A pharmaceutical composition for reducing uric acid and its preparation method

Abstract

The present invention relates to a pharmaceutical composition for reducing uric acid and a preparation method thereof, belonging to the field of biotechnology. The pharmaceutical composition contains earthworm peptides, fungal fermentation products, fenugreek seed extract, chicory extract, and plantain extract; the earthworm peptides contain products obtained by successively subjecting earthworms to hierarchical enzymatic hydrolysis with trypsin, lumbrokinase, and nattokinase; the fungal fermentation products contain products obtained by jointly fermenting the pulp of cornel and smilax glabra rhizome with bacillus subtilis and bifidobacterium adolescentis; the fenugreek seed extract contains products obtained by successively subjecting fenugreek seed kernels to hierarchical enzymatic hydrolysis with ficin, trypsin, and papain; the chicory extract is an ethanol percolation extract of chicory roots; the plantain extract is an ethanol percolation extract of plantain. The combined use of each component has good uric acid reducing and anti-inflammatory effects, can quickly and efficiently relieve inflammation and reduce the blood uric acid content, and is safe and reliable.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a pharmaceutical composition for reducing uric acid 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 generation and excretion of uric acid are in a balanced state, maintaining the stability of the uric acid level in the body. However, when purine metabolism is disordered or uric acid excretion is reduced, the blood uric acid level will increase. Long-term hyperuricemia may lead to various serious health problems such as gout, uric acid nephropathy, and cardiovascular diseases.

[0003] With the improvement of living standards and the change of dietary structure, the prevalence of hyperuricemia and gout has been increasing year by year globally, which makes the research and development and application of uric acid-lowering drugs become 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 uric acid synthesis by inhibiting the activity of xanthine oxidase; however, some patients will have severe allergic reactions, including exfoliative dermatitis, etc., which to a certain extent limits its use. Drugs that promote uric acid excretion, such as benzbromarone, can inhibit the reabsorption of uric acid by the renal tubules, thereby increasing uric acid excretion; but these drugs may cause adverse reactions such as gastrointestinal discomfort, liver and kidney function damage, and the formation of uric acid kidney stones, especially when used in patients with renal insufficiency, it needs to be carefully evaluated. In addition, there are also some traditional Chinese medicine therapies, but there are problems such as a long cycle and slow short-term efficacy.

[0004] Currently, the research and development of uric acid-lowering drugs still face many challenges, and it is necessary to continuously optimize the components of the drugs, improve the efficacy and safety, reduce the occurrence of adverse reactions, and shorten the treatment course; 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] Aiming at the problems of poor safety, easy side effects, slow efficacy, and long treatment course existing in the existing uric acid-lowering drugs, the present invention provides a pharmaceutical composition for reducing uric acid and a preparation method thereof. The pharmaceutical composition contains earthworm peptides obtained by specific enzymatic hydrolysis, fermentation products prepared by fermentation of specific substrates and bacteria, fenugreek seed extracts obtained by specific enzymatic hydrolysis, chicory extracts obtained by ethanol percolation extraction, and plantain extracts obtained by ethanol percolation extraction. The combined use of each component has good uric acid-lowering and anti-inflammatory effects, can quickly and efficiently relieve inflammation and reduce the blood uric acid content, and is safe and reliable. The specific technical solutions are as follows:

[0006] A pharmaceutical composition for reducing uric acid, comprising raw materials in the following parts by mass: 1 to 3 parts of earthworm peptide, 1 to 3 parts of fungal fermentation product, 0.2 to 0.5 part of fenugreek seed extract, 0.5 to 1.5 parts of chicory extract, 1 to 3 parts of plantain extract; the earthworm peptide contains the products obtained by sequentially fractionating and enzymolyzing earthworms with trypsin, lumbrokinase, and nattokinase; the fungal fermentation product contains the product obtained by jointly fermenting the pulp of cornel and glabrous greenbrier with Bacillus subtilis and Bifidobacterium adolescentis; the fenugreek seed extract contains the products obtained by sequentially fractionating and enzymolyzing fenugreek seed meat with ficin, trypsin, and papain; the chicory extract is an ethanol percolation extract of chicory root; the plantain extract is an ethanol percolation extract of plantain.

[0007] In the above pharmaceutical composition, the preparation method of the earthworm peptide comprises the following steps: selecting fresh and healthy earthworms, washing them, and performing freeze-drying treatment to make the water content less than 5%; pulverizing the dried earthworms into powder, passing through a 60-mesh to 80-mesh sieve, and taking the powder under the sieve; according to the mass ratio, powder:Tris-HCl buffer solution = 1:(8 - 12), adding the powder into the Tris-HCl buffer solution with a pH of 7.5 - 8.0, mixing evenly to form a suspension; adding 0.5% - 1.0% of the mass of the powder of trypsin to the suspension, enzymolyzing at 35°C - 38°C for 50 min - 70 min, raising the temperature to 50°C - 55°C, adding 0.5% - 1.0% of the mass of the powder of lumbrokinase, enzymolyzing at 50°C - 55°C for 60 min - 90 min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 6.5 - 7.0, adding 0.5% - 1.0% of the mass of the powder of nattokinase, enzymolyzing at 50°C - 55°C for 60 min - 90 min, heating to inactivate the enzyme, cooling to room temperature, centrifuging at 4000 r / min - 6000 r / min for 10 min - 20 min, and collecting the supernatant; ultrafiltering and separating the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, collecting the permeate to obtain a crude earthworm peptide extract; loading the crude earthworm peptide extract onto a macroporous adsorption resin column chromatography, first rinsing the resin column with deionized water to remove unadsorbed impurities, then eluting with an ethanol aqueous solution with a volume concentration of 20% - 30%, collecting the eluate, concentrating, and freeze-drying to a water content less than 5% to obtain the earthworm peptide.

[0008] In the above method for preparing earthworm peptides, the macroporous resin type of the macroporous adsorption resin column is D101 type; the sample loading flow rate of the crude earthworm peptide extract is 1 mL / min to 3 mL / min; the amount of deionized water used is 1 to 2 resin column volumes, and the rinsing flow rate is 3 mL / min to 5 mL / min; the amount of ethanol aqueous solution used is 2 to 4 resin column volumes, and the elution flow rate is 1 mL / min to 3 mL / min; the concentration is carried out by rotary evaporation at 40 °C to 50 °C and 0.08 MPa to 0.1 MPa, and the eluate is concentrated to 10% to 20% of the volume.

[0009] In the above method for preparing the bacterial ferment, the preparation method of the bacterial ferment includes the following steps: pulverize the fructus corni pulp, pulverize the rhizoma smilacis glabrae, and prepare a basal solution according to the mass ratio of water: fructus corni 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), perform sterilization treatment, and after cooling to room temperature, obtain a culture solution; inoculate 2% v / v to 5% v / v of Bacillus subtilis and 2% v / v to 5% v / v of Bifidobacterium adolescentis into the culture solution, mix evenly, first carry out aerobic fermentation at 30 °C to 37 °C for 24 h to 36 h to promote the growth and metabolism of Bacillus subtilis, and then carry out anaerobic fermentation at 35 °C to 37 °C for 48 h to 72 h to promote the growth and metabolism of Bifidobacterium adolescentis, obtain a fermentation broth, centrifuge at 4000 r / min to 6000 r / min for 10 min to 20 min, collect the supernatant, and concentrate and dry at 100 °C to 120 °C until the water content is below 5% to obtain the bacterial ferment.

