CN117695359A - Compound fermentation liquor of four-mulch pill as well as preparation method and application thereof - Google Patents
Compound fermentation liquor of four-mulch pill as well as preparation method and application thereof Download PDFInfo
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- CN117695359A CN117695359A CN202311491996.6A CN202311491996A CN117695359A CN 117695359 A CN117695359 A CN 117695359A CN 202311491996 A CN202311491996 A CN 202311491996A CN 117695359 A CN117695359 A CN 117695359A
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Abstract
The invention discloses a compound fermentation liquor of four wonderful pills, a preparation method and application thereof, and a powder mixture of achyranthes, coix seeds, rhizoma atractylodis and phellodendron is taken according to a preset proportion; preparing a liquid culture medium; adding the powder mixture into a liquid culture medium to obtain a fermentation medium, steaming at high temperature, and cooling; inoculating lactobacillus on the fermentation medium, and starting fermentation culture; terminating fermentation at high temperature; filtering, centrifuging, collecting supernatant, and making into compound fermentation liquor of four-effect pill, which can be used for reducing uric acid level of human body. The in-vivo research results of mice show that the uric acid reducing effect and the kidney protecting effect of the compound fermentation liquor of the four-mulation pill are superior to those of the compound fermentation liquor of the unfermented four-mulation pill, and the high-dose group of the fermented four-mulation pill can effectively reduce the uric acid level in the mice with hyperuricemia and play a role in protecting the kidney; the hepatotoxicity test result shows that the compound fermented four-mulation pill does not produce toxicity to the liver of the mice.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine fermentation, and relates to a fermentation broth of a compound of four wonderful pills, a fermentation method of the compound of four wonderful pills and application of the fermentation broth.
Background
Because the prescription mode of the traditional Chinese medicine is very flexible, the traditional Chinese medicine can be matched according to specific illness states and individual differences, and the compound quantity of the traditional Chinese medicine is very huge. At present, statistics show that various Chinese herbal medicine compounds in China are over ten thousand, and the number is continuously increasing. The continuous enrichment and development of the traditional Chinese medicine compound also provides rich choices for the clinical application of the traditional Chinese medicine.
The principle of modern traditional Chinese medicine fermentation technology is to screen and utilize proper probiotics and/or enzyme specific bioconversion capability, and change chemical components and biological activity of Chinese herbal medicine raw materials under specific environmental conditions (air, temperature, moisture and the like).
During fermentation of traditional Chinese medicine microorganisms, the microorganisms produce various metabolites including acids, bases, alcohols, ketones, esters, enzymes, etc. These metabolites may transform, degrade or enhance the active ingredients in the raw materials and produce new compounds. Such transformation processes tend to be complex, involving multiple microorganisms, multiple enzymes, and multiple reaction pathways, and thus difficult to predict and control entirely.
In addition, the complexity of the traditional Chinese medicine raw materials itself also increases the unpredictability of microbial fermentation. Traditional Chinese medicine is usually a compound comprising a plurality of plants or animals, and each raw material contains a plurality of active ingredients. There may be interactions and synergistic effects between these active ingredients, and metabolic changes during microbial fermentation may further alter such interactions and synergistic effects, resulting in changes in the activity and chemical composition of the final product.
The inventor makes a great deal of researches on compound microbial fermentation of four wonderful pills by a microbial fermentation technology, and in the process of realizing the invention, the inventor finds that at least one of the following technical problems exists in the prior art:
1. the four-effect pill compound is a pair of ancient Fang Fu prescription in Chinese traditional medicines, is composed of 4 traditional Chinese medicines of rhizoma atractylodis, cortex phellodendri, achyranthes and coix seed, has wide pharmacological effects, and clinical practice of the traditional Chinese medicine proves that the four-effect pill compound can effectively treat various types of arthritis, such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gouty arthritis and the like, but the prior art does not have reference materials for microbial fermentation of the four-effect pill compound.
2. The prior art does not disclose research results about the related influence of microbial fermentation of a compound prescription of the four wonderful pills and biological conversion products thereof, can not provide pharmacological action change direction guidance of the biological conversion products after the compound fermentation of the four wonderful pills compared with the compound fermentation of the four wonderful pills, and has great technical barriers on how to screen target fermentation microorganisms and how to realize effective fermentation.
Disclosure of Invention
In view of the above, one of the purposes of the present invention is to provide a compound fermentation broth of four wonderful pills with more pharmacological actions.
The second purpose of the invention is to provide a preparation method of the compound fermentation liquor of the four-ingredient pill.
The invention further aims to provide an application of the compound fermentation liquor of the four wonderful pills in reducing uric acid level.
The inventor continuously reforms and innovates through long-term exploration and trial and repeated experiments and efforts, and in order to solve the technical problems, the invention provides a preparation method of a four-wonderful compound fermentation liquor, which comprises the following steps:
s1, taking a powder mixture of achyranthes root, coix seed, rhizoma atractylodis and phellodendron according to a preset proportion;
or extracting Achyranthis radix, coicis semen, rhizoma Atractylodis and cortex Phellodendri with ethanol, and recovering ethanol to obtain ethanol extract;
or extracting Achyranthis radix, coicis semen, rhizoma Atractylodis, and cortex Phellodendri with water to obtain water extract;
s2, preparing a liquid culture medium;
s3, adding the powder mixture obtained in the step S1 into the liquid culture medium obtained in the step S2 to obtain a fermentation medium, and cooling after high-temperature cooking; or adding the alcohol extract or the water extract into a sterilized liquid culture medium to obtain a fermentation medium;
s4, inoculating lactic acid composite bacteria on the fermentation substrate in the S3, and starting fermentation culture;
s5, fermenting at a height of Wen Zhongzhi;
s6, filtering, centrifuging, and taking supernatant to obtain the compound fermentation liquor of the four-effect pill.
According to one embodiment of the preparation method of the compound fermentation liquor of the four-effect pill, the powder mixture comprises the following components in parts by weight: 2-8 parts of achyranthes root, 4-12 parts of coix seed, 2-8 parts of rhizoma atractylodis and 4-12 parts of amur corktree bark.
Preferably, in the powder mixture, the components are respectively as follows in parts by weight: 4 parts of achyranthes root, 4 parts of rhizoma atractylodis, 8 parts of coix seed, and 8 parts of amur corktree bark.
According to one embodiment of the preparation method of the compound fermentation liquor of the four-effect pill, the liquid culture medium is PDA liquid culture medium; 150-250 g of potato, 15-35 g of dextrose monohydrate, 2-4 g of magnesium sulfate, 3-5 g of monopotassium phosphate and vitamin B are added into every 1500ml of culture medium 1 20-40 mg, no agar is added.
According to one embodiment of the preparation method of the compound four-effect pill fermentation liquor, the liquid culture medium is soybean meal liquid culture medium, and each 250ml of the culture medium is added with 4-6 g of soybean meal, 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 dipotassium hydrogen phosphate.
According to one embodiment of the preparation method of the compound fermentation liquor of the four-effect pill, the liquid culture medium is milk.
Preferably, the liquid medium is PDA liquid medium; 200g of potato, 30g of dextrose monohydrate, 2.25g of magnesium sulfate, 4.5g of monopotassium phosphate and vitamin B are added into every 1500ml of culture medium 1 30mg, no agar was added.
According to one embodiment of the preparation method of the compound fermentation liquor of the four-effect pill, the feed liquid ratio of the powder mixture to the liquid culture medium is 1:7-70.
According to one embodiment of the preparation method of the compound four-effect pill fermentation liquor, the high-temperature cooking is carried out by placing a fermentation base into a closed container, and placing the closed container into an autoclave for cooking at 121 ℃ for 25-30 min.
According to one embodiment of the preparation method of the compound fermentation liquor of the four wonderful pills, the lactic acid composite bacteria comprise one or more of Lactobacillus fermentum, lactobacillus plantarum Lactobacillus plantarum, lactobacillus reuteri Lactobacillus reuteri, lactobacillus salivarius Lactobacillus salivarius and Lactobacillus acidophilus Lactobacillus acidophilus, and the inoculation amount is 0.1-10 multiplied by 10 6 CFU/ml。
According to one embodiment of the preparation method of the compound four-effect pill fermentation liquor, the fermentation culture is specifically as follows: culturing in shaking flask machine at 200-300 rpm and 37 deg.c for 7-10 days.
According to one embodiment of the preparation method of the compound four-effect pill fermentation liquor, the step S5 is specifically that a fermented product is placed in a device with the temperature of 95-100 ℃ and boiled for 10-20 min.
According to one embodiment of the preparation method of the compound fermentation liquor of the four wonderful pills, the step S6 specifically comprises the following steps: filtering the fermented product, discarding residues, centrifuging at 5000-10000 rpm for 15-30 min, and collecting supernatant to obtain compound fermentation liquor of four-mulch pill.
The invention also provides a compound fermentation liquor of the four-mulch pill, which is prepared by the method.
The invention also provides application of the compound fermentation liquor of the four-mulation pill in reducing uric acid level of human bodies.
Compared with the prior art, one of the technical schemes has the following advantages:
a) The invention utilizes the microbial fermentation technology, particularly uses lactic acid composite bacteria to carry out microbial fermentation on the compound of the four wonderful pills for the first time, carries out component analysis on the fermented compound of the four wonderful pills and the fingerprint spectrum of the compound of the unfermented four wonderful pills, and utilizes fermentation liquor to explore the effect of microbial fermentation products generated by the microbial fermentation of the compound of the four wonderful pills on uric acid level and kidney protection in mice.
b) In one embodiment of the preparation method of the compound fermentation liquor of the four-ingredient pill, the compound lactic acid bacteria are adopted to carry out microbial fermentation on the compound of the four-ingredient pill, the compound lactic acid bacteria are edible probiotics, 3 traditional Chinese medicines in the compound of the four-ingredient pill are medicinal and edible homologous traditional Chinese medicines, and a research foundation is laid for the subsequent research and development of the compound health-care food of the four-ingredient pill with higher nutritive value.
c) The inventor analyzes that the chemical components of the compound non-fermented four-mulation pill and the compound fermented four-mulation pill have great differences based on HPLC fingerprint detection.
d) The in-vivo research results of mice show that the uric acid reducing effect and the kidney protecting effect of the compound fermentation liquor of the four-mulation pill are better than those of the compound fermentation liquor of the unfermented four-mulation pill, and the high-dose group of the fermented four-mulation pill can effectively reduce the uric acid level in the mice with hyperuricemia and play a role in protecting the kidney; the hepatotoxicity test result shows that the compound fermented four-mulation pill does not produce toxicity to the liver of the mice.
The principle of uric acid reduction of four wonderful pills:
although the four wonderful pills have the effect of reducing uric acid of organisms, individual differences are remarkable. After the human body takes the traditional Chinese medicine, the traditional Chinese medicine needs digestion, absorption and metabolism of the gastrointestinal tract. The inconsistency of the flora of the gastrointestinal tract in the body is an important factor for the difference in absorption of the body due to individual differences. Lactic acid bacteria can promote absorption of gastrointestinal tract, and improve gastrointestinal flora. Fermentation can change the activity of lactobacillus and increase the amount of lactobacillus. The lactic acid composite bacteria and the four wonderful pills are adopted for fermentation, which simulates the in-vivo mode of human-machine gastrointestinal tract absorption.
After fermentation, the microorganism absorbs macromolecules such as cellulose, sugar, protein and the like in the four-effect pill plant body, and damages the cell wall of the plant to release the active ingredients of the medicine; the active ingredients excite the active substances in the four wonderful pills under the action of microbial enzymes, so that the active substance ingredients in the four wonderful pills are obviously increased. Animal experiments prove that the secondary metabolite produced by fermentation is beneficial to the absorption of the four-effect pill by the organism, improves the individual absorption difference caused by insufficient absorption and metabolism of the organism, improves the utilization rate of the four-effect pill, and enhances the effect of the four-effect pill on reducing uric acid of the organism. The difference in therapeutic effects between unfermented and fermented four-mulation pills results therefrom.
The compound four-effect pill prepared by lactic acid compound bacteria fermentation can inhibit the generation of purine in vivo and reduce the content of blood uric acid. The chemical components of the fermented four-ingredient pill are changed after fermentation, so that the fermented four-ingredient pill has higher uric acid reducing effect than the unfermented four-ingredient pill. The fermented four wonderful pills promote the excretion of uric acid salts of organisms, reduce the deposition of uric acid salts in the kidneys of the organisms and reduce inflammation, thereby playing a role in protecting kidney injury.
Therefore, compared with the non-fermented four-effect pill, the biological utilization rate of the four-effect pill is greatly improved, the expected effect of synergy of chemical components of the traditional Chinese medicine after biological conversion is fully reflected, and the basis is provided for developing the medicine and food homologous large health product of the four-effect pill.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a HPLC comparison chart of the compound supernatant of the four wonderful pills fermented by the lactic acid composite bacteria and the unfermented compound supernatant.
FIG. 2 is the effect of each experimental group on xanthine dehydrogenase (XO) protein levels in the liver of mice.
FIG. 3 is the effect of each experimental group on kidney histopathology in mice (20X).
FIG. 4 is the effect of each experimental group on liver histopathology in mice (20X).
Detailed Description
The following description is of one embodiment with reference to the accompanying drawings.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus, once an item is defined in one figure, it may not be further defined and explained in the following figures.
The four-effect pill compound is a pair of ancient Fang Fu prescriptions in Chinese traditional medicines, is carried in Qing dynasty Zhang Bingcheng for convenience in reading, is prepared by adding 2 flavors of achyranthes and coix seed on the basis of Ermiao powder in Danxi Xin method of Yuan Zhu Danxi, and is prepared by processing and cutting 4 traditional Chinese medicines of rhizoma atractylodis, cortex phellodendri, achyranthes and coix seed. The compound prescription of the four wonderful pills has wide pharmacological action, and clinical practice of the traditional Chinese medicine proves that the compound prescription of the four wonderful pills can effectively treat various types of arthritis, such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gouty arthritis and the like. In the recipe, rhizoma atractylodis has the effects of strengthening spleen and eliminating dampness, phellodendron has the effects of clearing heat and eliminating dampness, achyranthes root has the effects of tonifying liver and kidney, strengthening tendons and bones, and coix seed has the effects of strengthening spleen, clearing heat and promoting diuresis. The achyranthes root in the four-effect pill compound has various physiological activities of activating blood, resisting tumor, resisting inflammation, resisting arthritis, resisting oxidation, resisting aging, and the like, and is the most effective traditional Chinese medicine component for treating hyperuricemia in the four-effect pill compound. The coix seed is rich in various nutrient substances such as protein, carbohydrate, fat, crude fiber, vitamins, minerals, amino acids and the like, has high nutritive value, and has various biological activities such as immunoregulation, blood sugar reduction, tumor resistance, virus resistance, blood pressure reduction and the like.
Promoting uric acid excretion is one of the main means for treating hyperuricemia, and a representative drug such as benzbromarone takes kidneys as target organs, and promotes renal uric acid excretion by inhibiting renal tubule reabsorption, but side effects of the drug can continue to strengthen renal load, and cause adverse reactions such as chronic kidney diseases.
Compared with western medicines, the traditional Chinese medicine has the advantages of natural and green properties, small side effect and high safety. Along with the development of natural products such as traditional Chinese medicines, the development of natural products such as traditional Chinese medicines for reducing uric acid is gradually developed by researchers. In recent years, fermentation technology is widely adopted by scholars in traditional Chinese medicines, but the application research of the fermentation technology in the compound prescription of the four wonderful pills has not been developed yet.
In the course of completing the present invention, the inventors screened various microorganisms to ferment the four wonderful pill compound, and this example discloses a part of microorganism screening data to illustrate the importance of fermenting microorganism selection in the method of the present invention.
The optimal fermentation temperature of each bacterium is selected for fermentation: the fungus is fermented by microorganism at 26 ℃ and 34-37 ℃ of lactic acid composite bacteria and 26-30 ℃ of sweet wine yeast, and the bacteria are screened after 6 days of fermentation respectively. After fermentation, filtering, carrying out ultrasonic treatment on methanol, filtering again, carrying out HPLC detection, and analyzing whether the chemical components have obvious differences.
Compared with the compound of the non-fermented four wonderful pills, the compound of the fermented four wonderful pills has the advantages that the peak area of berberine hydrochloride for 36min serving as a reference substance of the compound of the fermented four wonderful pills is reduced, and the peak area of other chemical components is integrally reduced.
Compared with the compound without fermentation, the compound with the lactic acid compound bacteria has the advantages that the peak area of the berberine hydrochloride 36min in the compound with the fermentation is obviously increased, and the peak areas before and after the fermentation of the target peak 21min and the target peak 27min are obviously changed.
Compared with the compound of the four wonderful pills without fermentation, the compound of the four wonderful pills with the fermentation of the sweet distiller's yeast has the advantages that the peak area of the berberine hydrochloride is increased within 36min, but the peak area difference between the target peak area of 21min and the target peak area of 27min is smaller, which is not the same as that of the compound lactic acid bacteria fermented compound four wonderful pills.
The strain source of the lactic acid composite bacteria is China general microbiological culture collection center (CGMCC) (China, beijing). The lactic acid composite bacteria are compounded by 5 kinds of lactobacillus, and are respectively lactobacillus fermentum (CGMCC 1.15608), lactobacillus plantarum (CGMCC 1.12934), lactobacillus reuteri (CGMCC 1.2838), lactobacillus salivarius (CGMCC 1.1881) and lactobacillus acidophilus (CGMCC 1.3342). The four wonderful pill compound has 3 medicines which are listed in the medicine-food homologous catalogue, can promote the proliferation of lactic acid composite bacteria, and has the function of protecting the intestinal balance in the organism. Therefore, the inventor selects lactic acid composite bacteria which are beneficial to human bodies and are edible and probiotics as the fermentation inoculant of the four-effect pill compound.
In the process of completing the invention, the inventor tests the influence of different compound pretreatment modes of the four wonderful pills on the method, and verifies that high-temperature cooking has obvious influence on the preparation method of the compound fermentation liquor of the four wonderful pills. The inventors have extracted some of the experimental process data as follows:
each part of the Chinese medicinal composition comprises 4g of achyranthes root, 4g of rhizoma atractylodis, 8g of coix seed, 8g of amur corktree bark,crushing by a crusher, sieving with a 40-mesh sieve, and mixing to obtain a four-effect pill powder mixture. Preparing 250ml PDA liquid culture medium, potato 50g, dextrose monohydrate 5g, magnesium sulfate 0.4g, potassium dihydrogen phosphate 0.75g, vitamin B 1 1 mg, agar was not added. The pH of PDA culture medium is not required, natural pH is achieved, and the four wonderful pill powder mixture is added into 250ml PDA liquid culture based sealed shake flask without cooking.
Each part of achyranthes root 4g, rhizoma atractylodis 4g, coix seed 8g and phellodendron bark 8g are taken, crushed by a crusher, screened by a 40-mesh screen, and mixed to obtain a four-ingredient pill powder mixture. 250ml PDA liquid culture medium, 50g potato, 5g dextrose monohydrate, 0.4g magnesium sulfate, 0.75g potassium dihydrogen phosphate and vitamin B are prepared 1 1 mg, agar was not added. The pH of the PDA culture medium is not required, the natural pH is achieved, the four wonderful pill powder mixture is added into 250ml PDA liquid culture shake flask based on sealing, and the prepared shake flask is further placed into an autoclave for high-temperature steaming and boiling for 30min at 121 ℃.
Compared with no-cooking, the color of the four wonderful pill solution is changed visually, and the no-cooking color is lighter and vivid. The four wonderful pill materials after being steamed are brown and have darker color. And the two are subjected to content measurement, the sample to be measured liquid before and after steaming is treated under the same condition, the content measurement change difference is not obvious, and the chemical components of the four wonderful pills are not greatly influenced by steaming and non-steaming.
The components and the structure of the compound powder of the four wonderful pills are changed after high-temperature stewing, so that the active ingredients are easier to release, the active ingredients in the herbal medicine are more easily extracted, the solubility and the bioavailability of the active ingredients are increased, and more favorable conditions are created for the fermentation of the lactic acid composite bacteria. The inventors selected to use the sealed shake flask, and also avoided the possible loss of components due to high temperature, for example, the volatile oil of rhizoma Atractylodis could decrease with increasing temperature, and used the sealed shake flask, so that the components susceptible to high temperature, such as volatile oil of rhizoma Atractylodis, were not substantially affected.
Based on the results of the studies by the inventors, the following preferred embodiments were obtained.
Example 1
The preparation method of the compound four-effect pill fermentation liquor described in the embodiment comprises the following specific steps:
Step S1: according to the compound formula proportion of four wonderful pills in the pharmacopoeia of the people's republic of China, 4g of achyranthes root, 4g of rhizoma atractylodis, 8g of coix seed and 8g of amur corktree bark are taken per part, crushed by a crusher, screened by a 40-mesh screen and mixed to obtain a powder mixture. 6 parts of the powder mixture are prepared for use.
And S2, preparing a liquid culture medium.
PDA liquid culture medium is selected, and lactobacillus grows independently of carbon source, nitrogen source, microelements, vitamins, moisture and other growth factors, which are nutrients for lactobacillus growth and fermentation product production.
Preparing 1500ml of culture medium, 200g of potato, 30g of dextrose monohydrate, 2.25g of magnesium sulfate, 4.5g of monopotassium phosphate and vitamin B 1 30mg, no agar was added. The pH of the PDA culture medium is not required, the natural pH is achieved, and 250ml of PDA liquid culture medium is added into a shake flask. 1500ml of medium was dispensed into 6 shake flasks. The potato in PDA liquid culture medium can provide natural nitrogen source for lactobacillus, glucose provides carbon source, and magnesium sulfate and potassium dihydrogen phosphate play a role in providing microelements. The liquid culture medium can better lead the lactobacillus thallus to sufficiently shake and proliferate. Seal all shake flasks.
And S3, adding the powder mixture obtained in the step S1 into the liquid culture medium obtained in the step S2 to obtain a fermentation medium, and cooling to room temperature after high-temperature cooking.
To each shake flask of step S2 was added 1 part of a powder mixture having a feed liquid ratio to liquid medium of 1:10. The prepared shake flask was then put into an autoclave, steamed at 121℃for 30min, and cooled to room temperature. High-temperature cooking, on one hand, has a sterilization effect; on the other hand, the components and the structure of the compound powder of the four wonderful pills are changed, the active ingredients are easier to release, the active ingredients in the herbal medicine are more easily extracted, the solubility and the bioavailability of the active ingredients are increased, and more favorable conditions are created for the fermentation of the lactic acid composite bacteria.
And S4, inoculating lactic acid composite bacteria on the fermentation substrate in the step S3, wherein the ratio of the lactobacillus fermentum to the lactobacillus plantarum to the lactobacillus reuteri to the lactobacillus salivarius to the lactobacillus acidophilus is 1:1:1:1.
3 parts of the fermentation base steamed and boiled at the high temperature in the step S3 are used as treatment groups to be inoculated with lactic acid composite bacteria, and the inoculation amount is 10 6 CFU/ml. The other 3 points are used as a control group, and the microbial agent is not inoculated.
6 shake flasks were placed in a shake flask apparatus and incubated at 200rpm for 8 days at 37 ℃.
And S5, fermenting at a height of Wen Zhongzhi.
After the completion of the culture, the shake flask was placed in a 98℃apparatus and boiled for 15min. Killing microorganism in the culture solution, and stopping fermentation.
And S6, filtering, centrifuging, and taking supernatant to obtain the compound fermentation liquor of the four-effect pill.
Filtering the culture solution sterilized in the step S5, discarding residues, centrifuging at 8000rpm for 20min, and collecting supernatant to obtain compound fermentation broth of four Miao pills and non-fermentation supernatant of four Miao pills respectively.
The pH value of the non-fermented supernatant of the compound four-effect pill is 6, and the pH value of the compound fermented liquid of the four-effect pill is 4.12.
Example 2
As an alternative implementation way, 4g of achyranthes root, 4g of rhizoma atractylodis, 8g of coix seed and 8g of amur corktree bark are taken for each part, reflux extraction is carried out for 2 hours at 70 ℃ with 1000ml of distilled water, filtration is carried out, the medicine residues are reflux extracted for 1.5 hours with 600ml of distilled water, filtration is carried out, and the two filtrates are combined, thus obtaining the four-effect pill medicine material water extract.
Preparing a soybean meal liquid culture medium, weighing 5g of soybean meal, 1.25g of starch, 2.5g of sucrose, 0.5g of yeast meal, 0.5g of sodium chloride and 0.12g of dipotassium hydrogen phosphate, adding into 250ml of distilled water or deionized water, heating and boiling until the soybean meal liquid culture medium is completely dissolved, wherein the pH value of the culture medium is 7.3+/-0.1, subpackaging, putting the fermentation medium into a closed container, and placing into a pressure cooker for steaming at 121 ℃ for 25-30 min.
After the temperature is reduced to below 40 ℃, the bacteria are inoculated on a sterile operating table which is sterilized in advance for 30min.
Adding lactic acid composite bacteria 0.1-10×10 into shake flask 6 CFU/ml, recommended inoculum size is 1X 10 6 CFU/ml. The shake flask was placed at 37℃constantFermenting in a warm incubator for 8 days. After fermentation, the fermented product is filtered, the residue is removed, and the residue is centrifuged at 6000rpm for 20min, and the supernatant is taken.
Example 3
As an alternative embodiment, 4g of achyranthes root, 4g of rhizoma atractylodis, 8g of coix seed, 8g of phellodendron bark, 1000ml of 60% ethanol, reflux-extracting for 2h at 70 ℃, filtering, reflux-extracting residues with 600ml of 40% ethanol for 1.5h, filtering, combining the two filtrates, and recovering the ethanol in vacuum to obtain the four-ingredient pill material ethanol extract.
250ml of pure milk is added and stirred uniformly.
Under normal temperature, adding lactic acid complex bacteria 0.1X10 to shake flask 6 CFU/ml. The shake flask was placed in a constant temperature incubator at 37℃for fermentation for 8 days.
After fermentation, the fermented product is filtered, the residue is removed, and the mixture is centrifuged at 5000rpm for 15min, and the supernatant is taken.
Experimental example 1
For the technical effect of the compound fermentation liquor of the four wonderful pills prepared in the embodiment 1 of the specification, the experimental example is continuously developed.
1.1 HPLC measurements were performed on samples obtained in the control and treatment groups of example 1.
Performing HPLC (high Performance liquid chromatography) detection pretreatment on compound fermentation liquid of the four-effect pills and unfermented supernatant of the four-effect pills, respectively taking 5ml of compound fermentation liquid of the four-effect pills and unfermented supernatant of the four-effect pills, placing the compound fermentation liquid of the four-effect pills into 3 25ml volumetric flasks, and fixing the volume of methanol to the scale. The temperature is 33 ℃, the ultrasonic wave is carried out at 100HZ for 30min, the cooling is carried out, a proper amount of filter membrane with the volume of 0.22 mu m is taken for filtration, and 1.5ml of the filter membrane is filled in an HPLC sample bottle to wait for loading.
Chromatographic conditions: agilent 1260 index II HPLC chromatograph (configuration: 1260 differential refractive detector, 1260 diode array detector, agilent OpenLAB CDS software, GPC data analysis software), column SWELL C18 (250 mm. Times.4.6 mm. Times.5 μm, chromaplus) TM ) The method comprises the steps of carrying out a first treatment on the surface of the Column temperature is 30 ℃; the flow rate is 1ml/min; the sample injection volume is 10ul; the detection wavelength is 330nm. Mobile phase: phase A (acetonitrile, v/v), phase B (0.1% phosphoric acid, v/v); mobile phase elution gradient: 0-30 min, 7-24% (A); 30-40 min, 24-51% (A); 40 to 60min,51 percent～70％(A)；60～90min，90％(A)。
The measurement results are shown in Table 1 and FIG. 1. Table 1 shows the variation of the peak area of the compound for each retention time before and after fermentation of the four-mular pellet; in fig. 1, a is a control group, namely, a compound unfermented supernatant of the four-mulch pill, and B is a treatment group, namely, a compound fermented liquid of the four-mulch pill. Recording peak areas of corresponding chromatographic peaks under the retention time of each compound of the compound fermentation liquor of the compound of the four miao pills.
Table 1: variation of compound peak area under each retention time before and after compound fermentation of four wonderful pills
As can be seen from table 1 and fig. 1, the 36min retention time is the standard berberine hydrochloride, and the detection report shows that the peak area of berberine hydrochloride in the compound unfermented supernatant of the four-time pill is 3293.59; the peak with retention time of 16.2min is magnolol, and the peak area is 51.8; the peak area of the target peak with retention time 20.450min was 3206.97; the retention time of 24.634min was 62.59 as the peak area, which can represent the level of the analyte.
35.903min retention time is the control berberine hydrochloride, and the detection report shows that the peak area of the berberine hydrochloride in the compound fermentation liquor of the four-Miao pill is 3965.86 under the same detection condition; the peak with retention time of 15.9min is magnolol, and the peak area is 166.7; peak area for retention time 20.033min was 1819.98; the peak area for 24.256min retention time was 913.40. According to the target peak area change before and after fermentation treatment, the peak area of berberine hydrochloride in the compound fermentation liquid of the four-effect pill is increased by 20.41 percent compared with that of berberine hydrochloride in the compound unfermented supernatant liquid of the four-effect pill; the magnolol is increased by 221% after fermentation treatment; compared with the peak area of the compound non-fermented supernatant of the compound four-effect pill, the peak area of the target peak with the retention time of 20min is reduced by 43.25%, and the peak area of the target peak with the retention time of 24min is increased by 1359.34%.
1.2 in vivo uric acid lowering experiments in mice
The compound fermentation broth of the four wonderful pills obtained in the example 1 is subjected to in vivo uric acid reduction verification study. The method takes the factors of short period, low cost and the like into consideration, and adopts 200mg/kg of potassium oxazinate and 75mg/kg of adenine to perform combined gastric lavage for 28 days to form a mouse HUA model, so that the model can better reflect uric acid reaction of a mouse body and simulate hyperuricemia of the human body and renal diseases caused by the hyperuricemia. Therefore, the experiment adopts a method of combining potassium oxazinate with adenine modeling to evaluate the uric acid reducing and kidney protecting effects of the compound fermentation liquor of the four wonderful pills prepared according to the example 1.
Uric acid reducing experiment: taking compound fermentation liquid of the four-effect pills and unfermented supernatant of the compound fermentation liquid of the four-effect pills as a comparison experiment sample.
The experimental animals used in this experiment were: SPF-class C57BL/6 male mice 60, 6-8 weeks old, weight 20+ -22 g, purchased from Chengdu laboratory animal Co., ltd (China), license number: SCXK 2020-030. The raising environment temperature is (24+/-1)%, the humidity is (50+/-60)%, and 12 hours of light and shade alternate illumination are carried out, and the poplar padding is replaced periodically. Animals remained free to drink and eat during the experiment, and were fed adaptively for 7 days prior to the experiment.
The reagents used in this experiment were all commercially available, and Uric Acid (UA) kit was purchased from Nanjing institute of biological engineering, lot number (C012-1-1). The urea nitrogen (BUN) kit was purchased from Nanjing's institute of biological engineering, lot number (C013-2-1). Potassium oxazinate was purchased from Chemicals, inc., lot number (I2130045), and purine was purchased from Chemicals, inc., gebromarone was purchased from physical pharmaceutical shops.
The specific scheme of the animal experiment is as follows:
and (3) establishing an animal model: the mice HUA model was developed with 200mg/kg potassium oxazinate in combination with 75mg/kg adenine for 1 time/d. In order to ensure the balance of the concentration of the medicine in the centrifuge tube, the suspension adopts 0.5 percent CMC-Na solution to suspend the medicine, and the continuous molding is carried out for 35d. After the 7 th day of molding, 1ml of blood is taken from the eyeballs of the mice, the mice are placed at normal temperature for 1 to 2 hours, after the blood is coagulated, the mice are centrifuged for 10 minutes at 4 ℃ and 3000r, the supernatant is taken, and the uric acid value is measured by referring to the instruction book of a Uric Acid (UA) kit. A 20% increase in SUA levels above normal is considered successful in molding. According to the determination of the kit, the uric acid value of the model group is 471.73 (mu mol/L), the normal value of the mice is 41-126 mu mol/L, and the obtained value is 4 times of the maximum normal value, so that the HUA model of the mice is successful due to the combination of the potassium oxazinate and adenine for gastric lavage. The mice experimental groups are as follows:
Table 2: grouping of mice
Experimental grouping: normal control mice were gavaged with 0.5% cmc-Na solution, fed normal feed, and the remaining mice built the HUA model. After modeling was successful, mice were randomized into 6 groups with 10 doses each. The compound low dose group and the compound high dose group of the non-fermented four wonderful pill compound group and the fermented four wonderful pill compound are respectively a blank control group, a model group, a positive benzbromarone control group, a non-fermented four wonderful pill compound group and a fermented four wonderful pill compound low dose group. Administration was started on day 8 of molding, and after 3 hours of induction of hyperuricemia by daily gastric lavage, administration was continued for 21d. Modeling in the morning, performing dosing intervention on a positive afternoon group, a compound high-dose and low-dose fermented four-mul pill group and a compound unfermented four-mul pill group, and performing equal-volume physiological saline on a model group. Mice serum and tissue were taken for detection after day 28 of gavage.
Blank (NC): mice in the blank group were given normal free drinking water and feed supply throughout the experiment. The seventh day mice were perfused with 0.5% CMC-Na solution at the same time period as the other group hours, 0.2ml/d per mouse.
Model group (HUA): mice were given normal free drinking water and feed supply throughout the experiment. Modeling is carried out at 10 am, the concentration is obtained through a conversion method, and each mouse is irrigated with 0.08ml of potassium oxazinate/kg/d, 0.075ml of adenine/kg/d and 1 pm of physiological saline with the same volume.
Tribromone group (BEN): mice were given normal free drinking water and feed supply throughout the experiment. The dosing amount provided according to the reference was 6.5mg/kg, formulated at a concentration of 0.1ml/kg/d per mouse lavage.
Unfermented four-ingredient pill compound group (W): taking the compound unfermented supernatant of the four-mulch pill prepared in example 1 as an example, the mice were given normal free drinking water and feed supply throughout the experiment. Mice were modeled for hyperuricemia at 10 am and given intervention at 1 pm. The dosage of the four-effect pill is 12g/kg for adults per day according to the regulations of the pharmacopoeia of the people's republic of China in 2022, and the dosage of the pill converted by referring to the body surface areas of human bodies and mice is 1.48g/kg. And (3) concentrating 250ml of the centrifuged unfermented four-effect pill compound supernatant to 84ml through concentration calculation, and carrying out gastric lavage on each mouse by 0.1ml/d.
Fermented four wonderful compound low dose group (ZL): taking the compound fermentation broth of the four wonderful pills prepared in the example 1 as an example, the mice are given normal free drinking water and feed supply during the whole experimental period. According to the four wonderful pill set by the pharmacopoeia of the people's republic of China in 2022, the daily dosage of the four wonderful pill is 12g/kg, the low dosage group of the mice is set to be 3g/kg, and the dosage converted by referring to the body surface areas of the human body and the mice is 0.35g/kg. The supernatant was not concentrated by concentration calculation and each mouse was perfused with 0.73ml/d.
Fermented four-mulch pill compound high dose group (ZH): taking the compound fermentation broth of the four wonderful pills prepared in the example 1 as an example, the mice are given normal free drinking water and feed supply during the whole experimental period. According to the four wonderful pill set by the pharmacopoeia of the people's republic of China in 2022, the daily dosage of the four wonderful pill is 12g/kg, the low dosage group of the mice is set to be 12g/kg, and the dosage converted by referring to the body surface areas of the human body and the mice is 1.48g/kg. And (3) through concentration calculation, 250ml of the centrifuged compound supernatant of the fermented four-mul pill is concentrated to 84ml, and each mouse is irrigated with 0.1ml/d.
Collecting a tissue specimen: fasted for 12 hours before the mice are sacrificed, the following day of empty stomach modeling is performed, dosing intervention is performed after 1 hour, the mice are anesthetized by adopting pentobarbital sodium (the dosage is 80 mg/kg) after 1 hour, blood is taken from eyes of the mice after 10 minutes, the mice are placed at normal temperature for 1-2 hours, after the blood is coagulated, centrifugation is performed at 4 ℃ for 10 minutes at 3000r, supernatant is taken, and the supernatant is preserved at-80 ℃ for standby; and then taking kidney and liver tissues at two sides of the mouse, flushing the kidney and the liver tissues by using PBS buffer solution sterilized in advance, putting the completed kidney and liver tissues into 4% paraformaldehyde fixing solution prepared in advance for fixing, and putting the other half kidney tissues and liver tissues into a refrigerator at the temperature of minus 80 ℃ for preservation when pathological sections are used. Mice were observed for motor status, hair, diet, urine, and body mass measured 1 time per week during the experiment.
Western blot detection: the liver tissue was removed in a refrigerator at-80℃and 20mg of liver was placed in an EP tube. 200ul of cell lysate is added into an EP tube, 2 to 3 grinding beads (magnetic beads) sterilized by 75 percent ethanol are placed on ice, the mixture is centrifuged for 15min under the condition of 4 ℃ and 12000rpm, and the supernatant is taken to be the total tissue protein. And then measuring the absorbance value of the protein by using a 96-well plate, making a standard curve to obtain the protein concentration, quantifying the protein, and heating for denaturation. Preparing 10% of separating gel and 4% of concentrating gel, placing the gel plate in an electrophoresis tank with electrophoresis liquid, carefully horizontally pulling out the comb in the gel, and avoiding influencing sample loading. And (3) placing the rubber plate in an electrophoresis clamping plate for fixation, and taking a proper amount of electrophoresis liquid to be injected into an electrophoresis tank. And setting power to start electrophoresis after sample application, transferring a membrane, incubating the primary antibody and the secondary antibody, detecting protein expression bands by using a chemiluminescent instrument, performing gray level analysis, and calculating the relative content of the protein. The experiment uses beta-actin immune protein as internal reference correction.
HE staining: the kidneys of mice were removed from the fixative and left at room temperature for 24h, and the kidney tissue was dehydrated by ethanol gradient (75%, 85%, 95%, 100% twice) according to conventional methods, paraffin-encapsulated, and the kidneys were sectioned in longitudinal sections to a thickness of 4 μm. The sections were then placed in a 60 ℃ oven for fixation. The sections were taken out and immersed in xylene solution for 20min (one solution was replaced halfway) for dewaxing, then ethanol (100%, 95% twice, 75%) was added to each of the different concentrations, followed by immersing and washing in Phosphate Buffer (PBS). Washing, staining with hematoxylin for 10min, washing with PBS, washing with excess dye solution, dripping 1% hydrochloric acid alcohol to differentiate tissue for 10s, and washing with PBS twice. Finally, dyeing for 3min by using eosin dye liquor, removing redundant dye liquor, and then respectively dehydrating for 2min by using ethanol solutions (80%, 95% and 100%) with different concentrations, wherein the dehydration is carried out on dimethylbenzene for 2 times and 5min each time. After sealing with neutral resin, the film was observed under a microscope and photographed.
1.2.1 analysis of Effect on serum uric acid (HUA) levels in hyperuricemia mice
The end product of purine metabolism is uric acid, and uric acid elevation is a characteristic feature that causes hyperuricemia. In experiments, uric acid, urea nitrogen in serum is often used to more intuitively express uric acid in vivo.
Table 3: influence of each experimental group on hyperuricemia mice blood uric acid
Note that: comparing the compound high dose group of the four wonderful fermented pills with the blank group: * P <0.0001, compared to model group: * ** a P <0.001, compared with the unfermented compound four-ingredient pill group b P＜0.01。
Referring to table 3, after molding, the serum uric acid content of mice in the hyperuricemia model group (HUA) was significantly increased (P < 0.0001) compared to the blank group (NC), with significant differences, indicating successful replication of the hyperuricemia model. Compared with the model group, the blood uric acid content of the compound high-dose group (ZH) of the fermented four wonderful pill is obviously reduced (P < 0.001); the small amount of serum uric acid reduction of the benzbromarone group (BEN) has no significant difference, and is presumably related to the factors generating hepatotoxicity in mice subjected to long-time administration. Compared with the unfermented compound tetra-mulch pill group (W), the blood uric acid content of the compound high-dose group (ZH) of the fermented tetra-mulch pill is obviously reduced (P is less than 0.01), and the compound low-dose group (ZL) of the fermented tetra-mulch pill has no obvious difference, thus indicating that the compound high-dose group (ZH) of the fermented tetra-mulch pill has good uric acid reducing effect.
1.2.2 analysis of Effect on Urea Nitrogen (BUN) levels in serum of acute hyperuricemia mice
Urea nitrogen is a metabolic end product of body proteins, is an index of glomerular filtration function, and can be used to evaluate whether body urine excretion function is normal. The method is mainly used for judging various diseases such as chronic and acute renal failure caused by primary and secondary glomerulonephritis, renal tumor and the like in experiments and clinic.
Table 4: influence of each experimental group on blood urea nitrogen of hyperuricemia mice
Note that: the fermented four wonderful compound high dose group (ZH) was compared with the blank group: * P <0.0001, compared to model group: * ** a P <0.001, compared with the unfermented compound four-ingredient pill group b P＜0.01。
Referring to table 4, there was a significant difference in urea nitrogen index in the serum of mice in hyperuricemia model group (P < 0.0001) compared to the blank group, indicating that the renal function of mice was impaired and the glomerular filtration function was poor. Compared with the model group phase and the unfermented four-effect pill compound group, the urea nitrogen content in serum of the mice in the fermented four-effect pill compound high-dose group (ZH) is obviously reduced, and the urea nitrogen content are respectively obviously different (P <0.0001 and P < 0.001), which indicates that the high-dose of the fermented four-effect pill has an effect of promoting glomerular filtration of the mice with hyperuricemia.
1.2.3 analysis of Effect on Xanthine Oxidase (XO) protein levels in mouse liver
Xanthine Oxidase (XO) is a liver enzyme for the synthesis of UA by purine metabolism, purine synthesizes uric acid in the body by liver metabolism, and the reaction of Xanthine Oxidase (XO) to oxidize xanthine to uric acid is a two-step reaction. First, XO oxidizes xanthine to xanthine ketone. Then, xanthine ketone is further oxidized to uric acid. The influence of the drug on the liver of each experimental group of mice can be judged by detecting the change of the relative expression quantity of the XO protein in the liver.
FIG. 2 is the effect of each experimental group on xanthine dehydrogenase (XO) protein levels in the liver of mice.
Referring to fig. 2, the elevated levels of protein expression in the hyperuricic model group (HUA) XO relative to the blank group (NC) indicate increased XO activity in the liver of the model group. Compared with a model group (HUA) and an unfermented four-effect pill compound group (W), the fermented four-effect pill compound low-dose group (ZL) and the fermented four-effect pill compound high-dose group (ZH) have different degrees of down-regulation on protein expression levels, the down-regulation on the protein expression levels of the fermented four-effect pill compound high-dose group (ZH) is more obvious, and the difference has significance (P < 0.0001), so that the fermented four-effect pill compound can inhibit XO activity in the liver of a mouse and reduce uric acid level of an organism.
1.2.4 analysis of the Effect of acute hyperuricemia on renal histopathology in mice
Hyperuricemia can lead to renal function change, long-term uric acid is high, and urinary acid salt can directly cause urinary acid renal injury in kidney deposition, so as to cause gouty kidney disease, thereby leading to renal insufficiency of the organism.
FIG. 3 is a 20-fold magnification of the effect of each experimental group on kidney histopathology in mice.
Referring to fig. 3, no significant abnormalities were seen in the renal cortex and medulla of the kidneys of the mice in the blank (NC). The arrangement of the renal tubules and glomeruli in the renal cortex is normal, round and smooth, and the structure is clear; tubular epithelial cells are full, evenly colored and normal in morphology; the kidney interstitium has no obvious hyperplasia of hoof tissue and no obvious inflammatory cell infiltration. The glomerulus, the tubular and the tubular epithelial cells of the model group (HUA) are arranged in disorder, the glomerulus is hardened and the tubular is expanded, so that the vacuolated glomerulus and the tubular are obviously infiltrated by inflammatory cells, a large number of vacuolated changes of cells occur, and the kidney structure of the model group is indicated to be changed. Compared with the model group, the kidney lesions of the benzbromarone group (BEN) mice are not obviously improved, the kidney tubules are still expanded, the tube cavities are enlarged, the arrangement is disordered, obvious inflammatory cell infiltration exists, and the complete tube shape of the kidney tubules is even seen. Compared with NC group, the unfermented four wonderful pill compound group (W) is slightly improved, the tubular dilatation lesion is lightened, the complete tubular arrangement is visible, inflammatory cell infiltration is still present, tubular epithelial cell necrosis is still present, and the arrangement is not neat. The compound low dose group (ZL) of the fermented four wonderful pills and the compound high dose group (ZH) of the fermented four wonderful pills both have different degrees of alleviating kidney lesions, and compared with the compound group (W) of the non-fermented four wonderful pills and the model group (HUA), the compound low dose group (ZL) of the fermented four wonderful pills still has obvious renal tubule and glomerular expansion. The improvement degree of the kidney of the compound high-dose group (ZH) of the four-mulation-fermentation pill is more obvious, the glomerulus and the tubular are occasionally atrophic or vacuolated, the tubular are arranged orderly, the expansion is weakened, a small amount of inflammatory cells infiltrate, and the arrangement of epithelial cells is obviously improved. The compound high-dose group (ZH) of the four wonderful pills by conversion fermentation is suggested to improve the renal injury accompanied by hyperuricemia.
1.2.5 analysis of the Effect of liver histopathology on acute hyperuricemia mice
The liver is an important place for metabolism of substances, and participates in metabolism of drugs and poisons, and is one of target organs for toxic action. The four-component pill is a compound of four medicines, and whether the compound has liver toxicity effect on organisms after fermentation is considered, so that liver toxicity verification is required for mice with hyperuricemia.
FIG. 4 is a 20-fold magnification of the effect of each experimental group on liver histopathology in mice.
As shown in fig. 4, the stem cells of the mice in the blank group (NC) showed vacuoles, and fatty liver lesions were present. The blank group was considered to be in a free drinking and eating state during the experiment, and the mass was increased. Liver cell proliferation of model group (HUA) and benzbromarone group (BEN), unclear liver lobule morphology, liver cable arrangement disorder, and abundant red blood cells in liver sinus, and the red blood cells in model group (HUA) are obvious. The liver lobule of the unfermented compound pill group (W) and the liver lobule of the fermented compound pill group with different dosages are clear, the hepatic cable is orderly arranged, and no obvious erythrocyte is filled in the liver sinus. The compound fermented four-mulation pill is proved to have no toxicity to the liver of the mouse body.
The benzbromarone can inhibit the reabsorption of uric acid, promote uric acid to excrete into urine, and finally reduce serum uric acid level, and is a clinically common uric acid reducing medicament. But tribromone can cause dysfunction of liver cell mitochondria of the liver of an organism, thereby causing toxic effects of the liver. This was found to be true in the later stage of the experimental animal experiment, and the benzbromarone group (BEN) resulted in an increase in mortality of the other 5 mice in the later stage, indicating that benzbromarone has hepatotoxicity to hyperuricemia acute kidney injury mice.
The animal experiment results can be reasonably deduced, and the compound fermentation liquor of the four wonderful pills prepared by the preparation method of the compound fermentation liquor of the four wonderful pills can be used for reducing uric acid level of human bodies. In the subsequent drug development application, the active ingredients in the compound fermentation liquor of the four wonderful pills can be used for preparing drugs for reducing uric acid level of organisms, in particular drugs for treating gout and hyperuricemia, and the drugs can be powder, tablets, pills, capsules, oral liquid or injection and any carrier form in the prior art.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. The preparation method of the compound fermentation liquor of the four wonderful pills is characterized by comprising the following steps:
s1, taking a powder mixture of achyranthes root, coix seed, rhizoma atractylodis and phellodendron according to a preset proportion;
Or extracting Achyranthis radix, coicis semen, rhizoma Atractylodis and cortex Phellodendri with ethanol, and recovering ethanol to obtain ethanol extract;
or extracting Achyranthis radix, coicis semen, rhizoma Atractylodis, and cortex Phellodendri with water to obtain water extract;
s2, preparing a liquid culture medium;
s3, adding the powder mixture obtained in the step S1 into the liquid culture medium obtained in the step S2 to obtain a fermentation medium, and cooling after high-temperature cooking; or adding the alcohol extract or the water extract into a sterilized liquid culture medium to obtain a fermentation medium;
s4, inoculating lactic acid composite bacteria on the fermentation substrate in the S3, and starting fermentation culture;
s5, fermenting at a height of Wen Zhongzhi;
s6, filtering, centrifuging, and taking supernatant to obtain the compound fermentation liquor of the four-effect pill.
2. The method according to claim 1, wherein the powder mixture comprises the following components in parts by weight: 2-8 parts of achyranthes root, 4-12 parts of coix seed, 2-8 parts of rhizoma atractylodis and 4-12 parts of amur corktree bark.
3. The method of claim 1, wherein the liquid medium is PDA liquid medium; every 1500150-250 g of potato, 15-35 g of dextrose monohydrate, 2-4 g of magnesium sulfate, 3-5 g of monopotassium phosphate and vitamin B are added into the ml culture medium 1 20-40 mg, no agar is added;
or the liquid culture medium is soybean meal liquid culture medium, wherein each 250ml of the culture medium is added with 4-6 g of soybean meal, 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 dipotassium hydrogen phosphate;
Alternatively, the liquid medium is milk.
4. The method of claim 1, wherein the ratio of feed liquid of the powder mixture to the liquid medium is 1:7-70.
5. The method according to claim 1, wherein the high temperature cooking is to put the fermentation substrate into a closed container and put into an autoclave to cook at 121 ℃ for 25 to 30 minutes.
6. The method according to claim 1, wherein the lactic acid composite bacteria comprise one or more of Lactobacillus fermentum, lactobacillus plantarum Lactobacillus plantarum, lactobacillus reuteri Lactobacillus reuteri, lactobacillus salivarius Lactobacillus salivarius and Lactobacillus acidophilus Lactobacillus acidophilus, and the inoculation amount is 0.1-10×10 6 CFU/ml。
7. The method according to claim 1, characterized in that the fermentation culture is in particular: culturing in shaking flask machine at 200-300 rpm and 37 deg.c for 7-10 days.
8. The method according to claim 1, wherein the step S6 is specifically: filtering the fermented product, discarding residues, centrifuging at 5000-10000 rpm for 15-30 min, and collecting supernatant to obtain compound fermentation liquor of four-mulch pill.
9. A compound fermentation broth of four wonderful pills, which is characterized by being prepared by the method of any one of claims 1-8.
10. Use of the compound fermentation broth of four wonderful pills according to claim 9 for reducing uric acid level of human body.
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