because when the pressure reaches a certain value, part of the raw materials condense into blocks as the pressure increases, resulting in a decrease in the extraction rate. Therefore, the optimal extraction pressure is determined to be 35MPa.

[0076] At an extraction temperature of 50°C, an extraction pressure of 25 MPa, and an entrainer of 70% ethanol, the effects of different extraction times (30 min, 60 min, 90 min, 120 min, and 150 min) on the yields of total flavonoids, total alkaloids, total triterpenes, and volatile oil were investigated. Figure 3 As shown in the figure, the extraction rate gradually increases with extraction time between 30 and 150 minutes, with a clear upward trend. The maximum extraction rate is achieved at 150 minutes. However, after the extraction time reaches 120 minutes, further extension of the extraction time does not significantly increase the extraction rate. Therefore, the extraction time of 120 minutes was selected.

[0077] At an extraction temperature of 50°C, an extraction pressure of 25 MPa, and an extraction time of 120 min, ethanol was used as an entrainer to investigate the effects of different concentrations (10%, 30%, 50%, 70%, and 90%) on the yields of total flavonoids, total alkaloids, total triterpenes, and volatile oil. Figure 4 As shown, comprehensive analysis of various components shows that when 70% ethanol is used as the entrainer, the total effective substance extraction rate is the highest, and the entrainer is determined to be 70% ethanol.

[0078] The optimal extraction process was 70% ethanol as the entrainer, 35 MPa as the extraction pressure, 50°C as the extraction temperature, and 120 min as the extraction time. Using these extraction conditions, the above-described Chinese medicinal composition of this example was subjected to supercritical CO extraction. The freeze-dried powder masses of the Chinese medicinal composition obtained in three experiments were 178.04 g, 171.55 g, and 200.30 g, respectively, with a final average extraction yield of 19.77%.

[0079] Example 2

[0080] 1. Preparation and quality assessment of sustained-release tablets

[0081] 9.8 g of the lyophilized powder of the Chinese medicine composition of Example 1, 1.0 g of dextrin, 3.2 g of microcrystalline cellulose and 6 g of the sustained-release matrix material HPMC were weighed and mixed uniformly. 95% ethanol was used as a wetting agent to prepare a soft material until it was lightly grasped and crushed when lightly pressed. 20 mesh sieve granules were made, 40° C. dried for 30 min, 18 mesh sieve granules were added, 0.5% talc was added as a lubricant, and after mixing uniformly, they were pressed into sustained-release tablets weighing about 0.25 g. The sustained-release tablets had uniform color, smooth surface, moderate hardness (50-60 N), friability less than 1%, good formulation and process reproducibility, stable and feasible process, and met the quality requirements related to tablets.

[0082] 2. Investigation of the methodological basis for the determination of total flavonoids

[0083] 2.1 Drawing of Rutin Standard Curve

[0084] Accurately weigh the rutin standard solution and place it into a 25mL volumetric flask. Dose to volume with 70% ethanol, shake well, and refrigerate to cool. Use a pipette to accurately pipette 0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0mL of the rutin standard solution into a 25mL colorimetric tube. Add 70% ethanol to 10.0mL. Add 1mL of 5% NaNO₂, shake well, and let stand for 6 minutes. Then, add 1mL of 10% Al(NO₃)₃·9H₂O, shake well, and let stand for 6 minutes. Then, take 10mL of 4% NaOH, dilute to volume with purified water, shake well, and let stand for 15 minutes. Add these solutions sequentially into a cuvette and place in a UV spectrometer. Measure the absorbance A at a wavelength of 510nm. Regress the absorbance against the concentration to obtain the equation: The results show a good linear relationship between rutin and absorbance over the μg / mL concentration range.

