CN109337953B - Inonotus obliquus D extract and preparation method and detection method thereof - Google Patents
Inonotus obliquus D extract and preparation method and detection method thereof Download PDFInfo
- Publication number
- CN109337953B CN109337953B CN201811292267.7A CN201811292267A CN109337953B CN 109337953 B CN109337953 B CN 109337953B CN 201811292267 A CN201811292267 A CN 201811292267A CN 109337953 B CN109337953 B CN 109337953B
- Authority
- CN
- China
- Prior art keywords
- inonotus obliquus
- extract
- fermentation
- solution
- inonotus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of biology, and relates to a inonotus obliquus D extract, and a preparation method and a detection method thereof. The inonotus obliquus D extract is obtained by the steps of preparing inonotus obliquus coarse powder, preparing saccharomycete fermentation liquor, preparing inonotus obliquus fermentation extract, preparing inonotus obliquus D extract and the like. The content of inonotus obliquus D in the extract is high. The invention also provides a method for determining the content of the inonotus obliquus D in the inonotus obliquus extract by adopting the high performance liquid chromatography.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to an inonotus obliquus D extract, and a preparation method and a detection method thereof.
Technical background:
inonotus obliquus, Latin name: inonotubelliquus (Fr.) Pilat belongs to Basidiomycotina, Hymenomycetes, Hymenochaetales, Hymenochaetaceae, Aphelophorales (Inonotus). China also refers to Fuscoporia obliqua, Leptoporus obliquus or Chaba, etc.
The mycelium of inonotus obliquus is extremely cold-resistant, and the mycelium living in wood can resist low temperature of minus 40 ℃. Mainly distributed in regions of 45-50 degrees north latitude of northern hemisphere, such as North America, Finland, Poland, Russian West Siberian, far east part region, Japanese Hokkaido region, China's Heilongjiang province, Xinghanganling province, Jilin province Changbai mountain region, etc.
Modern researches show that inonotus obliquus has various pharmacological effects of resisting tumor and oxidation, reducing blood sugar, resisting virus, protecting liver, resisting aging, regulating immunity, preventing and treating AIDS, and has certain therapeutic effects on emesis, diarrhea, gastrointestinal dysfunction, gastric and duodenal ulcer, hepatitis, gastritis and nephritis.
The research on the anti-cancer components, the research on the immunoregulation action and the pharmacological experiments show that the inonotus obliquus has obvious anti-cancer action. The specific components of beta-D mannose and antioxidant substances contained in the sclerotium of the inonotus obliquus are obviously higher than those of other mushrooms. The liquid submerged fermentation culture of the inonotus obliquus mycelium and the extract of the active ingredients of the inonotus obliquus sclerotium can be used as main raw materials of health food, and can be used for developing and producing various green health foods such as health food, medicinal beverage and the like.
Modern researches show that the inonotus obliquus fermentation product and wild resources thereof have the same or similar pharmacological activity, and the medicinal curative effect of the fermentation product is even better than that of a fruiting body and sclerotium in some aspects, which has important significance for further research and development of the inonotus obliquus.
The invention discloses a method for culturing inonotus obliquus by fermentation, which is applied to Xuzhou university at 2008/12/02, and the patent name is 200810227890.4, and the publication number is CN 101748159B.
The invention discloses a Chinese patent invention, which is applied to the research institute of microorganisms of the academy of sciences of Heilongjiang province at 2010, 12 months and 16 days, and the patent name of the Chinese patent application is a submerged fermentation medium of inonotus obliquus and a submerged fermentation method of the inonotus obliquus, the application number is 201010591235.4, and the authorization notice number is CN 102115350B. The submerged fermentation culture medium is prepared from carboxymethyl cellulose, glucose, soybean flour, yeast extract, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1, and water. Deep fermentation: firstly, preparing a submerged fermentation culture medium of inonotus obliquus; secondly, inoculating inonotus obliquus; thirdly, deep fermentation is carried out in two stages. The submerged fermentation culture medium and the submerged fermentation method of inonotus obliquus can obtain higher mycelium yield and intracellular and extracellular polysaccharides.
The Inonotus obliquus contains various chemical components, mainly including various triterpenes, lignans, melanin, folic acid derivatives, polysaccharide, etc. Among them, inonotus obliquus D (3-lanosta-8, 24-diene-21-aldehyde) is a very important active ingredient in inonotus obliquus, and has antitumor activity, and also has activities of resisting mutation, resisting oxidation, etc. The structural formula is as follows:
zhang Shi Bao, a institute of medicine of Chengdu Chinese medicine university, Sichuan province, academyelination of medical science, found Inonotus obliquus D in Inonotus obliquus for the first time, and published a related paper, research on the chemical components of triterpenes of Inonotus obliquus (recorded in herbal 2015, volume 46, phase 16, page 2355, 2360) in journal of Chinese herbal medicine in 2015.
However, under natural conditions, the content of the inonotus obliquus D in the inonotus obliquus is very low, about 0.0003%, and how to improve the content of the inonotus obliquus D in the inonotus obliquus and obtain an extract of the inonotus obliquus D with higher content is a technical problem which needs to be solved at present.
The invention content is as follows:
the invention aims to provide a preparation method of a fuscoporin D extract.
The invention also aims to provide a method for measuring the content of the inonotus obliquus D extract.
