CN108186693B - Preparation method of paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia, active substance prepared by method and application thereof - Google Patents
Preparation method of paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia, active substance prepared by method and application thereof Download PDFInfo
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- CN108186693B CN108186693B CN201711452741.3A CN201711452741A CN108186693B CN 108186693 B CN108186693 B CN 108186693B CN 201711452741 A CN201711452741 A CN 201711452741A CN 108186693 B CN108186693 B CN 108186693B
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- paecilomyces hepiali
- active substance
- uric acid
- hepiali
- paecilomyces
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Abstract
The invention relates to a preparation method of paecilomyces hepiali active substances, the prepared active substances and application thereof, wherein the method comprises the following steps: pulverizing dried fermented Paecilomyces hepiali mycelia, preferably micronizing to obtain Paecilomyces hepiali micropowder; placing the crushed paecilomyces hepiali mycelia in an extraction solvent, extracting for 1-3 times at 60-100 ℃ for 1-3 hours, filtering the filtrate, and combining the filtrates; concentrating the obtained filtrate, and drying to obtain Paecilomyces hepiali extract, wherein the obtained Paecilomyces hepiali superfine powder or Paecilomyces hepiali extract or mixture (complex) of the extracts is Paecilomyces hepiali active substance. The preparation process of the paecilomyces hepiali active substance has the advantages of high extraction rate, high active substance content and low cost; has obvious effect of reducing uric acid, has obvious treatment effect and produces low side effect; solves the problem of scarcity of natural Cordyceps resources.
Description
Technical Field
The invention relates to a preparation method of a paecilomyces hepiali active substance, the active substance prepared by the preparation method and the application of the active substance, and relates to the technical field of medicines or health care products.
Background
In recent years, with the improvement of living standard and the change of dietary structure of people, the incidence of hyperuricemia and gout in China is on a trend of rising year by year. Hyperuricemia is a disease in which blood uric acid exceeds a normal level due to purine metabolic disorder and/or uric acid excretion disorder, and does not clinically manifest any symptoms. Under the condition that a human body is in hyperuricemia for a long time, uric acid in the body is precipitated in joints, soft tissues, cartilages and kidneys in a sodium salt form, so that organ and tissue diseases of the human body are caused, gout is caused, and serious complications such as gouty arthritis, gouty kidney diseases, gouty kidney stones, gouty heart diseases, gouty hypertension and the like are caused.
Therefore, how to effectively reduce uric acid, delay the process of inducing gout, and improve kidney damage caused by hyperuricemia is a major problem of current clinical treatment.
At present, the medicines for treating hyperuricemia and gout mainly comprise febuxostat, allopurinol, colchicine, benzbromarone, probenecid and the like. Gout is divided into acute and chronic. Clinically, acute gout is usually treated by adopting medicaments such as anti-inflammatory drugs, colchicine, corticotropin and the like through inhibiting inflammatory factors. For chronic gout, effective control of blood uric acid levels is critical, and the drugs for reducing uric acid concentration in human bodies mainly include: allopurinol, febuxostat (inhibiting uric acid synthesis), benzbromarone, probenecid (promoting uric acid excretion) and the like. Clinical application proves that the medicines have strong side effects on liver and kidney metabolic organs, are easy to cause gastrointestinal tract and cardiovascular diseases, and limit the clinical application of the medicines.
Therefore, it is important to develop safe and effective uric acid-lowering drugs or foods from abundant natural resources, improve the treatment quality of patients with hyperuricemia and gout, and reduce the treatment burden.
More and more researches are carried out on the preparation and development of uric acid reducing medicines or functional products by adopting Chinese herbal medicines or food raw materials, and the application has good prospects. The patent publication No. CN104758920A discloses a health product which takes ocean fish oligopeptide powder, tuckahoe and lophatherum gracile as raw materials and is prepared by reasonable proportion to effectively inhibit hyperuricemia, remove tophus and enhance immunity; the patent publication No. CN201510529008.1 discloses a composition comprising a perilla leaf extract or perilla, coix seed, kudzu root, sophora flower bud, corn stigma and the like, which can effectively inhibit the synthesis of uric acid and promote the excretion of uric acid and can be used for dietary therapy of patients with hyperuricemia; the patent publication No. CN103637105A discloses that the uric acid reducing capsule product prepared from cherry fruit powder and cherry extract as raw materials has good effect on rheumatic lumbocrural pain, and is suitable for patients with hyperuricemia and gout.