[0085] 2.2 Recovery rate experiment

[0086] Accurately weigh 3 portions of 24.24 g (low concentration), 30.30 g (medium concentration), and 36.26 mg (high concentration) of the Chinese medicine composition freeze-dried powder of Example 1, add appropriate amounts of 70% ethanol to each auxiliary material according to the prescription ratio of 2.1, ultrasonically extract for 30 min, centrifuge, take the supernatant as the extract solution to be tested, pipette 1 mL into a 25 mL colorimetric tube, prepare according to the method described in 2.1, measure the absorbance, and calculate the sample concentration. The experimental results are shown in Table 1. The extraction recovery of total flavonoids is within the range of 95%-105% at low, medium, and high concentrations, and the RSD is less than 2%, which meets the requirements of methodological validation. This shows that the auxiliary materials used do not affect the accuracy of drug content determination.

[0087] Table 1 Recovery determination results

[0088]

[0089] 3 Determination of drug release in vitro

[0090] 3.1 Drug content determination

[0091] Take 10 tablets, grind them into powder, accurately weigh 0.1g, add appropriate amount of 70% ethanol, ultrasonically extract for 30 minutes, centrifuge, and use the supernatant as the extract solution to be tested. Pipette 1mL into a 25mL colorimetric tube and prepare according to the method described in 2.2.1. Measure the absorbance at 510nm and calculate the sample concentration.

[0092] 3.2 Cumulative release percentage

[0093] According to the release determination method (basket method) of the 2020 edition of the "Pharmacopoeia of the People's Republic of China", the dissolution medium was 900 mL of 0.1 mol / L hydrochloric acid solution, the rotation speed was 100 r·min, and the temperature was (37±5)°C. 5 mL of samples were taken at 1, 2, 3, 4, 5, 6, 7, and 8 h, and an equal amount of blank solution was added. The solution was filtered through a 0.45 μm microporous filter membrane, and 2 mL was taken and placed in a 10 mL volumetric flask. The volume was adjusted to the scale, and the absorbance was measured at 510 nm to calculate the cumulative release percentage.

[0094] 4. Prescription screening of sustained-release tablets of pharmaceutical compositions

[0095] 4.1 Effect of HPMC Specifications on Drug Release

[0096] The drug release rate of HPMC hydrophilic gel matrix tablets decreases with the increase of the viscosity of the matrix material. In this experiment, HPMC specifications K4M, K15M, and K100M were selected for comparison. Other excipients were the same. The tablets were prepared according to the method under 2.1 of this example, and the cumulative release rate of total flavonoids was determined according to 3.2. The cumulative release curve of total flavonoids is shown in Figure 5 .Depend on Figure 5 It can be seen that HPMC of different specifications has a great influence on the drug release of the sustained-release tablets of the pharmaceutical composition, and the different HPMC viscosities have great differences in the blocking ability of the main drug release. The results show that the order of total flavonoids release speed in the sustained-release tablets of the pharmaceutical composition is K4M>K15M>K100M. K4M has a very weak blocking ability on the main drug and does not play a certain role in delaying the release. The sustained-release effects of K15M and K100M are significantly better than that of K4M. The cumulative release percentages in 8 hours are 91.22% and 78.6%, respectively. Both HPMC specifications K15M and K100M can be used as sustained-release materials for sustained-release tablets of the pharmaceutical composition.

[0097] 4.2 Effect of fillers on drug release

[0098] The main factor that determines the drug release effect is the solubility of the filler in water. Chinese herbal medicine extract powder or freeze-dried powder has a certain viscosity and hygroscopicity. Selecting a suitable filler is crucial to reducing hygroscopicity, formulation molding and drug release. Therefore, the effects of three fillers, lactose, pregelatinized starch and microcrystalline cellulose, on the release rate of sustained-release tablets were compared. The dosage was 20% of the tablet weight, and other excipients were the same. The tablets were prepared according to the method under Item 1 of this embodiment, and the cumulative release rate of total flavonoids was determined according to the method under Item 3.2. The cumulative release curves of total flavonoids under different fillers are shown in Figure 6 .like Figure 6As shown in the figure, different fillers have a certain impact on drug release. Lactose is a water-soluble filler, commonly found at a water content of 5%. This can be an unstable factor, especially when used with moisture-sensitive drugs. Its pore-forming effect increases the porosity of sustained-release tablets, thereby reducing drug diffusion resistance and increasing drug diffusion pathways, accelerating drug release. However, the effects of dextrin and microcrystalline cellulose on drug release are similar. Microcrystalline cellulose has good compressibility and strong binding properties, and disintegrates after absorption and swelling. It can enhance drug release in the middle and late stages while having little effect on early release. Therefore, both dextrin and microcrystalline cellulose can be used as fillers in sustained-release tablets.