The purpose of the invention is realized by the following modes:
an extract of inonotus obliquus D is prepared by fermenting and extracting by biological fermentation method.
The inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus, and pulverizing to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean powder in purified water, sterilizing, cooling, inoculating grape juice yeast, adding xylose, and culturing to obtain yeast fermentation liquid;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1) with the yeast fermentation liquid obtained in the step (2) and purified water, and performing fermentation culture to obtain inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: adding an ethanol solution into the inonotus obliquus fermentation liquid obtained in the step (3), carrying out continuous ultrasonic countercurrent extraction, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on the dregs of a decoction under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform and then adding NaHCO3Mixing the water solution, soaking, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting a macroporous adsorption resin column, washing with water, discarding eluent, and adding acetone-0.25 mol.L-1Eluting with phosphoric acid solution mixed solution as eluent, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
The inonotus obliquus D extract is preferably prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus at 35-45 ℃, crushing, and sieving with a 20-30-mesh sieve to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water, wherein the ratio of the defatted soybean flour to the purified water is 1 g: 8-12 mL, sterilizing at 110-130 ℃ for 20-30 min, cooling to room temperature, inoculating grape juice yeast, wherein the inoculation amount is 13-17%, adding xylose accounting for 0.03-0.05% of the weight of the grape juice yeast, and culturing at 36-38 ℃ for 25-35 h to obtain yeast fermentation liquor for later use;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 0.5-1.5 mL: 25-35 mL, and performing fermentation culture at 36-38 ℃ for 32-40 h to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 3-5 times of 70-80% ethanol solution, carrying out continuous ultrasonic countercurrent extraction for 40-50 min at the temperature of 35-45 ℃ and at the frequency of 25-35 KHz, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on herb residues under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the chloroform into the inonotus obliquus fermentation extract with the concentration of 0.4-0.6 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3The ratio of the aqueous solution to the three is 1 g: 10-20 mL: 0.5-1.5 mL, the three are fully mixed and soaked for 20-30 h, chloroform extraction liquid is collected, the residual substance is extracted once again according to the method, the two extraction liquid are combined and filtered, the filtrate is evaporated to dry the chloroform, and the filtrate is dried to obtain the inonotus obliquus fermentation extract for later use;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 7-9, the sample loading volume is 1-2 BV, washing with water for 3-7 BV, the flow rate is 0.5-1.5 BV/H, discarding eluent, and adding 0.25moL L.L.of acetone-1Eluting 7-9 BV with an eluent with the ratio of 1: 0.5-1.5 of phosphoric acid solution at the flow rate of 0.5-1.5 BV/h, collecting eluent, concentrating the eluent, and drying to obtain the inonotus obliquus D extract.
The inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3Mixing the aqueous solution at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L of acetone-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
A method for measuring content of inonotus obliquus D extract comprises measuring content of inonotus obliquus D in inonotus obliquus extract by high performance liquid chromatography; the inonotus obliquus D extract is prepared by fermenting and extracting by a biological fermentation method.
The method for measuring the content of the inonotus obliquus D extract adopts a high performance liquid chromatography to measure the content of the inonotus obliquus D in the inonotus obliquus extract, and comprises the following steps:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol mixed solution; detection wavelength: 410-430 nm; flow rate: 0.5 to 2.0 mL/min-1(ii) a The column temperature is 20-40 ℃; sample introduction amount: 5-20 mu L;
(2) preparation of control solutions: precisely weighing inonotus obliquus D reference substance, placing in a measuring flask, adding acetonitrile-water mixed solution, dissolving to scale, and shaking to obtain reference substance solution;
(3) preparation of a test solution: precisely weighing Fuscoporia obliqua extract, placing in a measuring flask, adding acetonitrile-water mixed solution, performing ultrasonic treatment, adding acetonitrile-water mixed solution to scale, shaking, filtering, precisely weighing subsequent filtrate, placing in a measuring flask, adding acetonitrile-water mixed solution to scale, and shaking to obtain sample solution;
(4) and (3) determination: precisely sucking the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and measuring;
the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus, and pulverizing to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean powder in purified water, sterilizing, cooling, inoculating grape juice yeast, adding xylose, and culturing to obtain yeast fermentation liquid;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1) with the yeast fermentation liquid obtained in the step (2) and purified water, and performing fermentation culture to obtain inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: adding an ethanol solution into the inonotus obliquus fermentation liquid obtained in the step (3), carrying out continuous ultrasonic countercurrent extraction, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on the dregs of a decoction under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform and then adding NaHCO3Mixing the water solution, soaking, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting a macroporous adsorption resin column, washing with water, discarding eluent, and adding acetone-0.25 mol.L-1Eluting with phosphoric acid solution mixed solution as eluent, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
The method for measuring the content of the inonotus obliquus D extract adopts a high performance liquid chromatography to measure the content of the inonotus obliquus D in the inonotus obliquus extract, and preferably comprises the following steps:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, gauge: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol in a volume ratio of 10: 38-42: 28-32: 18-22; detection wavelength: 418-426 nm; flow rate: 0.5 to 1.5 mL/min-1(ii) a The column temperature is 25-30 ℃; sample introduction amount: 5-20 mu L;
