CN109337953A - A kind of inonotus obliquus D extract and preparation method thereof and detection method - Google Patents

A kind of inonotus obliquus D extract and preparation method thereof and detection method Download PDF

Info

Publication number
CN109337953A
CN109337953A CN201811292267.7A CN201811292267A CN109337953A CN 109337953 A CN109337953 A CN 109337953A CN 201811292267 A CN201811292267 A CN 201811292267A CN 109337953 A CN109337953 A CN 109337953A
Authority
CN
China
Prior art keywords
inonotus obliquus
extract
prepared
spare
fermentation liquid
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.)
Granted
Application number
CN201811292267.7A
Other languages
Chinese (zh)
Other versions
CN109337953B (en
Inventor
焦月华
刘飞
张爽
王淑梅
韩雪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heilongjiang University of Chinese Medicine
Original Assignee
Heilongjiang University of Chinese Medicine
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Heilongjiang University of Chinese Medicine filed Critical Heilongjiang University of Chinese Medicine
Priority to CN201811292267.7A priority Critical patent/CN109337953B/en
Publication of CN109337953A publication Critical patent/CN109337953A/en
Application granted granted Critical
Publication of CN109337953B publication Critical patent/CN109337953B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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 field of biotechnology, it is related to a kind of inonotus obliquus D extract and preparation method thereof and detection method.By Inonotus obliquus coarse powder preparation, saccharomycetes to make fermentation liquid prepare, Inonotus obliquus fermentation liquid preparation, Inonotus obliquus extractive from fermentative preparation, Inonotus obliquus fermentation, extraction object preparation, inonotus obliquus D extract preparation and etc. obtain inonotus obliquus D extract.The content of inonotus obliquus D is higher in inonotus obliquus D extract.The present invention also provides the methods using inonotus obliquus D content in high effective liquid chromatography for measuring Inonotus obliquus extract.

Description

A kind of inonotus obliquus D extract and preparation method thereof and detection method
Technical field:
The invention belongs to field of biotechnology, and in particular to a kind of inonotus obliquus D extract and preparation method thereof and inspection Survey method.
Technical background:
Inonotus obliquus, latin name: Inonotusobliquus (Fr.) Pilat belongs to Basidiomycotina (Basidiomycotina), Hymenomycetes (Hymeno-mycetes), rust leather hole Zoopagales (Hymenochaetales), rust lead fungi section (Hymenochaetaceae), brown transverse hole fungus category (fine hole Pseudomonas) (In-onotus).China be also called the brown pore fungi of birch cancer, tiltedly Pipe fibre pore fungi or inonotus obliquus etc..
The mycelium of Inonotus obliquus is extremely cold-resistant, and mycelium in wood of living is resistant to subzero 40 DEG C of low temperature.Mainly It is distributed 45 °~50 ° of north latitude on the Northern Hemisphere of area, such as the north of North America, Finland, Poland, the West Siberia of Russia, the Far East Some areas, Hokkaido area of Japan, the big Xiaoxinanlin Mountains in Heilongjiang Province in China, Jilin Province Changbaishan area etc..
Modern research shows that Inonotus obliquus has antitumor, antioxidation, hypoglycemic effect, antivirus action, liver protection Effect, anti-aging effects, immunological regulation, prevention and treatment AIDS etc. multiple pharmacological effect, to vomiting, diarrhea, gastrointestinal dysfunction, Gastric and duodenal ulcer, hepatitis, gastritis and ephritis have certain therapeutic effect.
By carrying out the extraction of effective component to inonotus obliquus sclerotium, the research of anticancer component, immunoregulation effect are ground Study carefully and pharmacological evaluation shows that Inonotus obliquus has apparent antitumaous effect.The special efficacy composition β-D sweet dew that inonotus obliquus sclerotium contains Sugar, antioxidant are apparently higher than other mushroom classes.And the mycelial liquid deep layer fermenting culture of Inonotus obliquus and the brown hole of birch The extract of bacterium sclerotium effective component can be used as the primary raw material of healthy food, be used to development and production health food, medicinal drink A variety of green and healthy food such as material.
Modern study shows that Inonotus obliquus tunning and its wild resource have the same or similar pharmacological activity, The medicinal curative effect of some aspects tunning is even better than fructification and sclerotium, this further research and development to Inonotus obliquus Have great importance.
Xuzhou Normal University has applied for that Chinese invention patent, patent name are a kind of fermented and cultured on December 02nd, 2008 The method of Inonotus obliquus, application No. is 200810227890.4, Authorization Notice No. is CN101748159 B, the invention pass through to Rod method fungal cell wall fragment is added in the fermentation liquid of the fermented and cultured of Inonotus obliquus to cultivate Inonotus obliquus, the birch of acquisition is brown The accumulation of pore fungi phenolic compound intracellular and extracellular and antioxidant activity significantly improve.