[0099] 5. Prescription Optimization

[0100] On the basis of single factor experiments, the factors that significantly affected the in vitro release rate, namely, the amount of sustained-release matrix material HPMC (K15M), the amount of dextrin and the amount of microcrystalline cellulose, were selected as the research objects. Three levels were determined for each factor (see Table 2). 4 ) The experiment was arranged using an orthogonal experimental table, and the comprehensive score Y was used as the evaluation index: Y = ︱(15% - F1)︱+︱(F8 - 80%)|, where F1 is the cumulative release percentage in 1 h, and F8 is the cumulative release percentage in 8 h. The dosage of sustained-release excipients and the effects of other excipients on drug release were investigated, as shown in Tables 2 and 3.

[0101] Table 2 Orthogonal test factors and levels

[0102]

[0103] Table 3 Orthogonal test scheme and results

[0104]

[0105] The evaluation results were based on the degree to which the sustained-release tablet release percentage approached 15% and 80% at the 1- and 8-hour time points, respectively. The closer the percentage, the better the sustained-release tablet's release rate. In other words, the smaller the comprehensive score (Y), the better the sustained-release tablet's release effect. The experimental results in Table 3 show that the order of influence of the various factors on release effect is A > B > C. The comprehensive score (Y) is minimized when the factor levels are A2B1C3. This corresponds to an HPMC dosage of 30%, a dextrin dosage of 5%, and a microcrystalline cellulose dosage of 16%.

[0106] After the prescription was optimized through orthogonal test, the preparation process of the sustained-release tablets of the Chinese medicine composition was as follows: 49% of the Chinese medicine composition freeze-dried powder, 30% of HPMCK15M or HPMCK100M, 5% of dextrin, 16% of microcrystalline cellulose, and 95% ethanol was used as a wetting agent to prepare a soft material to a state where it could be easily squeezed into a mass and crushed by light pressure, sieved with 20 mesh, dried at 40°C for 30 minutes, sieved with 18 mesh, and 0.5% of talcum powder was added as a lubricant. After mixing evenly, the tablets were pressed into sustained-release tablets with a weight of about 0.25g. The sustained-release tablets prepared by wet granulation using the optimal prescription were uniform and smooth in color without spots. Three batches of sustained-release tablets of the Chinese medicine composition were prepared to investigate the release uniformity and process reproducibility of the sustained-release tablets. The cumulative release curve of total flavonoids in the sustained-release tablets is shown in the figure below. Figure 7 As shown, the release curves of the three batches of sustained-release tablets are basically the same, the curves are relatively smooth, and there is no significant difference. Therefore, the optimal formulation and process for the sustained-release tablets of the pharmaceutical composition are reproducible. The prepared sustained-release tablets slowly release in vitro, and the drug release can reach about 80% 8 hours after administration.

[0107] Example 3

[0108] experimental animals

[0109] Male SPF mice, weighing 20 ± 2 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. under animal license SCXK (Beijing) 2020-0004. During the experiment, the animals were housed in a barrier animal room and maintained adaptively with free access to food and water, and maintained a normal circadian rhythm. This study was approved by the Laboratory Animal Ethics Committee of the Medical Experimental Center of the China Academy of Chinese Medical Sciences (approval number: ERCCACMS11-2302-08).

[0110] Drugs and reagents

[0111] The Chinese medicine composition was prepared according to the optimal method of Example 1 (the entrainer was 70% ethanol, the extraction pressure was 35 MPa, the extraction temperature was 50° C., and the extraction time was 120 min).