(2) preparation of control solutions: precisely weighing 5-20 mg of inonotus obliquus D reference substance, placing the reference substance into a 10-20 mL measuring flask, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15, dissolving to a scale, and shaking up to obtain a reference substance solution;
(3) preparation of a test solution: precisely weighing 90-110 mg of inonotus obliquus extract, placing the inonotus obliquus extract in a 10-20 mL measuring flask, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15, carrying out ultrasonic treatment for 10-20 min, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15 to a scale, shaking up, filtering, precisely weighing 1-2 mL of subsequent filtrate, placing the subsequent filtrate in the 10-20 mL measuring flask, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15 to the scale, and shaking up to obtain a sample solution;
(4) and (3) determination: precisely sucking 5-20 mu L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;
the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus at 35-45 ℃, crushing, and sieving with a 20-30-mesh sieve to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water, wherein the ratio of the defatted soybean flour to the purified water is 1 g: 8-12 mL, sterilizing at 110-130 ℃ for 20-30 min, cooling to room temperature, inoculating grape juice yeast, wherein the inoculation amount is 13-17%, adding xylose accounting for 0.03-0.05% of the weight of the grape juice yeast, and culturing at 36-38 ℃ for 25-35 h to obtain yeast fermentation liquor for later use;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 0.5-1.5 mL: 25-35 mL, and performing fermentation culture at 36-38 ℃ for 32-40 h to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 3-5 times of 70-80% ethanol solution, carrying out continuous ultrasonic countercurrent extraction for 40-50 min at the temperature of 35-45 ℃ and at the frequency of 25-35 KHz, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on herb residues under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the chloroform into the inonotus obliquus fermentation extract with the concentration of 0.4-0.6 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3The ratio of the aqueous solution to the three is 1 g: 10-20 mL: 0.5-1.5 mL, the three are fully mixed and soaked for 20-30 h, chloroform extraction liquid is collected, the residual substance is extracted once again according to the method, the two extraction liquid are combined and filtered, the filtrate is evaporated to dry the chloroform, and the filtrate is dried to obtain the inonotus obliquus fermentation extract for later use;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 7-9, the sample loading volume is 1-2 BV, washing with water for 3-7 BV, the flow rate is 0.5-1.5 BV/H, discarding eluent, and adding 0.25moL L.L.of acetone-1Eluting 7-9 BV with an eluent with the ratio of 1: 0.5-1.5 of phosphoric acid solution at the flow rate of 0.5-1.5 BV/h, collecting eluent, concentrating the eluent, and drying to obtain the inonotus obliquus D extract.
The method for measuring the content of the inonotus obliquus D extract adopts a high performance liquid chromatography to measure the content of the inonotus obliquus D in the inonotus obliquus extract, and the most preferable steps are as follows:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, gauge: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol in a volume ratio of 10: 40: 30: 20; detection wavelength: 422 nm; flow rate: 1.0 mL/min-1(ii) a The column temperature is 28 ℃; sample introduction amount: 10 mu L of the solution;
(2) preparation of control solutions: precisely weighing 10mg of fuscoporin D reference substance, placing in a 10mL measuring flask, adding acetonitrile-water solution at volume ratio of 80: 20, dissolving to scale, and shaking to obtain the final product with concentration of 1.0 mg/mL-1The control stock solution of (a);
(3) preparation of a test solution: precisely weighing 100mg of Fuscoporia obliqua extract, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20, performing ultrasonic treatment for 15min, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, shaking up, filtering, precisely weighing 1mL of subsequent filtrate, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, and shaking up to obtain sample solution;
(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;
the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3Mixing the aqueous solution at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L of acetone-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
The technical effect of the invention is verified by the following experimental research:
experiment one: HPLC method for determining content of inonotus obliquus D in inonotus obliquus D extract
1 materials and methods
1.1 materials and reagents
Inonotus obliquus is purchased from Special products processing and distribution Co., Ltd in Hongfeng mountain of Daxing' an mountain of Heilongjiang, dried and crushed at 40 ℃ and sieved by a 40-mesh sieve. The inonotus obliquus D reference substance (the purity is more than or equal to 98%) is prepared by the laboratory; methanol, formic acid, isopropanol (all chromatographically pure), produced by Kancoded science and technology Limited, Tianjin, purchased from Harbin Deshi, Inc.; the rest reagents are analytically pure.
1.2 instruments and devices
Waters hplc; an electronic balance provided by Shanghai precision instruments and meters Co., Ltd; an electric heating forced air drying oven provided by Nanjing Huaao drying equipment Co.Ltd; an electric heating digital display constant temperature water bath kettle is provided by Xian Taikang biological science and technology limited company.
2 method
2.1 chromatographic conditions
A chromatographic column: c18Column (specification: 4.6 mm. times.250 mm, 5 μm); mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol in a volume ratio of 10: 40: 30: 20; detection wavelength: 422 nm; flow rate: 1.0 mL/min-1(ii) a Column temperature: 28 ℃; sample introduction amount: 10 μ L.
2.2 preparation of Inonotus obliquus D extract
(1) Preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus 1.00Kg at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3Mixing the aqueous solution at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L of acetone-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating, and drying to obtain 300.32mg of Inonotus obliquus D extract.