Institute of Microbiology, Heilongjiang Academy of Sciences has applied for Chinese invention patent, patent name on December 16th, 2010 For a kind of birch fuscoporia ferrugineo-fusca teng submerged fermentation culture medium and the submerged fermentation method of Inonotus obliquus, application No. is 201010591235.4, Authorization Notice No. is 102115350 B of CN, which will solve that existing birch fuscoporia ferrugineo-fusca teng submerged fermentation mycelial yield is low and deep layer The low problem of fermentation effective component Inonotus obliquus intracellular polyse, yield of extracellular polysaccharide.Submerged fermentation culture medium is by carboxymethyl cellulose Element, glucose, soy meal, yeast extract, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and surplus water be made.Submerged fermentation: one, Prepare birch fuscoporia ferrugineo-fusca teng submerged fermentation culture medium；Two, it is inoculated with Inonotus obliquus；Three, submerged fermentation in two stages.The submerged fermentation used Culture medium and birch fuscoporia ferrugineo-fusca teng submerged fermentation method, can get higher mycelial yield and intracellular, exocellular polysaccharide.
Inonotus obliquus chemical component containing there are many, mainly spreads out including a variety of triterpenes, lignanoids, melanin class, folic acid Biology, polysaccharide etc..Wherein, inonotus obliquus D (3- wool steroid -8,24- diene -21- aldehyde) is a kind of very heavy in Inonotus obliquus The active constituent wanted has anti-tumor activity, also has anti-mutation and anti-oxidant isoreactivity.Its structural formula is as follows:
Pharmaceutical college of Chengdu University of Traditional Chinese Medicine Zhang Shijin, Sichuan Academy of Medical Sciences thank to fortune and fly to find for the first time in Inonotus obliquus Inonotus obliquus D, and in 2015 correlative theses " Inonotus obliquus triterpenes chemical component has been delivered on " Chinese herbal medicine " magazine Research " (being loaded in " Chinese herbal medicine " magazine the 16th the 2355-2360 pages of phase of volume 46 in 2015).
But under field conditions (factors), content of the inonotus obliquus D in Inonotus obliquus is very low, and about 0.0003%, how to mention The content of inonotus obliquus D in high Inonotus obliquus, and the extract of the higher inonotus obliquus D of content is obtained, it is currently to need The technical issues of solution.
Summary of the invention:
The object of the present invention is to provide the preparation methods of inonotus obliquus D extract.
It is a further object of the present invention to provide inonotus obliquus D extractive content measuring methods.
The purpose of the present invention is what is be accomplished by the following way:
A kind of inonotus obliquus D extract is fermented using biological fermentation process, extracts preparation.
The inonotus obliquus D extract, prepares with the following method:
(1) prepared by Inonotus obliquus coarse powder: it takes Inonotus obliquus to dry, crushes, obtain Inonotus obliquus coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour is dissolved in pure water, is sterilized, it is cooling, and then it is inoculated with grape juice Yeast adds xylose, then cultivates, and obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, fermented and cultured, obtain Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), ethanol solution is added, even Filtrate is collected in continuous ultrasound adverse current extraction, filtration, and the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract once, twice Extraction filtrate merge, by after merging filtrate be concentrated, vacuum drying, obtain Inonotus obliquus extractive from fermentative, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then NaHCO is added3Aqueous solution after being sufficiently mixed immersion, collects chloroform extract liquor, and surplus materials according to the method described above, then extracts one It is secondary, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, Inonotus obliquus fermentation, extraction object is obtained, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography Large pore resin absorption column is selected in separation, and washing, eluent discards, with acetone -0.25moLL-1Phosphoric acid solution mixed solution is Eluant, eluent is eluted, and eluent is collected, and eluent concentration is dry to get inonotus obliquus D extract.
The inonotus obliquus D extract, preferably prepares with the following method:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus to dry through 35~45 DEG C, crushes, crosses 20~30 meshes, obtain birch Brown pore fungi coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water For 1g: 8~12mL, sterilize 20~30min at 110~130 DEG C, is cooled to room temperature, is then inoculated with saccharomyces uvarum, inoculum concentration It is 13~17%, adds the xylose of saccharomyces uvarum weight 0.03%~0.05%, is then trained under conditions of 36~38 DEG C 25~35h is supported, saccharomycetes to make fermentation liquid is obtained, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three be 1g: 0.5~ 1.5mL: 25~35mL, at 36~38 DEG C, 32~40h of fermented and cultured obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 3~5 times of amounts are added 70%~80% ethanol solution, continuous ultrasound adverse current extracts 40~50min, filter under conditions of 35~45 DEG C, 25~35KHz It crosses, collects filtrate, the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract primary, the filtrate merging of extraction twice, By the filtrate concentration after merging, vacuum drying obtains Inonotus obliquus extractive from fermentative, spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.4~0.6moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1's NaHCO3The ratio of aqueous solution three is 1g: 10~20mL: 0.5~1.5mL, after being sufficiently mixed 20~30h of immersion, collects chloroform Extract liquor, surplus materials according to the method described above, then extract once, extract liquor twice are merged, and filtration, filtrate volatilizes chloroform, It is dry, Inonotus obliquus fermentation, extraction object is obtained, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography H-30 type large pore resin absorption column is selected in separation, and it is 1: 7~9 that resin column diameter height, which compares, 1~2BV of loading volume, washes 3~7BV, Flow velocity is 0.5~1.5BV/h, and eluent discards, with acetone: 0.25moLL-1Phosphoric acid solution=1: 0.5~1.5 eluant, eluent 7~9BV of elution is carried out, flow velocity is 0.5~1.5BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D Extract.
The inonotus obliquus D extract, most preferably prepares with the following method:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus to dry through 40 DEG C, crushes, crosses 24 meshes, it is thick to obtain Inonotus obliquus Powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water It is 1g: 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, then it is inoculated with saccharomyces uvarum, inoculum concentration 15%, then plus Enter the xylose of saccharomyces uvarum weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtain saccharomycetes to make fermentation liquid, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, and Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, at 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 4 times of amounts 75% are added Ethanol solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are in above-mentioned item It repeats continuous ultrasound adverse current under part to extract once, the filtrate of extraction twice merges, and the filtrate after merging is concentrated, and vacuum is dry It is dry, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3 The ratio of aqueous solution three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to upper Method to be stated, then is extracted once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation Extract, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography H-30 type large pore resin absorption column is selected in separation, and it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, and flow velocity is 1BV/h, eluent discard, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, and flow velocity is 1BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D extract.
A kind of content assaying method of inonotus obliquus D extract, is mentioned using high effective liquid chromatography for measuring Inonotus obliquus Take the content of inonotus obliquus D in object；Inonotus obliquus D extract is to be fermented using biological fermentation process, extracted preparation 's.
The content assaying method of the inonotus obliquus D extract, using high effective liquid chromatography for measuring Inonotus obliquus The content of inonotus obliquus D in extract, steps are as follows:
(1) chromatographic condition: chromatographic column: C18Column, mobile phase: -0.15% phosphoric acid solution of acetonitrile-water-isopropyl alcohol mixture； Detection wavelength: 410~430nm；Flow velocity: 0.5~2.0mLmin-1；20~40 DEG C of column temperature；Sample volume: 5~20 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D reference substance is placed in measuring bottle, the acetonitrile-water added Mixed solution is dissolved to scale, shakes up to get reference substance solution；
(3) preparation of test solution: accurately weighed Inonotus obliquus extract is placed in measuring bottle, adds acetonitrile-water mixing molten Liquid, ultrasound, then plus acetonitrile-water mixed solution to scale, shake up, filter, precision measures subsequent filtrate and is placed in measuring bottle, adds acetonitrile- Water mixed solution shakes up to scale to get test solution；
(4) measure: precision draws reference substance solution, test solution injects high performance liquid chromatograph, is measured；
The inonotus obliquus D extract is prepared with the following method:
(1) prepared by Inonotus obliquus coarse powder: it takes Inonotus obliquus to dry, crushes, obtain Inonotus obliquus coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour is dissolved in pure water, is sterilized, it is cooling, and then it is inoculated with grape juice Yeast adds xylose, then cultivates, and obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, fermented and cultured, obtain Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), ethanol solution is added, even Filtrate is collected in continuous ultrasound adverse current extraction, filtration, and the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract once, twice Extraction filtrate merge, by after merging filtrate be concentrated, vacuum drying, obtain Inonotus obliquus extractive from fermentative, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then NaHCO is added3Aqueous solution after being sufficiently mixed immersion, collects chloroform extract liquor, and surplus materials according to the method described above, then extracts one It is secondary, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, Inonotus obliquus fermentation, extraction object is obtained, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography Large pore resin absorption column is selected in separation, and washing, eluent discards, with acetone -0.25moLL-1Phosphoric acid solution mixed solution is Eluant, eluent is eluted, and eluent is collected, and eluent concentration is dry to get inonotus obliquus D extract.
The content assaying method of the inonotus obliquus D extract, using high effective liquid chromatography for measuring Inonotus obliquus The content of inonotus obliquus D, preferred steps are as follows in extract:
(1) chromatographic condition: chromatographic column: C18Column, specification: 4.6mm × 250mm, 5 μm；Mobile phase: volume ratio 10: 38~ 42: 28~32: 18~22-0.15% phosphoric acid solution of acetonitrile-water-isopropanol；Detection wavelength: 418~426nm；Flow velocity: 0.5 ~1.5mLmin-1；25~30 DEG C of column temperature；Sample volume: 5~20 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D 5~20mg of reference substance is placed in 10~20mL measuring bottle In, adding volume ratio is that 75~85: 25~15 acetonitrile-aqueous solution is dissolved to scale, is shaken up to get reference substance solution；
(3) preparation of test solution: 90~110mg of accurately weighed Inonotus obliquus extract is placed in 10~20mL measuring bottle In, add volume ratio to be 75~85: 25~15 acetonitrile-aqueous solution, 10~20min of ultrasound, then plus volume ratio for 75~85: 25~ 15 acetonitrile-aqueous solution shakes up, filters, precision measures 1~2mL of subsequent filtrate and is placed in 10~20mL measuring bottle, adds volume to scale Than the acetonitrile-aqueous solution for 75~85: 25~15 to scale, shake up to get test solution；
(4) measure: precision draws reference substance solution, each 5~20 μ L of test solution, injects high performance liquid chromatograph, into Row measurement；
The inonotus obliquus D extract is prepared with the following method:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus to dry through 35~45 DEG C, crushes, crosses 20~30 meshes, obtain birch Brown pore fungi coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water For 1g: 8~12mL, sterilize 20~30min at 110~130 DEG C, is cooled to room temperature, is then inoculated with saccharomyces uvarum, inoculum concentration It is 13~17%, adds the xylose of saccharomyces uvarum weight 0.03%~0.05%, is then trained under conditions of 36~38 DEG C 25~35h is supported, saccharomycetes to make fermentation liquid is obtained, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three be 1g: 0.5~ 1.5mL: 25~35mL, at 36~38 DEG C, 32~40h of fermented and cultured obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 3~5 times of amounts are added 70%~80% ethanol solution, continuous ultrasound adverse current extracts 40~50min, filter under conditions of 35~45 DEG C, 25~35KHz It crosses, collects filtrate, the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract primary, the filtrate merging of extraction twice, By the filtrate concentration after merging, vacuum drying obtains Inonotus obliquus extractive from fermentative, spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.4~0.6moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1's NaHCO3The ratio of aqueous solution three is 1g: 10~20mL: 0.5~1.5mL, after being sufficiently mixed 20~30h of immersion, collects chloroform Extract liquor, surplus materials according to the method described above, then extract once, extract liquor twice are merged, and filtration, filtrate volatilizes chloroform, It is dry, Inonotus obliquus fermentation, extraction object is obtained, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography H-30 type large pore resin absorption column is selected in separation, and it is 1: 7~9 that resin column diameter height, which compares, 1~2BV of loading volume, washes 3~7BV, Flow velocity is 0.5~1.5BV/h, and eluent discards, with acetone: 0.25moLL-1Phosphoric acid solution=1: 0.5~1.5 eluant, eluent 7~9BV of elution is carried out, flow velocity is 0.5~1.5BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D Extract.