[0010] In the above method for preparing the bacterial ferment, 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.

[0011] In the above-mentioned pharmaceutical composition, the preparation method of the fenugreek seed extract comprises the following steps: Take fenugreek seed kernels, crush them, add distilled water with a mass 8 to 12 times that of the fenugreek seed kernels, add ficin with a mass of 0.5% to 1.5% of the fenugreek seed kernels, enzymatically hydrolyze at 60°C to 65°C for 60 min to 90 min, raise the temperature to inactivate the enzyme, after cooling to room temperature, adjust the pH to 8.0 to 8.5, add trypsin with a mass of 0.5% to 1.0% of the fenugreek seed kernels, enzymatically hydrolyze at 35°C to 40°C for 50 min to 70 min, raise the temperature to inactivate the enzyme, after cooling to room temperature, adjust the pH to 5.0 to 7.0, add papain with a mass of 0.5% to 1.0% of the fenugreek seed kernels, enzymatically hydrolyze at 55°C to 65°C for 50 min to 70 min, raise the temperature to inactivate the enzyme, after cooling to room temperature, centrifuge at 4000 r / min to 6000 r / min for 10 min to 20 min, and collect the supernatant; the supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, the permeate is collected, and concentrated and dried under reduced pressure at 40°C to 50°C until the water content is below 5% to obtain the fenugreek seed extract.

[0012] In the above-mentioned pharmaceutical composition, the preparation method of the chicory extract comprises the following steps: Take chicory roots, crush them, percolate with ethanol with a mass 8 to 10 times that of the chicory roots to obtain an extract, and concentrate and dry at 60°C to 80°C until the water content is below 5% to obtain the chicory extract.

[0013] In the above-mentioned pharmaceutical composition, the preparation method of the plantain extract comprises the following steps: Take the whole plant of plantain, crush it, percolate with ethanol with a mass 8 to 10 times that of the whole plant of plantain to obtain an extract, and concentrate and dry at 60°C to 80°C until the water content is below 5% to obtain the plantain extract.

[0014] In the preparation method of the above raw materials, the temperature for raising the temperature to inactivate the enzyme is 90°C to 95°C, and the time for raising the temperature to inactivate the enzyme is 20 min to 30 min.

[0015] The preparation method of the above-mentioned pharmaceutical composition for reducing uric acid comprises the following steps: By mass parts, mix the fungal fermentation product, fenugreek seed extract, chicory extract and plantain extract evenly, and then add earthworm peptides and mix evenly to obtain the pharmaceutical composition.

[0016] A pharmaceutical composition for reducing uric acid and its preparation method provided by the present invention have the following beneficial effects:

[0017] 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.

[0018] 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.

[0019] III. The pulp of Cornus officinalis contains abundant flavonoid compounds. During the fermentation process by bacteria, these flavonoid compounds will be transformed into more bioactive forms by microorganisms (Bacillus subtilis and Bifidobacterium adolescentis). For example, the enzymes of microorganisms will modify the flavonoid compounds, such as glycosylation or methylation, changing their chemical structures and making them more easily absorbed by the human body. These transformed flavonoid compounds work synergistically with the microbial metabolites to exert antioxidant effects. The antioxidant effect is important for reducing uric acid because oxidative stress can damage the cells of organs such as the kidneys, affecting the normal metabolism and excretion of uric acid. Flavonoid compounds indirectly facilitate uric acid metabolism and reduce the accumulation of uric acid in the body by scavenging free radicals and alleviating oxidative stress. The polysaccharide component in the pulp of Cornus officinalis provides a carbon source for microorganisms. At the initial stage of fermentation, Bacillus subtilis utilizes these polysaccharides for growth and reproduction. The massive reproduction of microorganisms will produce more enzymes and metabolites, which can further decompose other components in the pulp of Cornus officinalis, releasing more substances beneficial for reducing uric acid. For example, the enzymes produced by microorganisms will decompose some proteins in the pulp of Cornus officinalis, releasing nutrients such as amino acids. These nutrients will interact with other components during the subsequent fermentation process or be further transformed by microorganisms into substances with the function of regulating uric acid metabolism.

[0020] IV. Smilax glabra Roxb. contains various components such as steroidal saponins and alkaloids. During the fermentation process, these components will be decomposed or transformed by microorganisms. For example, the enzymes of microorganisms will hydrolyze steroidal saponins to produce bioactive secondary metabolites. These secondary metabolites have the function of regulating the intestinal flora, changing the composition of the intestinal flora in a direction beneficial to uric acid decomposition and excretion. The alkaloid components will be transformed into substances that promote uric acid excretion after fermentation. For example, by affecting the transport of uric acid by renal tubular cells in the kidneys, the excretion of uric acid is increased. Smilax glabra Roxb. itself has a certain diuretic effect, and its active components are enhanced after fermentation. The diuretic effect is very important for reducing 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 Roxb., making its diuretic effect more significant. For example, microorganisms will transform some components in Smilax glabra Roxb. into substances that can regulate the glomerular filtration rate or tubular reabsorption function of the kidneys, thereby promoting urine production and enabling more uric acid to be excreted with urine.

[0021] V. During the preparation of fenugreek seed extract, ficin, trypsin and papain are successively used for enzymatic hydrolysis, which can specifically decompose proteins and generate peptide segments with specific amino acid sequences. These peptide segments contain sequences that can inhibit the activity of uric acid-producing enzymes (such as xanthine oxidase), thereby reducing the production of uric acid. Moreover, they are easily absorbed by the human body and can enter cells faster to play their roles. For example, they can regulate the activity of uric acid metabolism-related enzymes in cells, affect the uric acid transport process in cells, and thus play a synergistic role in reducing uric acid. In addition, by improving blood circulation in the kidneys, they can promote the transport and excretion of uric acid. Fenugreek seed extract has a protective effect on kidney tissues and anti-inflammatory properties, reducing the accumulation of serum creatinine and blood urea nitrogen, and decreasing the synthesis and release of TNF-α and IL-6.

[0022] VI. Chicory extract contains components 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 multiple transporters. Chicory extract promotes the secretion of uric acid from renal tubular cells into the lumen, increasing the excretion of uric acid. In addition, chicory extract also has a certain diuretic effect, indirectly promoting the excretion of uric acid by increasing urine volume, thereby reducing the level of blood uric acid. Chicory extract can reduce inflammation and oxidative stress damage in kidney tissues and maintain the normal filtration function of the kidneys.

[0023] VII. Plantain extract mainly promotes uric acid excretion by increasing urine production, regulating the glomerular filtration rate and the reabsorption function of renal tubules, so that more uric acid is excreted from the body with urine. At the same time, plantain extract also contains some components that can inhibit the reabsorption of uric acid in renal tubules, further increasing the excretion of uric acid, thereby reducing the level of blood uric acid.