2.3 preparation of control solutions
Precisely weighing 10mg of fuscoporin D reference substance, placing in a 10mL measuring flask, adding acetonitrile-water solution at volume ratio of 80: 20, dissolving to scale, and shaking to obtain the final product with concentration of 1.0 mg/mL-1The control solution of (4).
2.4 preparation of test solutions
Precisely weighing 100mg of Fuscoporia obliqua extract, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20, performing ultrasonic treatment for 15min, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, shaking up, filtering, precisely weighing 1mL of subsequent filtrate, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, and shaking up to obtain the sample solution.
2.5 preparation of negative control solution
Acetonitrile-water solution with volume ratio of 80: 20 is used as sample solution to obtain negative control solution.
3 results and analysis
3.1 preparation of Standard Curve
Precisely measuring 1.0mL, 1.5mL, 2.0mL, 2.5 mL and 3.0mL of the reference solutions, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, and mixing to obtain standard solutions of different levels.
Taking each level of standard solution, carrying out sample injection analysis according to the chromatographic conditions provided by the invention, and carrying out regression analysis by taking the mass concentration of the reference solution as a horizontal coordinate and the peak area of a chromatographic peak as a vertical coordinate.
The regression equation: y is 1.6852X-18.264 and r is 0.9998.
The result shows that the inonotus obliquus D content is 35.9-298.6 mu g/mL-1Within the range, the linear relationship is good.
3.2 specificity experiments
Precisely sucking 10 microliter of reference substance solution, 10 microliter of test substance solution and 10 microliter of negative reference solution, and determining according to the chromatographic conditions and sample injection provided by the invention, wherein the result shows that the separation degree is better and the negative reference is not interfered, and the reference is shown in the attached figure 1, the attached figure 2 and the attached figure 3 of the specification.
3.3 precision test
And sampling the inonotus obliquus D reference substance solution for 6 times, measuring and recording the peak area, and calculating the Relative Standard Deviation (RSD) to be 0.28 percent to show that the precision of the instrument is good.
3.4 stability test
Sampling the same sample solution for 1 min, 10min, 20min, 30min, 40 min and 50min respectively, measuring, recording peak area, calculating RSD to be 0.42%, and indicating that the sample solution is stable within 50 min.
3.5 repeatability test
Taking 6 parts of the inonotus obliquus D extract, preparing a test solution according to the method provided by the invention, and measuring according to the chromatographic conditions provided by the invention, wherein the average content of the test solution is 35.6 percent, and the RSD is 1.24 percent. The repeatability is good.
3.6 sample application recovery test
Taking 6 parts of the fuscoporin D extract with known content, respectively taking 20.00mg of each, precisely weighing, respectively precisely adding precisely weighed fuscoporin D reference substances, preparing a test solution according to the method provided by the invention, measuring according to the chromatographic conditions provided by the invention, and calculating the recovery rate, wherein the results are shown in table 1.
Table 1 sample recovery test (n ═ 9)
3.7 measurement of sample content
The method established by the invention is adopted to determine the content of the inonotus obliquus D in 6 batches of inonotus obliquus D extracts, and the results are shown in table 2.
TABLE 26 Inonotus obliquus D content of samples of the batches
4.1 examination of ultrasonic extraction time in preparation of test solution
The invention inspects the ultrasonic extraction time in the preparation of the test solution, and respectively performs experiments on the extraction effects of ultrasonic for 10min, 15min, 20min and 25min, and the result shows that the ultrasonic extraction effect of 15min is the best.
4.2 selection of the Mobile phase
The invention respectively considers systems of methanol-water, methanol-water-formic acid, methanol-water-phosphoric acid, acetonitrile-water-0.15 percent phosphoric acid solution-isopropanol and the like, finally determines acetonitrile-water-0.15 percent phosphoric acid solution-isopropanol solution with the volume ratio of 10: 40: 30: 20 as a mobile phase, and has proper peak time and no tailing.
In conclusion, the HPLC method for determining the content of the inonotus obliquus D in the inonotus obliquus extract is simple and easy to implement, the detection method is high in precision, good in repeatability and good in stability, and the HPLC method can be used for detecting the product.
Experiment two: comparative study on purity and content of extract of fuscoporin D prepared by different extraction and purification methods
1 Inonotus obliquus D extract prepared by different extraction and purification methods
1.1 extract I:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus 1Kg at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3Mixing the aqueous solution at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of extract I: the birch brown hole obtained in the step (5)Separating the fermented extract by column chromatography, selecting H-30 type macroporous adsorbent resin column with height ratio of 1: 8, sample volume of 1BV, washing with water of 5BV and flow rate of 1BV/H, discarding eluate, and eluting with acetone of 0.25 mol.L-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating, and drying to obtain extract I300.61 mg.
1.2 extract II:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus 1Kg at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing an extract II: taking the inonotus obliquus coarse powder obtained in the step (1), adding chloroform, and then adding the coarse powder with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, inonotus obliquus coarse powder, chloroform, 0.5 mol.L-1NaHCO of3Mixing the aqueous solutions at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extract, extracting the rest materials again according to the above method, mixing the two extracts, filtering, volatilizing chloroform from the filtrate, and drying to obtain extract II 20265.62 mg.
1.3 extract III:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus 1Kg at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparation of extract III: performing column chromatography separation on the inonotus obliquus coarse powder obtained in the step (1), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter to the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L to acetone-1Eluting with eluent 1: 1 of phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluent, concentrating the eluent, and drying to obtain extract III 23286.63 mg.