However, most of the studies in the prior art focus on how to inhibit uric acid synthesis, promote excretion and reduce uric acid level in vivo, but the studies are less concerned with how to improve the problem of kidney damage caused by long-term hyperuricemia or taking uric acid-reducing drugs.
The edible fungi (such as lucid ganoderma, cordyceps sinensis and the like) are important natural resources in China, have a long edible history, are used for clinically treating diseases such as tumors, diabetes, hyperlipidemia and the like by modern medicine, and have obvious effects. The inventor prepares the cordyceps militaris active substance and finds that the cordyceps militaris active substance has good effect of preventing and treating hyperuricemia and also has good protection effect on kidney injury (CN 201410660797.8).
Meanwhile, Paecilomyces hepiali (Paecilomyces hepiali) is a ubiquitous anamorph of cordyceps sinensis, the effective components of the Paecilomyces hepiali are similar to those of the cordyceps sinensis, the fermentation mycelia of the Paecilomyces hepiali can be used as a substitute of the natural cordyceps sinensis, and large-scale production is realized. Modern pharmacological studies show that the paecilomyces hepiali contains active substances such as cordyceps polysaccharide, cordycepin and cordycepic acid, has the functions of resisting tumors, inflammation and fatigue, is listed as functional health-care food for developing and regulating immunity in 'a fungus strain list for health-care food' in 2001, and researches prove that the paecilomyces hepiali is safe and has no toxic or side effect.
At present, the application of paecilomyces hepiali and the preparation thereof in reducing the blood uric acid and treating gout is not reported in the prior art.
Disclosure of Invention
In view of the defects, the invention provides a preparation method of a paecilomyces hepiali active substance, the active substance prepared by the method and application of the active substance in reducing uric acid and treating gout.
The invention achieves the above purposes through the following scheme:
the inventor finds that the prepared paecilomyces hepiali active substance has good effects of inhibiting uric acid synthesis, promoting uric acid excretion and reducing uric acid through in-vivo hyperuricemia animal model evaluation for the first time, and has a strong protection effect on kidney injury.
On the basis, the inventor provides a preparation method of the paecilomyces hepiali active substance, and the prepared active substance is used for the treatment of clinical hyperuricemia or related diseases, the food therapy of hyperuricemia crowds and the like.
In the first aspect of the invention, the invention provides a preparation method of a paecilomyces hepiali active substance.
A method for preparing a paecilomyces hepiali active substance, which comprises the following steps:
(1) pulverizing dried fermented Paecilomyces hepiali mycelia, preferably micronizing to obtain Paecilomyces hepiali micropowder;
(2) placing the crushed paecilomyces hepiali mycelia in an extraction solvent, extracting for 1-3 times at 60-100 ℃ for 1-3 hours, filtering the filtrate, preferably filtering by using a 200-400 mesh filter screen, and combining the filtrates;
(3) concentrating the filtrate obtained in the step (2), and drying to obtain the paecilomyces hepiali extract, wherein the obtained paecilomyces hepiali superfine powder or the obtained paecilomyces hepiali extract or the mixture (compound) of the extracts is the active substance of the paecilomyces hepiali.
In the preparation method, the paecilomyces hepiali extract is obtained by the extraction solvent, and is used as an active substance of the paecilomyces hepiali; or a plurality of paecilomyces hepiali extracts and a compound (compound) of the plurality of paecilomyces hepiali extracts which are respectively prepared by a plurality of extraction solvents are used as the active substances of the paecilomyces hepiali.
Preferably, the dried paecilomyces hepiali mycelium powder is subjected to superfine grinding in the step (1) to obtain the paecilomyces hepiali superfine powder.
Preferably, the ultrafine pulverization time is 5 to 30 minutes, and more preferably 8 to 20 minutes each time.
Preferably, the number of the ultrafine pulverization is 1 to 3, and more preferably 2.
Preferably, the superfine grinding is grinding medium type grinding, mechanical shearing type superfine grinding or airflow type superfine grinding.
Preferably, the powder particle size of the paecilomyces hepiali submicron powder is 1-50 μm, and more preferably 5-20 μm.
Preferably, the extraction solvent is water or aqueous ethanol, and the weight ratio of the extraction solvent to the paecilomyces hepiali mycelium is 6-40: 1.
Preferably, the hydrous ethanol is 12-90% by volume, and more preferably 30-65% by volume.
Preferably, when the solvent is hydrous ethanol, the weight ratio of the hydrous ethanol to the paecilomyces hepiali mycelia is 8-30: 1.
Further preferably, the ethanol is food grade ethanol.
Preferably, the extraction mode of the step (2) is heating reflux, microwave extraction or ultrasonic-assisted extraction.
Preferably, the extraction time is 2 hours each time;
preferably, the number of extractions is 2.
Preferably, the drying in step (3) is air-blast drying, or freeze-drying or spray-drying, and more preferably spray-drying.
A method for preparing a paecilomyces hepiali active substance, which comprises the following steps:
(1) carrying out superfine grinding on dried fermented paecilomyces hepiali mycelia, wherein the superfine grinding is grinding medium type grinding, mechanical shearing type superfine grinding or airflow type superfine grinding, the superfine grinding time is 5-30 minutes, preferably 8-20 minutes each time, the superfine grinding frequency is 1-3 times, preferably 2 times, and paecilomyces hepiali superfine powder is obtained, and the particle size of the paecilomyces hepiali superfine powder is 1-50 micrometers, preferably 5-20 micrometers;
(2) placing the crushed paecilomyces hepiali mycelia in an extraction solvent, wherein the extraction solvent is water or aqueous ethanol, the weight ratio of the extraction solvent to the paecilomyces hepiali mycelia is 6-40:1, extracting for 1-3 hours, preferably for 2 hours, at 60-100 ℃, extracting for 1-3 times, preferably for 2 times, filtering the filtrate, preferably filtering by using a 200-400 mesh filter screen, and combining the filtrates;
(3) concentrating the filtrate obtained in the step (2), and drying, preferably spray drying to obtain the paecilomyces hepiali extract, wherein the extract or the mixture (compound) of the extracts is the paecilomyces hepiali active substance.
In the preparation method, the paecilomyces hepiali extract is obtained by the extraction solvent, and is used as an active substance of the paecilomyces hepiali; or a plurality of paecilomyces hepiali extracts and a compound (compound) of the plurality of paecilomyces hepiali extracts which are respectively prepared by a plurality of extraction solvents are used as the active substances of the paecilomyces hepiali.
A preparation method of a paecilomyces hepiali active substance, namely paecilomyces hepiali superfine powder, comprises the following steps: carrying out superfine grinding on dried fermented paecilomyces hepiali mycelia, wherein the superfine grinding is grinding medium type grinding, mechanical shearing type superfine grinding or airflow type superfine grinding, the superfine grinding time is 5-30 minutes, preferably 8-20 minutes each time, the superfine grinding frequency is 1-3 times, preferably 2 times, the particle size of the paecilomyces hepiali superfine powder is 1-50 micrometers, preferably 5-20 micrometers, and the paecilomyces hepiali superfine powder is obtained.
A preparation method of a paecilomyces hepiali active substance which is a paecilomyces hepiali water extract comprises the following steps:
(1) pulverizing dried fermented paecilomyces hepiali mycelia;
(2) placing the crushed paecilomyces hepiali mycelia in water, extracting for 1-3 times at 60-100 ℃ with a water-paecilomyces hepiali mycelium weight ratio of 6-40:1, filtering the filtrate, preferably with a 200-400 mesh filter screen, and combining the filtrates;
(3) concentrating the filtrate obtained in the step (2), and drying to obtain the paecilomyces hepiali water extract which is the active substance of the paecilomyces hepiali.
A preparation method of a paecilomyces hepiali active substance which is an aqueous ethanol extract of the paecilomyces hepiali comprises the following steps:
(1) pulverizing dried fermented paecilomyces hepiali mycelia;
(2) placing the crushed paecilomyces hepiali mycelia in 12-90% by volume of aqueous ethanol, preferably 30-65% by volume of aqueous ethanol, wherein the weight ratio of the aqueous ethanol to the paecilomyces hepiali mycelia is 8-30:1, extracting for 1-3 hours at 60-100 ℃, filtering the filtrate, preferably filtering by using a 200-400 mesh filter screen, extracting for 1-3 times, and combining the filtrates;
(3) concentrating the filtrate obtained in the step (2), and drying to obtain the paecilomyces hepiali aqueous ethanol extract which is the active substance of the paecilomyces hepiali.
Further preferably, the preparation method of the paecilomyces hepiali active substance comprises the step of mixing any two or three of the superfine powder, the water extract and the aqueous ethanol extract of the paecilomyces hepiali.
Preferably, in the preparation method, the active substances comprise 0-100% of paecilomyces hepiali water extract, 0-100% of paecilomyces hepiali aqueous ethanol extract and 0-100% of paecilomyces hepiali superfine powder in percentage by weight.
Preferably, in the preparation method, the active substances comprise 12-48% of the paecilomyces hepiali water extract and 20-62% of the paecilomyces hepiali superfine powder in percentage by weight.
Preferably, in the preparation method, the active substance comprises 5-32% of paecilomyces hepiali aqueous ethanol extract and 30-74% of paecilomyces hepiali superfine powder in percentage by weight.
Preferably, in the preparation method, the active substances comprise 7-30% of paecilomyces hepiali water extract, 5-35% of paecilomyces hepiali aqueous ethanol extract and 45-88% of paecilomyces hepiali superfine powder in percentage by weight.
In a second aspect of the invention, the paecilomyces hepiali active substance prepared by the preparation method is provided.
A Paecilomyces hepiali active substance comprises one or a mixture of two or three of a water extract or an aqueous ethanol extract of Paecilomyces hepiali and superfine powder of Paecilomyces hepiali.
Preferably, the active substances comprise 0-100% of paecilomyces hepiali water extract, 0-100% of paecilomyces hepiali aqueous ethanol extract and 0-100% of paecilomyces hepiali superfine powder in percentage by weight.
Further preferably, the active substances comprise 12-48% of paecilomyces hepiali water extract and 20-62% of paecilomyces hepiali superfine powder in percentage by weight.
More preferably, the active substances comprise 5-32% of paecilomyces hepiali aqueous ethanol extract and 30-74% of paecilomyces hepiali superfine powder in percentage by weight.
Further preferably, the active substances comprise 7-30% of paecilomyces hepiali water extract, 5-35% of paecilomyces hepiali aqueous ethanol extract and 45-88% of paecilomyces hepiali superfine powder in percentage by weight.
In a preferred embodiment, the active substance consists of the following components in percentage by weight: 14 percent of paecilomyces hepiali aqueous ethanol extract and 86 percent of paecilomyces hepiali superfine powder.
In a preferred embodiment, the active substance consists of the following components in percentage by weight: 35% of paecilomyces hepiali aqueous extract and 65% of paecilomyces hepiali superfine powder.
In a preferred embodiment, the active substance consists of the following components in percentage by weight: 28 percent of paecilomyces hepiali aqueous extract, 22 percent of paecilomyces hepiali aqueous ethanol extract and 50 percent of paecilomyces hepiali superfine powder.
By compounding, the content of effective substances in the active substance, such as polysaccharide content and adenosine content, can be increased, and the uric acid reducing effect of the active substance can be improved.
In a third aspect of the present invention, there is provided a pharmaceutical or food product comprising the above paecilomyces hepiali active substance.
Preferably, the food comprises common food, health food, formula food for special medical application and the like.
Preferably, the medicine comprises the active substance of the paecilomyces hepiali or pharmaceutically acceptable auxiliary materials.
Preferably, the medicament comprises tablets, granules, powder, capsules and oral liquid.
In a fourth aspect of the invention, there is provided a use of the above paecilomyces hepiali active substance or the above medicine or food.
Wherein, the use comprises the use in the aspect of reducing uric acid.
Further, the application comprises the treatment or prevention of hyperuricemia or gout or related complications and the improvement of the problem of kidney damage caused by long-term hyperuricemia or the administration of uric acid lowering drugs.
Further, the gout includes, but is not limited to, gouty arthritis, gouty kidney disease, gouty kidney stones, gouty heart disease, gouty hypertension, and the like.
The evaluation of an in-vivo hyperuricemia animal model proves that the paecilomyces hepiali active substance prepared by the invention has the pharmacological activities of remarkably reducing the synthesis of blood uric acid in a mouse body and promoting the excretion of uric acid, and further finds that the paecilomyces hepiali active substance has a good protection effect on kidney injury caused by hyperuricemia and uric acid reducing medicines.
The invention has the beneficial effects that:
(1) the preparation process of the paecilomyces hepiali active substance has the advantages of high extraction rate, high active substance content and low cost;
(2) the paecilomyces hepiali active substance has a remarkable uric acid reducing effect, can be used for preparing medicines or foods for relieving hyperuricemia or gout, has a good protection effect on kidney injury caused by hyperuricemia and uric acid reducing medicines, and has a remarkable treatment effect and low side effect;
(3) the invention adopts the fermented paecilomyces hepiali mycelia which are produced in a large scale, solves the problem of scarcity of natural cordyceps sinensis resources, and can promote the deep processing level development of the paecilomyces hepiali.
Drawings
FIG. 1 is an adenosine chromatogram of the active substance determined as described in example 6;
wherein A is an adenosine standard substance; B. c is paecilomyces hepiali active substance.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
Taking 100g of paecilomyces hepiali mycelium, crushing by using a crusher, adding into an erlenmeyer flask, adding 1000mL of distilled water, mixing, extracting under reflux at 100 ℃ for 2 hours, and filtering by using a 400-mesh filter screen. Extracting repeatedly by the above process once, mixing the obtained filtrates, distilling under reduced pressure, concentrating, and spray drying to obtain Paecilomyces hepiali water extract to obtain Paecilomyces hepiali active substance.
Premixing the paecilomyces hepiali water extract and a filler (the weight percentage of microcrystalline cellulose to isomaltitol is 1:2) according to the weight percentage of 1:1, adding a lubricant and a glidant in equal amount, gradually diluting, uniformly mixing, and performing a powder direct tabletting process to obtain the paecilomyces hepiali tablet.
Example 2
Taking 100g of paecilomyces hepiali mycelia, crushing by using a crusher, adding into a round-bottom flask, adding 2000mL of 60% ethanol water, mixing, extracting under reflux at 80 ℃ for 3 hours, and filtering by using a 200-mesh filter screen. Extracting repeatedly by the above process for one time, mixing the obtained filtrates, vacuum distilling, concentrating, and freeze drying to obtain Paecilomyces hepiali aqueous ethanol extract.
Taking 100g of paecilomyces hepiali mycelia, crushing by using a crusher, carrying out mechanical shearing type superfine crushing for 15 minutes, and repeating superfine crushing for 1 time to obtain the paecilomyces hepiali superfine powder with the powder particle size of 5-20 microns.
Respectively mixing 14% and 86% of the aqueous ethanol extract of the paecilomyces hepiali and the paecilomyces hepiali superfine powder by weight percentage to obtain the active substance of the paecilomyces hepiali, adding a filler, a lubricant and a glidant, uniformly mixing, and filling into hard capsules to obtain the paecilomyces hepiali capsules.
Example 3
Taking 100g of paecilomyces hepiali mycelia, crushing by using a crusher, carrying out mechanical shearing type superfine crushing for 15 minutes, repeating the superfine crushing for 1 time to obtain paecilomyces hepiali superfine powder with the powder particle size of 6-18 mu m, namely the paecilomyces hepiali active substance, and quantitatively packaging to obtain the paecilomyces hepiali powder.
Example 4
Taking 100g of paecilomyces hepiali mycelium, crushing by using a crusher, adding into an erlenmeyer flask, adding 1500mL of distilled water, mixing, extracting under reflux at 100 ℃ for 3 hours, and filtering by using a 200-mesh filter screen. Extracting repeatedly according to the above process for one time, mixing the obtained filtrates, vacuum distilling, concentrating, and spray drying to obtain Paecilomyces hepiali water extract.
Taking 100g of paecilomyces hepiali mycelia, crushing by using a crusher, carrying out mechanical shearing type superfine crushing for 15 minutes, and repeating superfine crushing for 1 time to obtain the paecilomyces hepiali superfine powder with the powder particle size of 5-18 mu m.
Respectively mixing 35% and 65% of the aqueous extract of the paecilomyces hepiali and the paecilomyces hepiali superfine powder by weight percentage to obtain the active substance of the paecilomyces hepiali, adding a filler, a lubricant and a glidant, uniformly mixing, and filling into hard capsules to obtain the paecilomyces hepiali capsules.
Example 5
Taking 100g of paecilomyces hepiali mycelium, crushing by using a crusher, adding into an erlenmeyer flask, adding 800mL of distilled water, mixing, extracting under reflux at 100 ℃ for 2 hours, and filtering by using a 400-mesh filter screen. Extracting repeatedly according to the above process for one time, mixing the obtained filtrates, vacuum distilling, concentrating, and spray drying to obtain Paecilomyces hepiali water extract.
Taking 100g of paecilomyces hepiali mycelia, crushing by using a crusher, adding into a round-bottom flask, adding 2500mL of 40% ethanol water, mixing, extracting under reflux at 80 ℃ for 3 hours, and filtering by using a 400-mesh filter screen. Extracting repeatedly by the above process for one time, mixing the obtained filtrates, vacuum distilling, concentrating, and freeze drying to obtain Paecilomyces hepiali aqueous ethanol extract.