[0024] VIII. Earthworm peptides mainly reduce uric acid by regulating the enzyme activity and kidney function in the body, while the fungal ferment focuses on regulating the balance of intestinal flora and metabolism. When they work together, the fungal ferment regulates the intestinal flora, reduces the absorption of purines, and decreases 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 uric acid production from both the source and the metabolic process. In terms of uric acid excretion, the fungal ferment improves the intestinal environment and promotes the excretion of uric acid with feces, while earthworm peptides regulate kidney function and promote the excretion of uric acid from urine. The combined action of the two enhances the excretion effect of uric acid and more effectively reduces the blood uric acid level. Earthworm peptides have a kidney-protecting effect and can reduce the damage of kidney tissue caused by urate crystals. The fungal ferment reduces the entry of harmful substances into the blood by regulating the intestinal flora and alleviates the kidney burden. Their combined action can better maintain the normal structure and function of the kidneys and reduce the increase of blood creatinine and blood urea nitrogen. Both earthworm peptides and the fungal ferment 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. The fungal ferment inhibits the over-activation of inflammatory cells and reduces the production of inflammatory factors by regulating the immune system function. Their combined action can more effectively reduce the damage of the inflammatory response to the kidneys and other organs, creating a good environment for uric acid metabolism and kidney function maintenance. Detailed implementation mode

[0025] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0026] Example 1: A pharmaceutical composition for reducing uric acid, comprising the following raw materials in parts by mass: 2 parts of earthworm peptides, 2 parts of fungal ferment, 0.3 part of fenugreek seed extract, 1 part of chicory extract, and 2 parts of plantain extract.

[0027] Among them, the preparation method of earthworm peptides includes the following steps: Select fresh and healthy earthworms, wash them, and perform freeze-drying treatment to make the water content 4.2%; crush the dried earthworms into powder, pass through an 80-mesh sieve, and take the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:10, add the powder to 0.08 mol / L Tris-HCl buffer with a pH of 7.8, mix evenly to form a suspension; add 0.8% of the mass of the powder of trypsin to the suspension, enzymatically hydrolyze at 37 °C for 60 min, raise the temperature to 52 °C, add 0.8% of the mass of the powder of lumbrokinase, enzymatically hydrolyze at 52 °C for 80 min, raise the temperature to 92 °C to inactivate the enzyme for 25 min, after cooling to room temperature, adjust the pH to 6.8, add 0.8% of the mass of the powder of nattokinase, enzymatically hydrolyze at 53 °C for 80 min, raise the temperature to 94 °C to inactivate the enzyme for 25 min, after cooling to room temperature, centrifuge at 5000 r / min for 15 min, and collect the supernatant; ultrafilter and separate the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, collect the permeate, and obtain a crude extract of earthworm peptides; load the crude extract of earthworm peptides onto a macroporous adsorption resin column chromatography at a flow rate of 2 mL / min. The macroporous resin model is D101. First, wash the resin column with 1 resin column volume of deionized water at a flow rate of 4 mL / min to remove unadsorbed impurities, and then elute with 3 resin column volumes of 25% (v / v) ethanol aqueous solution at a flow rate of 2 mL / min. Collect all the eluates of the ethanol aqueous solution, perform rotary evaporation and concentration to 15% (v / v) at 45 °C and 0.09 MPa, and freeze-dry to a water content of 4.5% to obtain earthworm peptides.

[0028] Among them, the preparation method of the bacterial ferment includes the following steps: Crush the fructus corni pulp and crush the smilax glabra. According to the mass ratio of water: fructus corni pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 95:5:6:2:1.5:0.2:0.08, prepare the base liquid, and perform sterilization treatment at 123 °C for 30 min. After cooling to room temperature, obtain the culture solution; inoculate the culture solution with 3% (v / v) of an activated bacterial solution of Bacillus subtilis with a concentration of 500 million CFU / mL and 3% (v / v) of an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 300 million CFU / mL, mix evenly, first perform aerobic fermentation at 35 °C for 30 h to promote the growth and metabolism of Bacillus subtilis, and then perform anaerobic fermentation at 36 °C for 60 h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain a fermentation broth. Centrifuge at 5000 r / min for 15 min, collect the supernatant, and concentrate and dry at 110 °C to reach a water content of 4.6% to obtain the bacterial ferment.

[0029] Among them, the preparation method of the fenugreek seed extract includes the following steps: Take fenugreek seed kernels and crush them, add distilled water with a mass 10 times that of the fenugreek seed kernels, add ficin with a mass of 1% of the fenugreek seed kernels, enzymatically hydrolyze at 62°C for 80 min, raise the temperature to 92°C to inactivate the enzyme for 25 min, after cooling to room temperature, adjust the pH to 8.2, add trypsin with a mass of 0.8% of the fenugreek seed kernels, enzymatically hydrolyze at 37°C for 60 min, raise the temperature to 93°C to inactivate the enzyme for 25 min, after cooling to room temperature, adjust the pH to 6.2, add papain with a mass of 0.6% of the fenugreek seed kernels, enzymatically hydrolyze at 60°C for 60 min, raise the temperature to 92°C to inactivate the enzyme for 25 min, after cooling to room temperature, centrifuge at 5000 r / min for 15 min, and collect the supernatant; the supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, the permeate is collected, and concentrated and dried under reduced pressure at 45°C until the water content reaches 4.3% to obtain the fenugreek seed extract.

[0030] Among them, the preparation method of the chicory extract includes the following steps: Take chicory roots and crush them, percolate with ethanol with a mass 9 times that of the chicory roots to obtain an extract, and concentrate and dry at 70°C until the water content is 3.8% to obtain the chicory extract.

[0031] Among them, the preparation method of the plantain extract includes the following steps: Take the whole plant of plantain and crush it, percolate with ethanol with a mass 9 times that of the whole plant of plantain to obtain an extract, and concentrate and dry at 70°C until the water content is 4.3% to obtain the plantain extract.

[0032] The preparation method of the above-mentioned anti-hyperuricemic pharmaceutical composition includes the following steps: By mass fraction, mix the fungal ferment, fenugreek seed extract, chicory extract and plantain extract evenly, and then add earthworm peptide and mix evenly to obtain the pharmaceutical composition.

[0033] Example 2: An anti-hyperuricemic pharmaceutical composition, comprising the following raw materials by mass fraction: 1 part of earthworm peptide, 1 part of fungal ferment, 0.2 part of fenugreek seed extract, 0.5 part of chicory extract, and 1 part of plantain extract.