1.4 extract IV:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus 1Kg at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparation of inonotus obliquus extract: taking the inonotus obliquus coarse powder obtained in the step (1), and adding chloroformThen adding the mixture to the mixture with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, inonotus obliquus coarse powder, chloroform, 0.5 mol.L-1NaHCO of3Mixing the aqueous solutions at a ratio of 1 g: 15 mL: 1mL, soaking for 24 hr, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, drying, and collecting Inonotus obliquus extract.
(2) Preparing an extract IV: performing column chromatography separation on the inonotus obliquus coarse powder obtained in the step (1), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter to the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L to acetone-1Eluting with eluent 1: 1 of phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluent, concentrating the eluent, and drying to obtain extract III 2284.69 mg.
2. Measurement of
The HPLC method provided by the experiment I of the invention is adopted to determine the content of the inonotus obliquus D in the inonotus obliquus D extract.
3. Measurement results
The results are shown in Table 3.
TABLE 3 Inonotus obliquus D content in extracts
4. Conclusion
As can be seen from the experimental results in Table 3, the extract of inonotus obliquus D obtained by the preparation method provided by the invention not only has high quality of the extracted inonotus obliquus D, but also has high content in the extract.
Description of the drawings:
FIG. 1 is a chromatogram of a control solution, wherein peak No. 1 is a chromatographic peak of inonotus obliquus D.
FIG. 2 is a chromatogram of a test solution, wherein the peak No. 1 is a chromatographic peak of inonotus obliquus D.
FIG. 3 is a negative control solution chromatogram.
The specific implementation mode is as follows:
example 1:
1. inonotus obliquus D extract and its preparation method
Preparation of Inonotus obliquus D extract
(1) Preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus 1Kg at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3The ratio of the aqueous solution to the three is 1 g: 15 mL: 1mL, and the three are fully mixed and soakedSoaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L of acetone-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating, and drying to obtain 299.12mg of Inonotus obliquus D extract.
2. Content determination of inonotus obliquus D
The method for determining the content of inonotus obliquus D in the inonotus obliquus extract by adopting high performance liquid chromatography comprises the following steps:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, gauge: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol in a volume ratio of 10: 40: 30: 20; detection wavelength: 422 nm; flow rate: 1.0 mL/min-1(ii) a The column temperature is 28 ℃; sample introduction amount: 10 mu L of the solution;
(2) preparation of control solutions: precisely weighing 10mg of fuscoporin D reference substance, placing in a 10mL measuring flask, adding acetonitrile-water solution at volume ratio of 80: 20, dissolving to scale, and shaking to obtain the final product with concentration of 1.0 mg/mL-1The control stock solution of (a);
(3) preparation of a test solution: precisely weighing 100mg of Fuscoporia obliqua extract, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20, performing ultrasonic treatment for 15min, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, shaking up, filtering, precisely weighing 1mL of subsequent filtrate, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, and shaking up to obtain sample solution;
(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;
(5) as a result: the weight of the inonotus obliquus D is 101.82mg, and the content is 34.04%.
Claims (6)
1. A preparation method of an inonotus obliquus D extract is characterized in that the inonotus obliquus D extract is prepared by the following steps:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus, and pulverizing to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean powder in purified water, sterilizing, cooling, inoculating grape juice yeast, adding xylose, and culturing to obtain yeast fermentation liquid;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1) with the yeast fermentation liquid obtained in the step (2) and purified water, and performing fermentation culture to obtain inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: adding an ethanol solution into the inonotus obliquus fermentation liquid obtained in the step (3), carrying out continuous ultrasonic countercurrent extraction, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on the dregs of a decoction under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform and then adding NaHCO3Mixing the water solution, soaking, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting a macroporous adsorption resin column, washing with water, discarding eluent, and adding acetone-0.25 mol.L-1Eluting with phosphoric acid solution mixed solution as eluent, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
2. The method of preparing the inonotus obliquus D extract according to claim 1, wherein the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus at 35-45 ℃, crushing, and sieving with a 20-30-mesh sieve to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water, wherein the ratio of the defatted soybean flour to the purified water is 1 g: 8-12 mL, sterilizing at 110-130 ℃ for 20-30 min, cooling to room temperature, inoculating grape juice yeast, wherein the inoculation amount is 13-17%, adding xylose accounting for 0.03-0.05% of the weight of the grape juice yeast, and culturing at 36-38 ℃ for 25-35 h to obtain yeast fermentation liquor for later use;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 0.5-1.5 mL: 25-35 mL, and performing fermentation culture at 36-38 ℃ for 32-40 h to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 3-5 times of 70-80% ethanol solution, carrying out continuous ultrasonic countercurrent extraction for 40-50 min at the temperature of 35-45 ℃ and at the frequency of 25-35 KHz, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on herb residues under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the chloroform into the inonotus obliquus fermentation extract with the concentration of 0.4-0.6 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3The ratio of the aqueous solution to the aqueous solution is 1 g: 10-20 mL: 0.5-1.5 mL, the chloroform extract is collected after fully mixing and soaking for 20-30 h, the residual substance is extracted once again according to the method, the two extracts are combined, filtered, the filtrate is dried by volatilizing chloroform and dried,obtaining inonotus obliquus fermentation extract for later use;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 7-9, the sample loading volume is 1-2 BV, washing with water for 3-7 BV, the flow rate is 0.5-1.5 BV/H, discarding eluent, and adding 0.25moL L.L.of acetone-1Eluting 7-9 BV with an eluent with the ratio of 1: 0.5-1.5 of phosphoric acid solution at the flow rate of 0.5-1.5 BV/h, collecting eluent, concentrating the eluent, and drying to obtain the inonotus obliquus D extract.
