A traditional Chinese medicine composition for treating hyperuricemia and its application

Abstract

This invention discloses a traditional Chinese medicine composition for treating hyperuricemia and its application, belonging to the field of traditional Chinese medicine technology. The preparation method of the traditional Chinese medicine composition includes: mixing Ganoderma lucidum spore powder and Vernicia veitchii fruit powder at a mass ratio of 10-90:90-10, then extracting the extract using supercritical CO2 fluid extraction technology to obtain the extract phase, followed by depressurization to precipitate the extract, and drying to obtain the traditional Chinese medicine composition powder. This invention provides a traditional Chinese medicine composition composed of Ganoderma lucidum spore oil and Vernicia veitchii oil. Cell experiments and animal experiments have demonstrated that the traditional Chinese medicine composition can significantly inhibit uric acid synthesis and reabsorption, thereby reducing serum uric acid levels and improving renal function damage caused by hyperuricemia. It can be used as a functional raw material for preparing products for treating hyperuricemia or improving renal damage caused by hyperuricemia; moreover, the raw material is readily available, low in cost, and highly safe, possessing significant development and application value.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a traditional Chinese medicine composition for treating hyperuricemia or improving kidney damage caused by hyperuricemia, and its application. Background Technology

[0002] Hyperuricemia (HUA) is a chronic metabolic disease characterized by abnormally elevated serum uric acid levels and is a fundamental factor leading to gout. Uric acid is the final product of purine metabolism in the body, and elevated uric acid levels may be related to diet, genetics, and kidney function. With improved living conditions, abnormal purine metabolism due to diet has increased, leading to a year-on-year increase in the incidence of HUA and a trend towards younger age of onset. Furthermore, long-term HUA exacerbates kidney metabolic function and easily causes kidney damage. Therefore, effectively controlling HUA is of great significance for improving the health of the population.

[0003] Based on the mechanism of action of HUA formation, a series of uric acid-lowering drugs have been developed clinically, such as drugs that inhibit uric acid production (allopurinol, febuxostat), drugs that inhibit uric acid reabsorption (benzbromarone and probenecid), adjuvant drugs for lowering uric acid (sodium bicarbonate), and drugs that relieve symptoms during acute gout attacks (colchicine and nonsteroidal anti-inflammatory drugs).

[0004] Although these drugs can effectively lower serum uric acid levels or relieve gout symptoms, they have limitations such as single target, liver and kidney toxicity due to long-term use, and the need for long-term or even lifelong medication. For example, allopurinol can cause gastrointestinal reactions and acute renal failure, febuxostat can cause abnormal liver function and increase the risk of cardiovascular events, benzbromarone and ricinard can increase the risk of stone formation, precatorium can easily induce acute gout attacks, and colchicine can cause liver and kidney damage and bone marrow transplantation, making the treatment of hyperuricemia (HUA) less than ideal.

[0005] Therefore, finding a drug with low side effects and proven efficacy for treating HUA remains a pressing public health issue that needs to be addressed in this field.

[0006] Traditional Chinese medicine (TCM) theory considers hyperuricemia to be a condition of deficiency in the root and excess in the branch, caused by spleen and kidney deficiency. TCM treatment of hyperuricemia is based on the etiology and pathogenesis, and alleviates the patient's symptoms and reduces organ damage through syndrome differentiation and treatment. Clinically, the main treatment methods are to tonify the liver and kidney, strengthen the spleen and stomach, remove dampness and blood stasis, clear heat and promote diuresis and eliminate turbidity, and activate blood circulation and remove blood stasis. For example, the decoction for removing dampness and eliminating turbidity is often used to promote the excretion of serum uric acid and reduce serum uric acid levels. TCM treatment of hyperuricemia has good efficacy, high safety and few adverse reactions. The holistic approach of syndrome differentiation and treatment can better guide clinical medication (Liu Xuan. Pathogenesis and research progress of TCM treatment of hyperuricemia and related diseases. Asia-Pacific Traditional Medicine, 2025, 21(3): 251-256.).

[0007] Ganoderma lucidum spore powder consists of tiny reproductive cells ejected from the cap folds during the growth and maturation of Ganoderma lucidum. Ganoderma lucidum spore oil, composed of triterpenoids, fatty acids, and sterols, is one of its active ingredients and has effects such as regulating immunity, anti-tumor, liver protection, anti-aging, and cardiovascular protection.

[0008] The Chinese privet (Vernicia fordii) is a woody oilseed tree species with a high oil content. Its fruits and seeds contain various beneficial chemical components, such as oleic acid, linoleic acid, vitamin E, flavonoids, and phytosterols, making it valuable for development in the food and healthcare fields. Modern pharmacological studies have shown that the Chinese privet possesses anti-inflammatory, antioxidant, lipid-lowering, cardiovascular-protective, and anti-obesity effects.

[0009] There is currently no substantial evidence to suggest that Ganoderma lucidum spore oil or tung oil has the function of lowering uric acid, nor have there been any reports of combining the two to prepare products that lower uric acid. Summary of the Invention

[0010] The purpose of this invention is to provide a traditional Chinese medicine composition for treating hyperuricemia with low side effects and definite efficacy, and to apply it to the development of related products for treating hyperuricemia and improving kidney damage caused by hyperuricemia.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a traditional Chinese medicine composition for treating hyperuricemia. The preparation method of the traditional Chinese medicine composition includes: mixing Ganoderma lucidum spore powder and tung oil fruit powder in a mass ratio of 10-90:90-10, extracting the extract using supercritical CO2 fluid extraction technology, precipitating the extract under reduced pressure, and drying the extract to obtain the traditional Chinese medicine composition powder.