Taking 100g of paecilomyces hepiali mycelia, crushing by using a crusher, carrying out mechanical shearing type superfine crushing for 8 minutes, and repeating superfine crushing for 3 times to obtain the paecilomyces hepiali superfine powder with the powder particle size of 8-16 mu m.
Mixing the paecilomyces hepiali aqueous extract, the paecilomyces hepiali aqueous ethanol extract and the paecilomyces hepiali superfine powder respectively according to the weight percentage of 28%, 22% and 50% to obtain the paecilomyces hepiali active substance, and quantitatively packaging to obtain the paecilomyces hepiali powder.
Example 6
Qualitative-quantitative analysis of paecilomyces hepiali active substance
1. The determination basis is as follows:
(1) adenosine: high performance liquid chromatography for determining cordycepin and adenosine in NY/T2116-2012 cordyceps sinensis product
(2) Crude polysaccharide: chineseme et al, Chineseme spectrophotometry for wolfberry polysaccharide content, chemistry of health food and its detection technology [ M ], published by the Chinese light industry, 1998.
2. Test samples: paecilomyces hepiali active substance prepared from example 1-example 5
3. Instruments and reagents: an Agilent100 high performance liquid chromatograph (Agilent corporation, usa), a UV-1750 UV-visible spectrophotometer (shimadzu corporation, japan), and the like; adenosine control, glucose, etc
4. Test method
4.1 adenosine
(1) Chromatographic conditions are as follows: agilent prep-C18 chromatographic column (250mm × 4.6mm,5 μm), column temperature 30 deg.C, ultraviolet detector, wavelength 260nm, mobile phase 10% methanol-water solution, flow rate 1mL/min, sample size 20 μ L.
(2) And (3) preparing a standard curve: accurately weighing 10mg (accurate to 0.0001g) adenosine standard, dissolving in 25mL volumetric flask with ultrapure water, respectively preparing adenosine standard solutions of 2.00. mu.g/mL, 4.00. mu.g/mL, 10.0. mu.g/mL, 20.0. mu.g/mL and 60.0. mu.g/mL, performing liquid chromatography under given instrument conditions, qualitatively analyzing peak appearance time, preparing standard curve by sample peak area to standard solution concentration, and calculating linear regression equation as y-44.607 x-1.174, R20.9999. The linear range is 2.0. mu.g/mL to 60. mu.g/mL.
(3) Sample detection: taking pure water as an extraction solvent, accurately weighing 0.2g of Paecilomyces hepiali active substance, pouring into a 25mL volumetric flask, adding about 20mL of pure water, performing ultrasonic treatment for 60min (300W and 40kHz), cooling, supplementing water to full scale, shaking up fully, centrifuging at 8000r/min for 5min, taking a proper amount of supernatant, filtering through a 0.22 mu m microporous filter membrane, injecting a sample, detecting, and calculating the content.
The adenosine chromatograms of the different samples obtained are shown in FIG. 1.
4.2 crude polysaccharide
(1) And (3) preparing a standard curve: 50mg of analytically pure glucose (dried to constant weight at 105 ℃) is accurately weighed, and is dissolved with distilled water to be constant volume to 500mL, so that 100 mu g/mL of standard glucose solution is obtained. 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL are respectively sucked into 6 colorimetric tubes, 1.00mL, 0.80mL, 0.60mL, 0.40mL, 0.20mL, 0.00mL deionized water are sequentially added, 1.5mL of 5% phenol solution and 7.0mL of concentrated sulfuric acid are respectively added, the mixture is shaken and then is kept stand for half an hour at room temperature, after cooling, the mixture is zeroed by a reagent blank under the condition of 488nm wavelength, and the A488 value is respectively determined. Plotting glucose standard curve according to the ratio of concentration to absorbance as y ═ 15.608x +0.0005, R2＝0.9999。
(2) Sample detection: accurately weighing 0.25g of paecilomyces hepiali active substance, adding 10mL of distilled water, shaking up, boiling water bath for 2 hours, cooling, supplementing water to 10mL, centrifuging for 15min at 4000r/min, taking supernatant, adding water to the precipitate again, shaking up, water bath for 1 hour in boiling water, centrifuging for 15min at 4000r/min, taking supernatant, adding 5mL of water to the precipitate, washing, centrifuging for 15min at 4000r/min, mixing the supernatant for three times, fixing the volume to 25mL, accurately taking 1.0mL of absolute ethyl alcohol 5mL, shaking up uniformly, precipitating overnight, centrifuging for 15min at 4000r/min, discarding supernatant, taking precipitate, adding 5mL of absolute ethyl alcohol, washing twice, dissolving the precipitate in 10mL of deionized water, sucking 1mL of solution into a half an hour, adding 1.5mL of phenol and 7.0mL of concentrated sulfuric acid, shaking up, reacting, cooling, and reacting at 488nm wavelength, the reagent blank was zeroed and the A488 values were determined. And comparing with a glucose standard sample to obtain the content of crude polysaccharide.
The results of measuring the adenosine and polysaccharide contents of the various samples obtained are shown in Table 1.
TABLE 1 content of adenosine, crude polysaccharide active ingredient in Paecilomyces hepiali active substance
Note: table 1 shows the paecilomyces hepiali active substance prepared in example 1; 2 is the aqueous ethanol extract of paecilomyces hepiali prepared in example 2; 3, the micro powder of the paecilomyces hepiali prepared in the example 3; 4 is the paecilomyces hepiali active substance prepared in example 2; 5 is the paecilomyces hepiali active substance prepared in example 4; 6 shows the paecilomyces hepiali active substance prepared in example 5. Wherein the measured masses of the samples 1 to 6 were the same.
As shown in FIG. 1, the chromatographic conditions can be used for accurately detecting adenosine in the Paecilomyces hepiali active substance.
From table 1, it can be seen that paecilomyces hepiali is prepared by different processes and reasonably compounded to obtain the paecilomyces hepiali active substance with higher content of active ingredients adenosine and crude polysaccharide, and the effect is better.
Example 7
Evaluation test of in vivo uric acid lowering function of paecilomyces hepiali
1. Experimental Material
Test animals: 80 SPF-grade KM mice, male, with the weight of 18 +/-5 g, provided by Guangdong provincial medical animal experiment center, and the license number: SYXK (Yue 2012 & 0122)
2. Test samples: 6 groups of paecilomyces hepiali actives determined in table 1; positive control: allopurinol.
3. Instruments and reagents: hitachi 7220 full-automatic biochemical analyzer; potassium oxonate and hypoxanthine.
4. Preparation of dosing solutions
Preparing a test sample: the paecilomyces hepiali active substances of the examples 1-5 are prepared by taking 6 groups (respectively corresponding to the 1-6 groups of samples in the content determination in the example 6) of the paecilomyces hepiali active substances, setting the administration dose to be 500mg/kg, and dissolving the dose with distilled water.
Positive control solution: 5mg/kg of allopurinol is prepared by distilled water.
5. Experimental methods
Taking 90 male Kunming mice, randomly dividing into 9 groups (n is 10): a normal control group, a hyperuricemic acid model group, a positive group, and a paecilomyces hepiali active substance 6 group (500mg/kg, 6 groups measured in table 1, respectively).
Except the normal group, the other groups are injected with hypoxanthine in the stomach and potassium oxonate in the abdominal cavity for molding every day, and water is not forbidden for one hour before molding; groups were gavaged after l hours: the normal control group and the model group are respectively administered by intragastric administration of sterile distilled water with equal gastric perfusion volume, the tested sample according to low set dose, and the yang medicine group is administered by intragastric administration of allopurinol with 5mg/(kg.d), and the continuous 7 days are carried out. And (3) taking blood from the eyeball of the animal after l hours after the last day of intragastric administration, centrifuging at 4000rpm for 10min, separating to obtain serum, and detecting the content of uric acid, creatinine and urea nitrogen in the serum.
Measuring serum uric acid content: the operation and the determination are carried out by adopting a tungstic acid method and strictly according to the instruction of a kit, and the operation and the determination of the content of creatinine and urea nitrogen are strictly carried out according to the instruction of the kit.
Statistical treatment: all data are as follows
This is shown processed using the sps 20.0 statistical software. Results tables 1, 2 and 3 show that the differences (P) were significant compared with the normal control group<0.01); # denotes significant difference compared to the model control group (P)<0.01))。
The results of the effect of the paecilomyces hepiali active substance on the content of uric acid, creatinine and urea nitrogen in the serum of the mice with hyperuricemia are shown in tables 2, 3 and 4.
TABLE 2 influence of Paecilomyces hepiali active substances on the serum uric acid content of hyperuricemia mice
Note: (1) p compared to normal control group<0.05,**p<0.01; in comparison to the set of models,#p<0.05，##p<0.01.
(2) in the table, 1-6 groups of paecilomyces hepiali active substances correspond to 1-6 groups of samples for content determination in example 6 respectively.
TABLE 3 influence of Paecilomyces hepiali active substances on Urea Nitrogen (BUN) in hyperuricemic mice
Note: p compared to normal control group<0.05,**p<0.01; in comparison to the set of models,#p<0.05，##p<0.01.
TABLE 4 influence of Paecilomyces hepiali active on creatinine (Cr) in hyperuricemia mice
Note: p compared to normal control group<0.05,**p<0.01; in comparison to the set of models,#p<0.05，##p<0.01.
as can be seen from Table 2, the hepialus armoricanus enzymatic active substance has a good uric acid reducing effect, and the uric acid reducing effect can reduce the blood uric acid concentration of a hyperuricemia model group to the uric acid level of a normal mouse. Urea nitrogen and creatinine levels are used as indexes for measuring kidney functions, and if the urea nitrogen and creatinine levels are high, the occurrence of kidney injury is prompted.
From tables 3 and 4, it can be seen that hyperuricemia and the harm of renal injury caused by allopurinol which is a uric acid reducing drug exist, and the paecilomyces hepiali active substance can effectively reduce urea nitrogen and creatinine to normal levels, so that a certain protection effect on renal injury is provided, and the paecilomyces hepiali active substance can be used for preparing medicines or foods for reducing uric acid and improving gout.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.
Claims (14)
1. A preparation method of paecilomyces hepiali active substances for reducing uric acid or treating hyperuricemia is characterized in that the active substances comprise 12-48% of paecilomyces hepiali water extract and 20-62% of paecilomyces hepiali superfine powder in percentage by weight;
or the active substances comprise 7 to 30 percent of paecilomyces hepiali water extract, 5 to 35 percent of paecilomyces hepiali aqueous ethanol extract and 45 to 88 percent of paecilomyces hepiali superfine powder according to the weight percentage;
the paecilomyces hepiali superfine powder is obtained by superfine grinding of dried fermented paecilomyces hepiali mycelia;
the paecilomyces hepiali water extract is obtained by crushing dried fermented paecilomyces hepiali mycelia, putting the crushed paecilomyces hepiali mycelia in water, extracting at 60-100 ℃ for 1-3 hours, filtering the filtrate, extracting for 1-3 times, combining the filtrates, concentrating the filtrate, and drying;
the paecilomyces hepiali aqueous ethanol extract is prepared by crushing dried fermented paecilomyces hepiali mycelia, putting the crushed paecilomyces hepiali mycelia into 12-90% by volume of aqueous ethanol, extracting the aqueous ethanol and the paecilomyces hepiali mycelia at a weight ratio of 8-30:1 at 60-100 ℃ for 1-3 hours, filtering the filtrate, extracting for 1-3 times, combining the filtrates, concentrating the filtrate, and drying.
2. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the aqueous extract of Paecilomyces hepiali and the aqueous ethanol extract of Paecilomyces hepiali are extracted at 60-100 ℃ for 2 hours.
3. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the aqueous extract of Paecilomyces hepiali and the aqueous ethanol extract of Paecilomyces hepiali are extracted at 60-100 ℃ for 2 times.
4. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the filtrate is filtered by a 200-400 mesh filter screen.
5. The method for preparing Paecilomyces hepiali Chen & Shen active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the micronization is milling medium type micronization, mechanical shearing type micronization or air stream type micronization.
6. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the micronization time is 5-30 minutes each time; and/or the number of times of the superfine grinding is 1-3.
7. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 6, wherein the micronization time is 8-20 minutes each time.
8. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 6, wherein the number of micronization is 2.
9. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the powder particle size of the Paecilomyces hepiali superfine powder is 1-50 μm.
10. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 9, wherein the powder particle size of the Paecilomyces hepiali superfine powder is 5-20 μm.
11. The method for preparing Paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 1, wherein the hydrous ethanol is 30-65% by volume.
12. A paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia, prepared by the preparation method of the paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to any one of claims 1 to 11.
13. A medicament for reducing uric acid or treating hyperuricemia, which is prepared from the paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 12.
14. Use of the paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia according to claim 12 in the preparation of a medicament for reducing uric acid or treating hyperuricemia.
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