3. The method of preparing the inonotus obliquus D extract according to claim 2, wherein the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3Mixing the aqueous solution at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L of acetone-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
4. A method for measuring the content of an inonotus obliquus D extract is characterized in that the content of the inonotus obliquus D in the inonotus obliquus extract is measured by adopting a high performance liquid chromatography, and the method comprises the following steps:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol mixed solution; detection wavelength: 410-430 nm; flow rate: 0.5 to 2.0 mL/min-1(ii) a The column temperature is 20-40 ℃; sample introduction amount: 5-20 mu L;
(2) preparation of control solutions: precisely weighing inonotus obliquus D reference substance, placing in a measuring flask, adding acetonitrile-water mixed solution, dissolving to scale, and shaking to obtain reference substance solution;
(3) preparation of a test solution: precisely weighing Fuscoporia obliqua extract, placing in a measuring flask, adding acetonitrile-water mixed solution, performing ultrasonic treatment, adding acetonitrile-water mixed solution to scale, shaking, filtering, precisely weighing subsequent filtrate, placing in a measuring flask, adding acetonitrile-water mixed solution to scale, and shaking to obtain sample solution;
(4) and (3) determination: precisely sucking the reference solution and the sample solution, injecting into a high performance liquid chromatograph, and measuring;
the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus, and pulverizing to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean powder in purified water, sterilizing, cooling, inoculating grape juice yeast, adding xylose, and culturing to obtain yeast fermentation liquid;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1) with the yeast fermentation liquid obtained in the step (2) and purified water, and performing fermentation culture to obtain inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: adding an ethanol solution into the inonotus obliquus fermentation liquid obtained in the step (3), carrying out continuous ultrasonic countercurrent extraction, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on the dregs of a decoction under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform and then adding NaHCO3Mixing the water solution, soaking, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting a macroporous adsorption resin column, washing with water, discarding eluent, and adding acetone-0.25 mol.L-1Eluting with phosphoric acid solution mixed solution as eluent, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
5. The method for measuring the content of inonotus obliquus D extract according to claim 4, wherein the content of inonotus obliquus D in the inonotus obliquus extract is measured by high performance liquid chromatography, comprising the steps of:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, gauge: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol in a volume ratio of 10: 38-42: 28-32: 18-22; detection wavelength: 418-426 nm; flow rate: 0.5 to 1.5 mL/min-1(ii) a The column temperature is 25-30 ℃; sample introduction amount: 5-20 mu L;
(2) preparation of control solutions: precisely weighing 5-20 mg of inonotus obliquus D reference substance, placing the reference substance into a 10-20 mL measuring flask, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15, dissolving to a scale, and shaking up to obtain a reference substance solution;
(3) preparation of a test solution: precisely weighing 90-110 mg of inonotus obliquus extract, placing the inonotus obliquus extract in a 10-20 mL measuring flask, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15, carrying out ultrasonic treatment for 10-20 min, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15 to a scale, shaking up, filtering, precisely weighing 1-2 mL of subsequent filtrate, placing the subsequent filtrate in the 10-20 mL measuring flask, adding acetonitrile-water solution with the volume ratio of 75-85: 25-15 to the scale, and shaking up to obtain a sample solution;
(4) and (3) determination: precisely sucking 5-20 mu L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;
the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: drying inonotus obliquus at 35-45 ℃, crushing, and sieving with a 20-30-mesh sieve to obtain inonotus obliquus coarse powder for later use;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water, wherein the ratio of the defatted soybean flour to the purified water is 1 g: 8-12 mL, sterilizing at 110-130 ℃ for 20-30 min, cooling to room temperature, inoculating grape juice yeast, wherein the inoculation amount is 13-17%, adding xylose accounting for 0.03-0.05% of the weight of the grape juice yeast, and culturing at 36-38 ℃ for 25-35 h to obtain yeast fermentation liquor for later use;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 0.5-1.5 mL: 25-35 mL, and performing fermentation culture at 36-38 ℃ for 32-40 h to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 3-5 times of 70-80% ethanol solution, carrying out continuous ultrasonic countercurrent extraction for 40-50 min at the temperature of 35-45 ℃ and at the frequency of 25-35 KHz, filtering, collecting filtrate, repeating continuous ultrasonic countercurrent extraction once again on herb residues under the conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the chloroform into the inonotus obliquus fermentation extract with the concentration of 0.4-0.6 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3The ratio of the aqueous solution to the three is 1 g: 10-20 mL: 0.5-1.5 mL, the three are fully mixed and soaked for 20-30 h, chloroform extraction liquid is collected, the residual substance is extracted once again according to the method, the two extraction liquid are combined and filtered, the filtrate is evaporated to dry the chloroform, and the filtrate is dried to obtain the inonotus obliquus fermentation extract for later use;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 7-9, the sample loading volume is 1-2 BV, washing with water for 3-7 BV, the flow rate is 0.5-1.5 BV/H, discarding eluent, and adding 0.25moL L.L.of acetone-1Eluting 7-9 BV with an eluent with the ratio of 1: 0.5-1.5 of phosphoric acid solution at the flow rate of 0.5-1.5 BV/h, collecting eluent, concentrating the eluent, and drying to obtain the inonotus obliquus D extract.
