Pharmaceutical composition for reducing uric acid and preparation method thereof

Abstract

The invention relates to a pharmaceutical composition for reducing uric acid and a preparation method thereof, and belongs to the technical field of biology, the pharmaceutical composition contains earthworm peptide, a bacterium fermentation product, a fenugreek seed extract, a chicory extract and a plantain herb extract; the earthworm peptide contains a product obtained by sequentially carrying out graded enzymolysis on earthworms by virtue of trypsin, lumbrukinase and nattokinase; the bacterial fermentation product contains bacillus subtilis and bifidobacterium adolescentis which are used for jointly fermenting the cornus officinalis pulp and the rhizoma smilacis glabrae to obtain a product; the fenugreek seed extract contains a product obtained by sequentially performing graded enzymolysis on fenugreek seed meat through fig protease, trypsin and papain; the chicory extract is an ethanol percolation extract of chicory roots; the plantain herb extract is an ethanol percolation extract of plantain herb. The components are matched for use, so that good uric acid reducing and anti-inflammatory effects are achieved, inflammation can be quickly and efficiently relieved, the blood uric acid content is reduced, and safety and reliability are achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a uric acid-lowering pharmaceutical composition and a preparation method thereof. Background Art

[0002] Uric acid is the end product of purine metabolism in the human body. Under normal circumstances, the production and excretion of uric acid are in a balanced state, maintaining the stability of uric acid levels in the body. However, when purine metabolism is disordered or uric acid excretion is reduced, blood uric acid levels will increase. Long-term hyperuricemia may lead to a variety of serious health problems such as gout, uric acid nephropathy and cardiovascular disease.

[0003] With the improvement of living standards and changes in dietary structure, the prevalence of hyperuricemia and gout has been increasing year by year worldwide, which makes the research and development and application of uric acid-lowering drugs increasingly important. Traditional uric acid-lowering drugs mainly include drugs that inhibit uric acid production and drugs that promote uric acid excretion. Drugs that inhibit uric acid production, such as allopurinol, reduce the synthesis of uric acid by inhibiting the activity of xanthine oxidase; however, some patients will experience severe allergic reactions, including exfoliative dermatitis, which limits their use to a certain extent. Drugs that promote uric acid excretion, such as benzbromarone, can inhibit the reabsorption of uric acid by the renal tubules, thereby increasing the excretion of uric acid; however, such drugs may cause adverse reactions such as gastrointestinal discomfort, liver and kidney damage, and uric acid kidney stone formation, especially in patients with renal insufficiency, and need to be carefully evaluated when used. In addition, there are some traditional Chinese medicine therapies, but there are problems with long cycles and no obvious short-term effects.

[0004] At present, the research and development of uric acid-lowering drugs still faces many challenges. It is necessary to continuously optimize the composition of the drugs, improve efficacy and safety, reduce the occurrence of adverse reactions, and shorten the course of treatment in order to improve the quality of life of patients with hyperuricemia and gout and reduce the risk of related complications. Summary of the invention

[0005] In view of the problems of poor safety, easy side effects, slow effect and long course of treatment of existing uric acid lowering drugs, the present invention provides a uric acid lowering pharmaceutical composition and its preparation method, the pharmaceutical composition contains earthworm peptides obtained by specific enzymatic hydrolysis, bacterial fermentation prepared by specific substrate and bacterial fermentation, fenugreek seed extract obtained by specific enzymatic hydrolysis, chicory extract extracted by ethanol percolation, and plantain extract extracted by ethanol percolation. The combination of the components has good uric acid lowering and anti-inflammatory effects, can quickly and efficiently relieve inflammation and reduce blood uric acid content, and is safe and reliable. Its specific technical scheme is as follows: A pharmaceutical composition for lowering uric acid comprises the following raw materials in parts by mass: 1-3 parts of earthworm peptide, 1-3 parts of bacterial fermentation product, 0.2-0.5 parts of fenugreek seed extract, 0.5-1.5 parts of chicory extract, and 1-3 parts of plantain extract; the earthworm peptide comprises a product obtained by sequentially graded enzymolysis of earthworms with trypsin, lumbrokinase, and nattokinase; the bacterial fermentation product comprises a product obtained by fermenting cornus officinalis pulp and smilax glabra with Bacillus subtilis and Bifidobacterium adolescentis; the fenugreek seed extract comprises a product obtained by sequentially graded enzymolysis of fenugreek seed pulp with ficin, trypsin, and papain; the chicory extract is an ethanol percolation extract of chicory root; and the plantain extract is an ethanol percolation extract of plantain.

[0006] In the above-mentioned pharmaceutical composition, the preparation method of the earthworm peptide comprises the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to make the moisture content less than 5%; crushing the dried earthworms into powder, passing them through a 60-mesh to 80-mesh sieve, and taking the powder under the sieve; according to the mass ratio of powder:Tris-HCl buffer = 1: (8-12), adding the powder to Tris-HCl buffer with a pH of 7.5-8.0, mixing them evenly to form a suspension; adding 0.5%-1.0% of the mass of trypsin to the suspension, enzymolyzing at 35°C to 38°C for 50min to 70min, heating to 50°C to 55°C, adding 0.5%-1.0% of the mass of lumbrokinase, and enzymolyzing at 50°C to 55°C for 60min to 90m in, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 6.5-7.0, adding 0.5%-1.0% nattokinase by weight of the powder, performing enzymatic hydrolysis at 50°C-55°C for 60min-90min, heating to inactivate the enzyme, cooling to room temperature, centrifuging at 4000r / min-6000r / min for 10min-20min, and collecting the supernatant; the supernatant is subjected to ultrafiltration separation through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the permeate is collected to obtain a crude earthworm peptide extract; the crude earthworm peptide extract is loaded onto a macroporous adsorption resin column for chromatography, the resin column is first rinsed with deionized water to remove unabsorbed impurities, and then eluted with an ethanol aqueous solution with a volume concentration of 20%-30%, the eluate is collected, concentrated, and freeze-dried to a moisture content of less than 5% to obtain earthworm peptide.

[0007] In the above-mentioned preparation method of earthworm peptide, the macroporous resin model of the macroporous adsorption resin column is D101; the loading flow rate of the earthworm peptide crude extract is 1mL / min~3mL / min; the amount of deionized water is 1~2 resin column volumes, and the flushing flow rate is 3mL / min~5mL / min; the amount of ethanol aqueous solution is 2~4 resin column volumes, and the elution flow rate is 1mL / min~3mL / min; the concentration is carried out by rotary evaporation at 40℃~50℃ and 0.08MPa~0.1MPa to concentrate the eluate to 10%~20% volume.

[0008] In the above-mentioned pharmaceutical composition, the preparation method of the bacterial fermentation product comprises the following steps: crushing the fruit pulp of Cornus officinalis, crushing the Rhizoma Smilacis Glabrae, preparing a base solution according to the mass ratio of water: cornus officinalis fruit pulp: Rhizoma Smilacis Glabrae: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = (90-100): (3-6): (5-8): (1-3): (1-2): (0.1-0.3): (0.05-0.1), sterilizing the base solution, cooling the base solution to room temperature, and obtaining a culture solution; inoculating the culture solution with 2% v / v to 5% v / v of subtilis buds Bacillus subtilis and 2% v / v ~ 5% v / v of Bifidobacterium adolescentis are mixed evenly, and aerobic fermentation is first carried out at 30°C ~ 37°C for 24h ~ 36h to promote the growth and metabolism of Bacillus subtilis, and then anaerobic fermentation is carried out at 35°C ~ 37°C for 48h ~ 72h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain a fermentation liquid, which is centrifuged at 4000r / min ~ 6000r / min for 10min ~ 20min, the supernatant is collected, and concentrated and dried at 100°C ~ 120°C to achieve a water content of less than 5% to obtain a bacterial fermentation product.