[0034] Among them, the preparation method of earthworm peptides includes the following steps: Select fresh and healthy earthworms, wash them, and perform freeze-drying treatment to make the water content 3%; crush the dried earthworms into powder, pass through a 60-mesh sieve, and take the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:8, add the powder to 0.08 mol / L Tris-HCl buffer with a pH of 7.5, mix evenly to form a suspension; add 0.5% of the mass of the powder of trypsin to the suspension, enzymatically hydrolyze at 35°C for 50 min, raise the temperature to 50°C, add 0.5% of the mass of the powder of earthworm kinase, enzymatically hydrolyze at 50°C for 60 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, after cooling to room temperature, adjust the pH to 6.5, add 0.5% of the mass of the powder of nattokinase, enzymatically hydrolyze at 50°C for 60 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, after cooling to room temperature, centrifuge at 4000 r / min for 10 min, and collect the supernatant; ultrafilter and separate the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, collect the permeate to obtain a crude extract of earthworm peptides; load the crude extract of earthworm peptides onto a macroporous adsorption resin column chromatography at a flow rate of 1 mL / min. The macroporous resin model is D101. First, wash the resin column with 1 resin column volume of deionized water at a flow rate of 3 mL / min to remove unadsorbed impurities, then elute with 2 resin column volumes of 20% (v / v) ethanol aqueous solution at a flow rate of 1 mL / min, collect the eluate of all ethanol aqueous solutions, perform rotary evaporation and concentration to 10% (v / v) at 40°C and 0.08 MPa, and freeze-dry to a water content of 3.2% to obtain earthworm peptides.

[0035] Among them, the preparation method of the bacterial ferment includes the following steps: Crush the pulp of Cornus officinalis, crush Smilax glabra, and prepare a basal solution according to the mass ratio of water:pulp of Cornus officinalis:Smilax glabra:glucose:soy peptone:potassium dihydrogen phosphate:magnesium sulfate = 90:3:5:1:1:0.1:0.05. Sterilize at 121°C for 20 min, and after cooling to room temperature, obtain a culture solution; inoculate the culture solution with 2% (v / v) of an activated bacterial solution of Bacillus subtilis with a concentration of 100 million CFU / mL and 2% (v / v) of an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 200 million CFU / mL, mix evenly, first perform aerobic fermentation at 30°C for 24 h to promote the growth and metabolism of Bacillus subtilis, and then perform anaerobic fermentation at 35°C for 48 h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain a fermentation broth. Centrifuge at 4000 r / min for 10 min, collect the supernatant, concentrate and dry at 100°C to reach a water content of 3.5% to obtain the bacterial ferment.

[0036] Among them, the preparation method of the fenugreek seed extract includes the following steps: Take fenugreek seed kernels and crush them, add distilled water with a mass 8 times that of the fenugreek seed kernels, add ficin with a mass of 0.5% of the fenugreek seed kernels, enzymatically hydrolyze at 60°C for 60 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, after cooling to room temperature, adjust the pH to 8.0, add trypsin with a mass of 0.5% of the fenugreek seed kernels, enzymatically hydrolyze at 35°C for 50 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, after cooling to room temperature, adjust the pH to 5.0, add papain with a mass of 0.5% of the fenugreek seed kernels, enzymatically hydrolyze at 55°C for 50 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, after cooling to room temperature, centrifuge at 4000 r / min for 10 min, and collect the supernatant; The supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, the permeate is collected, and concentrated and dried under reduced pressure at 40°C until the water content reaches 3.8% to obtain the fenugreek seed extract.

[0037] Among them, the preparation method of the chicory extract includes the following steps: Take chicory roots and crush them, percolate with ethanol with a mass 8 times that of the chicory roots to obtain an extract, and concentrate and dry at 60°C until the water content is 4.1% to obtain the chicory extract.

[0038] Among them, the preparation method of the plantain extract includes the following steps: Take the whole plant of plantain and crush it, percolate with ethanol with a mass 8 times that of the whole plant of plantain to obtain an extract, and concentrate and dry at 60°C until the water content is 5% to obtain the plantain extract.

[0039] The preparation method of the above-mentioned anti-hyperuricemic pharmaceutical composition includes the following steps: By mass, mix the fungal fermentation product, fenugreek seed extract, chicory extract and plantain extract evenly, and then add earthworm peptides and mix evenly to obtain the pharmaceutical composition.

[0040] Example 3: An anti-hyperuricemic pharmaceutical composition, comprising the following raw materials in parts by mass: 1 part of earthworm peptides, 3 parts of fungal fermentation product, 0.2 part of fenugreek seed extract, 1.5 parts of chicory extract, and 1 part of plantain extract.

[0041] Among them, the preparation method of earthworm peptides includes the following steps: select fresh and healthy earthworms, wash them, and perform freeze-drying treatment to make the water content 5%; crush the dried earthworms into powder, pass through a 60-mesh sieve, and take the powder under the sieve; according to the mass ratio, powder: Tris-HCl buffer = 1:12, add the powder to 0.08 mol / L Tris-HCl buffer with a pH of 7.5, mix evenly to form a suspension; add 1.0% of the mass of the powder of trypsin to the suspension, enzymatically hydrolyze at 35 °C for 70 min, raise the temperature to 50 °C, add 0.5% of the mass of the powder of earthworm kinase, enzymatically hydrolyze at 50 °C for 90 min, raise the temperature to 90 °C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 6.5, add 1.0% of the mass of the powder of nattokinase, enzymatically hydrolyze at 50 °C for 90 min, raise the temperature to 90 °C to inactivate the enzyme for 30 min, after cooling to room temperature, centrifuge at 4000 r / min for 20 min, and collect the supernatant; ultrafilter and separate the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, collect the permeate to obtain a crude extract of earthworm peptides; load the crude extract of earthworm peptides onto a macroporous adsorption resin column chromatography at a flow rate of 1 mL / min. The macroporous resin model is D101. First, wash the resin column with 2 resin column volumes of deionized water at a flow rate of 3 mL / min to remove unadsorbed impurities, then elute with 4 resin column volumes of 20% (v / v) ethanol aqueous solution at a flow rate of 3 mL / min, collect the eluate of all ethanol aqueous solutions, perform rotary evaporation and concentration to 10% (v / v) at 40 °C and 0.1 MPa, and freeze-dry to a water content of 4.8% to obtain earthworm peptides.

[0042] Among them, the preparation method of the bacterial ferment includes the following steps: crush the fructus corni pulp and crush the smilax glabra; prepare a basal solution according to the mass ratio of water: fructus corni pulp: smilax glabra: glucose: soy peptone: potassium dihydrogen phosphate: magnesium sulfate = 90:6:5:3:1:0.3:0.05, perform sterilization treatment at 125 °C for 20 min, and after cooling to room temperature, obtain a culture solution; inoculate the culture solution with an activated bacterial solution of Bacillus subtilis with a concentration of 800 million CFU / mL at 5% (v / v) and an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 500 million CFU / mL at 2% (v / v), mix evenly, first perform aerobic fermentation at 30 °C for 36 h to promote the growth and metabolism of Bacillus subtilis, and then perform anaerobic fermentation at 35 °C for 72 h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain a fermentation broth, centrifuge at 4000 r / min for 20 min, collect the supernatant, and concentrate and dry at 100 °C to reach a water content of 3% to obtain the bacterial ferment.

[0043] Among them, the preparation method of the fenugreek seed extract includes the following steps: Take fenugreek seed kernels and crush them, add distilled water with a mass 8 times that of the fenugreek seed kernels, add ficin with a mass of 1.5% of the fenugreek seed kernels, enzymatically hydrolyze at 60 °C for 90 min, raise the temperature to 90 °C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 8.0, add trypsin with a mass of 1.0% of the fenugreek seed kernels, enzymatically hydrolyze at 35 °C for 70 min, raise the temperature to 90 °C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 5.0, add papain with a mass of 1.0% of the fenugreek seed kernels, enzymatically hydrolyze at 55 °C for 70 min, raise the temperature to 90 °C to inactivate the enzyme for 30 min, after cooling to room temperature, centrifuge at 4000 r / min for 20 min, and collect the supernatant; The supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, the permeate is collected, and concentrated and dried under reduced pressure at 40 °C until the water content reaches 5% to obtain the fenugreek seed extract.

[0044] Among them, the preparation method of the chicory extract includes the following steps: Take chicory roots and crush them, percolate with ethanol with a mass 8 times that of the chicory roots to obtain an extract, and concentrate and dry at 80 °C until the water content is 3.6% to obtain the chicory extract.

[0045] Among them, the preparation method of the plantain extract includes the following steps: Take the whole plant of plantain and crush it, percolate with ethanol with a mass 10 times that of the whole plant of plantain to obtain an extract, and concentrate and dry at 60 °C until the water content is 3% to obtain the plantain extract.

[0046] The preparation method of the above-mentioned anti-hyperuricemic pharmaceutical composition includes the following steps: By mass fraction, mix the fungal fermentation product, fenugreek seed extract, chicory extract, and plantain extract evenly, and then add earthworm peptides and mix evenly to obtain the pharmaceutical composition.

[0047] Example 4: An anti-hyperuricemic pharmaceutical composition, comprising the following raw materials by mass fraction: 1.5 parts of earthworm peptides, 2.5 parts of fungal fermentation product, 0.4 part of fenugreek seed extract, 1.2 parts of chicory extract, and 2.6 parts of plantain extract.

[0048] Among them, the preparation method of earthworm peptides includes the following steps: Select fresh and healthy earthworms, wash them, and perform freeze-drying treatment to make the water content 4.5%; crush the dried earthworms into powder, pass through an 80-mesh sieve, and take the powder under the sieve; according to the mass ratio of powder:Tris-HCl buffer solution = 1:9, add the powder to 0.08 mol / L Tris-HCl buffer solution with a pH of 7.6, mix evenly to form a suspension; add trypsin at 0.6% of the mass of the powder to the suspension, enzymatically hydrolyze at 36 °C for 65 min, raise the temperature to 55 °C, add earthworm kinase at 0.9% of the mass of the powder, enzymatically hydrolyze at 55 °C for 70 min, raise the temperature to 91 °C to inactivate the enzyme for 25 min, after cooling to room temperature, adjust the pH to 6.6, add nattokinase at 0.6% of the mass of the powder, enzymatically hydrolyze at 53 °C for 80 min, raise the temperature to 90 °C to inactivate the enzyme for 20 min, after cooling to room temperature, centrifuge at 6000 r / min for 15 min, and collect the supernatant; ultrafilter and separate the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, collect the permeate to obtain a crude extract of earthworm peptides; load the crude extract of earthworm peptides onto a macroporous adsorption resin column chromatography at a flow rate of 2.5 mL / min. The macroporous resin model is D101. First, wash the resin column with 2 resin column volumes of deionized water at a flow rate of 4.5 mL / min to remove unadsorbed impurities, then elute with 3 resin column volumes of 25% (v / v) ethanol aqueous solution at a flow rate of 2.5 mL / min, collect the eluate of all ethanol aqueous solutions, perform rotary evaporation and concentration at 50 °C and 0.08 MPa to 18% (v / v), and freeze-dry to a water content of 3% to obtain earthworm peptides.

[0049] Among them, the preparation method of the bacterial ferment includes the following steps: Crush the fructus corni pulp and crush the smilax glabra. According to the mass ratio of water:fructus corni pulp:smilax glabra:glucose:soy peptone:potassium dihydrogen phosphate:magnesium sulfate = 96:5:7:2.5:1.5:0.3:0.06, prepare the basal liquid, and perform sterilization treatment at 121 °C for 35 min. After cooling to room temperature, obtain the culture solution; inoculate the culture solution with an activated bacterial solution of Bacillus subtilis with a concentration of 1 billion CFU / mL at 3% (v / v) and an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 200 million CFU / mL at 3% (v / v), mix evenly, first perform aerobic fermentation at 35 °C for 32 h to promote the growth and metabolism of Bacillus subtilis, and then perform anaerobic fermentation at 37 °C for 65 h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain the fermentation broth. Centrifuge at 6000 r / min for 20 min, collect the supernatant, and concentrate and dry at 120 °C to reach a water content of 5% to obtain the bacterial ferment.

[0050] Among them, the preparation method of the fenugreek seed extract comprises the following steps: Take fenugreek seed kernels, crush them, add distilled water with a mass 11 times that of the fenugreek seed kernels, add ficin with a mass of 1.2% of the fenugreek seed kernels, enzymatically hydrolyze at 63 °C for 85 min, raise the temperature to 90 °C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 8.1, add trypsin with a mass of 0.8% of the fenugreek seed kernels, enzymatically hydrolyze at 40 °C for 50 min, raise the temperature to 95 °C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 5.8, add papain with a mass of 0.6% of the fenugreek seed kernels, enzymatically hydrolyze at 62 °C for 60 min, raise the temperature to 95 °C to inactivate the enzyme for 30 min, after cooling to room temperature, centrifuge at 5000 r / min for 20 min, and collect the supernatant; the supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, the permeate is collected, concentrated and dried under reduced pressure at 50 °C until the water content reaches 3%, and the fenugreek seed extract is obtained.

[0051] Among them, the preparation method of the chicory extract comprises the following steps: Take chicory roots, crush them, percolate with ethanol with a mass 10 times that of the chicory roots to obtain an extract, concentrate and dry at 70 °C until the water content is 3%, and the chicory extract is obtained.

[0052] Among them, the preparation method of the plantain extract comprises the following steps: Take the whole plant of plantain, crush it, percolate with ethanol with a mass 10 times that of the whole plant of plantain to obtain an extract, concentrate and dry at 80 °C until the water content is 4.2%, and the plantain extract is obtained.

[0053] The preparation method of the above-mentioned anti-hyperuricemic pharmaceutical composition comprises the following steps: By mass, mix the fungal fermentation product, fenugreek seed extract, chicory extract and plantain extract evenly, and then add earthworm peptides and mix evenly to obtain the pharmaceutical composition.

[0054] Example 5: An anti-hyperuricemic pharmaceutical composition, comprising the following raw materials in parts by mass: 3 parts of earthworm peptides, 3 parts of fungal fermentation product, 0.5 part of fenugreek seed extract, 1.5 parts of chicory extract, and 3 parts of plantain extract.

[0055] Among them, the preparation method of earthworm peptides includes the following steps: Select fresh and healthy earthworms, wash them, and perform freeze-drying treatment to make the moisture content 3.8%; crush the dried earthworms into powder, pass through an 80-mesh sieve, and take the powder under the sieve; according to the mass ratio, powder:Tris-HCl buffer solution = 1:12, add the powder to 0.08 mol / L Tris-HCl buffer solution with a pH of 8.0, mix evenly to form a suspension; add 1.0% of the mass of the powder of trypsin to the suspension, enzymatically hydrolyze at 38 °C for 70 min, raise the temperature to 55 °C, add 1.0% of the mass of the powder of earthworm kinase, enzymatically hydrolyze at 55 °C for 90 min, raise the temperature to 95 °C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 7.0, add 1.0% of the mass of the powder of nattokinase, enzymatically hydrolyze at 55 °C for 90 min, raise the temperature to 95 °C to inactivate the enzyme for 30 min, after cooling to room temperature, centrifuge at 6000 r / min for 20 min, and collect the supernatant; the supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and the permeate is collected to obtain a crude extract of earthworm peptides; at a flow rate of 3 mL / min, the crude extract of earthworm peptides is loaded onto a macroporous adsorption resin column chromatography, the macroporous resin model is D101 type, first wash the resin column with 2 resin column volumes of deionized water at a flow rate of 5 mL / min to remove unadsorbed impurities, then elute with 4 resin column volumes of 30% (v / v) ethanol aqueous solution at a flow rate of 3 mL / min, collect the eluate of all ethanol aqueous solutions, perform rotary evaporation and concentration at 50 °C and 0.1 MPa to 20% (v / v), and freeze-dry to a moisture content of 5% to obtain earthworm peptides.

[0056] Among them, the preparation method of the bacterial ferment includes the following steps: Crush the fructus corni pulp and crush the smilax glabra; according to the mass ratio of water:fructus corni pulp:smilax glabra:glucose:soy peptone:potassium dihydrogen phosphate:magnesium sulfate = 100:6:8:3:2:0.3:0.1, prepare the base solution, perform sterilization treatment at 125 °C for 40 min, and after cooling to room temperature, obtain the culture solution; inoculate the culture solution with an activated bacterial solution of Bacillus subtilis with a concentration of 600 million CFU / mL at 5% (v / v) and an activated bacterial solution of Bifidobacterium adolescentis with a concentration of 100 million CFU / mL at 5% (v / v), mix evenly, first perform aerobic fermentation at 37 °C for 36 h to promote the growth and metabolism of Bacillus subtilis, and then perform anaerobic fermentation at 37 °C for 72 h to promote the growth and metabolism of Bifidobacterium adolescentis to obtain a fermentation broth, centrifuge at 6000 r / min for 20 min, collect the supernatant, and concentrate and dry at 120 °C to reach a water content of 3.1% to obtain the bacterial ferment.

[0057] Among them, the preparation method of the fenugreek seed extract includes the following steps: Take fenugreek seed kernels, crush them, add distilled water with a mass 12 times that of the fenugreek seed kernels, add ficin with a mass of 1.5% of the fenugreek seed kernels, enzymatically hydrolyze at 65°C for 90 min, raise the temperature to 95°C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 8.5, add trypsin with a mass of 1.0% of the fenugreek seed kernels, enzymatically hydrolyze at 40°C for 70 min, raise the temperature to 95°C to inactivate the enzyme for 30 min, after cooling to room temperature, adjust the pH to 7.0, add papain with a mass of 1.0% of the fenugreek seed kernels, enzymatically hydrolyze at 65°C for 70 min, raise the temperature to 95°C to inactivate the enzyme for 30 min, after cooling to room temperature, centrifuge at 6000 r / min for 20 min, and collect the supernatant; the supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, the permeate is collected, and concentrated and dried under reduced pressure at 50°C until the water content reaches 4.3% to obtain the fenugreek seed extract.

[0058] Among them, the preparation method of the chicory extract includes the following steps: Take chicory roots, crush them, percolate with ethanol with a mass 10 times that of the chicory roots to obtain an extract, and concentrate and dry at 80°C until the water content is 5% to obtain the chicory extract.

[0059] Among them, the preparation method of the plantain extract includes the following steps: Take the whole plant of plantain, crush it, percolate with ethanol with a mass 10 times that of the whole plant of plantain to obtain an extract, and concentrate and dry at 80°C until the water content is 4.7% to obtain the plantain extract.

[0060] The preparation method of the above-mentioned anti-hyperuricemic pharmaceutical composition includes the following steps: By mass, mix the fungal fermentation product, fenugreek seed extract, chicory extract and plantain extract evenly, and then add earthworm peptides and mix evenly to obtain the pharmaceutical composition.

[0061] In the above-mentioned examples, the pH regulator is an aqueous hydrochloric acid solution with a concentration of 0.5 mol / L and an aqueous sodium hydroxide solution with a concentration of 0.5 mol / L.

[0062] In the above-mentioned examples: The earthworm is Pheretima guillelmi. The trypsin is porcine trypsin, sourced from Shanxi Zhongnuo Biotechnology Co., Ltd., with an enzyme activity of 4000 U / g; the earthworm kinase is sourced from Xi'an Muguo Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; the nattokinase is sourced from Shaanxi Yunhe Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; the Bacillus subtilis is sourced from Jinan Jinyuyuan Biotechnology Co., Ltd.; the Bifidobacterium adolescentis is sourced from Shanxi HeTai Biotechnology Co., Ltd.; the ficin is sourced from Xuzhou Shengyi Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g; the papain is sourced from Shandong Jishuo Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g; the D101 macroporous resin is sourced from Langfang Miaoyang Chemical Industry Co., Ltd.

[0063] Comparative Example 1

[0064] The earthworm peptide was replaced with a sterilized freeze-dried powder obtained by directly pulverizing earthworms; other parameters and methods were the same as in Example 1.

[0065] Comparative Example 2

[0066] In the preparation method of the earthworm peptide, trypsin was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0067] Comparative Example 3

[0068] In the preparation method of the earthworm peptide, lumbrokinase was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0069] Comparative Example 4

[0070] In the preparation method of the earthworm peptide, nattokinase was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0071] Comparative Example 5

[0072] In the preparation method of the bacterial ferment, fructus corni flesh was not added; other parameters and methods were the same as in Example 1.

[0073] Comparative Example 6

[0074] In the preparation method of the bacterial ferment, glabrous greenbrier rhizome was not added; other parameters and methods were the same as in Example 1.

[0075] Comparative Example 7

[0076] In the preparation method of the bacterial ferment, fructus corni flesh was not added, and aerobic fermentation with Bifidobacterium adolescentis was not carried out; other parameters and methods were the same as in Example 1.

[0077] Comparative Example 8

[0078] In the preparation method of the fenugreek seed extract, ficin was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0079] Comparative Example 9

[0080] In the preparation method of the fenugreek seed extract, trypsin was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0081] Comparative Example 10

[0082] In the preparation method of the fenugreek seed extract, papain was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0083] Comparative Example 11

[0084] The fenugreek seed extract was obtained by ethanol percolation extraction: The fenugreek seed kernels were crushed, and percolated with ethanol at 9 times the mass of the fenugreek seed kernels to obtain an extract solution, which was concentrated and dried at 70 °C until the water content was 4.1% to obtain the fenugreek seed extract. Other parameters and methods were the same as those in Example 1.

[0085] Comparative Example 12

[0086] In the pharmaceutical composition, earthworm peptides and fungal fermentates were not added at the same time, and the mass parts of earthworm peptides and fungal fermentates were replaced with starch. Other parameters and methods were the same as those in Example 1.

[0087] The pharmaceutical compositions prepared in the above examples and comparative examples were subjected to safety and efficacy tests.

[0088] I. Safety test

[0089] (1) Acute toxicity test: 180 SPF-grade adult Kunming mice, half male and half female, weighing 18 - 22 g, were randomly divided into 18 groups (10 mice in each group). One group was the blank control group, which was intragastrically administered with normal saline, and the remaining groups were intragastrically administered with the pharmaceutical composition at 2 g / kg body weight. Observation was continuously carried out for 14 days. The general state of the mice (including mental state, activity, diet, respiration, etc.) was observed, and the death situation and weight change of the mice were recorded. The results showed that there was no death in each group, the diet, activity, and mental state were normal, the weight continued to increase, and there was no significant difference compared with the blank control group, indicating that the pharmaceutical composition had no obvious acute toxicity even at high doses.

[0090] (2) Long-term toxicity test: 180 SPF-grade adult male SD rats, weighing 230 - 280 g, half male and half female, were randomly divided into 18 groups (10 rats in each group). One group was the blank control group, which was intragastrically administered with normal saline, and the remaining groups were intragastrically administered with the pharmaceutical composition at 500 mg / kg body weight once a day for 90 consecutive days. The general condition of the rats during the drug administration period, including mental state, activity, diet, respiration, etc., was observed. After the test ended, the rats were sacrificed, and the main organs of the rats (including heart, liver, spleen, lung, kidney) were dissected. The results showed that the daily behavior of the rats was normal and there were no lesions in each organ, indicating that the long-term safety of each composition was good.

[0091] (3) Guinea pig active systemic anaphylaxis test: Select 54 healthy adult guinea pigs, randomly divided into 18 groups (3 in each group). Sensitization was carried out on the 0th, 7th, and 14th days, that is, each composition sample (3 mL, composition concentration 0.1 mg / ml, solvent water) was given by gavage, and the blank control group was given an equal amount of normal saline by gavage; on the 21st day, challenge was carried out, that is, each composition sample (3 mL, composition concentration 1.0 mg / ml) was given by gavage, and the blank control group was given an equal amount of normal saline by gavage; 24 hours and 72 hours after challenge administration, the general reactions of the guinea pigs were observed. The results showed that no symptoms such as restlessness, abnormal defecation, dyspnea, convulsions, etc. occurred in each group of guinea pigs. It shows that the components of the pharmaceutical composition are safe and there is no systemic allergic reaction.

[0092] II. Efficacy detection

[0093] (1)Lowering blood uric acid level and improving renal function indexes: Select 200 healthy adult SD rats (blood uric acid value between 140 μmol / L and 180 μmol / L), randomly divided into 20 groups (10 in each group). Among them, 1 group is the blank control group, 1 group is the negative control group, and 1 group is the positive control group. Except for the blank control group, the rats in each group were intraperitoneally injected with potassium oxonate sodium chloride solution (potassium oxonate dosage 300 mg / kg), and the blank control group was intraperitoneally injected with an equal amount of normal saline. After continuous administration for 7 days, after confirming the successful establishment of the model (blood uric acid value between 400 μmol / L and 460 μmol / L), each comparative example and each example group were given the composition by gavage at 15 mg / kg body weight, the blank control group and the negative control group were given an equal amount of normal saline by gavage, and the positive control group was given allopurinol by gavage at 10 mg / kg body weight, once a day for 14 days. After the test: ① Blood was collected from the orbital venous plexus of the rats, serum was separated, and the blood uric acid level was measured by the uricase method; ② Blood was collected from the abdominal aorta of the rats, serum was separated, and the levels of serum creatinine and blood urea nitrogen were detected by an automatic biochemical analyzer. The results are shown in Table 1 below.

[0094] (2)Inhibiting inflammatory response: Mouse macrophages (RAW264.7) cultured in vitro were seeded into 96-well plates at a density of 5×10³ cells per well and cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) until the logarithmic growth phase. Then, the medium was replaced with a cell culture medium containing the drug composition (composition concentration: 0.1 mg / ml). At the same time, a blank control group, a negative control group (cultured only with high-glucose DMEM cell culture medium), and a positive control group (containing dexamethasone at a volume concentration of 0.1% in high-glucose DMEM cell culture medium) were set up and continued to be cultured. After 2 h, except for the blank control group, lipopolysaccharide LPS (1 μg / mL) was added to the other groups and cultured for another 24 h. The cell culture supernatant was collected, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 1 below.

[0095] Table 1 Results of efficacy detection (mean values)

[0096]

[0097] From the above experimental results, it can be seen that the drug compositions prepared in Examples 1 to 5 have safe components, no toxicity and allergic reactions; 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.

[0098] From the results of Comparative Example 1, it can be seen that earthworm peptides were replaced with freeze-dried powder obtained by directly crushing earthworms. The complex components contained in the earthworm freeze-dried powder are not easily absorbed by the body. The earthworm peptides obtained by specific enzymatic extraction are specific small-molecule peptides, which have better effects of inhibiting xanthine oxidase activity and promoting uric acid excretion, and can better inhibit uric acid production.

[0099] From the results of Comparative Examples 2 to 4, it can be seen that in the preparation method of earthworm peptides, trypsin, earthworm kinase or nattokinase is not used for enzymatic hydrolysis, and the components of the obtained products change, especially the small-molecule peptide products are different, and the effects of earthworm peptides in inhibiting uric acid, improving renal function and inflammatory response are weakened.

[0100] From the results of Comparative Examples 5 to 7, it can be seen that the bacterial ferment has the effect of regulating the balance of intestinal flora and improving the intestinal microecological environment. A healthy intestinal flora contributes to the decomposition and excretion of uric acid, and can assist in reducing the uric acid level. In addition, the bacterial ferment prepared from specific fermentation substrates has different therapeutic effects; in the preparation method of the bacterial ferment, if cornel fruit is not added, or coix seed is not added, or cornel fruit is not added and Bifidobacterium adolescentis is not added for aerobic fermentation, the components of the obtained products change, and the effects of the drug composition in inhibiting uric acid, improving renal function and inflammatory response are weakened.

[0101] From the results of Comparative Examples 8 to 11, it can be seen that the fenugreek seed extract contains rich chemical components, can regulate the hormone levels in the body, enhance the filtration and excretion functions of the kidneys for uric acid, reduce uric acid synthesis. In the preparation method of the fenugreek seed extract, ficin, trypsin or papain is not used for enzymatic hydrolysis, and the components of the obtained product change. In particular, the structure of the functional small molecule peptide product changes, and the therapeutic effect changes, and the effects of the pharmaceutical composition in inhibiting uric acid, improving renal function and inflammatory response are weakened.

[0102] From the results of Comparative Example 11, it can be seen that the fenugreek seed extract is extracted by ethanol percolation, and the pharmacological effects of the components of the obtained product are insufficient, and the therapeutic effect is weakened.

[0103] From the results of Comparative Example 12, it can be seen that when both earthworm peptides and fungal fermentations are not added to the pharmaceutical composition, and the mass fractions of earthworm peptides and fungal fermentations are both replaced by starch, the effects of the pharmaceutical composition in inhibiting uric acid, improving renal function and inflammatory response are greatly weakened, and the combined use of the two has a good synergistic effect.

Claims

1. A pharmaceutical composition for reducing uric acid, characterized in that, The pharmaceutical composition comprises raw materials in the following parts by mass: 1 to 3 parts of earthworm peptide, 1 to 3 parts of bacterial ferment, 0.2 to 0.5 part 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 products obtained by sequentially subjecting earthworms to hierarchical enzymatic hydrolysis with trypsin, earthworm kinase, and nattokinase; the bacterial ferment contains the products obtained by co-fermenting the pulp of cornel and glabrous greenbrier with Bacillus subtilis and Bifidobacterium adolescentis; the fenugreek seed extract contains the products obtained by sequentially subjecting fenugreek seed kernels to hierarchical enzymatic hydrolysis with ficin, trypsin, and papain; the chicory extract is an ethanol percolation extract of chicory roots; the plantain extract is an ethanol percolation extract of plantain.

2. The pharmaceutical composition for reducing uric acid according to claim 1, wherein The preparation method of the earthworm peptide comprises the following steps: Select fresh and healthy earthworms, wash them, and perform freeze-drying treatment to make the water content less than 5%; crush the dried earthworms into powder, pass through a 60-mesh to 80-mesh sieve, and take the powder under the sieve; according to the mass ratio, powder:Tris-HCl buffer solution = 1:(8 - 12), add the powder to the Tris-HCl buffer solution with a pH of 7.5 - 8.0, mix evenly to form a suspension; add 0.5% - 1.0% of the mass of the powder of trypsin to the suspension, enzymatically hydrolyze at 35°C - 38°C for 50 min - 70 min, raise the temperature to 50°C - 55°C, add 0.5% - 1.0% of the mass of the powder of earthworm kinase, enzymatically hydrolyze at 50°C - 55°C for 60 min - 90 min, heat to inactivate the enzyme, after cooling to room temperature, adjust the pH to 6.5 - 7.0, add 0.5% - 1.0% of the mass of the powder of nattokinase, enzymatically hydrolyze at 50°C - 55°C for 60 min - 90 min, heat to inactivate the enzyme, after cooling to room temperature, centrifuge at 4000 r / min - 6000 r / min for 10 min - 20 min, and collect the supernatant; ultrafilter and separate the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, collect the permeate to obtain a crude earthworm peptide extract; load the crude earthworm peptide extract onto a macroporous adsorption resin column for chromatography, first wash the resin column with deionized water to remove unadsorbed impurities, then elute with an ethanol aqueous solution with a volume concentration of 20% - 30%, collect the eluate, concentrate, and freeze-dry to a water content less than 5% to obtain the earthworm peptide.

3. The pharmaceutical composition for reducing uric acid according to claim 2, wherein The macroporous resin type of the macroporous adsorption resin column is D101 type; the loading flow rate of the crude earthworm peptide extract is 1 mL / min - 3 mL / min; the dosage of deionized water is 1 to 2 resin column volumes, and the washing flow rate is 3 mL / min - 5 mL / min; the dosage of the ethanol aqueous solution is 2 to 4 resin column volumes, and the elution flow rate is 1 mL / min - 3 mL / min; the concentration is carried out by rotary evaporation at 40°C - 50°C and 0.08 MPa - 0.1 MPa to concentrate the eluate to 10% - 20% by volume.

4. The pharmaceutical composition for reducing uric acid according to claim 1, characterized in that, The preparation method of the bacterial ferment includes the following steps: pulverize the fructus corni pulp, pulverize the smilax glabra rhizome, and prepare a basal solution according to the mass ratio of water: fructus corni pulp: smilax glabra rhizome: 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). After sterilization treatment and cooling to room temperature, a culture solution is obtained; inoculate 2% v / v - 5% v / v of bacillus subtilis and 2% v / v - 5% v / v of bifidobacterium adolescentis into the culture solution, mix evenly, first perform aerobic fermentation at 30°C - 37°C for 24h - 36h to promote the growth and metabolism of bacillus subtilis, and then perform anaerobic fermentation at 35°C - 37°C for 48h - 72h to promote the growth and metabolism of bifidobacterium adolescentis to obtain a fermentation broth. Centrifuge at 4000r / min - 6000r / min for 10min - 20min, collect the supernatant, and concentrate and dry it at 100°C - 120°C until the water content is below 5% to obtain the bacterial ferment.

5. The pharmaceutical composition for reducing uric acid according to claim 4, wherein The said bacillus subtilis is an activated bacterial solution of bacillus subtilis with a concentration of 100 million CFU / mL - 1 billion CFU / mL; the said bifidobacterium adolescentis is an activated bacterial solution of bifidobacterium adolescentis with a concentration of 100 million CFU / mL - 500 million CFU / mL; the temperature of the said sterilization treatment is 121°C - 125°C, and the time of the said sterilization treatment is 20min - 40min.

6. The pharmaceutical composition for reducing uric acid according to claim 1, wherein The preparation method of the fenugreek seed extract includes the following steps: pulverize the fenugreek seed kernel, add distilled water with a mass 8 - 12 times that of the fenugreek seed kernel, add ficin with a mass of 0.5% - 1.5% of the fenugreek seed kernel, enzymolyze at 60°C - 65°C for 60min - 90min, heat up to inactivate the enzyme, and after cooling to room temperature, adjust the pH to 8.0 - 8.

5. Then add trypsin with a mass of 0.5% - 1.0% of the fenugreek seed kernel, enzymolyze at 35°C - 40°C for 50min - 70min, heat up to inactivate the enzyme, and after cooling to room temperature, adjust the pH to 5.0 - 7.

0. Next, add papain with a mass of 0.5% - 1.0% of the fenugreek seed kernel, enzymolyze at 55°C - 65°C for 50min - 70min, heat up to inactivate the enzyme, and after cooling to room temperature, centrifuge at 4000r / min - 6000r / min for 10min - 20min to collect the supernatant; the supernatant is ultrafiltered and separated through an ultrafiltration membrane with a molecular weight cut-off of 5kDa, the permeate is collected, and it is concentrated and dried under reduced pressure at 40°C - 50°C until the water content is below 5% to obtain the fenugreek seed extract.

7. The pharmaceutical composition for reducing uric acid according to claim 1, wherein, The preparation method of the chicory extract includes the following steps: pulverize the chicory root, percolate with ethanol with a mass 8 - 10 times that of the chicory root to obtain an extraction solution, and concentrate and dry it at 60°C - 80°C until the water content is below 5% to obtain the chicory extract.

8. A pharmaceutical composition for reducing uric acid according to claim 1, characterized in that, The preparation method of the plantain extract comprises the following steps: taking the whole plant of plantain, pulverizing it, percolating with ethanol 8 to 10 times the mass of the whole plant of plantain to obtain an extract, and concentrating and drying it at 60 °C to 80 °C until the water content is below 5% to obtain the plantain extract.

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

10. A method for preparing a pharmaceutical composition for reducing uric acid according to claim 1, characterized in that, The preparation method comprises the following steps: mixing the bacterial fermentation product, fenugreek seed extract, chicory extract and plantain extract evenly by mass parts, and then adding earthworm peptide and mixing evenly to obtain the pharmaceutical composition.

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