6. The method for measuring the content of inonotus obliquus D extract according to claim 5, wherein the content of inonotus obliquus D in the inonotus obliquus extract is measured by high performance liquid chromatography, comprising the steps of:
(1) chromatographic conditions are as follows: a chromatographic column: c18Column, gauge: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-water-0.15% phosphoric acid solution-isopropanol in a volume ratio of 10: 40: 30: 20; detection wavelength: 422 nm; flow rate: 1.0 mL/min-1(ii) a The column temperature is 28 ℃; sample introduction amount: 10 mu L of the solution;
(2) preparation of control solutions: precisely weighing 10mg of fuscoporin D reference substance, placing in a 10mL measuring flask, adding acetonitrile-water solution at volume ratio of 80: 20, dissolving to scale, and shaking to obtain the final product with concentration of 1.0 mg/mL-1The control stock solution of (a);
(3) preparation of a test solution: precisely weighing 100mg of Fuscoporia obliqua extract, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20, performing ultrasonic treatment for 15min, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, shaking up, filtering, precisely weighing 1mL of subsequent filtrate, placing in a 10mL measuring flask, adding acetonitrile-water solution with volume ratio of 80: 20 to scale, and shaking up to obtain sample solution;
(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;
the inonotus obliquus D extract is prepared by the following method:
(1) preparing inonotus obliquus coarse powder: oven drying Inonotus obliquus at 40 deg.C, pulverizing, and sieving with 24 mesh sieve to obtain Inonotus obliquus coarse powder;
(2) preparing yeast fermentation liquor: dissolving defatted soybean flour in purified water at a ratio of 1 g: 10mL, sterilizing at 120 deg.C for 25min, cooling to room temperature, inoculating grape juice yeast with an inoculum size of 15%, adding xylose in an amount of 0.04% of the weight of grape juice yeast, and culturing at 37 deg.C for 30h to obtain yeast fermentation broth;
(3) preparing inonotus obliquus fermentation liquor: mixing the inonotus obliquus coarse powder obtained in the step (1), the yeast fermentation liquid obtained in the step (2) and purified water, wherein the ratio of the inonotus obliquus coarse powder to the yeast fermentation liquid to the purified water is 1 g: 1 mL: 30mL, and performing fermentation culture at 37 ℃ for 36 hours to obtain the inonotus obliquus fermentation liquid for later use;
(4) preparation of the inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation liquor obtained in the step (3), adding 75% ethanol solution in an amount which is 4 times that of the inonotus obliquus fermentation liquor, carrying out continuous ultrasonic countercurrent extraction for 45min at 40 ℃ under 30KHz, filtering, collecting filtrate, carrying out continuous ultrasonic countercurrent extraction on the medicine residue once again under the above conditions, combining the two extracted filtrates, concentrating the combined filtrate, and carrying out vacuum drying to obtain an inonotus obliquus fermentation extract for later use;
(5) preparing a inonotus obliquus fermentation extract: taking the inonotus obliquus fermentation extract obtained in the step (4), adding chloroform, and then adding the solution with the concentration of 0.5 mol.L-1NaHCO of3Aqueous solution, Inonotus obliquus fermented extract, chloroform, and 0.5 mol.L-1NaHCO of3Mixing the aqueous solution at a ratio of 1 g: 15 mL: 1mL, soaking for 24h, collecting chloroform extractive solution, extracting the rest materials according to the above method, mixing the two extractive solutions, filtering, volatilizing chloroform from the filtrate, and drying to obtain Inonotus obliquus fermented extract;
(6) preparation of inonotus obliquus D extract: performing column chromatography separation on the inonotus obliquus fermentation extract obtained in the step (5), selecting an H-30 type macroporous adsorption resin column, wherein the height ratio of the resin column diameter is 1: 8, the sample loading volume is 1BV, washing with water for 5BV, the flow rate is 1BV/H, discarding the eluent, and adding 0.25moL L.L of acetone-1Eluting with eluent of 1: 1 phosphoric acid solution at flow rate of 1BV/h for 8BV, collecting eluate, concentrating the eluate, and drying to obtain Inonotus obliquus D extract.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811292267.7A CN109337953B (en) | 2018-11-01 | 2018-11-01 | Inonotus obliquus D extract and preparation method and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811292267.7A CN109337953B (en) | 2018-11-01 | 2018-11-01 | Inonotus obliquus D extract and preparation method and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109337953A CN109337953A (en) | 2019-02-15 |
| CN109337953B true CN109337953B (en) | 2021-11-26 |
Family
ID=65312890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811292267.7A Active CN109337953B (en) | 2018-11-01 | 2018-11-01 | Inonotus obliquus D extract and preparation method and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109337953B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825540B (en) * | 2019-03-28 | 2020-05-08 | 青岛农业大学 | Preparation method and application of inonotus obliquus non-saccharide extract |