[0009] In the preparation method of the above-mentioned bacterial fermentation product, the Bacillus subtilis is an activated bacterial solution of Bacillus subtilis with a concentration of 100 million CFU / mL to 1 billion CFU / mL; the Bifidobacterium adolescentis is an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 100 million CFU / mL to 500 million CFU / mL; the temperature of the sterilization treatment is 121°C to 125°C, and the time of the sterilization treatment is 20min to 40min.

[0010] In the above-mentioned pharmaceutical composition, the preparation method of the fenugreek seed extract comprises the following steps: taking fenugreek seed meat and crushing it, adding distilled water of 8 to 12 times the mass of the fenugreek seed meat, adding 0.5% to 1.5% of the mass of fenugreek seed meat of ficin, enzymolysis at 60°C to 65°C for 60min to 90min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 8.0 to 8.5, adding 0.5% to 1.0% of the mass of fenugreek seed meat of trypsin, enzymolysis at 35°C to 40°C for 50min to 70min, heating to inactivate the enzyme, After cooling to room temperature, adjusting the pH to 5.0-7.0, adding papain in an amount of 0.5%-1.0% by weight of fenugreek seed pulp, performing enzymolysis at 55-65° C. for 50-70 min, heating to inactivate the enzyme, cooling to room temperature, centrifuging at 4000-6000 r / min for 10-20 min, and collecting the supernatant; performing ultrafiltration separation on the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, collecting the permeate, concentrating and drying under reduced pressure at 40-50° C. to achieve a water content of less than 5%, and obtaining a fenugreek seed extract.

[0011] In the above-mentioned pharmaceutical composition, the preparation method of the chicory extract comprises the following steps: taking chicory roots for crushing, using ethanol with a mass of 8 to 10 times that of the chicory roots for percolation to obtain an extract, concentrating and drying at 60° C. to 80° C. to a water content of less than 5%, to obtain the chicory extract.

[0012] In the above-mentioned pharmaceutical composition, the preparation method of the plantain extract comprises the following steps: taking the whole plant of plantain and crushing it, using ethanol with a mass of 8 to 10 times that of the whole plant of plantain for filtration to obtain an extract, concentrating and drying at 60°C to 80°C to a water content of less than 5%, to obtain the plantain extract.

[0013] In the preparation method of the above raw materials, the temperature for heating to inactivate the enzyme is 90°C to 95°C, and the time for heating to inactivate the enzyme is 20min to 30min.

[0014] The preparation method of the above-mentioned uric acid-lowering pharmaceutical composition comprises the following steps: uniformly mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to mass proportions, and then adding earthworm peptide and mixing uniformly to obtain the pharmaceutical composition.

[0015] The present invention provides a uric acid-lowering pharmaceutical composition and a preparation method thereof, and the beneficial effects are as follows: 1. Earthworm peptide contains various small molecule peptides and small water-soluble active substances obtained by graded enzymatic hydrolysis of earthworms by trypsin, lumbrokinase and nattokinase, which can regulate blood uric acid through various pathways. Some active ingredients can inhibit key enzymes in the purine metabolism process and reduce the production of uric acid. Some active ingredients can act on the renal tubular cells of the kidney, regulate the function of these transporters, promote the transport of uric acid from the blood to the renal tubular lumen, increase the excretion of uric acid, and thus reduce the level of blood uric acid. Earthworm peptide has a certain kidney protection effect. When the blood uric acid level increases, it will cause kidney damage and lead to the accumulation of metabolic wastes such as blood creatinine and urea nitrogen in the body. Earthworm peptide reduces the damage of urate crystals to kidney tissue and maintains the normal filtration and excretion function of the kidney, thereby reducing the increase of blood creatinine and urea nitrogen. Some active substance peptides of earthworms inhibit the activation of inflammatory cells, reduce the release of TNF-α and IL-6, interfere with these signaling pathways, prevent inflammatory cells from producing and releasing inflammatory factors, and reduce the adverse effects of inflammatory response on the body.

[0016] 2. Bacillus subtilis and Bifidobacterium adolescentis in bacterial fermentation products produce a variety of metabolites during the fermentation process. Beneficial bacterial metabolites can regulate the composition and function of intestinal flora. Intestinal flora participates in the purine metabolism of the human body. Healthy intestinal flora can decompose purine in the intestine and reduce the absorption of purine, thereby reducing blood uric acid levels. In addition, some metabolites can directly or indirectly affect the excretion of uric acid, for example, by regulating the osmotic pressure of the intestine and promoting the excretion of uric acid with feces. The regulation of intestinal flora by bacterial fermentation products helps maintain the barrier function of the intestine. When the intestinal barrier function is impaired, harmful substances such as endotoxins can enter the blood circulation, increase the burden on the kidneys, and lead to increased blood creatinine and urea nitrogen. By improving the balance of intestinal flora, bacterial fermentation products can reduce the absorption of harmful substances, reduce the burden on the kidneys, and help maintain normal blood creatinine and urea nitrogen levels. Some bacterial fermentation products can also regulate the function of the immune system and inhibit the excessive activation of inflammatory cells. In addition, some components in the fermentation product can directly act on the inflammatory signaling pathway, inhibiting the synthesis and release of inflammatory factors such as TNF-α and IL-6, thereby reducing the damage of inflammatory response to the kidneys and other organs.

[0017] 3. Cornus officinalis pulp is rich in flavonoids. During the bacterial fermentation process, these flavonoids are converted into more biologically active forms by microorganisms (Bacillus subtilis and Bifidobacterium adolescentis). For example, microbial enzymes modify flavonoids, such as glycosylation or methylation, to change their chemical structure and make them more easily absorbed by the human body. These converted flavonoids work synergistically with microbial metabolites to exert antioxidant effects. Antioxidant effects are important for lowering uric acid because oxidative stress can damage cells in organs such as the kidneys and affect the normal metabolism and excretion of uric acid. Flavonoids indirectly benefit the metabolism of uric acid and reduce the accumulation of uric acid in the body by scavenging free radicals and reducing oxidative stress. The polysaccharide components in cornus officinalis pulp provide a carbon source for microorganisms. In the early stages of fermentation, Bacillus subtilis uses these polysaccharides for growth and reproduction. The massive reproduction of microorganisms will produce more enzymes and metabolites, which can further decompose other components in cornus officinalis pulp and release more substances that are beneficial to lowering uric acid. For example, the enzymes produced by microorganisms will break down some proteins in the Cornus officinalis pulp, releasing nutrients such as amino acids. These nutrients will interact with other ingredients in the subsequent fermentation process, or be further converted by microorganisms into substances that have the function of regulating uric acid metabolism.

[0018] 4. Smilax glabra contains a variety of steroidal saponins, alkaloids and other ingredients. During the fermentation process, these ingredients will be decomposed or transformed by microorganisms. For example, microbial enzymes will hydrolyze steroidal saponins to produce biologically active secondary metabolites. These secondary metabolites have the function of regulating intestinal flora, changing the composition of intestinal flora in a direction that is conducive to the decomposition and excretion of uric acid. After fermentation, alkaloid components will be converted into substances that promote uric acid excretion, such as increasing uric acid excretion by affecting the transport of uric acid by renal tubular cells in the kidneys. Smilax glabra itself has a certain diuretic effect, and its active ingredients are enhanced after fermentation. The diuretic effect is very important for lowering uric acid, because increasing urine volume can promote the excretion of uric acid. During the fermentation process, microorganisms will modify or transform the diuretic components in Smilax glabra, making its diuretic effect more significant. For example, microorganisms will convert some components in Smilax glabra into substances that can regulate the glomerular filtration rate or tubular reabsorption function of the kidneys, thereby promoting urine production and allowing more uric acid to be excreted from the body with urine.

[0019] 5. During the preparation of fenugreek seed extract, ficin, trypsin and papain are used for enzymatic hydrolysis, which can specifically decompose proteins and produce peptides with specific amino acid sequences. These peptides contain sequences that can inhibit the activity of uric acid-producing enzymes (such as xanthine oxidase), thereby reducing the production of uric acid, and are easily absorbed by the human body and can enter cells more quickly to exert their effects. For example, it regulates the activity of uric acid metabolism-related enzymes in cells, affects the uric acid transport process of cells, thereby playing a synergistic role in the process of lowering uric acid, and promotes the transport and excretion of uric acid by improving the blood circulation of the kidneys. Fenugreek seed extract has protective effects and anti-inflammatory properties on renal tissue, reduces the accumulation of blood creatinine and urea nitrogen, and reduces the synthesis and release of TNF-α and IL-6.

[0020] 6. Chicory extract contains ingredients such as chicoric acid, which can regulate the function of uric acid transporters. In the kidneys, the excretion and reabsorption of uric acid are related to a variety of transporters. Chicory extract promotes the secretion of uric acid from renal tubular cells to the lumen, increasing the excretion of uric acid. In addition, chicory extract also has a certain diuretic effect, which indirectly promotes the excretion of uric acid by increasing urine volume, thereby reducing blood uric acid levels. Chicory extract can reduce inflammation and oxidative stress damage in renal tissue and maintain the normal filtration function of the kidney.

[0021] 7. Plantain extract mainly promotes uric acid excretion by increasing urine production, regulating the glomerular filtration rate and tubular reabsorption function, allowing more uric acid to be excreted from the body with urine. At the same time, plantain extract also contains some ingredients that can inhibit the reabsorption of uric acid in the tubules, further increasing the excretion of uric acid, thereby reducing blood uric acid levels.

[0022] 8. Earthworm peptides mainly reduce uric acid by regulating enzyme activity and kidney function in the body, while bacterial fermentation products focus on regulating the balance and metabolism of intestinal flora. When the two work together, bacterial fermentation products regulate intestinal flora, reduce the absorption of purine, and reduce the raw materials for uric acid production entering the blood. At the same time, earthworm peptides inhibit the activity of uric acid-producing enzymes in the body, reducing the production of uric acid from the source and metabolic process. In terms of uric acid excretion, bacterial fermentation products improve the intestinal environment and promote the excretion of uric acid with feces, while earthworm peptides regulate kidney function and promote the excretion of uric acid from urine. The two work together to enhance the excretion effect of uric acid and more effectively reduce blood uric acid levels. Earthworm peptides have a renal protective effect and can reduce the damage of urate crystals to renal tissue. Bacterial fermentation products reduce the burden on the kidneys by regulating intestinal flora and reducing the entry of harmful substances into the blood. The synergistic effect of the two can better maintain the normal structure and function of the kidneys and reduce the increase of blood creatinine and urea nitrogen. Both earthworm peptides and bacterial fermentation products have anti-inflammatory effects. Earthworm peptides reduce the release of TNF-α and IL-6 by inhibiting the activation of inflammatory cells and inflammatory signaling pathways. Bacterial fermentation products regulate the function of the immune system, inhibit the overactivation of inflammatory cells, and reduce the production of inflammatory factors. Together, they can more effectively reduce the damage of inflammatory reactions to the kidneys and other organs, creating a good environment for uric acid metabolism and kidney function maintenance. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0024] Example 1: A pharmaceutical composition for lowering uric acid, comprising the following raw materials in parts by weight: 2 parts of earthworm peptide, 2 parts of bacterial fermentation product, 0.3 parts of fenugreek seed extract, 1 part of chicory extract, and 2 parts of plantain extract.

[0025] The preparation method of earthworm peptide includes the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to make the moisture content 4.2%; crushing the dried earthworms into powder, passing them through an 80-mesh sieve, and taking the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:10, adding the powder to a 0.08 mol / L concentration Tris-HCl buffer at pH 7.8, mixing evenly to form a suspension; adding 0.8% of the mass of trypsin to the suspension, enzymolyzing at 37°C for 60 minutes, heating to 52°C, adding 0.8% of the mass of lumbrokinase, enzymolyzing at 52°C for 80 minutes, heating to 92°C to inactivate the enzyme for 25 minutes, cooling to room temperature, adjusting the pH to 6.8, adding 0.8% of the mass of nattokinase, and enzymolyzing at 53°C for 80 minutes. in, heating to 94°C to inactivate the enzyme for 25 minutes, cooling to room temperature, centrifuging at 5000r / min for 15 minutes, and collecting the supernatant; the supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the permeate was collected to obtain a crude extract of earthworm peptide; at a flow rate of 2mL / min, the crude extract of earthworm peptide was loaded on a macroporous adsorption resin column for chromatography, the macroporous resin model was D101, and the resin column was first rinsed with 1 resin column volume of deionized water at a flow rate of 4mL / min to remove unadsorbed impurities, and then 3 resin column volumes of 25% volume concentration of ethanol aqueous solution were eluted at a flow rate of 2mL / min, and all the eluates of ethanol aqueous solution were collected, concentrated to 15% volume by rotary evaporation at 45°C and 0.09MPa, and freeze-dried to a moisture content of 4.5% to obtain earthworm peptide.

[0026] The preparation method of the bacterial fermentation product includes the following steps: crushing the cornus officinalis pulp and the smilax glabra, preparing a base solution according to the mass ratio of water: cornus officinalis pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 95:5:6:2:1.5:0.2:0.08, sterilizing at 123°C for 30 minutes, cooling to room temperature, and obtaining a culture solution; inoculating 3% v / v of 500 million CFU / mL of Bacillus subtilis spores into the culture solution The activated bacterial liquid of the bacteria was inoculated with 3% v / v activated bacterial liquid of Bifidobacterium adolescentis with a concentration of 300 million CFU / mL, the mixture was evenly mixed, and aerobic fermentation was first performed at 35°C for 30 hours to promote the growth and metabolism of Bacillus subtilis, and then anaerobic fermentation was performed at 36°C for 60 hours to promote the growth and metabolism of Bifidobacterium adolescentis to obtain fermentation liquid, which was centrifuged at 5000 r / min for 15 minutes, the supernatant was collected, and concentrated and dried at 110°C to achieve a water content of 4.6% to obtain a bacterial fermentation product.

[0027] The preparation method of fenugreek seed extract comprises the following steps: crushing fenugreek seed flesh, adding distilled water 10 times the mass of fenugreek seed flesh, adding 1% of fig protease by mass of fenugreek seed flesh, performing enzymolysis at 62°C for 80 minutes, heating to 92°C to inactivate the enzyme for 25 minutes, cooling to room temperature, adjusting the pH to 8.2, adding 0.8% of trypsin by mass of fenugreek seed flesh, performing enzymolysis at 37°C for 60 minutes, heating to 93°C to inactivate the enzyme for 25 minutes, cooling to room temperature, adjusting the pH to 6.2, adding 0.6% of papain by mass of fenugreek seed flesh, performing enzymolysis at 60°C for 60 minutes, heating to 92°C to inactivate the enzyme for 25 minutes, cooling to room temperature, centrifuging at 5000r / min for 15 minutes, and collecting the supernatant; performing ultrafiltration separation on the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5kDa, collecting the permeate, concentrating and drying under reduced pressure at 45°C to achieve a water content of 4.3%, and obtaining the fenugreek seed extract.

[0028] The preparation method of chicory extract comprises the following steps: crushing chicory root, filtering with ethanol 9 times the mass of the chicory root to obtain an extract, concentrating and drying at 70° C. to a water content of 3.8%, and obtaining the chicory extract.

[0029] The preparation method of the plantain extract comprises the following steps: crushing the whole plant of plantain, filtering with ethanol 9 times the mass of the whole plant of plantain to obtain an extract, concentrating and drying at 70°C to a water content of 4.3%, and obtaining the plantain extract.

[0030] The preparation method of the above-mentioned uric acid-lowering pharmaceutical composition comprises the following steps: uniformly mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to mass proportions, and then adding earthworm peptide and mixing uniformly to obtain the pharmaceutical composition.

[0031] Example 2: A pharmaceutical composition for lowering uric acid, comprising the following raw materials in parts by weight: 1 part of earthworm peptide, 1 part of bacterial fermentation product, 0.2 parts of fenugreek seed extract, 0.5 parts of chicory extract, and 1 part of plantain extract.

[0032] Among them, the preparation method of earthworm peptide includes the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to make the moisture content of 3%; crushing the dried earthworms into powder, passing them through a 60-mesh sieve, and taking the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:8, adding the powder to a 0.08 mol / L concentration Tris-HCl buffer at pH 7.5, mixing evenly to form a suspension; adding 0.5% of the mass of trypsin to the suspension, enzymatically hydrolyzing at 35°C for 50 minutes, heating to 50°C, adding 0.5% of the mass of lumbrokinase, enzymatically hydrolyzing at 50°C for 60 minutes, heating to 90°C to inactivate the enzyme for 20 minutes, cooling to room temperature, adjusting the pH to 6.5, adding 0.5% of the mass of nattokinase, and enzymatically hydrolyzing at 50°C for 60 minutes. , heat to 90℃ to inactivate the enzyme for 20min, cool to room temperature, centrifuge at 4000r / min for 10min, and collect the supernatant; the supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the permeate is collected to obtain a crude extract of earthworm peptide; at a flow rate of 1mL / min, the crude extract of earthworm peptide is loaded on a macroporous adsorption resin column for chromatography, the macroporous resin model is D101, first rinse the resin column with 1 resin column volume of deionized water at a flow rate of 3mL / min to remove unadsorbed impurities, and then elute with 2 resin column volumes of 20% volume concentration of ethanol aqueous solution at a flow rate of 1mL / min, collect all the eluates of ethanol aqueous solution, concentrate to 10% volume by rotary evaporation at 40℃ and 0.08MPa, and freeze-dry to a moisture content of 3.2% to obtain earthworm peptide.

[0033] The preparation method of the bacterial fermentation product includes the following steps: crushing the cornus officinalis pulp and the smilax glabra, preparing a base solution according to the mass ratio of water: cornus officinalis pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 90:3:5:1:1:0.1:0.05, sterilizing at 121°C for 20 minutes, cooling to room temperature, and obtaining a culture solution; inoculating 2% v / v of Bacillus subtilis with a concentration of 100 million CFU / mL into the culture solution. The activated bacterial liquid was inoculated with 2% v / v activated bacterial liquid of Bifidobacterium adolescentis with a concentration of 200 million CFU / mL, the mixture was evenly mixed, and aerobic fermentation was carried out at 30°C for 24 hours to promote the growth and metabolism of Bacillus subtilis. Then, anaerobically fermentation was carried out at 35°C for 48 hours to promote the growth and metabolism of Bifidobacterium adolescentis to obtain fermentation liquid. The mixture was centrifuged at 4000 r / min for 10 minutes, the supernatant was collected, and concentrated and dried at 100°C to achieve a water content of 3.5% to obtain a bacterial fermentation product.

[0034] The preparation method of fenugreek seed extract comprises the following steps: crushing fenugreek seed flesh, adding distilled water 8 times the mass of fenugreek seed flesh, adding 0.5% of fig protease by mass of fenugreek seed flesh, performing enzymolysis at 60°C for 60 minutes, heating to 90°C to inactivate the enzyme for 20 minutes, cooling to room temperature, adjusting the pH to 8.0, adding 0.5% of trypsin by mass of fenugreek seed flesh, performing enzymolysis at 35°C for 50 minutes, heating to 90°C to inactivate the enzyme for 20 minutes, cooling to room temperature, adjusting the pH to 5.0, adding 0.5% of papain by mass of fenugreek seed flesh, performing enzymolysis at 55°C for 50 minutes, heating to 90°C to inactivate the enzyme for 20 minutes, cooling to room temperature, centrifuging at 4000r / min for 10 minutes, and collecting the supernatant; performing ultrafiltration separation on the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5kDa, collecting the permeate, concentrating and drying under reduced pressure at 40°C to achieve a water content of 3.8%, and obtaining the fenugreek seed extract.

[0035] The preparation method of chicory extract comprises the following steps: crushing chicory root, filtering with ethanol 8 times the mass of the chicory root to obtain an extract, concentrating and drying at 60° C. to a water content of 4.1%, and obtaining the chicory extract.

[0036] The preparation method of the plantain extract comprises the following steps: crushing the whole plant of plantain, filtering with ethanol 8 times the mass of the whole plant of plantain to obtain an extract, concentrating and drying at 60°C to a water content of 5%, and obtaining the plantain extract.

[0037] The preparation method of the above-mentioned uric acid-lowering pharmaceutical composition comprises the following steps: uniformly mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to mass proportions, and then adding earthworm peptide and mixing uniformly to obtain the pharmaceutical composition.

[0038] Example 3: A pharmaceutical composition for lowering uric acid, comprising the following raw materials in parts by weight: 1 part of earthworm peptide, 3 parts of bacterial fermentation product, 0.2 parts of fenugreek seed extract, 1.5 parts of chicory extract, and 1 part of plantain extract.

[0039] Among them, the preparation method of earthworm peptide includes the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to make the moisture content of 5%; crushing the dried earthworms into powder, passing them through a 60-mesh sieve, and taking the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:12, adding the powder to a 0.08 mol / L concentration Tris-HCl buffer at pH 7.5, mixing evenly to form a suspension; adding 1.0% of the mass of trypsin to the suspension, enzymatically hydrolyzing at 35°C for 70 minutes, heating to 50°C, adding 0.5% of the mass of lumbrokinase, enzymatically hydrolyzing at 50°C for 90 minutes, heating to 90°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 6.5, adding 1.0% of the mass of nattokinase, enzymatically hydrolyzing at 50°C for 90 minutes n, heating to 90°C to inactivate the enzyme for 30 minutes, cooling to room temperature, centrifuging at 4000r / min for 20 minutes, and collecting the supernatant; the supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the permeate was collected to obtain a crude extract of earthworm peptide; at a flow rate of 1mL / min, the crude extract of earthworm peptide was loaded on a macroporous adsorption resin column for chromatography, the macroporous resin model was D101, and the resin column was first rinsed with 2 resin column volumes of deionized water at a flow rate of 3mL / min to remove unadsorbed impurities, and then 4 resin column volumes of 20% volume concentration ethanol aqueous solution were used for elution at a flow rate of 3mL / min, and all the eluates of the ethanol aqueous solution were collected, concentrated to 10% volume by rotary evaporation at 40°C and 0.1MPa, and freeze-dried to a moisture content of 4.8% to obtain earthworm peptide.

[0040] The preparation method of the bacterial fermentation product includes the following steps: crushing the cornus officinalis pulp and the smilax glabra, preparing a base solution according to the mass ratio of water: cornus officinalis pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 90:6:5:3:1:0.3:0.05, sterilizing at 125°C for 20 minutes, cooling to room temperature, and obtaining a culture solution; inoculating 5% v / v of 800 million CFU / mL of Bacillus subtilis spores into the culture solution The activated bacterial liquid of the bacteria was inoculated with 2% v / v activated bacterial liquid of Bifidobacterium adolescentis with a concentration of 500 million CFU / mL, the mixture was evenly mixed, and aerobic fermentation was first performed at 30°C for 36 hours to promote the growth and metabolism of Bacillus subtilis, and then anaerobically fermented at 35°C for 72 hours to promote the growth and metabolism of Bifidobacterium adolescentis to obtain fermentation liquid, which was centrifuged at 4000 r / min for 20 minutes, the supernatant was collected, and concentrated and dried at 100°C to achieve a water content of 3% to obtain a bacterial fermentation product.

[0041] The preparation method of fenugreek seed extract comprises the following steps: crushing fenugreek seed flesh, adding distilled water 8 times the mass of fenugreek seed flesh, adding 1.5% of fig protease by mass of fenugreek seed flesh, enzymolysis at 60°C for 90 minutes, heating to 90°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 8.0, adding 1.0% of trypsin by mass of fenugreek seed flesh, enzymolysis at 35°C for 70 minutes, heating to 90°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 5.0, adding 1.0% of papain by mass of fenugreek seed flesh, enzymolysis at 55°C for 70 minutes, heating to 90°C to inactivate the enzyme for 30 minutes, cooling to room temperature, centrifuging at 4000r / min for 20 minutes, and collecting the supernatant; ultrafiltration separation of the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5kDa, collecting the permeate, concentrating and drying under reduced pressure at 40°C to achieve a water content of 5%, and obtaining the fenugreek seed extract.

[0042] The preparation method of chicory extract comprises the following steps: crushing chicory root, filtering with ethanol 8 times the mass of the chicory root to obtain an extract, concentrating and drying at 80° C. to a water content of 3.6%, and obtaining the chicory extract.

[0043] The preparation method of the plantain extract comprises the following steps: crushing the whole plant of plantain, filtering with ethanol 10 times the mass of the whole plant of plantain to obtain an extract, concentrating and drying at 60°C to a water content of 3%, and obtaining the plantain extract.

[0044] The preparation method of the above-mentioned uric acid-lowering pharmaceutical composition comprises the following steps: uniformly mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to mass proportions, and then adding earthworm peptide and mixing uniformly to obtain the pharmaceutical composition.

[0045] Example 4: A pharmaceutical composition for lowering uric acid, comprising the following raw materials in parts by weight: 1.5 parts of earthworm peptide, 2.5 parts of bacterial fermentation product, 0.4 parts of fenugreek seed extract, 1.2 parts of chicory extract, and 2.6 parts of plantain extract.

[0046] The preparation method of earthworm peptide includes the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to make the moisture content 4.5%; crushing the dried earthworms into powder, passing them through an 80-mesh sieve, and taking the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:9, adding the powder to a 0.08 mol / L concentration Tris-HCl buffer at pH 7.6, mixing evenly to form a suspension; adding 0.6% of the mass of trypsin to the suspension, enzymolyzing at 36°C for 65 minutes, heating to 55°C, adding 0.9% of the mass of lumbrokinase, enzymolyzing at 55°C for 70 minutes, heating to 91°C to inactivate the enzyme for 25 minutes, cooling to room temperature, adjusting the pH to 6.6, adding 0.6% of the mass of nattokinase, and enzymolyzing at 53°C for 80 minutes, The enzyme was inactivated by heating to 90°C for 20 min, and the temperature was lowered to room temperature before centrifugation at 6000 r / min for 15 min to collect the supernatant. The supernatant was separated by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate was collected to obtain a crude extract of earthworm peptide. The crude extract of earthworm peptide was loaded on a macroporous adsorption resin column for chromatography at a flow rate of 2.5 mL / min. The macroporous resin model was D101. The resin column was first rinsed with 2 resin column volumes of deionized water at a flow rate of 4.5 mL / min to remove unadsorbed impurities, and then 3 resin column volumes of 25% volume concentration of ethanol aqueous solution were used for elution at a flow rate of 2.5 mL / min. All the eluates of ethanol aqueous solution were collected, concentrated to 18% volume by rotary evaporation at 50°C and 0.08 MPa, and freeze-dried to a moisture content of 3% to obtain earthworm peptide.

[0047] The preparation method of the bacterial fermentation product includes the following steps: crushing the cornus officinalis pulp and the smilax glabra, preparing a base solution according to the mass ratio of water: cornus officinalis pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 96:5:7:2.5:1.5:0.3:0.06, sterilizing at 121°C for 35 minutes, cooling to room temperature, and obtaining a culture solution; inoculating the culture solution with 3% v / v of 1 billion CFU / mL subtilis buds The activated bacterial liquid of Bacillus subtilis was inoculated with 3% v / v activated bacterial liquid of Bifidobacterium adolescentis with a concentration of 200 million CFU / mL, the mixture was evenly mixed, and aerobic fermentation was carried out at 35°C for 32 hours to promote the growth and metabolism of Bacillus subtilis. Then, anaerobically fermentation was carried out at 37°C for 65 hours to promote the growth and metabolism of Bifidobacterium adolescentis to obtain fermentation liquid, which was centrifuged at 6000 r / min for 20 minutes, the supernatant was collected, and concentrated and dried at 120°C to achieve a water content of 5% to obtain a fermentation product.

[0048] The preparation method of fenugreek seed extract comprises the following steps: crushing fenugreek seed flesh, adding distilled water 11 times the mass of fenugreek seed flesh, adding 1.2% of fig protease by mass of fenugreek seed flesh, performing enzymolysis at 63°C for 85 minutes, heating to 90°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 8.1, adding 0.8% of trypsin by mass of fenugreek seed flesh, performing enzymolysis at 40°C for 50 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 5.8, adding 0.6% of papain by mass of fenugreek seed flesh, performing enzymolysis at 62°C for 60 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, cooling to room temperature, centrifuging at 5000r / min for 20 minutes, and collecting the supernatant; performing ultrafiltration separation on the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5kDa, collecting the permeate, concentrating and drying under reduced pressure at 50°C to achieve a water content of 3%, and obtaining the fenugreek seed extract.

[0049] The preparation method of chicory extract comprises the following steps: crushing chicory root, filtering with ethanol 10 times the mass of the chicory root to obtain an extract, concentrating and drying at 70° C. to a water content of 3%, and obtaining the chicory extract.

[0050] The preparation method of the plantain extract comprises the following steps: crushing the whole plant of plantain, filtering with ethanol 10 times the mass of the whole plant of plantain to obtain an extract, concentrating and drying at 80° C. to a water content of 4.2%, and obtaining the plantain extract.

[0051] The preparation method of the above-mentioned uric acid-lowering pharmaceutical composition comprises the following steps: uniformly mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to mass proportions, and then adding earthworm peptide and mixing uniformly to obtain the pharmaceutical composition.

[0052] Example 5: A pharmaceutical composition for lowering uric acid, comprising the following raw materials in parts by weight: 3 parts of earthworm peptide, 3 parts of bacterial fermentation product, 0.5 parts of fenugreek seed extract, 1.5 parts of chicory extract, and 3 parts of plantain extract.

[0053] The preparation method of earthworm peptide includes the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to make the moisture content of 3.8%; crushing the dried earthworms into powder, passing them through an 80-mesh sieve, and taking the powder under the sieve; according to the mass ratio of powder: Tris-HCl buffer = 1:12, adding the powder to a 0.08 mol / L concentration Tris-HCl buffer at pH 8.0, mixing them evenly to form a suspension; adding 1.0% trypsin by weight of the powder to the suspension, enzymolyzing it at 38°C for 70 minutes, heating it to 55°C, adding 1.0% lumbrokinase by weight of the powder, enzymolyzing it at 55°C for 90 minutes, heating it to 95°C to inactivate the enzyme for 30 minutes, cooling it to room temperature, adjusting the pH to 7.0, adding 1.0% nattokinase by weight of the powder, enzymolyzing it at 55°C for 90 minutes, and then heating it to 95°C to inactivate the enzyme. min, heated to 95℃ to inactivate the enzyme for 30min, cooled to room temperature, centrifuged at 6000r / min for 20min, and collected the supernatant; the supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the permeate was collected to obtain a crude earthworm peptide extract; the crude earthworm peptide extract was loaded on a macroporous adsorption resin column for chromatography at a flow rate of 3mL / min, the macroporous resin model was D101, and the resin column was first rinsed with 2 resin column volumes of deionized water at a flow rate of 5mL / min to remove unadsorbed impurities, and then 4 resin column volumes of 30% volume concentration ethanol aqueous solution were eluted at a flow rate of 3mL / min, and all the eluates of the ethanol aqueous solution were collected, concentrated to 20% volume by rotary evaporation at 50℃ and 0.1MPa, and freeze-dried to a moisture content of 5% to obtain earthworm peptide.

[0054] The preparation method of the bacterial fermentation product includes the following steps: crushing the cornus officinalis pulp and the smilax glabra, preparing a base solution according to the mass ratio of water: cornus officinalis pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 100:6:8:3:2:0.3:0.1, sterilizing at 125°C for 40 minutes, cooling to room temperature, and obtaining a culture solution; inoculating 5% v / v of Bacillus subtilis with a concentration of 600 million CFU / mL into the culture solution. The activated bacterial liquid was inoculated with 5% v / v activated bacterial liquid of Bifidobacterium adolescentis with a concentration of 100 million CFU / mL, the mixture was mixed evenly, and aerobic fermentation was carried out at 37°C for 36 hours to promote the growth and metabolism of Bacillus subtilis. Then, anaerobically fermentation was carried out at 37°C for 72 hours to promote the growth and metabolism of Bifidobacterium adolescentis to obtain fermentation liquid. The mixture was centrifuged at 6000 r / min for 20 minutes, the supernatant was collected, and concentrated and dried at 120°C to achieve a water content of 3.1% to obtain a bacterial fermentation product.

[0055] The preparation method of fenugreek seed extract comprises the following steps: crushing fenugreek seed flesh, adding distilled water 12 times the mass of fenugreek seed flesh, adding 1.5% of fig protease by mass of fenugreek seed flesh, performing enzymolysis at 65°C for 90 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 8.5, adding 1.0% of trypsin by mass of fenugreek seed flesh, performing enzymolysis at 40°C for 70 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, cooling to room temperature, adjusting the pH to 7.0, adding 1.0% of papain by mass of fenugreek seed flesh, performing enzymolysis at 65°C for 70 minutes, heating to 95°C to inactivate the enzyme for 30 minutes, cooling to room temperature, centrifuging at 6000r / min for 20 minutes, and collecting the supernatant; performing ultrafiltration separation on the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5kDa, collecting the permeate, concentrating and drying under reduced pressure at 50°C to achieve a water content of 4.3%, and obtaining the fenugreek seed extract.

[0056] The preparation method of chicory extract comprises the following steps: crushing chicory root, filtering with ethanol 10 times the mass of the chicory root to obtain an extract, concentrating and drying at 80° C. to a water content of 5%, and obtaining the chicory extract.

[0057] The preparation method of the plantain extract comprises the following steps: crushing the whole plant of plantain, filtering with ethanol 10 times the mass of the whole plant of plantain to obtain an extract, concentrating and drying at 80°C to a water content of 4.7%, and obtaining the plantain extract.

[0058] The preparation method of the above-mentioned uric acid-lowering pharmaceutical composition comprises the following steps: uniformly mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to mass proportions, and then adding earthworm peptide and mixing uniformly to obtain the pharmaceutical composition.

[0059] In the above embodiments, the pH adjusters are 0.5 mol / L aqueous hydrochloric acid solution and 0.5 mol / L aqueous sodium hydroxide solution.

[0060] In the above embodiments: earthworm is Pheretima Williamii. Trypsin is porcine trypsin, which is sourced from Shanxi Zhongnuo Biotechnology Co., Ltd., with an enzyme activity of 4000 U / g; lumbrokinase is sourced from Xi'an Muguo Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; nattokinase is sourced from Shaanxi Yunhe Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; Bacillus subtilis is sourced from Jinan Jinyuyuan Biotechnology Co., Ltd.; Bifidobacterium adolescentis is sourced from Shanxi Hepeptide Biotechnology Co., Ltd.; Ficin is sourced from Xuzhou Shengyi Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g; papain is sourced from Shandong Jishuo Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; D101 macroporous resin is sourced from Langfang Miaoyang Chemical Co., Ltd.

[0061] Comparative Example 1 The earthworm peptide was replaced by sterilized freeze-dried powder obtained by directly crushing earthworms; other parameters and methods were the same as those in Example 1.

[0062] Comparative Example 2 In the preparation method of earthworm peptide, trypsin is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0063] Comparative Example 3 In the preparation method of earthworm peptide, lumbrokinase is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0064] Comparative Example 4 In the preparation method of earthworm peptide, nattokinase is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0065] Comparative Example 5 In the method for preparing the fermented product, no Cornus officinalis pulp is added; other parameters and methods are the same as in Example 1.

[0066] Comparative Example 6 In the preparation method of the bacterial fermentation product, no Smilax glabra is added; other parameters and methods are the same as in Example 1.

[0067] Comparative Example 7 In the preparation method of the bacterial fermentation product, no cornus fruit pulp and no Bifidobacterium adolescentis added for aerobic fermentation; other parameters and methods are the same as those in Example 1.

[0068] Comparative Example 8 In the preparation method of fenugreek seed extract, ficin is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0069] Comparative Example 9 In the preparation method of the fenugreek seed extract, trypsin is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0070] Comparative Example 10 In the preparation method of the fenugreek seed extract, papain is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0071] Comparative Example 11 Fenugreek seed extract is extracted by ethanol percolation: Fenugreek seed meat is crushed, and ethanol with a mass of 9 times that of the fenugreek seed meat is used for percolation to obtain an extract, which is concentrated and dried at 70° C. to a water content of 4.1% to obtain a fenugreek seed extract. Other parameters and methods are the same as in Example 1.

[0072] Comparative Example 12 The pharmaceutical composition does not contain earthworm peptide and bacterial fermentation product, and the mass fractions of earthworm peptide and bacterial fermentation product are replaced by starch. Other parameters and methods are the same as those in Example 1.

[0073] The safety and efficacy tests were performed on the pharmaceutical compositions prepared in the above-mentioned embodiments and comparative examples.

[0074] 1. Safety Testing (1) Acute toxicity test: 180 SPF-grade adult Kunming mice, half male and half female, weighing 18-22 g, were selected and randomly divided into 18 groups (10 mice in each group). One group was a blank control group, which was given normal saline by gavage. The remaining groups were given 2 g / kg body weight of the drug composition by gavage for 14 consecutive days. The general state of the mice (including mental state, activity, diet, breathing, etc.) was observed, and the death and weight changes of the mice were recorded. The results showed that there was no death in each group, and the diet, activity, and mental state were normal. The weight of the mice continued to increase, with no significant difference compared with the blank control group, indicating that the drug composition had no obvious acute toxicity even at high doses.

[0075] (2) Long-term toxicity test: 180 SPF adult male SD rats, weighing 230-280 g, half of which were male and half were female, were randomly divided into 18 groups (10 rats in each group), of which one group was a blank control group, which was given normal saline by gavage, and the other groups were given 500 mg / kg body weight of the drug composition by gavage, once a day, for 90 consecutive days. The general condition of the rats during the medication period, including mental state, activity, diet, breathing, etc., was observed. After the experiment, the rats were killed and the main organs of the rats (including heart, liver, spleen, lung, and kidney) were dissected. The results showed that the daily behavior of the rats was normal and there was no lesion in each organ, indicating that the long-term safety of each composition was good.

[0076] (3) Guinea pig active systemic allergy test: 54 healthy adult guinea pigs were selected and randomly divided into 18 groups (3 in each group). Sensitization was performed on the 0th, 7th and 14th days, i.e., 3 mL of each composition sample (composition concentration was 0.1 mg / ml, solvent was water) was administered by gavage, and the blank control group was administered an equal amount of normal saline by gavage; on the 21st day, stimulation was performed, i.e., 3 mL of each composition sample (composition concentration was 1.0 mg / ml) was administered by gavage, and the blank control group was administered an equal amount of normal saline by gavage; 24 hours and 72 hours after stimulation, the general reaction of the guinea pigs was observed, and the results showed that no guinea pigs in each group showed symptoms such as irritability, abnormal defecation, dyspnea, convulsions, etc. This indicates that the ingredients of the drug composition are safe and there is no systemic allergic reaction.

[0077] 2. Drug efficacy testing (1) Reducing blood uric acid levels and improving renal function indicators: 200 healthy adult SD rats (blood uric acid levels between 140 μmol / L and 180 μmol / L) were selected and randomly divided into 20 groups (10 rats in each group), of which 1 group was a blank control group, 1 group was a negative control group, and 1 group was a positive control group. Rats in all groups except the blank control group were intraperitoneally injected with potassium oxonate sodium chloride solution (potassium oxonate dosage of 300 mg / kg), and the blank control group was intraperitoneally injected with an equal amount of normal saline. The drugs were administered for 7 consecutive days. After the model was successfully established (blood uric acid level was 140 μmol / L-180 μmol / L), the rats were randomly divided into 20 groups (10 rats in each group), of which 1 group was a blank control group, 1 group was a negative control group, and 1 group was a positive control group. Uric acid value is 400μmol / L~460μmol / L), each comparative example and each embodiment group is gavaged with the composition 15mg / kg body weight, the blank control group and the negative control group are gavaged with the same amount of normal saline, and the positive control group is gavaged with allopurinol 10mg / kg body weight, once a day for 14 consecutive days. After the test: ① blood is collected from the orbital venous plexus of the rats, serum is separated, and the blood uric acid level is determined by uricase method; ② blood is collected from the abdominal aorta of the rats, serum is separated, and the blood creatinine and urea nitrogen levels are detected by automatic biochemical analyzer. The results are shown in Table 1 below.

[0078] (2) Inhibition of inflammatory response: Mouse macrophages (RAW264.7) cultured in vitro were inoculated in 96-well plates at 5×10³ cells per well, cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) until the logarithmic growth phase, and then replaced with a cell culture medium containing the drug composition (the concentration of the composition was 0.1 mg / ml). A blank control group, a negative control group (cultured only with high-glucose DMEM cell culture medium) and a positive control group (the volume concentration of dexamethasone in the high-glucose DMEM cell culture medium was 0.1%) were set up for continued culture. After 2 hours, lipopolysaccharide LPS (1 μg / mL) was added to all groups except the blank control group, and culture was continued for 24 hours. The cell culture supernatant was collected, and the content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 1 below.

[0079] Table 1 Efficacy test results (average value)

[0080] From the above experimental results, it can be seen that the ingredients of the pharmaceutical compositions prepared in Examples 1 to 5 are safe, non-toxic and non-allergic; compared with the negative control group, they can reduce the blood uric acid level of rats, improve renal function, and inhibit inflammatory response, and the effect is better than that of the positive control group, and can effectively control the uric acid level.

[0081] From the results of Comparative Example 1, it can be seen that earthworm peptide is replaced by freeze-dried powder of directly crushed earthworms. The complex components contained in the freeze-dried earthworm powder are not easily absorbed by the body. The earthworm peptide extracted by specific enzymatic hydrolysis is a specific small molecule peptide, which has the effect of better inhibiting the activity of xanthine oxidase and promoting the excretion of uric acid, and can better inhibit the production of uric acid.

[0082] It can be seen from the results of Comparative Examples 2 to 4 that the preparation method of earthworm peptide does not use trypsin, lumbrokinase or nattokinase for enzymatic hydrolysis, the composition of the extracted product changes, especially the small molecule peptide products are different, and the effect of earthworm peptide in inhibiting uric acid, improving renal function and inflammatory response is weakened.

[0083] It can be seen from the results of Comparative Examples 5 to 7 that the bacterial fermentation product has the function of regulating the balance of intestinal flora and improving the intestinal microecological environment. Healthy intestinal flora contributes to the decomposition and excretion of uric acid and can assist in lowering uric acid levels. In addition, the bacterial fermentation product prepared with specific fermentation substrates has different therapeutic effects. In the preparation method of the bacterial fermentation product, no Cornus officinalis pulp is added, or no Smilax glabra is added, or no Cornus officinalis pulp is added and no Bifidobacterium adolescentis is added for aerobic fermentation, the composition of the extracted product is changed, and the effect of the pharmaceutical composition in inhibiting uric acid, improving renal function and inflammatory response is weakened.

[0084] From the results of Comparative Examples 8 to 11, it can be seen that the fenugreek seed extract contains rich chemical components, can regulate hormone levels in the body, enhance the kidney's filtration and excretion function of uric acid, and reduce uric acid synthesis. The preparation method of the fenugreek seed extract does not use ficin, trypsin or papain for enzymatic hydrolysis, and the composition of the product obtained by extraction changes, especially the structure of the functional small molecule peptide product changes, the therapeutic effect changes, and the effect of the pharmaceutical composition in inhibiting uric acid, improving renal function and inflammatory response is weakened.

[0085] From the results of Comparative Example 11, it can be seen that the fenugreek seed extract is extracted by ethanol percolation, and the product components obtained by extraction have insufficient efficacy and weakened therapeutic effect.

[0086] From the results of Comparative Example 12, it can be seen that when earthworm peptide and bacterial fermentation product are not added to the pharmaceutical composition at the same time, and the mass fractions of earthworm peptide and bacterial fermentation product are replaced by starch, the effects of the pharmaceutical composition in inhibiting uric acid, improving renal function and inflammatory response are greatly weakened, and the combination of the two has a good synergistic effect.

Claims

1. A pharmaceutical composition for lowering uric acid, characterized in that: The pharmaceutical composition comprises the following raw materials in parts by weight: 1 to 3 parts of earthworm peptide, 1 to 3 parts of bacterial fermentation product, 0.2 to 0.5 parts of fenugreek seed extract, 0.5 to 1.5 parts of chicory extract, and 1 to 3 parts of plantain extract; The earthworm peptide contains the product obtained by graded enzymatic hydrolysis of earthworms with trypsin, lumbrokinase and nattokinase in sequence; the bacterial fermentation product contains the product obtained by fermenting Cornus officinalis pulp and Smilax glabra with Bacillus subtilis and Bifidobacterium adolescentis; the fenugreek seed extract contains the product obtained by graded enzymatic hydrolysis of fenugreek seed pulp with ficin, trypsin and papain in sequence; the chicory extract is an ethanol diafiltration extract of chicory roots; and the plantain extract is an ethanol diafiltration extract of plantain.

2. A uric acid-lowering pharmaceutical composition according to claim 1, characterized in that: The preparation method of earthworm peptide comprises the following steps: selecting fresh and healthy earthworms, washing them, and freeze-drying them to reduce the moisture content to less than 5%; crushing the dried earthworms into powder, passing them through a 60-mesh to 80-mesh sieve, and taking the powder under the sieve; adding the powder to a Tris-HCl buffer solution with a pH of 7.5 to 8.0 according to a mass ratio of powder:Tris-HCl buffer solution=1:(8-12), mixing them evenly to form a suspension; adding 0.5% to 1.0% of the mass of trypsin to the suspension, enzymolyzing them at 35°C to 38°C for 50min to 70min, heating them to 50°C to 55°C, adding 0.5% to 1.0% of the mass of lumbrokinase, enzymolyzing them at 50°C to 55°C for 60min to 90min, and heating them to 50°C to 55°C. The enzyme was inactivated by heating, and after the temperature was lowered to room temperature, the pH was adjusted to 6.5-7.0, 0.5%-1.0% of nattokinase by weight of the powder was added, and the enzyme was hydrolyzed at 50°C-55°C for 60min-90min, the enzyme was inactivated by heating, and after the temperature was lowered to room temperature, the enzyme was centrifuged at 4000r / min-6000r / min for 10min-20min, and the supernatant was collected; the supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the permeate was collected to obtain a crude earthworm peptide extract; the crude earthworm peptide extract was loaded onto a macroporous adsorption resin column for chromatography, the resin column was first rinsed with deionized water to remove unabsorbed impurities, and then eluted with an ethanol aqueous solution with a volume concentration of 20%-30%, the eluate was collected, concentrated, and freeze-dried to a moisture content of less than 5% to obtain earthworm peptide.

3. A uric acid-lowering pharmaceutical composition according to claim 2, characterized in that: The macroporous resin model of the macroporous adsorption resin column is D101; the loading flow rate of the earthworm peptide crude extract is 1mL / min~3mL / min; the amount of deionized water is 1~2 resin column volumes, and the flushing flow rate is 3mL / min~5mL / min; the amount of ethanol aqueous solution is 2~4 resin column volumes, and the elution flow rate is 1mL / min~3mL / min; the concentration is carried out by rotary evaporation at 40℃~50℃ and 0.08MPa~0.1MPa to concentrate the eluate to 10%~20% volume.

4. A uric acid-lowering pharmaceutical composition according to claim 1, characterized in that: The preparation method of the bacterial fermentation product comprises the following steps: crushing cornus officinalis pulp, crushing smilax glabra, preparing a base solution according to the mass ratio of water: cornus officinalis pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = (90-100): (3-6): (5-8): (1-3): (1-2): (0.1-0.3): (0.05-0.1), sterilizing, cooling to room temperature, and obtaining a culture solution; inoculating 2% v / v-5% v / v of Bacillus subtilis and 2% v / v to 5% v / v of Bifidobacterium adolescentis are mixed evenly, and firstly aerobically fermented at 30°C to 37°C for 24h to 36h to promote the growth and metabolism of Bacillus subtilis, and then anaerobically fermented at 35°C to 37°C for 48h to 72h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain a fermentation liquid, and centrifuged at 4000r / min to 6000r / min for 10min to 20min, and the supernatant is collected, and concentrated and dried at 100°C to 120°C to achieve a water content of less than 5% to obtain a bacterial fermentation product.

5. A uric acid-lowering pharmaceutical composition according to claim 4, characterized in that: The Bacillus subtilis is an activated bacterial solution of Bacillus subtilis with a concentration of 100 million CFU / mL to 1 billion CFU / mL; the Bifidobacterium adolescentis is an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 100 million CFU / mL to 500 million CFU / mL; the temperature of the sterilization treatment is 121° C. to 125° C., and the time of the sterilization treatment is 20 min to 40 min.

6. A uric acid-lowering pharmaceutical composition according to claim 1, characterized in that: The preparation method of the fenugreek seed extract comprises the following steps: taking fenugreek seed flesh and crushing it, adding distilled water of 8 to 12 times the mass of the fenugreek seed flesh, adding 0.5% to 1.5% of the mass of the fenugreek seed flesh of fig protease, enzymolyzing it at 60 to 65° C. for 60 to 90 minutes, heating it up to inactivate the enzyme, cooling it down to room temperature, adjusting the pH to 8.0 to 8.5, adding 0.5% to 1.0% of the mass of the fenugreek seed flesh of trypsin, enzymolyzing it at 35 to 40° C. for 50 to 70 minutes, heating it up to inactivate the enzyme, cooling it down to room temperature, , adjust the pH to 5.0-7.0, add 0.5%-1.0% papain by weight of fenugreek seed pulp, perform enzymolysis at 55°C-65°C for 50min-70min, raise the temperature to inactivate the enzyme, cool to room temperature, centrifuge at 4000r / min-6000r / min for 10min-20min, collect the supernatant; perform ultrafiltration separation on the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5kDa, collect the permeate, concentrate and dry under reduced pressure at 40°C-50°C to achieve a water content of less than 5%, and obtain a fenugreek seed extract.

7. A uric acid-lowering pharmaceutical composition according to claim 1, characterized in that: The preparation method of the chicory extract comprises the following steps: crushing chicory roots, using ethanol 8 to 10 times the mass of the chicory roots for percolation to obtain an extract, concentrating and drying at 60 to 80° C. to a water content of less than 5%, and obtaining the chicory extract.

8. A uric acid-lowering pharmaceutical composition according to claim 1, characterized in that: The preparation method of the plantain extract comprises the following steps: taking the whole plant of plantain and crushing it, using ethanol with a mass of 8 to 10 times that of the whole plant of plantain for percolation to obtain an extract, concentrating and drying at 60° C. to 80° C. to a water content of less than 5%, and obtaining the plantain extract.

9. A uric acid-lowering pharmaceutical composition according to claim 2 or 6, characterized in that: The temperature for heating and inactivating the enzyme is 90° C. to 95° C., and the time for heating and inactivating the enzyme is 20 min to 30 min.

10. A method for preparing a uric acid-lowering pharmaceutical composition, characterized in that: The preparation method comprises the following steps: uniformly mixing bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract according to weight proportions, then adding earthworm peptide and mixing uniformly to obtain a pharmaceutical composition.

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