[0112] Febuxostat (purchased from Shanghai McLean Biochemical Technology Co., Ltd.); uric acid (UA) test kit (purchased from BioAssay Systems, USA); potassium oxonate and adenine (purchased from Aladdin Holding Group Co., Ltd.); mouse interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kit, mouse tumor necrosis factor-α (TNF-α) ELISA kit (purchased from Invitrogen, USA); creatinine (Cr) and urea nitrogen (BUN) detection kits (purchased from Sangon Biotech (Shanghai) Co., Ltd.); xanthine oxidase (XOD) detection kit (purchased from Shanghai Shangbao Biotechnology Co., Ltd.).

[0113] instrument

[0114] MB100-4A microplate constant temperature oscillator (Hangzhou Aosheng Instrument Co., Ltd.), MS1003S10 analytical balance (Mettler Toledo, Switzerland), Fles Station 3 multifunctional microplate reader (Molecular Devices, USA).

[0115] Establishment of hyperuricemia mouse model and drug administration

[0116] After one week of adaptive feeding, 70 male mice were randomly divided into a normal group, a model group, an allopurinol group, a febuxostat group, and a low-, medium-, and high-dose group of the Chinese medicine composition, with 10 mice in each group. Each drug group was prepared as a suspension using 0.5% sodium hydroxycellulose (CMC-Na). The allopurinol and febuxostat groups were given a dose of 5 mg / kg. -1 The high, medium and low doses of the Chinese medicine composition are 2.314, 1.157 and 0.5785 g·kg -1 , equivalent to 2, 1, and 0.5 times the clinical equivalent dose, respectively.

[0117] Starting from the first day, except for the normal group, the other groups were gavaged with potassium oxonate (350 mg·kg -1 ) and adenine (80 mg kg -1 ) were used for modeling, once a day for 14 consecutive days. The corresponding drugs were given on the 7th day, and the Febuxostat group was given 5 mg·kg -1 Febuxostat and allopurinol groups were given 5 mg·kg -1 Allopurinol, the high, medium and low dose groups of the Chinese medicine composition were given 2.314, 1.157 and 0.5785 g·kg -1 The normal group was gavaged with 0.5% CMC-Na every day for 7 consecutive days. On the 13th day, the mice were fasted overnight and orbital blood was collected 1 hour after the last administration.

[0118] Blood was collected from the mouse orbits and allowed to stand at room temperature for 0.5 h at 4500 r·min -1 Centrifuge for 10 minutes, collect serum, and use the kit to detect UA, Cr, and BUN levels according to the instructions. Take the mouse liver, weigh 0.1g, mince it, add 1mL of tissue lysis solution, and homogenize it in an ice bath at 4℃ and 8000r / min. -1 Centrifuge for 10 minutes, take the supernatant, put it on ice, and use the kit to detect XOD activity according to the instructions. Detect the levels of TNF-α and IL-1β in serum according to the instructions of the ELISA kit. Statistical analysis was performed using SPSS 26.0. One-way analysis of variance was used for comparison among the groups, and P < 0.05 indicated a statistically significant difference.

[0119] A hyperuricemia mouse model was established using a combination of potassium oxonate and adenine. Potassium oxonate is a UA enzyme inhibitor, while adenine increases the source of UA synthesis. Their combined use resulted in elevated UA levels. After confirming elevated serum UA, the medicinal composition was administered at varying doses to verify its uric acid-lowering effect. As shown in Table 4, the experimental results show that after 14 days of continuous modeling, the model group mice had significantly elevated serum UA levels compared to the normal control group. However, after administration of the Chinese medicinal composition, serum UA levels in the treated mice decreased compared to the model group, demonstrating the Chinese medicinal composition's effective in vivo UA-lowering effect. If hyperuricemia is left untreated, it can recur, leading to a series of serious complications such as renal failure, hydronephrosis, and nephritis. Cr and BUN are key indicators of renal function, so their serum levels were measured. The experimental results showed that serum Cr and BUN levels in the model group mice were significantly elevated compared to the normal control group, while serum Cr and BUN levels in the Chinese medicinal composition groups decreased to varying degrees compared to the model group.

[0120] Table 4 UA, Cr and BUN levels in serum of mice in different groups

[0121]

[0122]

[0123] Note: Compared with the normal group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01

[0124] Uric acid levels in the body depend on the balance between liver synthesis and renal excretion. Uric acid production involves multiple enzymes, the most critical of which is xanthine oxidase (XOD), which catalyzes the conversion of purines to uric acid. As shown in Table 5, compared with the normal control group, the activity of XOD in the liver of mice in the model group was elevated; compared with the model group, the activity of XOD in the liver of mice in the treatment group was significantly decreased in a dose-dependent manner, indicating that the present composition can significantly reduce uric acid production in mice by inhibiting the activity of XOD in the liver.

[0125] Table 5 XOD activity in the liver of mice in different groups

[0126]

[0127] Note: Compared with the normal group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01

[0128] Uric acid can induce inflammation in vivo through both crystal-dependent and crystal-independent pathways, stimulating inflammasome activation, producing inflammatory factors such as IL-1β and TNF-α, and promoting T cells to produce more related pro-inflammatory factors. As shown in Table 6, serum IL-1β and TNF-α levels were significantly elevated in the model group compared with the normal group; however, the levels of IL-1β and TNF-α in the TCM composition group were significantly decreased compared with the model group, suggesting that the TCM composition has a certain anti-inflammatory effect.

[0129] Table 6 Levels of inflammatory factors TNF-α and IL-1β in the serum of mice in different groups

[0130]

[0131] Note: Compared with the normal group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01

[0132] In summary, this example shows that the traditional Chinese medicine composition of the present invention has a uric acid-lowering effect on hyperuricemia mice, which may be related to the composition inhibiting liver XOD, reducing uric acid synthesis and promoting uric acid excretion; at the same time, the composition can also reduce serum Cr, BUN, IL-1β, and TNF-α levels, and this effect may be related to the anti-inflammatory effect.

[0133] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine composition for treating hyperuricemia, characterized in that: The following steps are involved: Crush the Chinese medicine raw materials, pass them through a No. 4 sieve, and put them into the extraction kettle. Set the extraction temperature. When the set temperature is reached and the refrigeration temperature is below 5°C, open the plug and load the material. Adjust the valve to make the extraction pressure equal to the tank pressure. Turn on the CO2 pump, and the CO2 is compressed by the compressor and enters the extraction kettle. After the extraction pressure reaches the set value and stabilizes, add the entrainer for supercritical extraction. Collect the extracted liquid from the outlet every 30 minutes until the extracted liquid is completely collected. The extracted stock solution was concentrated under reduced pressure using a rotary evaporator until the volume no longer changed, and then freeze-dried to obtain a traditional Chinese medicine composition; The traditional Chinese medicine composition is composed of the following components and their contents in parts by weight: 53-54 parts of Atractylodes lancea, 107-108 parts of Honeysuckle Caulis, 53-54 parts of Earthworm, 80-81 parts of Forsythia suspensa, 53-54 parts of Phellodendron amurense, 107-108 parts of Coix seeds, 53-54 parts of Saposhnikovia divaricata, 80-81 parts of Cyathula capitata, 80-81 parts of Trichosanthes kirilowii, 107-108 parts of Morus alba, 80-81 parts of Stephania tetrandra, and 64-65 parts of Clematidis. The entrainer is 70% ethanol; The extraction temperature was 50 °C; The extraction pressure is 35 MPa; The extraction time was 120 min.

2. Use of the Chinese medicine composition prepared by the method for preparing the Chinese medicine composition for treating hyperuricemia as claimed in claim 1 in preparing a medicament for treating hyperuricemia.

3. A sustained-release tablet for treating hyperuricemia, characterized in that: It is composed of the following components and their weight proportions: The method for preparing a traditional Chinese medicine composition for treating hyperuricemia according to claim 1, wherein the traditional Chinese medicine composition comprises 49%, HPMC 30%, dextrin 5% and microcrystalline cellulose 16%; The HPMC is HPMCK15M or HPMCK100M.

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