| CN113069484B (en) * | 2021-03-12 | 2022-05-13 | 黑龙江中医药大学 | Preparation method of microcapsule preparation of anti-aging medicine |
| CN116120480B (en) * | 2023-01-06 | 2024-04-26 | 湖南杰萃生物技术有限公司 | Method for simultaneously extracting inonotus obliquus polysaccharide and inonotus obliquus element |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418325A (en) * | 2008-12-03 | 2009-04-29 | 王�琦 | Inonotus obliquus extracellular and incellular mixed exopolysaccharide and preparation method thereof and pharmaceutical use |
| CN106978465A (en) * | 2017-04-06 | 2017-07-25 | 杭州娃哈哈科技有限公司 | A kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield |
| CN107841523A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | Triterpene substance method is extracted from Inonotus obliquus using the induction of quorum sensing molecule |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9399050B2 (en) * | 2000-10-04 | 2016-07-26 | Paul Edward Stamets | Controlling insects and arthropods using preconidial mycelium and extracts of preconidial mycelium from entomopathogenic fungi |
-
2018
- 2018-11-01 CN CN201811292267.7A patent/CN109337953B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418325A (en) * | 2008-12-03 | 2009-04-29 | 王�琦 | Inonotus obliquus extracellular and incellular mixed exopolysaccharide and preparation method thereof and pharmaceutical use |
| CN106978465A (en) * | 2017-04-06 | 2017-07-25 | 杭州娃哈哈科技有限公司 | A kind of fermentation process for improving Inonotus obliquus total triterpene fermentation yield |
| CN107841523A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | Triterpene substance method is extracted from Inonotus obliquus using the induction of quorum sensing molecule |
Non-Patent Citations (4)
| Title |
|---|
| 张仕瑾.桦褐孔菌三萜类化学成分研究.《中草药》.2015,第46卷(第16期),第2355-2360页. * |
| 桦褐孔菌三萜类化学成分研究;张仕瑾;《中草药》;20150828;第46卷(第16期);第2355-2360页 * |
| 桦褐孔菌多糖的分离纯化及其组成分析;范柳萍;《食品工业科技》;20120215;第33卷(第11期);第101-103页 * |
| 高效液相色谱和超高效液相色谱在药物分析中的应用研究;郑勤琴;《上海畜牧兽医通讯》;20170420(第2期);第27-31页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109337953A (en) | 2019-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109337953B (en) | Inonotus obliquus D extract and preparation method and detection method thereof | |
| CN102977114B (en) | Inoscavin A as a monomeric component in phellinus as well as prepearation method and application thereof | |
| CN102058641A (en) | Angelica dahurica extract and quality detection method | |
| CN1683418A (en) | Process for preparing antrodia camphorata polysaccharide and antrodia camphorata triterpene with micro-prorous adsorptive resin and its product | |
| Gao-Sheng et al. | Accumulation of biomass and four triterpenoids in two-stage cultured Poria cocos mycelia and diuretic activity in rats | |
| CN109749941B (en) | Paecilomyces cicadae fermentation medium and method for preparing ergosterol by fermenting Paecilomyces cicadae | |
| CN104987429B (en) | A kind of Antrodia Camphorata compound component separating and extracting process | |
| CN106119333A (en) | A kind of method utilizing fatty acid-induced to extract betulic acid from Inonqqus obliquus | |
| CN101481379B (en) | Compound separated from acetic acid ethyl ester extract of ginko endogenetic fungal bacterial strain fermentation liquor | |
| CN107823235B (en) | Processing method for solid fermentation of ginseng, American ginseng and pseudo-ginseng | |
| CN115779001A (en) | Method for extracting alkaloid from Stephania delavayi Diels | |
| CN113105567B (en) | Paecilomyces cicadae mannan and preparation and application thereof | |
| CN109876021B (en) | Preparation method of Lyophyllum decastes extract rich in polyacetylene compounds and its blood sugar lowering application | |
| CN104262448B (en) | Method for extracting glycyrrhizic acid for licorice | |
| CN111875483B (en) | Method for extracting lignanoid compounds from schisandra chinensis | |
| CN110878259B (en) | Fermentation method of cordyceps sinensis mycelia | |
| CN106565444A (en) | Extraction method and application of phenanthrene compounds from overground part of Chinese yam | |
| CN113440551A (en) | Salvia miltiorrhiza residue extract with antioxidant activity and application thereof | |
| CN113564210B (en) | Method for fermenting industrial hemp by utilizing liquorice endophytic fungi | |
| CN110585221A (en) | Albizzia julibrissin new lignan compound for improving steatosis and application thereof | |
| CN109439697B (en) | Method for producing high-efficiency antioxidant active substance by utilizing microbial fermentation | |
| CN114028460B (en) | Application of green plum wine in blood sugar reduction, extraction method of blood sugar reducing active substance of green plum wine and detection method of blood sugar reducing active substance | |
| KR102529587B1 (en) | Method for Preparing Fermented Alcohol Beverage Using Non-saponin Derived From Ginseng | |
| CN112457285B (en) | Preparation method of naphthopyrone enrichment substance | |
| CN116570653B (en) | Alcohol extraction fermentation process of lipid-lowering traditional Chinese medicine composition and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |