A simiao pill compound fermentation liquor and a preparation method and application thereof

Abstract

The application discloses a Simiao pill compound fermentation liquor and a preparation method and application thereof, and comprises the following steps: taking a mixture of Radix Cyathulae, Coicis Semen, Atractylodis Rhizome and Cortex Phellodendri powder according to a preset proportion; preparing a liquid culture medium; adding the powder mixture into the liquid culture medium to obtain a fermentation base, and cooling after high-temperature cooking; inoculating Lactobacillus on the fermentation base to start fermentation culture; terminating the fermentation at high temperature; filtering, centrifuging, taking supernatant to obtain the Simiao pill compound fermentation liquor, and the fermentation liquor can be used for reducing the uric acid level of human body. The in-vivo research results of mice show that the Simiao pill compound fermentation liquor is superior to the unfermented Simiao pill compound in the effects of reducing uric acid and protecting kidney, and the high-dose group of the fermented Simiao pill can effectively reduce the uric acid level in the body of hyperuricemia mice and play a role in protecting the kidney; the liver toxicity experiment results show that the fermented Simiao pill compound does not produce toxicity to the liver of mice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of traditional Chinese medicine fermentation, and relates to a fermentation liquor of Simiao pill compound, and a fermentation method of the Simiao pill compound and application of the fermentation liquor. BACKGROUND

[0002] Due to the flexible prescription mode of traditional Chinese medicine, the prescription can be matched according to the specific condition and individual difference, so the number of traditional Chinese medicine prescriptions is very large. At present, according to statistics, the number of all kinds of traditional Chinese medicine prescriptions in China has exceeded ten thousand, and this number is still growing. The continuous enrichment and development of traditional Chinese medicine prescriptions also provide rich choices for the clinical application of traditional Chinese medicine.

[0003] The principle of modern traditional Chinese medicine fermentation technology is to screen and utilize appropriate probiotics and / or the specific biological transformation ability of enzymes to change the chemical composition and biological activity of Chinese herbal medicine raw materials under specific environmental conditions (air, temperature, moisture, etc.).

[0004] In the process of microbial fermentation of traditional Chinese medicine, microorganisms produce various metabolites, including acids, bases, alcohols, ketones, esters, enzymes, etc. These metabolites can transform, degrade or enhance the active ingredients in the raw materials and produce new compounds. This transformation process is often complex, involving multiple microorganisms, multiple enzymes and multiple reaction pathways, so it is difficult to completely predict and control.

[0005] In addition, the complexity of traditional Chinese medicine raw materials also increases the unpredictability of microbial fermentation. Traditional Chinese medicine is usually a compound composed of multiple plants or animals, and each raw material contains multiple active ingredients. There may be interactions and synergies between these active ingredients, and the metabolic changes during microbial fermentation may further change this interaction and synergy, leading to changes in the activity and chemical composition of the final product.

[0006] The inventors have conducted a lot of research on the microbial fermentation of Simiao pill compound through microbial fermentation technology, and in the process of realizing the present application, the inventors have found that at least one of the following technical problems exists in the prior art:

[0007] 1. Simiao pill compound is a traditional Chinese medicine compound prescription in China, which is composed of atractylodes, Chinese cork tree, Chinese milk vetch and coix seed, has a wide range of pharmacological effects, and traditional Chinese medicine clinical practice has proved that Simiao pill compound can effectively treat various types of arthritis, such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis and gouty arthritis, but there is no reference material on microbial fermentation of Simiao pill compound in the prior art.

[0008] 2. Existing technologies do not disclose research results on the effects of microbial fermentation of the Simiaowan compound formula and its biotransformation products. They cannot provide guidance on the changes in pharmacological effects of the biotransformation products of the fermented Simiaowan compound compared with the unfermented Simiaowan compound. There are significant technical obstacles in how to screen for target fermentation microorganisms and how to achieve effective fermentation. Summary of the Invention

[0009] Therefore, one of the objectives of this invention is to provide a compound fermentation liquid of Simiao Pill with more pharmacological effects.

[0010] The second objective of this invention is to provide a method for preparing the aforementioned Simiao Pill compound fermentation liquid.

[0011] The third objective of this invention is to provide a method for using the compound fermentation liquid of Simiao Pill to lower uric acid levels.

[0012] Through long-term exploration and experimentation, as well as numerous trials and efforts, the inventors have continuously reformed and innovated to solve the above-mentioned technical problems. The technical solution provided by this invention is a method for preparing the fermentation broth of the Simiao Pill compound, comprising the following steps:

[0013] S1. Take a mixture of powders of Achyranthes bidentata, Coix lacryma-jobi, Atractylodes lancea, and Phellodendron chinense according to the preset ratio;

[0014] Alternatively, Achyranthes bidentata, Coix lacryma-jobi, Atractylodes lancea, and Phellodendron chinense can be extracted with ethanol, and the ethanol can be recovered to obtain the alcohol extract;

[0015] Alternatively, water extracts can be obtained from Achyranthes bidentata, Coix lacryma-jobi, Atractylodes lancea, and Phellodendron chinense.

[0016] S2. Prepare liquid culture medium;

[0017] S3. Add the powder mixture described in S1 to the liquid culture medium described in S2 to obtain a fermentation medium, and then cool it after high-temperature cooking; or, add the alcohol extract or water extract to a sterile liquid culture medium to obtain a fermentation medium.

[0018] S4. Inoculate lactic acid bacteria onto the fermentation medium described in S3 and begin fermentation culture;

[0019] S5. High temperature terminates fermentation;

[0020] S6. Filter, centrifuge, and take the supernatant to obtain the Si Miao Wan compound fermentation broth.

[0021] According to one embodiment of the preparation method of the compound fermentation liquid of Simiao Pill of the present invention, the components in the powder mixture are as follows by weight: 2-8 parts of Achyranthes bidentata, 4-12 parts of Coix lacryma-jobi, 2-8 parts of Atractylodes lancea, and 4-12 parts of Phellodendron chinense.

[0022] Preferably, the powder mixture comprises, by weight parts, 4 parts of Radix Achyranthis Bidentatae, 4 parts of Rhizoma Atractylodis, 8 parts of Semen Coicis, and 8 parts of Cortex Phellodendri.

[0023] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the liquid culture medium is PDA liquid culture medium; 150-250 g of potato, 15-35 g of glucose monohydrate, 2-4 g of magnesium sulfate, 3-5 g of potassium dihydrogen phosphate, and 0-40 mg of vitamin B1 are added to each 1500 ml of the culture medium, and no agar is added.

[0024] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the liquid culture medium is soybean powder liquid culture medium; 4-6 g of soybean powder, 1-3 g of starch, 2-3 g of sucrose, 0.1-1 g of yeast extract powder, 0.1-1 g of sodium chloride, and 0.05-0.2 g of potassium phosphate dibasic are added to each 250 ml of the culture medium.

[0025] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the liquid culture medium is milk liquid.

[0026] Preferably, the liquid culture medium is PDA liquid culture medium; 200 g of potato, 30 g of glucose monohydrate, 2.25 g of magnesium sulfate, 4.5 g of potassium dihydrogen phosphate, and 30 mg of vitamin B1 are added to each 1500 ml of the culture medium, and no agar is added.

[0027] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the ratio of the powder mixture to the liquid culture medium is 1:7-70.

[0028] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the high-temperature cooking is performed by placing the fermentation base into a sealed container and placing the container in a pressure cooker for 25-30 min at 121 ℃.

[0029] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the lactic acid complex bacteria include one or more of Lactobacillus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus acidophilus, and the inoculation amount is 0.1-10×10 6 CFU / ml.

[0030] According to an embodiment of the method for preparing the compound fermentation liquor of Simiao Pills, the fermentation culture is performed in a shaking flask machine at 200-300 rpm and at 37 ℃ for 7-10 days.

[0031] According to one embodiment of the preparation method of the Simiao Pill compound fermentation liquor, the step S5 is specifically placing the fermentation product in a device at 95-100 DEG C and boiling for 10-20 min.

[0032] According to one embodiment of the preparation method of the Simiao Pill compound fermentation liquor, the step S6 is specifically filtering the fermentation product, discarding the residue, centrifuging at 5000-10000 rpm for 15-30 min, and taking the supernatant to obtain the Simiao Pill compound fermentation liquor.

[0033] The application further provides a Simiao Pill compound fermentation liquor prepared by the method.

[0034] The application further provides an application of the Simiao Pill compound fermentation liquor in reducing the uric acid level of human body.

[0035] Compared with the prior art, one of the above technical solutions has the following advantages:

[0036] a) The application first uses microbial fermentation technology, in particular, uses lactic acid composite bacteria to ferment the Simiao Pill compound, and through component analysis of the fingerprint of the fermented Simiao Pill compound and the unfermented Simiao Pill compound, the application explores the effect of the microbial fermentation product of the microbial fermentation Simiao Pill compound on the uric acid level and kidney protection of mice by using the fermentation liquor.

[0037] b) In one embodiment of the preparation method of the Simiao Pill compound fermentation liquor, lactic acid composite bacteria are used to ferment the Simiao Pill compound, the lactic acid composite bacteria are edible probiotics, and three Chinese medicines in the Simiao Pill compound are medicinal and edible medicinal materials, which lays a research foundation for subsequent research and development of the Simiao Pill compound health care food with higher nutritional value.

[0038] c) Based on HPLC fingerprint detection, the inventors analyze that the chemical components of the fingerprint of the unfermented Simiao Pill compound and the fingerprint of the fermented Simiao Pill compound are greatly different.

[0039] d) The in vivo research results of mice show that the uric acid lowering effect and kidney protection effect of the Simiao Pill compound fermentation liquor are better than those of the unfermented Simiao Pill compound, the high-dose group of the fermented Simiao Pill can effectively reduce the uric acid level of hyperuricemic mice and has the effect of kidney protection; the liver toxicity experiment results show that the fermented Simiao Pill compound does not produce toxicity to the liver of mice.

[0040] The principle of the Simiao Pill in reducing uric acid is as follows:

[0041] Although Simiao Pill has the effect of reducing body uric acid, individual differences are significant. After the human body takes traditional Chinese medicine, the traditional Chinese medicine needs to be digested and absorbed by the gastrointestinal tract and metabolized. Due to individual differences, the flora in the gastrointestinal tract of the body is inconsistent, which is an important factor leading to the difference in absorption of the body. Lactic acid bacteria can promote the absorption of the gastrointestinal tract of the body and improve the gastrointestinal flora. Fermentation can change the activity of lactic acid bacteria and increase the number of lactic acid bacteria. The lactic acid complex bacteria are fermented with Simiao Pill, which simulates the in vivo mode of absorption of the gastrointestinal tract of the human body.

[0042] After fermentation, microorganisms absorb cellulose, saccharides, proteins and other macromolecules in the plant body of Simiao Pill, destroy the cell wall of the plant and release the active ingredients of the medicine; under the action of microbial enzymes, the active ingredients stimulate the active substances in Simiao Pill, so that the active substance content in Simiao Pill is significantly increased. Animal experiments show that the secondary metabolites produced by fermentation are beneficial to the absorption of Simiao Pill by the body, improve the individual absorption difference caused by insufficient absorption and metabolism of the body, improve the utilization rate of Simiao Pill and enhance the effect of Simiao Pill on reducing uric acid in the body. The difference in curative effect between unfermented Simiao Pill and fermented Simiao Pill is thus generated.

[0043] The Simiao Pill compound fermented by lactic acid complex bacteria can inhibit the generation of purines in the body and reduce the content of blood uric acid. Due to the change of chemical components after fermentation of Simiao Pill, the fermented Simiao Pill has a higher effect on reducing uric acid than the unfermented Simiao Pill. Fermented Simiao Pill promotes the excretion of urate in the body, reduces the deposition of urate in the kidney of the body and reduces inflammation, thereby playing a role in protecting the kidney damage.

[0044] As can be seen, after fermentation, the biotransformed Simiao Pill greatly improves the bioavailability of Simiao Pill compared with unfermented Simiao Pill, fully embodies the expected effect of the increase of chemical components after biotransformation of traditional Chinese medicine and provides a basis for the development of Simiao Pill health products with the same origin of food and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0046] Figure 1 is the HPLC comparison chart of the supernatant of Simiao Pill compound fermented by lactic acid complex bacteria and unfermented Simiao Pill.

[0047] Figure 2 is the effect of each experimental group on the protein level of xanthine dehydrogenase (XO) in the liver of mice.

[0048] Figure 3 Figure 11 is the effect of each experimental group on the histopathology of the liver tissue of mice (20X).

[0049] Figure 4 Figure 11 is the effect of each experimental group on the histopathology of the liver tissue of mice (20X). DETAILED DESCRIPTION

[0050] The present application will be described in greater detail with reference to the accompanying drawings, in which:

[0051] In order to make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the following will be combined with the accompanying drawings and a specific embodiment to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application but not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0052] It should be noted that similar reference numerals and letters refer to similar items in the following drawings, and thus, once an item is defined in one drawing, it can not be further defined and explained in subsequent drawings.

[0053] Simiao pill compound is a traditional Chinese medicine compound prescription in China, which is recorded in Zhang Bingcheng's Shengfang Bie Du (A Handbook of Classical Prescriptions) in the Qing Dynasty. It is based on the Ermiao San (Two Wonderful Powder) of Zhu Danxi's Danxi Xinfa (Danxi's Heart Method) in the Yuan Dynasty, with the addition of two ingredients, i.e., Niuxi (Radix Achyranthis Bidentatae) and Yiyiren (Semen Coicis), and is made by panning and modifying. It is composed of four traditional Chinese medicines, i.e., Cangzhu (Rhizoma Atractylodis), Huangbai (Cortex Phellodendri), Niuxi (Radix Achyranthis Bidentatae), and Yiyiren (Semen Coicis). The Simiao pill compound has a wide range of pharmacological effects. Clinical practice of traditional Chinese medicine has confirmed that the Simiao pill compound can effectively treat various types of arthritis, such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, and gouty arthritis. In the prescription, Cangzhu (Rhizoma Atractylodis) can invigorate the spleen and dry dampness, Huangbai (Cortex Phellodendri) can clear heat and dry dampness, Niuxi (Radix Achyranthis Bidentatae) can tonify the liver and kidney and strengthen the tendons and bones, and Yiyiren (Semen Coicis) can invigorate the spleen, clear heat, and drain dampness. Niuxi (Radix Achyranthis Bidentatae) in the Simiao pill compound has multiple physiological activities, such as promoting blood circulation, resisting tumors, resisting arthritis, resisting oxidation, resisting aging, etc., and is the most effective traditional Chinese medicine ingredient in the Simiao pill compound for treating hyperuricemia. Yiyiren (Semen Coicis) is rich in nutrients such as protein, carbohydrates, fat, crude fiber, vitamins, minerals, and amino acids, and has a high nutritional value. It has multiple biological activities, such as immune regulation, blood glucose reduction, anti-tumor, anti-virus, and blood pressure reduction.

[0054] Promoting the excretion of uric acid is one of the main means for treating hyperuricemia. Representative drugs such as benzbromarone target the kidney and promote the excretion of uric acid from the kidney by inhibiting the reabsorption of the renal tubule, but their side effects continue to increase the load on the kidney, causing adverse reactions such as chronic kidney disease.

[0055] Traditional Chinese medicine has the advantage of being natural and green, and has less side effects and high safety compared with western medicine. With the development of natural products such as traditional Chinese medicine, natural products such as traditional Chinese medicine have been developed by researchers. In recent years, fermentation technology has been widely used in traditional Chinese medicine by scholars, but the use of fermentation technology in Simiao pill compound prescription has not been developed.

[0056] In the process of completing the present application, the inventors screened a variety of microorganisms to ferment the Simiao pill compound, and the present embodiment discloses part of the microbial screening data to illustrate the importance of the selection of fermentation microorganisms in the method of the present application.

[0057] The optimal fermentation temperature of each bacterium was selected for fermentation: mold 26℃, lactic acid complex bacteria 34-37℃, sweet wine starter 26-30℃ for microbial fermentation, and each bacterium was screened for 6 days. After fermentation, filtration, methanol ultrasonic treatment, and then filtration were performed for HPLC detection to analyze whether there was a significant difference in chemical composition.

[0058] Compared with the unfermented Simiao pill compound, the fermentation Simiao pill compound had a reduced peak area of berberine hydrochloride at 36 min, and the peak areas of other chemical components were reduced as a whole.

[0059] Compared with the unfermented Simiao pill compound, the fermentation Simiao pill compound had a significantly increased peak area of berberine hydrochloride at 36 min, and the peak areas of target peaks at 21 min and 27 min changed significantly before and after fermentation.

[0060] Compared with the unfermented Simiao pill compound, the fermentation Simiao pill compound had an increased peak area of berberine hydrochloride at 36 min, but the peak areas of target peaks at 21 min and 27 min had less difference, which was not as good as the fermentation Simiao pill compound of lactic acid complex bacteria.

[0061] The strain source of the lactic acid complex bacteria is the China General Microbiological Culture Collection Center (CGMCC) (China, Beijing). The lactic acid complex bacteria are composed of five kinds of lactobacillus, including Fermentum lactis (CGMCC 1.15608), Lactobacillus plantarum (CGMCC 1.12934), Lactobacillus reuteri (CGMCC 1.2838), Lactobacillus salivarius (CGMCC 1.1881), and Lactobacillus acidophilus (CGMCC 1.3342). Three of the components of the Simiao pill compound have been listed in the directory of food and medicine, which can promote the proliferation of lactic acid complex bacteria, and lactic acid complex bacteria also have the effect of protecting the balance of the body's intestinal tract. Therefore, the inventors selected lactic acid complex bacteria, which are beneficial to the human body and can be eaten and are probiotics, as the fermentation agent of the Simiao pill compound.

[0062] In the process of completing the present application, the inventors tested the effects of different pre-treatment methods of Simiao Pill compound on the method, and verified that high-temperature cooking has a significant effect on the preparation method of Simiao Pill compound fermentation broth. The inventors excerpted part of the test process data as follows:

[0063] Take 4g of Radix Cyathulae, 4g of Atractylodes, 8g of Coix seed, and 8g of Phellodendron for each portion, crush them with a crusher, pass them through a 40-mesh sieve, mix them, and obtain a Simiao Pill powder mixture. Prepare 250ml of PDA liquid medium, potato 50g, glucose 5g, magnesium sulfate 0.4g, potassium dihydrogen phosphate 0.75g, vitamin B1 1mg, and do not add agar. The pH of the PDA medium is not required, and the natural pH is acceptable. Add the Simiao Pill powder mixture to the 250ml PDA liquid medium in a sealed shake flask, and do not cook it.

[0064] Take 4g of Radix Cyathulae, 4g of Atractylodes, 8g of Coix seed, and 8g of Phellodendron for each portion, crush them with a crusher, pass them through a 40-mesh sieve, mix them, and obtain a Simiao Pill powder mixture. Prepare 250ml of PDA liquid medium, potato 50g, glucose 5g, magnesium sulfate 0.4g, potassium dihydrogen phosphate 0.75g, vitamin B1 1mg, and do not add agar. The pH of the PDA medium is not required, and the natural pH is acceptable. Add the Simiao Pill powder mixture to the 250ml PDA liquid medium in a sealed shake flask, and further place the prepared shake flask in a high-pressure sterilization device for high-temperature cooking at 121℃ for 30min.

[0065] Compared with non-cooking, the color of the Simiao Pill solution changes visibly, and the color of the non-cooked solution is lighter and brighter. The Simiao Pill medicinal materials after cooking are brown and have a deeper color. The contents of the two were determined, and the changes in the contents of the samples before and after cooking under the same conditions were not significant. Cooking and non-cooking have no significant interference with the chemical components of Simiao Pill.

[0066] After high-temperature cooking, the components and structure of the Simiao Pill compound powder change, the effective components are more easily released, it is more conducive to extracting active ingredients from herbs, and it increases the solubility and bioavailability, creating more favorable conditions for lactic acid complex fermentation. It should be noted that the inventors chose to use a sealed shake flask, which also avoids the loss of components due to high temperature, such as the reduction of atractylodes volatile oil with the increase of temperature. The inventors used a sealed shake flask, which did not substantially affect the components that are easily affected by high temperature, such as atractylodes volatile oil.

[0067] Based on the research results of the inventors, the following preferred embodiments are obtained.

[0068] Example 1

[0069] The preparation method of Simiao Pill compound fermentation broth described in this embodiment is as follows:

[0070] Step S1: According to the compound prescription proportion of Simiao Pill in the Chinese Pharmacopoeia, take 4g of Radix Cyathulae, 4g of Atractylodes, 8g of Coicis Semen and 8g of Phellodendri Cortex for each part, crush with a crusher, pass through a 40-mesh sieve, mix, and obtain a powder mixture. Prepare 6 portions of the powder mixture for use.

[0071] Step S2, prepare the liquid medium.

[0072] Select PDA liquid medium. Lactic acid bacteria cannot grow without carbon source, nitrogen source, trace elements, vitamins, and other growth factors such as moisture, which are nutrients for lactic acid bacteria growth and fermentation product production.

[0073] Prepare 1500ml of medium, 200g of potato, 30g of glucose, 2.25g of magnesium sulfate, 4.5g of potassium dihydrogen phosphate, and 30mg of vitamin B1 without agar. The pH of the PDA medium is not required, and the natural pH is acceptable. Add 250ml of PDA liquid medium to each flask. Divide the 1500ml medium into 6 flasks. The potato in the PDA liquid medium can provide natural nitrogen source for lactic acid bacteria, glucose provides carbon source, and magnesium sulfate and potassium dihydrogen phosphate provide trace elements. The liquid medium can better make the lactic acid bacteria cell fully shake and proliferate. Seal all the flasks.

[0074] Step S3, add the powder mixture in S1 to the liquid medium in S2 to obtain a fermentation base, and then cook at high temperature and cool to room temperature.

[0075] Add 1 portion of the powder mixture to each flask in step S2, and the solid-liquid ratio of the powder mixture to the liquid medium is 1:10. Then put the prepared flask into a pressure cooker, cook at 121℃ for 30min, and cool to room temperature. High temperature cooking can sterilize and change the composition and structure of the Simiao Pill compound powder, making it easier to release active ingredients, more conducive to extracting active ingredients from herbs, and increasing solubility and bioavailability, creating more favorable conditions for lactic acid complex fermentation.

[0076] Step S4, inoculate lactic acid complex bacteria on the fermentation base in S3, and the ratio of Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius and Lactobacillus acidophilus is 1:1:1:1:1.

[0077] After high-temperature cooking in step S3, 3 portions are used as the treatment group, inoculated with lactic acid complex bacteria, and the inoculation amount is 10 6 CFU / ml. The other 3 portions are used as the control group without inoculating microbial inoculants.

[0078] Put the 6 flasks into a flask machine and incubate at 200rpm and 37℃ for 8 days.

[0079] Step S5, high temperature termination of fermentation.

[0080] After the completion of the culture, the shake flask is placed in a device at 98°C and boiled for 15 min. The microorganisms in the culture solution are killed and the fermentation is terminated.

[0081] Step S6, filtration, centrifugation, and taking the supernatant to obtain the Simiao pill compound fermentation liquor.

[0082] The culture solution after sterilization in step S5 is filtered, the residue is discarded, and the supernatant is obtained by centrifugation at 8000 rpm for 20 min, to obtain the Simiao pill compound fermentation liquor and the Simiao pill compound non-fermentation supernatant, respectively.

[0083] According to the pH value determination, the pH value of the Simiao pill compound non-fermentation supernatant is 6, and the pH value of the Simiao pill compound fermentation liquor is 4.12.

[0084] Example 2

[0085] As an alternative embodiment, 4 g of Radix Cyathulae, 4 g of Rhizoma Atractylodis, 8 g of Semen Coicis, and 8 g of Cortex Phellodendri are taken in each portion, 1000 ml of distilled water is used for reflux extraction at 70°C for 2 h, filtration is performed, the residue is extracted with 600 ml of distilled water for reflux extraction for 1.5 h, filtration is performed, and the two filtrates are combined to obtain the Simiao pill medicinal material water extract.

[0086] The soybean powder liquid medium is prepared, 5 g of soybean powder, 1.25 g of starch, 2.5 g of sucrose, 0.5 g of yeast powder, 0.5 g of sodium chloride, and 0.12 g of potassium phosphate dibasic are weighed, added to 250 ml of distilled water or deionized water, heated and boiled until completely dissolved, the pH of the culture medium is 7.3±0.1, and the fermentation base is placed in a sealed container and placed in a pressure cooker at 121°C for 25-30 min.

[0087] After the temperature drops below 40°C, inoculation is performed on a sterile operation table sterilized 30 min in advance.

[0088] 0.1-10×10 6 CFU / ml of lactic acid complex bacteria is added to the shake flask, and the recommended inoculation amount is 1×10 6 CFU / ml. The shake flask is placed in a 37°C constant temperature incubator for fermentation, and the fermentation time is 8 days. After the fermentation is completed, the fermentation product is filtered, the residue is discarded, centrifugation is performed at 6000 rpm for 20 min, and the supernatant is taken.

[0089] Example 3

[0090] As an alternative embodiment, each of the Radix Cyathulae 4g, Rhizoma Atractylodis 4g, Semen Coicis 8g, and Cortex Phellodendri 8g is added to 1000ml of 60% ethanol, and refluxed at 70°C for 2h. The filtrate is collected, and the residue is extracted with 600ml of 40% ethanol for 1.5h. The filtrate is collected, and the ethanol is recovered under vacuum to obtain the alcohol extract of Simiao Pills.

[0091] Add 250ml of pure milk and stir well.

[0092] Under normal temperature conditions, 0.1x10 6 CFU / ml of lactic acid complex bacteria is added to the shake flask. The shake flask is placed in a 37°C constant temperature incubator for fermentation, and the fermentation time is 8 days.

[0093] After the fermentation is completed, the fermented product is filtered, the residue is discarded, and the supernatant is obtained by centrifugation at 5000rpm for 15min.

[0094] Experimental Example 1

[0095] To illustrate the technical effect of the Simiao Pills compound fermentation broth prepared in the first embodiment of the specification, this experimental example is continued.

[0096] 1.1 HPLC determination of the samples obtained from the control group and the treatment group in Example 1.

[0097] The Simiao Pills compound fermentation broth and the Simiao Pills compound non-fermented supernatant are pretreated before HPLC detection. 5ml of the Simiao Pills compound fermentation broth and the Simiao Pills compound non-fermented supernatant are each placed in three 25ml volumetric flasks, and methanol is added to the mark. The temperature is 33°C, and the ultrasonic time is 30min. After cooling, an appropriate volume is filtered through a 0.22μm filter membrane, and 1.5ml is loaded into an HPLC sample bottle for machine detection.

[0098] Chromatographic conditions: Agilent 1260infinity II HPLC chromatograph (configuration: 1260 differential refractometer detector, 1260 diode array detector, Agilent OpenLAB CDS software, GPC data analysis software), chromatographic column SWELL C18 (250mmx4.6mmx5μm, Chromplus TM ); column temperature 30°C; flow rate 1ml / min; injection volume 10ul; detection wavelength 330nm. Mobile phase: A phase (acetonitrile, v / v), B phase (0.1% phosphoric acid, v / v); mobile phase elution gradient: 0-30min, 7%-24%(A); 30-40min, 24%-51%(A); 40-60min, 51%-70%(A); 60-90min, 90%(A).

[0099] The determination results are shown in Table 1 and Figure 1The retention time of each compound is shown in Table 1. Table 1 shows the change of peak area of each compound before and after fermentation of Simiao Pill; Figure 1 In the experiment, A is the control group, i.e. the supernatant of Simiao Pill compound without fermentation, and B is the treatment group, i.e. the fermentation broth of Simiao Pill compound. Under the same injection amount, the peak area of each compound of the supernatant of Simiao Pill compound without fermentation and the fermentation broth of Simiao Pill compound was recorded.

[0100] Table 1: Change of peak area of each compound before and after fermentation of Simiao Pill compound

[0101]

[0102] From Table 1 and Figure 1 It can be seen that the retention time of 36 min is the standard berberine hydrochloride. According to the detection report, the peak area of berberine hydrochloride in the supernatant of Simiao Pill compound without fermentation is 3293.59. The peak with a retention time of 16.2 min is magnoflorine, and the peak area is 51.8. The peak area of the target peak with a retention time of 20.450 min is 3206.97. The peak area with a retention time of 24.634 min is 62.59. The peak area can represent the content of the detected substance.

[0103] The retention time of 35.903 min is the control berberine hydrochloride. According to the detection report, under the same detection conditions, the peak area of berberine hydrochloride in the fermentation broth of Simiao Pill compound is 3965.86. The peak with a retention time of 15.9 min is magnoflorine, and the peak area is 166.7. The peak area with a retention time of 20.033 min is 1819.98. The peak area with a retention time of 24.256 min is 913.40. According to the change of the peak area of the target peak before and after fermentation, the peak area of berberine hydrochloride in the fermentation broth of Simiao Pill compound is increased by 20.41% compared with that in the supernatant of Simiao Pill compound without fermentation. Magnoflorine is increased by 221% after fermentation. Compared with the peak area of the supernatant of Simiao Pill compound without fermentation, the peak area of the target peak with a retention time of 20 min is reduced by 43.25%, and the peak area of the target peak with a retention time of 24 min is increased by 1359.34%.

[0104] 1.2 Uric acid reduction experiment in mice

[0105] The fermented liquid of Simiao Pill compound obtained in Example 1 was subjected to in vivo uric acid reduction verification research. Considering the factors of establishing a stable model, short cycle, low cost and the like, a mouse HUA model was established by intragastrically administering 200 mg / kg of oxypurinol and 75 mg / kg of adenine for 28 days, which can better reflect the uric acid response of the mouse body and simulate the condition of hyperuricemia and the kidney disease caused thereby in the human body. Therefore, the method of establishing a model by combining oxypurinol and adenine was used to evaluate the uric acid reducing and kidney protecting effects of the fermented liquid of Simiao Pill compound prepared according to Example 1.

[0106] Uric acid reducing experiment: The fermented liquid of Simiao Pill compound and the supernatant of Simiao Pill compound without fermentation were taken as comparative experimental samples.

[0107] The experimental animals used in this experiment were 60 SPF C57BL / 6 male mice, 6-8 weeks old, weighing 20±22 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (China), license number: SCXK (Chuan) 2020-030. The feeding environment temperature was (24±1) °C, the humidity was (50±60) %, the light and dark alternation was 12 h, and the poplar bedding was changed regularly. The animals were allowed to drink water and eat freely during the experiment, and were adaptively fed for 7 days before the experiment.

[0108] The reagents used in this experiment were all commercially available. The uric acid (UA) kit was purchased from Nanjing Jianshen Biological Engineering Institute, batch number (C012-1-1). The urea nitrogen (BUN) kit was purchased from Nanjing Jianshen Biological Engineering Institute, batch number (C013-2-1). Oxypurinol was purchased from Chengdu Kolon Chemical Co., Ltd., batch number (I2130045), purine was purchased from Chengdu Kolon Chemical Co., Ltd., and benzbromarone was purchased from a physical pharmacy.

[0109] The specific scheme of the animal experiment was as follows:

[0110] Model establishment: A mouse HUA model was established by intragastrically administering 200 mg / kg of oxypurinol and 75 mg / kg of adenine, 1 time / d. To ensure the uniformity of the drug concentration in the centrifuge tube, the suspension agent was used to suspend the drug in 0.5% CMC-Na solution, and the modeling was continuously performed for 35 days. On the 7th day after modeling, 1 ml of blood was taken from the eyeball of the mouse, and was placed at room temperature for 1-2 h. After the blood coagulated, it was centrifuged at 4 °C and 3000 r for 10 minutes, and the supernatant was taken. The uric acid value was determined according to the uric acid (UA) kit instruction. The SUA level was considered to be successfully modeled if it was increased by more than 20% compared with the normal group. According to the determination by the kit, the uric acid value of the model group was 471.73 (μmol / L), the normal value of the mouse was 41-126 μmol / L, and the obtained value was 4 times the maximum normal value, so it was determined that the mouse HUA model was successfully established by intragastrically administering oxypurinol and adenine. The mouse experimental groups were as follows:

[0111] Table 2: Grouping of mice in the experiment

[0112]

[0113] Grouping of the experiment: The normal control group of mice was given 0.5% CMC-Na solution by gavage, and normal feed was fed. The rest of the mice were constructed into HUA models. After the modeling was successful, the mice were randomly divided into 6 groups, 10 mice in each group, for drug administration. They were blank control group, model group, positive benzbromarone control group, unfermented Simiao Pill compound group, fermented Simiao Pill compound low-dose group, and compound high-dose group. Drug administration started on the 8th day of modeling. After 3 hours of induction of hyperuricemia, the mice were given drugs by gavage every day for 21 days. In the morning, the positive group, fermented Simiao Pill compound high-dose group, low-dose group, and unfermented Simiao Pill compound group were given drug intervention in the afternoon, and the model group was given an equal volume of normal saline. After the 28th day of gavage, the mouse serum and tissues were taken for detection.

[0114] Blank group (NC): During the entire experiment, the blank group of mice was given normal free drinking water and feed supply. On the seventh day, the mice were given 0.5% CMC-Na solution by gavage at the same time as the other groups, 0.2 ml / d for each mouse.

[0115] Model group (HUA): During the entire experiment, the mice were given normal free drinking water and feed supply. Modeling was performed at 10 am, and the concentration was calculated. Each mouse was given 0.08 ml of oxypurinol potassium / kg / d and 0.075 ml of adenine / kg / d by gavage, and an equal volume of normal saline was given by gavage at 1 pm.

[0116] Benzbromarone group (BEN): During the entire experiment, the mice were given normal free drinking water and feed supply. According to the reference, the administration dose was 6.5 mg / kg, which was prepared according to the concentration as 0.1 ml / kg / d for each mouse by gavage.

[0117] Unfermented Simiao Pill compound group (W): Using the unfermented supernatant of the Simiao Pill compound prepared in Example 1 as an example, during the entire experiment, the mice were given normal free drinking water and feed supply. The mice were modeled for hyperuricemia in the morning at 10 am, and were given drug intervention in the afternoon at 1 pm. According to the Simiao Pill for adults in the People's Republic of China Pharmacopoeia 2022, the daily dosage is 12 g / kg, and the administration dose calculated by referring to the body surface area conversion of humans and mice is 1.48 g / kg. The unfermented Simiao Pill compound supernatant after centrifugation was concentrated from 250 ml to 84 ml, and each mouse was given 0.1 ml / d by gavage.

[0118] Fermented Simiao Pill Compound Low-dose Group (ZL): Take the fermented liquid of Simiao Pill Compound prepared in Example 1 as an example. During the entire experiment, the mice were given normal free drinking water and feed supply. According to the Simiao Pill adult daily dosage of 12 g / kg stipulated in the 2022 edition of the People's Republic of China Pharmacopoeia, the mouse low-dose group was set to 3 g / kg, and the dosage converted from the body surface area of human and mouse was 0.35 g / kg. After concentration calculation, the supernatant was not concentrated, and each mouse was given 0.73 ml / d by gavage.

[0119] Fermented Simiao Pill Compound High-dose Group (ZH): Take the fermented liquid of Simiao Pill Compound prepared in Example 1 as an example. During the entire experiment, the mice were given normal free drinking water and feed supply. According to the Simiao Pill adult daily dosage of 12 g / kg stipulated in the 2022 edition of the People's Republic of China Pharmacopoeia, the mouse low-dose group was set to 12 g / kg, and the dosage converted from the body surface area of human and mouse was 1.48 g / kg. After concentration calculation, 250 ml of centrifuged fermented Simiao Pill Compound supernatant was concentrated to 84 ml, and each mouse was given 0.1 ml / d by gavage.

[0120] Tissue specimen collection: The mice were fasted for 12 h before being sacrificed, and the next day they were given modeling on an empty stomach. After 1 h of drug intervention, the mice were anesthetized with sodium pentobarbital (dose of 80 mg / kg), and 10 minutes later, blood was taken from the eyes of the mice. The blood was placed at room temperature for 1-2 h, and after coagulation, it was centrifuged at 4°C and 3000 r for 10 minutes. The supernatant was collected and stored at -80°C for later use. The kidneys and liver tissues of the mice were then taken, washed with pre-sterilized PBS buffer, and the intact kidneys and liver tissues were fixed in pre-prepared 4% paraformaldehyde fixative for pathological sectioning. The other half of the kidney and liver tissues were stored in a -80°C freezer. During the experiment, the activity, hair, diet, and urine of the mice were observed, and the body weight of the mice was measured once a week.

[0121] Western blotting detection: The liver tissue was taken out from the -80°C freezer, and 20 mg of liver was placed in an EP tube. 200 ul of cell lysis solution was added to the EP tube, 2-3 sterilized ethanol grinding beads (magnetic beads) were placed in it, and it was placed on ice. It was centrifuged at 4°C and 12000 rpm for 15 min, and the supernatant was collected as the total protein of the tissue. Then the absorbance value of the protein was measured using a 96-well plate, a standard curve was established to obtain the protein concentration, and the protein was quantified and denatured by heating. Prepare 10% separating gel and 4% concentrated gel, and place the gel plate in the electrophoresis tank with electrophoresis solution. Carefully pull out the comb in the gel to avoid affecting the loading. Place the gel plate in the electrophoresis clamp and fix it. Add an appropriate amount of electrophoresis solution to the electrophoresis tank. After loading, set the power to start electrophoresis, then transfer the membrane, incubate the primary and secondary antibodies, and finally use a chemiluminescence instrument to detect the protein expression band and perform gray scale analysis to calculate the relative content of the protein. The beta-actin immune protein was used as an internal reference for correction.

[0122] HE staining: The mouse kidney was taken out from the fixative and placed at room temperature for 24 h. The kidney tissue was dehydrated by ethanol gradient (75%, 85%, 95%, 100% twice) according to the conventional method, and was sealed with paraffin. The kidney was cut into sections with a longitudinal section and a thickness of 4 μm. Then the sections were placed in a 60°C oven for fixation. After the sections were taken out, they were immersed in xylene solution for 20 min (the solution needed to be replaced once in the middle) to remove the wax, and then were immersed in different concentrations of ethanol (100%, 95% twice, 75%) and then phosphate buffer solution (PBS) for washing. After washing, the sections were stained with hematoxylin for 10 min, and the excess dye was washed with PBS. Then 1% hydrochloric acid alcohol was added and the tissue was differentiated for 10 s, and then was washed with PBS twice. Finally, the sections were stained with eosin solution for 3 min, and then were dehydrated with different concentrations of ethanol solution (80%, 95%, 100%) for 2 min, and were dehydrated with xylene twice for 5 min each time. After the sections were sealed with neutral gum, they were observed under a microscope and were photographed.

[0123] 1.2.1 Effect on serum uric acid (HUA) level of hyperuricemic mice

[0124] The final product of purine metabolism is uric acid, and high uric acid is a typical feature of hyperuricemia. In the experiment, the uric acid and urea nitrogen in the serum were usually used to more directly express the uric acid in vivo.

[0125] Table 3: Effect of each experimental group on serum uric acid of hyperuricemic mice

[0126]

[0127] Note: The fermented Simiao pill compound high-dose group was compared with the blank group: ****P<0.0001, and the model group: *** a P<0.001, compared with the unfermented Simiao pill compound group** b P<0.01.

[0128] See Table 3, after modeling, compared with the blank group (NC), the serum uric acid content of hyperuricemia model group (HUA) mice increased significantly (P<0.0001), with significant difference, indicating that the high uric acid model was successfully replicated. Compared with the model group, the high dose group of fermented Simiao pill compound (ZH) significantly reduced the content of blood uric acid (P<0.001); the serum uric acid of benzbromarone group (BEN) was slightly reduced, and there was no significant difference, which was related to the factors of liver toxicity in mice with long-term medication. Compared with the non-fermented Simiao pill compound group (W), the high dose group of fermented Simiao pill compound (ZH) significantly reduced the content of blood uric acid (P<0.01), and the low dose group of fermented Simiao pill compound (ZH) had no significant difference, indicating that the high dose group of fermented Simiao pill compound (ZH) had good uric acid reducing effect.

[0129] 1.2.2 Influence analysis of serum urea nitrogen (BUN) level in acute hyperuricemia mice

[0130] Urea nitrogen is the end product of protein metabolism in the body and an indicator of glomerular filtration function, which can be used to evaluate whether the body's urine excretion function is normal. It is mainly used in experiments and clinics to judge chronic and acute renal failure and other diseases caused by various primary and secondary glomerulonephritis, kidney tumors, etc.

[0131] Table 4: Influence of each experimental group on blood urea nitrogen of hyperuricemia mice

[0132]

[0133] Note: The high dose group of fermented Simiao pill compound (ZH) compared with the blank group: ****P<0.0001, compared with the model group: *** a P<0.001, compared with the non-fermented Simiao pill compound group b P<0.01.

[0134] See Table 4, compared with the blank group, the serum urea nitrogen index of hyperuricemia model group mice had significant difference (P<0.0001), indicating that the kidney function of mice was impaired and the glomerular filtration function was poor. Compared with the model group and the non-fermented Simiao pill compound group, the urea nitrogen content in the serum of the high dose group of fermented Simiao pill compound (ZH) mice decreased significantly, and had significant difference respectively (P<0.0001, P<0.001), indicating that the high dose of fermented Simiao pill had a promoting effect on the glomerular filtration of hyperuricemia mice.

[0135] 1.2.3 Influence analysis of xanthine oxidase (XO) protein level in mice liver

[0136] Xanthine oxidase (XO) is a liver enzyme that synthesizes UA from purine metabolism, purine is synthesized in the body by liver metabolism, and the reaction of xanthine oxidase (XO) oxidizing xanthine to uric acid is a two-step reaction. First, XO oxidizes xanthine to xanthone. Then, xanthone is further oxidized to uric acid. By detecting the changes in the relative expression of XO protein in the liver, the effects of drugs on the livers of mice in each experimental group can be determined.

[0137] Figure 2 The effects of each experimental group on the protein level of xanthine dehydrogenase (XO) in the liver of mice.

[0138] See Figure 2 Compared with the blank group (NC), the relative protein expression level of XO in the high uric acid model group (HUA) increased, indicating that the XO activity in the liver of the model group increased. Compared with the model group (HUA) and the unfermented Simiao pill compound group (W), the fermented Simiao pill compound low dose group (ZL) and the fermented Simiao pill compound high dose group (ZH) showed different degrees of down-regulation of protein expression level, and the fermented Simiao pill compound high dose group (ZH) showed more obvious down-regulation of protein expression level, and the difference was significant (P<0.0001), indicating that the fermented Simiao pill compound can inhibit the XO activity in the liver of mice and reduce the uric acid level in the body.

[0139] 1.2.4 Analysis of the effects of fermented Simiao pill compound on the histopathology of the kidney tissue of acute hyperuricemic mice

[0140] Hyperuricemia can cause changes in kidney function, and long-term high uric acid can directly cause uric acid nephropathy, causing gouty nephropathy, and thus leading to renal insufficiency in the body.

[0141] Figure 3 The effects of each experimental group on the histopathology of the kidney tissue of mice are shown in the 20-fold magnification image.

[0142] See Figure 3, no obvious abnormality was observed in the renal cortex and medulla of the kidney of the NC mice. The renal tubules and glomeruli in the renal cortex were arranged normally, round, and the structure was clear; the renal tubular epithelial cells were full, uniformly colored, and normal in shape; there was no obvious hyperplasia of the renal interstitium, and no obvious inflammatory cell infiltration was observed. The glomeruli, renal tubules, and renal tubular epithelial cells in the model group (HUA) were arranged in disorder, the glomeruli were sclerotic, the renal tubules were dilated, and vacuolar glomeruli and renal tubules were observed, with obvious inflammatory cell infiltration, and a large number of vacuolar changes in the cells, indicating that the structure of the kidney in the model group was changed. The renal lesions in the BEN group were not obviously improved compared with the model group, the renal tubules were still dilated, the lumen was enlarged, the arrangement was disorderly, and there was obvious inflammatory cell infiltration, and occasional complete tubular casts were observed. Compared with the NC group, the unfermented Simiao Pill compound group (W) was slightly improved, the renal tubular dilatation was reduced, and complete renal tubular arrangement was observed, but there was still inflammatory cell infiltration, renal tubular epithelial cell necrosis, and disordered arrangement. The fermented Simiao Pill compound low-dose group (ZL) and the fermented Simiao Pill compound high-dose group (ZH) all had different degrees of reduction of renal lesions, compared with the model group (HUA) and the unfermented Simiao Pill compound group (W), the fermented Simiao Pill compound low-dose group (ZL) still showed obvious renal tubular and glomerular dilatation. The fermented Simiao Pill compound high-dose group (ZH) had a more obvious improvement in the kidney, occasional glomerular and renal tubular atrophy or vacuolization was observed, the renal tubules were arranged in order, the dilatation was reduced, there was a small amount of inflammatory cell infiltration, and the epithelial cell arrangement was obviously improved. It is indicated that the fermented Simiao Pill compound high-dose group (ZH) can improve the renal injury accompanied by hyperuricemia.

[0143] 1.2.5 Analysis of the effects of acute hyperuricemia on liver histopathology in mice

[0144] The liver is an important place for material metabolism, participates in the metabolism of drugs and poisons, and is one of the target organs of toxic effects. Simiao Pill is a compound of four drugs, and it is necessary to verify the liver toxicity of hyperuricemic mice after fermentation.

[0145] Figure 4 is the effect of each experimental group on the liver histopathology of mice, 20 times magnification.

[0146] As Figure 4As shown, the blank group (NC) mouse stem cells present vacuoles, fatty liver lesions. Considered is that the blank group is in the free drinking water and food during the experiment, the reason for the increasing body weight. The hepatocyte proliferation of the model group (HUA) and the benzbromarone group (BEN) is unclear, the liver lobule morphology is unclear, the liver cord arrangement is disorder, there are a large number of red blood cells in the liver sinus, and the red blood cells in the model group (HUA) are more obvious. The liver lobule of the unfermented Simiao pill compound group (W) and the fermented Simiao pill compound different dose groups is clear, the liver cord arrangement is neat, and there is no obvious red blood cell filling in the liver sinus. It is shown that the fermented Simiao pill compound does not produce toxicity to the liver of the mouse body.

[0147] Benzbromarone can inhibit the reabsorption of uric acid, promote the excretion of uric acid into urine, and ultimately reduce the serum uric acid level, and is a commonly used uric acid-lowering drug in clinic. However, benzbromarone can cause dysfunction of liver cell mitochondria in the liver, thus leading to liver toxicity. It is indeed so that the benzbromarone group (BEN) leads to the increase of the mortality of the other 5 groups of mice in the later period of the experiment, which shows that benzbromarone has hepatotoxicity to the mice with hyperuricemia and acute kidney injury.

[0148] The above animal experiment results can be reasonably deduced that the fermented Simiao pill compound fermentation liquor prepared by the preparation method of the fermented Simiao pill compound fermentation liquor can be used to reduce the uric acid level of the human body. In subsequent drug development and application, the effective components in the fermented Simiao pill compound fermentation liquor can be used to manufacture drugs for reducing the uric acid level of the body, especially drugs for treating gout and hyperuricemia, and these drugs can be powders, powders, tablets, pills, capsules, oral liquids or injections, and any carrier form in the prior art.

[0149] The above is only the preferred embodiment of the present application, and it should be pointed out that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A preparation method of Simiao pill compound fermentation liquor, characterized in that, It comprises the following steps: S1, 2-8 parts of Radix Cyathulae, 4-12 parts of Radix Rehmanniae Preparata, 2-8 parts of Radix Atractylodis, and 4-12 parts of Cortex Phellodendri powder are mixed by weight; S2, a liquid medium is prepared; S3, the powder mixture of S1 is added into the liquid medium of S2 to obtain a fermentation base, which is high-temperature cooked and then cooled; S4, inoculating lactic acid complex bacteria on the fermentation base in S3 to start fermentation culture; the lactic acid complex bacteria are Lactobacillus fermentum Lactobacillus, Lactobacillus plantarum Lactobacillus plantarum Lactobacillus rapi Lactobacillus reuteri Lactobacillus salivarius Lactobacillus salivarius Lactobacillus acidophilus Lactobacillus acidophilus mixed bacteria agent S5, high-temperature termination of fermentation; S6, filtration, centrifugation, taking supernatant to obtain the Simiao pill compound fermentation liquor.

2. The method of claim 1, wherein, The liquid medium is PDA liquid medium; 150-250 g of potato, 15-35 g of glucose, 2-4 g of magnesium sulfate, 3-5 g of potassium dihydrogen phosphate, and 0-40 mg of vitamin B1 are added into 1500 ml of medium, without agar; Alternatively, the liquid medium is soybean powder liquid medium; 4-6 g of soybean powder, 1-3 g of starch, 2-3 g of sucrose, 0.1-1 g of yeast extract powder, 0.1-1 g of sodium chloride, and 0.05-0.2 g of potassium phosphate dibasic are added into 250 ml of medium; Alternatively, the liquid medium is milk liquid.

3. The method of claim 1, wherein, The ratio of the powder mixture to the liquid medium is 1:7-70.

4. The method of claim 1, wherein, The high-temperature cooking is to put the fermentation base into a sealed container and place it in a pressure cooker, and cook at 121℃ for 25-30 min.

5. The method of claim 1, wherein, The inoculation amount of the lactic acid complex bacteria is 0.1-10 x 10 6 CFU / ml.

6. The method of claim 1, wherein, The fermentation culture is to culture in a shaker at 200-300 rpm and 37℃ for 7-10 days.

7. The method of claim 1, wherein, The step S6 is specifically as follows: the fermentation product is filtered, the residue is discarded, centrifuged at 5000-10000 rpm for 15-30 min, and the supernatant is taken to obtain the Simiao pill compound fermentation liquor.

8. A compound fermentation liquor of Simiaodecoction for reducing uric acid, characterized in that, Prepared by the method of any one of claims 1-7.

9. Use of the Simiao pill compound fermentation liquor of claim 8 in the preparation of a medicine for reducing the uric acid level of human body.

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