[0012] This invention has found that a combination of Ganoderma lucidum spore powder and tung oil seeds, and then extracted using supercritical CO2 fluid extraction technology, can exert a synergistic effect in lowering uric acid through multiple targets and pathways, and alleviate kidney damage caused by hyperuricemia.

[0013] This invention validated the therapeutic effect of the traditional Chinese medicine composition on hyperuricemia in models of hyperuricemia induced by different factors. Mechanistic studies showed that the traditional Chinese medicine composition can lower uric acid levels through pathways such as inhibiting uric acid synthesis and inhibiting uric acid reabsorption.

[0014] The manifestations of hyperuricemia include a significant increase in at least one of the following indicators: serum uric acid, creatinine, and blood urea nitrogen concentrations.

[0015] The Ganoderma lucidum spore powder can be a commercially available broken-cell wall spore powder product.

[0016] The *Vernicia montana* fruit powder is a product obtained by pulverizing *Vernicia montana* fruit. The preparation method can be as follows: *Vernicia montana* fruit is dried, pulverized, and passed through a 60-80 mesh sieve to obtain *Vernicia montana* fruit powder. Alternatively, fresh *Vernicia montana* fruit can be used for extraction.

[0017] This invention combines Ganoderma lucidum spore powder with tung oil fruit powder and optimizes the ratio to enhance the uric acid-lowering activity of the extracted product.

[0018] Preferably, the Ganoderma lucidum spore powder and the fruit powder of the tung tree are mixed in a mass ratio of 20-50:50-80.

[0019] In a further preferred embodiment, the Ganoderma lucidum spore powder and the tung oil fruit powder are mixed at a mass ratio of 40:60.

[0020] This invention utilizes supercritical CO2 fluid extraction technology to extract an oil phase containing uric acid-lowering active substances from Ganoderma lucidum spore powder and Vernicia fordii fruit. Optimization of extraction conditions facilitates the extraction of active ingredients. Preferably, the extraction pressure is 25-35 MPa, the extraction temperature is 45-55℃, the amount of supercritical CO2 fluid used is 20-40 times the weight of the mixed powder, and the CO2 flow rate is 20 kg / h.

[0021] Specifically, Ganoderma lucidum spore powder and tung oil fruit powder are mixed and placed into an extraction vessel. The extraction vessel is sealed, and supercritical CO2 fluid, which accounts for 20-40 times the weight of the mixed powder, is pumped in at a pressure of 25-35 MPa. The CO2 flow rate is 20 kg / h, and the extraction temperature is 45-55℃.

[0022] Further optimization involves an extraction pressure of 25-30 MPa and an extraction temperature of 50°C; the amount of supercritical CO2 fluid used is 20-30 times the weight of the mixed powder.

[0023] After extraction, the extract phase is transferred to a separation vessel, and the pressure reducing valve is adjusted to precipitate the extract, yielding the eluent. Preferably, the eluent is extracted at a pressure of 4-10 MPa and at a temperature of 40-50°C.

[0024] Further optimization resulted in a desorption pressure of 6 MPa and a desorption temperature of 40-45℃.

[0025] After analysis, the herbal composition powder is obtained by drying. Preferably, the drying is performed by freeze drying for 48 hours.

[0026] This invention also provides the use of the aforementioned traditional Chinese medicine composition in the preparation of products for treating hyperuricemia or improving kidney damage caused by hyperuricemia. The products may be, but are not limited to, pharmaceuticals.

[0027] Furthermore, the medicine comprises an effective dose of the traditional Chinese medicine composition and a pharmaceutically acceptable carrier.

[0028] In this invention, the pharmaceutically acceptable carrier is any formulation or carrier medium capable of delivering an effective dose of the active substance of this invention, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject. The pharmaceutically acceptable carrier includes one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.

[0029] In this invention, the pharmaceutical preparation can be an oral formulation. Specific formulations can be, but are not limited to, capsules, microcapsules, oral liquids, pills, and drops.

[0030] The beneficial effects of this invention are as follows: (1) This invention provides a traditional Chinese medicine composition made of Ganoderma lucidum spore oil and tung oil. Cell experiments and animal experiments have shown that the traditional Chinese medicine composition can significantly inhibit uric acid synthesis and reabsorption, thereby reducing serum uric acid levels and improving renal function damage caused by hyperuricemia. It can be used as a functional raw material for preparing products for treating hyperuricemia or improving renal damage caused by hyperuricemia, and has great development and application value.

[0031] (2) The traditional Chinese medicine composition provided by this invention can exert a synergistic effect of lowering uric acid and alleviating kidney damage caused by hyperuricemia through the effects of multiple components, multiple targets and multiple pathways, which is in line with the characteristics of traditional Chinese medicine that treats both the symptoms and the root cause. The raw materials used, Ganoderma lucidum spore powder and tung oil fruit, are both traditional Chinese medicines with both medicinal and edible value. They have the advantages of easy availability of raw materials, low cost and high safety, and are suitable for industrial production. Attached Figure Description

[0032] Figure 1 Effects of Ganoderma lucidum spore oil, tung oil and traditional Chinese medicine composition 1-9 on HK-2 cell viability ( n = 6).

[0033] Figure 2 Effects of Ganoderma lucidum spore oil, tung oil and traditional Chinese medicine composition 1-9 on uric acid synthesis in HK-2 cells ( n =6), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, # indicates P <0.05, ## indicates P <0.01, ### indicates P <0.001.

[0034] Figure 3 The effect of traditional Chinese medicine compositions 6, 10-20 on HK-2 cell viability (n = 6).

[0035] Figure 4 The effects of traditional Chinese medicine compositions 6, 10-20 on uric acid synthesis in HK-2 cells ( n = 6), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, # indicates P <0.05, ## indicates P <0.01, ### indicates P <0.001.

[0036] Figure 5 Effects of traditional Chinese medicine compositions 6, 10-20 on HKC cell viability ( n = 6).

[0037] Figure 6 The effects of traditional Chinese medicine compositions 6, 10-20 on URAT1 gene expression in HKC cells ( n = 6), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, ## indicates P <0.01, ### indicates P <0.001.

[0038] Figure 7 The effects of traditional Chinese medicine compositions 6, 10-20 on GLUT9 gene expression in HKC cells ( n = 6), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, ## indicates P <0.01, ### indicates P <0.001.

[0039] Figure 8 The effects of traditional Chinese medicine compositions 6, 10, 11, 13, and 17 on serum uric acid concentration in rats with high-fructose diet-induced hyperuricemia ( n = 8), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, ### indicates P <0.001.

[0040] Figure 9 The effects of traditional Chinese medicine compositions 6, 10, 11, 13, and 17 on serum creatinine concentration in rats with high-fructose diet-induced hyperuricemia (n = 8), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, ## indicates P <0.01, ### indicates P <0.001.

[0041] Figure 10 The effects of traditional Chinese medicine compositions 6, 10, 11, 13, and 17 on serum urea nitrogen concentration in rats with high-fructose diet-induced hyperuricemia ( n = 8), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, # indicates P <0.05, ## indicates P <0.01, ### indicates P <0.001.

[0042] Figure 11 The effects of traditional Chinese medicine compositions 6, 10, 11, 13, and 17 on serum uric acid concentration in mice with potassium oxonate-induced acute hyperuricemia ( n = 8), in the figure * indicates a significant difference compared to group Con, *** indicates P <0.001; # indicates a significant difference compared to the Model group, ### indicates P <0.001. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0045] The Ganoderma lucidum spore powder used in the following examples is a commercially available broken-cell wall Ganoderma lucidum spore powder product, purchased from Zhejiang Wuyangtang Pharmaceutical Co., Ltd. The raw material for *Vernicia fordii* (mountain pine) is dried *Vernicia fordii* fruit, provided by the Subtropical Forestry Research Institute of the Chinese Academy of Forestry.

[0046] The extraction equipment used in the following examples is a supercritical extraction device (Jiangsu Huaan Scientific Instruments Co., Ltd., model: HA120-50-01).

[0047] Example 1 This embodiment utilizes supercritical CO2 fluid extraction technology to extract Ganoderma lucidum spore oil from Ganoderma lucidum spore powder. The method steps are as follows: (1) Take 100 portions of Ganoderma lucidum spore powder and put them into an extraction vessel. Seal the extraction vessel and pump in 20 times the amount of supercritical CO2 fluid at 30 MPa for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the Ganoderma lucidum spore powder extract phase. (2) The extract of Ganoderma lucidum spore powder obtained in step (1) is transferred to a separation vessel, the pressure is reduced to 6 MPa and the solution is analyzed at 45°C to obtain the extract of Ganoderma lucidum spore powder. (3) Take the Ganoderma lucidum spore powder solution obtained in step (2) and freeze-dry it for 48 h to obtain Ganoderma lucidum spore oil.

[0048] Example 2 This embodiment utilizes supercritical CO2 fluid extraction technology to extract tung oil from tung oil powder. The method steps are as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take 100 parts of tung oil powder and put it into an extraction vessel. Seal the extraction vessel and pump 20 times the amount of supercritical CO2 fluid at 30 MPa for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the tung oil extract phase. (3) The extract phase of the tung oil obtained in step (2) is transferred to a separation vessel, the pressure is reduced to 6 MPa and the solution is analyzed at 45°C to obtain the tung oil solution; (4) Take the elution solution of the tung oil obtained in step (3) and freeze-dry it for 48 h to obtain tung oil.

[0049] Example 3 In this embodiment, Ganoderma lucidum spore powder and tung oil powder are mixed in different mass ratios, and then a traditional Chinese medicine composition of Ganoderma lucidum spore oil and tung oil is prepared by supercritical CO2 fluid extraction technology. The specific method is as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take Ganoderma lucidum spore powder and mix it with tung oil powder in mass ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90 respectively to obtain traditional Chinese medicine composition powder 1-9; (3) Take the Chinese herbal medicine composition powder 1-9 obtained in step (2) and put them into the extraction vessel. Seal the extraction vessel and pump 20 times the amount of supercritical CO2 fluid at 30MPa for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the extract phase 1-9. (4) The extract phases 1-9 obtained in step (3) are transferred to the separation vessel, the pressure is reduced to 6 MPa and the solution is analyzed at 45°C to obtain the eluent 1-9 respectively; (5) Take the elution solution 1-9 obtained in step (4) and freeze-dry it for 48 h to obtain the traditional Chinese medicine composition 1-9.

[0050] Example 4 In this embodiment, Ganoderma lucidum spore powder and tung oil powder were mixed at a mass ratio of 40:60, and then extracted using supercritical CO2 fluid extraction technology under different pressure conditions to prepare a traditional Chinese medicine composition composed of Ganoderma lucidum spore oil and tung oil. The specific method is as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take 3 parts of Ganoderma lucidum spore powder and mix them with tung oil powder at a mass ratio of 40:60 to obtain 10-12 parts of traditional Chinese medicine composition powder; (3) Take 10-12 of the Chinese herbal medicine composition powder and put them into the extraction vessel. Seal the extraction vessel and pump 30 times the amount of supercritical CO2 fluid at 25 MPa, 30 MPa and 35 MPa respectively for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the extract phase 10-12. (4) The extract phase 10-12 obtained in step (3) is transferred to the separation vessel, the pressure is reduced to 6 MPa and the solution is analyzed at 40°C to obtain the eluent 10-12; (5) Take the 10-12 solution obtained in step (4) and freeze-dry it for 48 h to obtain the traditional Chinese medicine composition 10-12.

[0051] Example 5 In this embodiment, Ganoderma lucidum spore powder and tung oil powder were mixed at a mass ratio of 40:60, and then a traditional Chinese medicine composition of Ganoderma lucidum spore oil and tung oil was prepared by supercritical CO2 fluid extraction under different fluid volume conditions. The specific method is as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take 2 portions of Ganoderma lucidum spore powder and mix them with tung oil powder at a mass ratio of 40:60 to obtain 13-14 portions of traditional Chinese medicine composition powder; (3) Take the powder of the Chinese herbal medicine composition 13-14 and put it into the extraction vessel. Seal the extraction vessel and pump 20 times and 40 times the amount of supercritical CO2 fluid into it at 30 MPa respectively for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the extract phase 13-14. (4) The extract phases 13-14 obtained in step (3) are transferred to the separation vessel, the pressure is reduced to 6 MPa and the solution is analyzed at 40°C to obtain the eluent 13-14; (5) Take the elution solution 13-14 obtained in step (4) and freeze-dry it for 48 hours to obtain the traditional Chinese medicine composition 13-14.

[0052] Example 6 In this embodiment, Ganoderma lucidum spore powder and tung oil powder were mixed at a mass ratio of 40:60, and then a traditional Chinese medicine composition of Ganoderma lucidum spore oil and tung oil was prepared by supercritical CO2 fluid extraction under different extraction temperature conditions. The specific method is as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take 2 portions of Ganoderma lucidum spore powder and mix them with tung oil powder at a mass ratio of 40:60 to obtain 15-16 portions of traditional Chinese medicine composition powder; (3) Take 15-16 of the Chinese herbal medicine composition powder and put them into the extraction vessel. Seal the extraction vessel and pump 20 times the amount of supercritical CO2 fluid at 30 MPa for extraction. The CO2 flow rate is 20 kg / h and the extraction temperatures are 45℃ and 55℃ respectively to obtain the extract phase 15-16. (4) Transfer the extract phases 15-16 obtained in step (3) to the separation vessel, reduce the pressure to 6 MPa and perform analysis at 40°C to obtain the eluent 15-16; (5) Take the elution solution 15-16 obtained in step (4) and freeze-dry it for 48 hours to obtain the traditional Chinese medicine composition 15-16.

[0053] Example 7 In this embodiment, Ganoderma lucidum spore powder and tung oil powder were mixed at a mass ratio of 40:60, and then extracted using supercritical CO2 fluid extraction technology under different desorption pressure conditions to prepare a traditional Chinese medicine composition composed of Ganoderma lucidum spore oil and tung oil. The specific method is as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take 3 parts of Ganoderma lucidum spore powder and mix them with tung oil powder at a mass ratio of 40:60 to obtain 17-19 parts of traditional Chinese medicine composition powder; (3) Take the powder of the Chinese herbal medicine composition 17-19 and put it into the extraction vessel. Seal the extraction vessel and pump 20 times the amount of supercritical CO2 fluid at 30 MPa for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the extract phase 17-19. (4) The extract phases 17-19 obtained in step (3) are transferred to the separation vessel, and the pressure is reduced to 4, 8 and 10 MPa respectively and the solution is analyzed at 40°C to obtain the eluent 17-19. (5) Take the elution solution 17-19 obtained in step (4) and freeze-dry it for 48 hours to obtain the traditional Chinese medicine composition 17-19.

[0054] Example 8 In this embodiment, Ganoderma lucidum spore powder and tung oil powder were mixed at a mass ratio of 40:60, and then a traditional Chinese medicine composition of Ganoderma lucidum spore oil and tung oil was prepared by supercritical CO2 fluid extraction under different desorption temperature conditions. The specific method is as follows: (1) Take the raw material of *Vernicia fordii*, crush it, and pass it through a 60-mesh sieve to obtain *Vernicia fordii* powder; (2) Take Ganoderma lucidum spore powder and mix it with tung oil powder at a mass ratio of 40:60 to obtain 20g of traditional Chinese medicine composition powder; (3) Take 20 of the Chinese herbal medicine composition powder and put it into the extraction vessel. Seal the extraction vessel and pump 20 times the amount of supercritical CO2 fluid at 30 MPa for extraction. The CO2 flow rate is 20 kg / h and the extraction temperature is 50℃ to obtain the extract phase 20. (4) Transfer the extract phase 20 obtained in step (3) to the separation vessel, reduce the pressure to 6 MPa and perform analysis at 50°C to obtain the eluent 20; (5) Take the elution solution 20 obtained in step (4) and freeze-dry it for 48 h to obtain the traditional Chinese medicine composition 20.

[0055] Test Example 1: Inhibitory effect of Ganoderma lucidum spore oil, tung oil and traditional Chinese medicine combination on uric acid synthesis 1. Experimental Materials Cell line: Human renal tubular epithelial cells (HK-2) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Adherent cells were grown in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity, with medium changes and passages every 2-3 days.

[0056] Experimental samples: Ganoderma lucidum spore oil prepared in Example 1, tung oil prepared in Example 2, and traditional Chinese medicine compositions 1-9 prepared in Example 3 were dissolved in anhydrous ethanol to prepare a 50 mg / mL stock solution. After filtration and sterilization, the solution was stored at -20°C. Before use, the solution was diluted to the experimental concentration with DMEM culture medium.

[0057] 2. Experimental Methods (1) Effects of traditional Chinese medicine composition on HK-2 cell viability HK-2 cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / mL using DMEM. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates (excluding edge wells). 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to each edge well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0058] When the cells reached 50-60% confluence, they were divided into a control group, a Ganoderma lucidum spore oil group, a Vernicia galanga oil group, and groups of traditional Chinese medicine compositions 1-9. The control group received 200 μL of DMEM medium, while the Ganoderma lucidum spore oil group, Vernicia galanga oil group, and groups of traditional Chinese medicine compositions 1-9 received 200 μL of a 500 μg / mL sample solution. After culturing for another 24 h, the original medium was removed, and 90 μL of fresh DMEM medium and 10 μL of CCK-8 solution were added to each well. Simultaneously, 90 μL of fresh DMEM medium and 10 μL of CCK-8 solution were added to cell-free wells as a background control. The cells were incubated at 37°C in the dark for 2 h, and the absorbance (OD) at 450 nm was measured using a multi-mode microplate reader. The relative cell viability was calculated based on the OD values ​​of each well using the following formula: Relative cell viability (%) = [(OD)] 实验组 -OD 背景组 ) / (OD 对照组 -OD 背景组 )]×100%.

[0059] (2) Effects of traditional Chinese medicine composition on uric acid synthesis in HK-2 cells HK-2 cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / mL using DMEM. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates (excluding edge wells). 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to each edge well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0060] When the cells reached 50-60% confluence, they were divided into a control group, a model group, a Ganoderma lucidum spore oil group, a Vernicia galanga oil group, and traditional Chinese medicine composition groups 1-9. The control group received 200 μL of DMEM medium, the model group received 200 μL of DMEM medium containing xanthine (200 μM), and the Ganoderma lucidum spore oil group, Vernicia galanga oil group, and traditional Chinese medicine composition groups 1-12 received 200 μL of a sample solution (500 μg / mL) containing xanthine (200 μM). After culturing for another 24 h, the cell culture medium was harvested, and the concentration of uric acid in the culture medium was measured using a uric acid detection kit.

[0061] 3. Experimental Results The effects of each experimental sample on HK-2 cell viability are shown in the figure. Figure 1 Compared with the control group, Ganoderma lucidum spore oil, tung oil and traditional Chinese medicine composition 1-9 had no significant effect on the viability of HK-2 cells at 500 μg / mL.

[0062] The effects of each experimental sample on uric acid synthesis in HK-2 cells are shown in the figure. Figure 2 Compared with the control group, the uric acid concentration in the cell culture medium of the model group was significantly increased. Compared with the model group, all samples significantly reduced the uric acid concentration in the cell culture medium, with the traditional Chinese medicine composition 6 showing the best effect, indicating that the inhibitory effects of Ganoderma lucidum spore oil and tung oil on uric acid synthesis have a synergistic effect.

[0063] Test Example 2: Synergistic inhibitory effect of Ganoderma lucidum spore oil and tung oil on uric acid synthesis 1. Experimental Materials Cell line: Human renal tubular epithelial cells (HK-2) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Adherent cells were grown in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity, with medium changes and passages every 2-3 days.

[0064] Experimental samples: Ganoderma lucidum spore oil prepared in Example 1, tung oil prepared in Example 2, and traditional Chinese medicine composition 6 prepared in Example 3 were dissolved in anhydrous ethanol to prepare a 50 mg / mL stock solution. After filtration and sterilization, the solution was stored at -20℃. Before use, the solution was diluted with DMEM culture medium to the experimental concentration.

[0065] 2. Experimental Methods (1) Effects of traditional Chinese medicine composition on uric acid synthesis in HK-2 cells HK-2 cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / mL using DMEM. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates (excluding edge wells). 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to each edge well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0066] When the cells reached 50-60% confluence, they were divided into six groups: control group, model group, Ganoderma lucidum spore oil group, Vernicia galanga oil group, and traditional Chinese medicine composition group. The control group received 200 μL of DMEM medium, the model group received 200 μL of DMEM medium containing 200 μM xanthine, and the Ganoderma lucidum spore oil group, Vernicia galanga oil group, and traditional Chinese medicine composition group received 200 μL of sample solution containing 200 μM xanthine (100, 200, 300, 400, and 500 μg / mL, respectively). After culturing for another 24 h, the cell culture medium was harvested, and the concentration of uric acid in the medium was measured using a uric acid detection kit. The uric acid synthesis inhibition rate was calculated, and the synergistic coefficient of the synergistic effect of Ganoderma lucidum spore oil and Vernicia galanga oil in lowering uric acid was calculated using the Chou-Talalay combination index (CI) method. The calculation formula is as follows: Coefficient of Cooperation (CI) = [D1 / (Dx)1] + [D2 / (Dx)2]; Wherein, D1 and D2 refer to the concentrations of Ganoderma lucidum spore oil and Vernicia veitchii oil, respectively, when the inhibition rate of uric acid synthesis by the traditional Chinese medicine composition 6 reaches a certain level; (Dx)1 and (Dx)2 refer to the concentrations required for Ganoderma lucidum spore oil and Vernicia veitchii oil, when used alone, to achieve the same inhibition rate of uric acid synthesis. Generally, a CI < 1 is considered to indicate a synergistic effect, and the smaller the CI, the stronger the synergistic effect.

[0067] 3. Experimental Results The inhibition rates of each experimental sample on uric acid synthesis in HK-2 cells are shown in Table 1.

[0068] Table 1 Synergy Factor (CI): Since Ganoderma lucidum spore oil inhibits uric acid synthesis by 22.52% at its highest concentration, the synergy factor is calculated based on a 20% inhibition rate. Using GraphPad Prism (version 8.2.1), the concentrations of Ganoderma lucidum spore oil, *Vernicia veitchii* oil, and the traditional Chinese medicine combination 6 at a 20% inhibition rate for uric acid synthesis were calculated to be 473.61, 338.54, and 135.81 μg / mL, respectively. Based on the synergy factor (CI) calculation formula, the CI is 0.35. That is, when the uric acid synthesis inhibition rate is 20%, the synergy factor between Ganoderma lucidum spore oil and *Vernicia veitchii* oil is 0.35.

[0069] Test Example 3: Inhibitory Effect of Traditional Chinese Medicine Composition on Uric Acid Synthesis 1. Experimental Materials Cell line: Human renal tubular epithelial cells (HK-2) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Adherent cells were grown in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity, with medium changes and passages every 2-3 days.

[0070] Experimental samples: Traditional Chinese medicine composition 6 prepared in Example 3 and traditional Chinese medicine compositions 10-20 prepared in Examples 4-8 were dissolved in anhydrous ethanol to prepare a stock solution of 50 mg / mL. After filtration and sterilization, the solution was stored at -20℃. Before use, the solution was diluted with DMEM culture medium to the experimental concentration.

[0071] 2. Experimental Methods (1) Effects of traditional Chinese medicine composition on HK-2 cell viability HK-2 cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / mL using DMEM. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates (excluding edge wells). 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to each edge well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0072] When the cells reached 50-60% confluence, they were divided into a control group and three groups (groups 6 and 10-20) containing the traditional Chinese medicine composition. The control group received 200 μL of DMEM medium, while groups 6 and 10-20 received 200 μL of a 500 μg / mL sample solution. After culturing for 24 h, the original medium was removed, and 90 μL of fresh DMEM medium and 10 μL of CCK-8 solution were added to each well. Simultaneously, 90 μL of fresh DMEM medium and 10 μL of CCK-8 solution were added to cell-free wells as a background control. The cells were incubated at 37°C in the dark for 2 h, and the absorbance (OD) at 450 nm was measured using a multi-mode microplate reader. The relative cell viability was calculated based on the OD values ​​of each well using the following formula: Relative cell viability (%) = [(OD)] 实验组 -OD 背景组 ) / (OD 对照组 -OD 背景组 )]×100%.

[0073] (2) Effects of traditional Chinese medicine composition on uric acid synthesis in HK-2 cells HK-2 cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / mL using DMEM. 4Cells were seeded at a density of 100 μL / mL into 96-well plates (excluding edge wells). 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to each edge well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0074] When the cells reached 50-60% confluence, they were divided into a control group, a model group, and groups containing traditional Chinese medicine composition 6 and 10-20. The control group received 200 μL of DMEM medium, the model group received 200 μL of DMEM medium containing xanthine (200 μM), and groups containing traditional Chinese medicine composition 6 and 10-20 received 200 μL of a sample solution containing xanthine (200 μM) (500 μg / mL). After culturing for another 24 h, the cell culture medium was harvested, and the concentration of uric acid in the medium was measured using a uric acid detection kit.

[0075] 3. Experimental Results The effects of each experimental sample on HK-2 cell viability are shown in the figure. Figure 3 Compared with the control group, herbal composition 6 and herbal composition 10-20 had no significant effect on the viability of HK-2 cells at 500 μg / mL.

[0076] The effects of each experimental sample on uric acid synthesis in HK-2 cells are shown in the figure. Figure 4 Compared with the control group, the uric acid concentration in the cell culture medium of the model group was significantly increased. Compared with the model group, all samples significantly reduced the uric acid concentration in the cell culture medium, with the traditional Chinese medicine composition 6 showing the best effect.

[0077] Test Example 4: Inhibitory effect of traditional Chinese medicine composition on uric acid reabsorption 1. Experimental Materials Cell line: Human renal cortical proximal tubule epithelial cells (HKC) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Adherent cells were grown in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity, with medium changes and passages every 2-3 days.

[0078] Experimental samples: Traditional Chinese medicine composition 6 prepared in Example 3 and traditional Chinese medicine compositions 10-20 prepared in Examples 4-8 were dissolved in anhydrous ethanol to prepare a stock solution of 50 mg / mL. After filtration and sterilization, the solution was stored at -20℃. Before use, the solution was diluted with DMEM culture medium to the experimental concentration.

[0079] 2. Experimental Methods (1) Effects of traditional Chinese medicine composition on HKC cell viability HKC cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / mL using DMEM.4 Cells were seeded at a density of 100 μL / mL into 96-well plates (excluding edge wells). 100 μL of cell suspension was added to each well, and 100 μL of PBS was added to each edge well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0080] When the cells reached 50-60% confluence, they were divided into a control group, three groups containing the traditional Chinese medicine (TCM) composition (group 6), and three groups containing TCM composition (groups 10-20). The control group received 200 μL of DMEM medium, while the TCM composition groups (groups 6 and 10-20) received 200 μL of a 500 μg / mL sample solution. After culturing for 24 h, the original medium was removed, and 90 μL of fresh DMEM medium and 10 μL of CCK-8 solution were added to each well. Simultaneously, 90 μL of fresh DMEM medium and 10 μL of CCK-8 solution were added to cell-free wells as a background control. The cells were incubated at 37°C in the dark for 2 h, and the absorbance (OD) at 450 nm was measured using a multi-mode microplate reader. The relative cell viability was calculated based on the OD values ​​of each well using the following formula: Relative cell viability (%) = [(OD)] 实验组 -OD 背景组 ) / (OD 对照组 -OD 背景组 )]×100%.

[0081] (2) Effects of traditional Chinese medicine composition on the expression of URAT1 and GLUT9 genes in HKC cells HKC cells in logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 1×10⁻⁶ cells using DMEM. 5 Cells were seeded at a density of 1 cell / mL into 6-well plates, with 2 mL of cell suspension added to each well. The plates were then incubated in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0082] When the cells reached 50-60% confluence, they were divided into a control group, a model group, six groups of traditional Chinese medicine (TCM) compositions, and ten-twenty groups of TCM compositions. The control group received 2 mL of DMEM medium, the model group received 2 mL of DMEM medium containing 400 μM sodium urate, and the six and ten-twenty groups received 2 mL of sample solution (500 μg / mL) containing 400 μM sodium urate. After culturing for another 24 h, the original medium was aspirated, the cells were washed with PBS, and total RNA was extracted. The expression levels of URAT1 and GLUT9 genes were detected by qRT-PCR.

[0083] URAT1 and GLUT9 are two core urate transporters expressed on the proximal tubular epithelial cells of the kidney, and they work together to reabsorb urate. The primer sequences for URAT1, GLUT9, and the internal reference gene β-Actin are shown in Table 2.

[0084] Table 2. Primer sequences for qRT-PCR experiments 3. Experimental Results The effects of each experimental sample on HKC cell viability are shown in the figure. Figure 5 Compared with the control group, herbal composition 6 and herbal composition 10-20 had no significant effect on the viability of HKC cells at 500 μg / mL. The effects of each experimental sample on uric acid synthesis in HKC cells are shown in the figure. Figure 6 and Figure 7 Compared with the control group, the expression levels of URAT1 and GLUT9 genes in the model group cells were significantly increased. Compared with the model group, traditional Chinese medicine compositions 6, 10, 11, 12, 13, 14 and 17 significantly reduced the expression level of URAT1 gene in cells, and traditional Chinese medicine compositions 6, 10, 11, 13 and 17 significantly reduced the expression level of GLUT9 gene in cells, with traditional Chinese medicine composition 6 showing the best effect.

[0085] Test Example 5: Therapeutic effect of traditional Chinese medicine composition on hyperuricemia induced by high fructose diet 1. Experimental Materials Experimental animals: 56 clean-grade healthy male SD rats, weighing 200 ± 20 g, were provided by the Experimental Animal Center of Zhejiang Academy of Medical Sciences and housed at the Experimental Animal Center of Zhejiang Agriculture and Forestry University.

[0086] Experimental samples: Traditional Chinese medicine composition 6 prepared in Example 3 and traditional Chinese medicine compositions 10, 11, 13 and 17 prepared in Examples 4-8.

[0087] 2. Experimental Methods Establishment of a rat model of hyperuricemia: Rats were allowed to drink 10% fructose water freely for 8 consecutive weeks to induce a rat model of hyperuricemia.

[0088] Grouping and Treatment of Rats: SD rats were randomly divided into 7 groups: normal control group (Con), model group (Model), and traditional Chinese medicine composition group, with 8 rats in each group. Rats in the Model and Traditional Chinese Medicine composition groups had free access to 10% fructose water for 8 consecutive weeks. Rats in the Traditional Chinese Medicine composition group were administered the Traditional Chinese Medicine composition (200 mg / kg / day) by gavage from weeks 5 to 8. Rats in the Con group were administered an equal volume of physiological saline by gavage. Before the end of the experiment, rats were fasted but allowed free access to water for 12 hours. After anesthesia, blood was collected from the abdominal aorta, and serum was separated by centrifugation. Serum uric acid, creatinine, and blood urea nitrogen concentrations were measured. Serum creatinine and blood urea nitrogen levels were used as indicators of kidney injury.

[0089] 3. Experimental Results The effects of each experimental sample on serum uric acid concentration in rats with high-fructose diet-induced hyperuricemia are shown in the figure. Figure 8 Compared with the control group (serum uric acid concentration of 33.41 μM), the serum uric acid concentration in the model group rats increased to 186.78 μM. Compared with the model group, traditional Chinese medicine compositions 6, 10, 13, and 17 significantly reduced the serum uric acid concentration in rats. Among them, traditional Chinese medicine composition 6 had the best effect, reducing the serum uric acid concentration in rats to 45.37 μM.

[0090] The effects of each experimental sample on serum creatinine concentration in rats with high-fructose diet-induced hyperuricemia are shown in the figure. Figure 9 Compared with the control group (serum uric acid concentration of 22.89 μM), the serum creatinine concentration in the model group rats increased to 77.12 μM. Compared with the model group, traditional Chinese medicine compositions 6, 10, 13, and 17 significantly reduced the serum creatinine concentration in rats. Among them, traditional Chinese medicine composition 6 had the best effect, reducing the serum creatinine concentration in rats to 34.85 μM.

[0091] The effects of each experimental sample on serum urea nitrogen concentration in rats with high-fructose diet-induced hyperuricemia are shown in the figure. Figure 10 Compared with the control group (serum urea nitrogen concentration of 4.46 μM), the serum urea nitrogen concentration in the model group rats increased to 16.85 μM. Compared with the model group, traditional Chinese medicine compositions 6, 10, 13, and 17 significantly reduced the serum urea nitrogen concentration in rats. Among them, traditional Chinese medicine composition 6 had the best effect, reducing the serum urea nitrogen concentration in rats to 7.33 μM.

[0092] Test Example 6: Therapeutic Effect of Traditional Chinese Medicine Composition on Acute Hyperuricemia 1. Experimental Materials Experimental animals: 56 clean-grade healthy male ICR mice, weighing 20 ± 2 g, were provided by the Experimental Animal Center of Zhejiang Academy of Medical Sciences and housed at the Experimental Animal Center of Zhejiang A&F University.

[0093] Experimental samples: Traditional Chinese medicine composition 6 prepared in Example 3 and traditional Chinese medicine compositions 10, 11, 13 and 17 prepared in Examples 4-8.

[0094] 2. Experimental Methods Establishment of an acute hyperuricemia mouse model: Mice were injected intraperitoneally with potassium oxonate (PO, 250 mg / kg / day) for 7 consecutive days to induce an acute hyperuricemia mouse model.

[0095] Grouping and Treatment: ICR mice were randomly divided into 7 groups: a normal control group (Con), a model group (Model), and a traditional Chinese medicine (TCM) composition group, with 8 mice in each group. Mice in the TCM composition group were administered the TCM composition (200 mg / kg / day) by gavage for 4 consecutive weeks. In the third week of the experiment, 1 hour after gavage administration of the TCM composition, mice in the model and TCM composition groups were intraperitoneally injected with potassium oxonate (PO, 250 mg / kg / day) for 7 consecutive days. Mice in the Con group were administered an equal volume of physiological saline by gavage. Before the end of the experiment, mice were fasted but allowed free access to water for 12 hours. After anesthesia, blood was collected from the heart, and serum was separated by centrifugation to determine the concentration of serum uric acid.

[0096] 3. Experimental Results The effects of each experimental sample on serum uric acid concentration in mice with acute hyperuricemia are shown in the figure. Figure 11 Compared with the control group (serum uric acid concentration of 53.64 μM), the serum uric acid concentration of mice in the model group increased to 526.82 μM. Compared with the model group, traditional Chinese medicine compositions 6, 10, and 17 significantly reduced the serum uric acid concentration in mice. Among them, traditional Chinese medicine composition 6 had the best effect, reducing the serum uric acid concentration in mice to 61.33 μM.

[0097] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for treating hyperuricemia, characterized in that, The preparation method of the traditional Chinese medicine composition includes: mixing Ganoderma lucidum spore powder and tung tree fruit powder in a mass ratio of 20-50:50-80, then extracting the extract using supercritical CO2 fluid extraction technology to obtain the extract phase, then depressurizing the extract to precipitate the extract, and drying it to obtain the traditional Chinese medicine composition powder. The extraction pressure is 25-35 MPa, the amount of supercritical CO2 fluid used is 20-40 times the weight of the mixed powder, the CO2 flow rate is 20 kg / h, and the extraction temperature is 45-55℃. The analysis pressure is 4-10 MPa, and the analysis temperature is 40-50℃.

2. The traditional Chinese medicine composition of claim 1, wherein, The Ganoderma lucidum spore powder and the tung oil fruit powder were mixed in a mass ratio of 40:

60.

3. The traditional Chinese medicine composition of claim 1, wherein, The extraction pressure is 25-30 MPa, the amount of supercritical CO2 fluid used is 20-30 times the weight of the mixed powder, and the extraction temperature is 50℃.

4. The traditional Chinese medicine composition of claim 1, wherein, The desorption pressure is 6 MPa, and the desorption temperature is 40-45℃.

5. The traditional Chinese medicine composition of claim 1, wherein, The drying process employs freeze drying.

6. The use of the traditional Chinese medicine composition according to any one of claims 1-5 in the preparation of a medicament for treating hyperuricemia.

7. Use according to claim 6, wherein The drug comprises an effective dose of the traditional Chinese medicine composition as described in any one of claims 1-5 and a pharmaceutically acceptable carrier.

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