The content assaying method of the inonotus obliquus D extract, using high effective liquid chromatography for measuring Inonotus obliquus The content of inonotus obliquus D in extract, most preferred steps are as follows:
(1) chromatographic condition: chromatographic column: C18Column, specification: 4.6mm × 250mm, 5 μm；Mobile phase: volume ratio 10: 40: 30 : 20-0.15% phosphoric acid solution of acetonitrile-water-isopropanol；Detection wavelength: 422nm；Flow velocity: 1.0mLmin-1；28 DEG C of column temperature； Sample volume: 10 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D reference substance 10mg is placed in 10mL measuring bottle, adds body For product than being dissolved to scale for 80: 20 acetonitrile-aqueous solution, shaking up to get concentration is 1.0mgmL-1Reference substance stock solution；
(3) preparation of test solution: accurately weighed Inonotus obliquus extract 100mg is placed in 10mL measuring bottle, adds volume Than the acetonitrile-aqueous solution for 80: 20, ultrasonic 15min, then plus volume ratio be 80: 20 acetonitrile-aqueous solution to scale, shake up, filter It crosses, precision measures subsequent filtrate 1mL and is placed in 10mL measuring bottle, adds the acetonitrile-aqueous solution that volume ratio is 80: 20 to scale, shakes up, i.e., Obtain test solution；
(4) measure: precision draws reference substance solution, each 10 μ L of test solution, injects high performance liquid chromatograph, is surveyed It is fixed；
The inonotus obliquus D extract is prepared with the following method:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus to dry through 40 DEG C, crushes, crosses 24 meshes, it is thick to obtain Inonotus obliquus Powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water It is 1g: 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, then it is inoculated with saccharomyces uvarum, inoculum concentration 15%, then plus Enter the xylose of saccharomyces uvarum weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtain saccharomycetes to make fermentation liquid, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, and Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, at 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 4 times of amounts 75% are added Ethanol solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are in above-mentioned item It repeats continuous ultrasound adverse current under part to extract once, the filtrate of extraction twice merges, and the filtrate after merging is concentrated, and vacuum is dry It is dry, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3 The ratio of aqueous solution three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to upper Method to be stated, then is extracted once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation Extract, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography H-30 type large pore resin absorption column is selected in separation, and it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, and flow velocity is 1BV/h, eluent discard, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, and flow velocity is 1BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D extract.
Technical effect of the invention is verified in research through the following experiment:
Test the content of inonotus obliquus D in one: HPLC method measurement inonotus obliquus D extract
1 materials and methods
1.1 materials and reagent
Inonotus obliquus is purchased from the processing of the Greater Hinggan Mountains in Heilongjiang's Mo River flood peak mountain specialty and sells Co., Ltd, through 40 DEG C of ovendry powers Broken 40 mesh of mistake.Inonotus obliquus D reference substance (purity >=98%), is made by oneself by this laboratory；Methanol, formic acid, isopropanol (are Chromatographically pure), it is produced by Tianjin Concord Technology Co., Ltd., Co., Ltd when being purchased from Harbin De Li；Remaining reagent is point It analyses pure.
1.2 instrument and equipment
Waters high performance liquid chromatograph；Electronic balance is provided by Shanghai precision instrumentation Co., Ltd；Electric heating air blast Drying box is provided by Nanjing Hua Ao drying equipment Co., Ltd；Electric heating digital display thermostat water bath is had by Xi'an Taikan biotechnology Limit company provides.
2 methods
2.1 chromatographic condition
Chromatographic column: C18Column (specification: 4.6mm × 250mm, 5 μm)；Mobile phase: the acetonitrile-of volume ratio 10: 40: 30: 20 - 0.15% phosphoric acid solution of water-isopropanol；Detection wavelength: 422nm；Flow velocity: 1.0mLmin-1；Column temperature: 28 DEG C；Sample volume: 10 μ L。
The preparation of 2.2 inonotus obliquus D extracts
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus 1.00Kg to dry through 40 DEG C, crushes, crosses 24 meshes, it is brown to obtain birch Pore fungi coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water It is 1g: 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, then it is inoculated with saccharomyces uvarum, inoculum concentration 15%, then plus Enter the xylose of saccharomyces uvarum weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtain saccharomycetes to make fermentation liquid, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, and Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, at 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 4 times of amounts 75% are added Ethanol solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are in above-mentioned item It repeats continuous ultrasound adverse current under part to extract once, the filtrate of extraction twice merges, and the filtrate after merging is concentrated, and vacuum is dry It is dry, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3 The ratio of aqueous solution three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to upper Method to be stated, then is extracted once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation Extract, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography H-30 type large pore resin absorption column is selected in separation, and it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, and flow velocity is 1BV/h, eluent discard, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, and flow velocity is 1BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D extract 300.32mg.
2.3 the preparation of reference substance solution
Accurately weighed inonotus obliquus D reference substance 10mg, is placed in 10mL measuring bottle, and adding volume ratio is 80: 20 acetonitrile-water Solution is dissolved to scale, and shaking up to get concentration is 1.0mgmL-1Reference substance solution.
The preparation of 2.4 test solutions
Accurately weighed Inonotus obliquus extract 100mg, is placed in 10mL measuring bottle, and the acetonitrile-water for adding volume ratio to be 80: 20 is molten Liquid, ultrasonic 15min, then plus volume ratio be 80: 20 acetonitrile-aqueous solution to scale, shake up, filter, precision measures subsequent filtrate 1mL It is placed in 10mL measuring bottle, adds the acetonitrile-aqueous solution that volume ratio is 80: 20 to scale, shake up to get test solution.
The preparation of 2.5 negative control solutions
Use volume ratio be 80: 20 acetonitrile-aqueous solution as sample solution to get negative control solution.
3 results and analysis
The production of 3.1 standard curves
Precision measure reference substance solution it is each 1.0,1.5,2.0,2.5,3.0mL be placed in 10mL measuring bottle, adding volume ratio is 80: 20 acetonitrile-aqueous solution mixes to scale, obtains standard solution at different levels.
Standard solution at different levels are taken, sample introduction is analyzed by chromatographic condition provided by the invention, with the mass concentration of reference substance solution For abscissa, chromatographic peak peak area is ordinate, carries out regression analysis.
Regression equation: Y=1.6852X-18.264, r=0.9998.
The result shows that inonotus obliquus D is in 35.9~298.6 μ gmL-1In range, linear relationship is good.
The experiment of 3.2 specificities
Precision draws 10 μ L of reference substance solution, 10 μ L of test solution, 10 μ L of negative control solution, by provided by the invention Chromatographic condition, sample introduction measurement, the results show that separating degree is preferable, negative control is noiseless, sees Figure of description 1, attached drawing 2, attached drawing 3。
3.3 Precision Experiment
Inonotus obliquus D reference substance solution is taken, sample introduction 6 times, measures and records peak area, calculate relative standard deviation (RSD) It is 0.28%, shows that instrument precision is good.
3.4 stability experiment
Same test solution is taken, respectively in 1,10,20,30,40, the measurement of 50min sample introduction, peak area is recorded, calculates RSD It is 0.42%, shows that test solution is stablized in 50min.
3.5 repeated experiment
6 parts of inonotus obliquus D extract is taken, prepares test solution by method provided by the invention, is provided by the present invention Chromatographic condition be measured, average content 35.6%, RSD 1.24%.Illustrate repeated good.
The experiment of 3.6 sample recovery rates
6 parts, each 20.00mg of inonotus obliquus D extract of known content are taken respectively, it is accurately weighed, it is accurate respectively to be added Accurately weighed inonotus obliquus D reference substance prepares test solution by method provided by the invention, by chromatography provided by the invention Condition is measured, and is calculated the rate of recovery, be the results are shown in Table 1.
1 sample recovery rate of table tests (n=9)
The measurement of 3.7 sample sizes
The method measurement established using the present invention is to the inonotus obliquus D content in 6 batches of inonotus obliquus D extracts, knot Fruit is shown in Table 2.
The content of inonotus obliquus D in 26 batches of samples of table
4 discuss
The investigation of ultrasonic extraction time in the preparation of 4.1 test solutions
The present invention in test solution preparation in the ultrasonic extraction time investigated, respectively to ultrasonic 10min, The extraction effect of 15min, 20min, 25min are tested, the results show that the effect of ultrasonic extraction 15min is best.
The selection of 4.2 mobile phases
The present invention has investigated methanol-water, methanol-water-formic acid, methanol-water-phosphoric acid, -0.15% phosphoric acid of acetonitrile-water respectively The systems such as solution-isopropanol, it is final to determine that volume ratio is 10: 40: 30: 20-0.15% phosphoric acid solution of acetonitrile-water-isopropanol Solution is mobile phase, and appearance time is appropriate, no hangover.
To sum up, the method for the content of inonotus obliquus D is simple in HPLC method measurement Inonotus obliquus extract provided by the invention Single, easy, detection method precision is high, and reproducible, stability is good, can be used for the detection of this product.
Experiment two: the purity that different method for extraction and purification prepare the extraction of inonotus obliquus D extract is ground compared with content Study carefully
The inonotus obliquus D extract of 1 different method for extraction and purification preparations
1.1 extracts I:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus 1Kg to dry through 40 DEG C, crushes, crosses 24 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water It is 1g: 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, then it is inoculated with saccharomyces uvarum, inoculum concentration 15%, then plus Enter the xylose of saccharomyces uvarum weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtain saccharomycetes to make fermentation liquid, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, and Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, at 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 4 times of amounts 75% are added Ethanol solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are in above-mentioned item It repeats continuous ultrasound adverse current under part to extract once, the filtrate of extraction twice merges, and the filtrate after merging is concentrated, and vacuum is dry It is dry, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3 The ratio of aqueous solution three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to upper Method to be stated, then is extracted once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation Extract, it is spare；
(6) prepared by extract I: the resulting Inonotus obliquus fermentation, extraction object of step (5) being carried out column chromatography for separation, selects H- 30 type large pore resin absorption columns, it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, flow velocity 1BV/h, and eluent is abandoned It goes, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, flow velocity 1BV/h, collects eluent, Eluent concentration, it is dry to get I 300.61mg of extract.
1.2 extracts II:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus 1Kg to dry through 40 DEG C, crushes, crosses 24 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) prepared by extract II: taking Inonotus obliquus coarse powder obtained by step (1), chloroform is added, adding concentration is 0.5moL·L-1NaHCO3Aqueous solution, Inonotus obliquus coarse powder, chloroform, 0.5moLL-1NaHCO3The ratio of aqueous solution three It is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials according to the method described above, then extracts one It is secondary, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain II 20265.62mg of extract.
1.3 extracts III:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus 1Kg to dry through 40 DEG C, crushes, crosses 24 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) prepared by extract III: the resulting Inonotus obliquus coarse powder of step (1) being carried out column chromatography for separation, selects H-30 type Large pore resin absorption column, it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, flow velocity 1BV/h, and eluent discards, With acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, flow velocity 1BV/h, collects eluent, washes De- liquid concentration, it is dry to get III 23286.63mg of extract.
1.4 extracts IV:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus 1Kg to dry through 40 DEG C, crushes, crosses 24 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) prepared by Inonotus obliquus extract: taking Inonotus obliquus coarse powder obtained by step (1), chloroform is added, adding concentration is 0.5moL·L-1NaHCO3Aqueous solution, Inonotus obliquus coarse powder, chloroform, 0.5moLL-1NaHCO3The ratio of aqueous solution three It is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials according to the method described above, then extracts one It is secondary, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, dry, Inonotus obliquus extract.
(2) prepared by extract IV: the resulting Inonotus obliquus coarse powder of step (1) being carried out column chromatography for separation, selects H-30 type Large pore resin absorption column, it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, flow velocity 1BV/h, and eluent discards, With acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, flow velocity 1BV/h, collects eluent, washes De- liquid concentration, it is dry to get III 2284.69mg of extract.
2. measurement
Contained using inonotus obliquus D in HPLC method measurement inonotus obliquus D extract provided by present invention experiment one The method of amount is measured.
3. measurement result
Measurement result is shown in Table 3.
The content of inonotus obliquus D in 3 extract of table
4. conclusion
By 3 experimental result of table as it can be seen that the inonotus obliquus D extract obtained using preparation method provided by the invention, no Only extract obtain inonotus obliquus D quality it is more, and content in extract is high.
Detailed description of the invention:
Fig. 1 is reference substance solution chromatogram, wherein No. 1 peak is inonotus obliquus D chromatographic peak.
Fig. 2 is test solution chromatogram, wherein No. 1 peak is inonotus obliquus D chromatographic peak.
Fig. 3 is negative control solution chromatogram.
Specific embodiment:
Embodiment 1:
1, inonotus obliquus D extract and its preparation
The preparation of inonotus obliquus D extract
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus 1Kg to dry through 40 DEG C, crushes, crosses 24 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water It is 1g: 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, then it is inoculated with saccharomyces uvarum, inoculum concentration 15%, then plus Enter the xylose of saccharomyces uvarum weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtain saccharomycetes to make fermentation liquid, it is spare；
(3) prepared by Inonotus obliquus fermentation liquid: saccharomycete obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2) is sent out Zymotic fluid and pure water are mixed, and Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, at 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), 4 times of amounts 75% are added Ethanol solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are in above-mentioned item It repeats continuous ultrasound adverse current under part to extract once, the filtrate of extraction twice merges, and the filtrate after merging is concentrated, and vacuum is dry It is dry, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: Inonotus obliquus extractive from fermentative obtained by step (4) is taken, chloroform is added, then Addition concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3 The ratio of aqueous solution three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to upper Method to be stated, then is extracted once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation Extract, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is carried out column chromatography H-30 type large pore resin absorption column is selected in separation, and it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, and flow velocity is 1BV/h, eluent discard, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, and flow velocity is 1BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D extract 299.12mg.
2, the assay of inonotus obliquus D
Using the content of inonotus obliquus D in high effective liquid chromatography for measuring Inonotus obliquus extract, steps are as follows:
(1) chromatographic condition: chromatographic column: C18Column, specification: 4.6mm × 250mm, 5 μm；Mobile phase: volume ratio 10: 40: 30 : 20-0.15% phosphoric acid solution of acetonitrile-water-isopropanol；Detection wavelength: 422nm；Flow velocity: 1.0mLmin-1；28 DEG C of column temperature； Sample volume: 10 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D reference substance 10mg is placed in 10mL measuring bottle, adds body For product than being dissolved to scale for 80: 20 acetonitrile-aqueous solution, shaking up to get concentration is 1.0mgmL-1Reference substance stock solution；
(3) preparation of test solution: accurately weighed Inonotus obliquus extract 100mg is placed in 10mL measuring bottle, adds volume Than the acetonitrile-aqueous solution for 80: 20, ultrasonic 15min, then plus volume ratio be 80: 20 acetonitrile-aqueous solution to scale, shake up, filter It crosses, precision measures subsequent filtrate 1mL and is placed in 10mL measuring bottle, adds the acetonitrile-aqueous solution that volume ratio is 80: 20 to scale, shakes up, i.e., Obtain test solution；
(4) measure: precision draws reference substance solution, each 10 μ L of test solution, injects high performance liquid chromatograph, is surveyed It is fixed；
(5) result: inonotus obliquus D mass 101.82mg, content 34.04%.

Claims (8)

1. a kind of inonotus obliquus D extract, which is characterized in that inonotus obliquus D extract be using biological fermentation process into Preparation is extracted in row fermentation.
2. inonotus obliquus D extract as described in claim 1, which is characterized in that inonotus obliquus D extract is to use Following method preparation:
(1) prepared by Inonotus obliquus coarse powder: it takes Inonotus obliquus to dry, crushes, obtain Inonotus obliquus coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour is dissolved in pure water, is sterilized, it is cooling, and then it is inoculated with grape juice ferment Mother adds xylose, then cultivates, and obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: by saccharomycetes to make fermentation liquid obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2), And pure water is mixed, fermented and cultured, obtains Inonotus obliquus fermentation liquid, it is spare；
(4) prepared by Inonotus obliquus extractive from fermentative: Inonotus obliquus fermentation liquid obtained by step (3) is taken, ethanol solution is added, it is continuous super Sound adverse current is extracted, filtration, collects filtrate, and the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract primary, mentioning twice The filtrate taken merges, and the filtrate after merging is concentrated, and vacuum drying obtains Inonotus obliquus extractive from fermentative, spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: taking Inonotus obliquus extractive from fermentative obtained by step (4), chloroform is added, adds NaHCO3Aqueous solution after being sufficiently mixed immersion, collects chloroform extract liquor, and surplus materials according to the method described above, then extracts once, will Extract liquor twice merges, and filtration, filtrate volatilizes chloroform, dry, obtains Inonotus obliquus fermentation, extraction object, spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is subjected to column chromatography for separation, Large pore resin absorption column is selected, washing, eluent discards, with acetone -0.25moLL-1Phosphoric acid solution mixed solution is eluant, eluent It is eluted, collects eluent, eluent concentration is dry to get inonotus obliquus D extract.
3. inonotus obliquus D extract as claimed in claim 2, which is characterized in that inonotus obliquus D extract is to use Following method preparation:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus to dry through 35~45 DEG C, crushes, crosses 20~30 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water is 1g : 8~12mL, sterilize 20~30min at 110~130 DEG C, is cooled to room temperature, is then inoculated with saccharomyces uvarum, inoculum concentration 13 ~17%, the xylose of saccharomyces uvarum weight 0.03%~0.05% is added, then cultivates 25 under conditions of 36~38 DEG C ~35h obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: by saccharomycetes to make fermentation liquid obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2), And pure water is mixed, Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 0.5~1.5mL: 25~35mL, at 36~38 DEG C, 32~40h of fermented and cultured obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: take Inonotus obliquus fermentation liquid obtained by step (3), be added 3~5 times of amounts 70%~ 80% ethanol solution, continuous ultrasound adverse current extracts 40~50min under conditions of 35~45 DEG C, 25~35KHz, filters, and receives Collect filtrate, the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract once, and the filtrate of extraction twice merges, and will merge Filtrate concentration afterwards, vacuum drying obtains Inonotus obliquus extractive from fermentative, spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: taking Inonotus obliquus extractive from fermentative obtained by step (4), chloroform is added, adds Concentration is 0.4~0.6moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1's NaHCO3The ratio of aqueous solution three is 1g: 10~20mL: 0.5~1.5mL, after being sufficiently mixed 20~30h of immersion, collects chloroform Extract liquor, surplus materials according to the method described above, then extract once, extract liquor twice are merged, and filtration, filtrate volatilizes chloroform, It is dry, Inonotus obliquus fermentation, extraction object is obtained, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is subjected to column chromatography for separation, H-30 type large pore resin absorption column is selected, it is 1: 7~9 that resin column diameter height, which compares, 1~2BV of loading volume, washes 3~7BV, flow velocity For 0.5~1.5BV/h, eluent is discarded, with acetone: 0.25moLL-1Phosphoric acid solution=1: 0.5~1.5 eluant, eluent carries out 7~9BV is eluted, flow velocity is 0.5~1.5BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D extraction Object.
4. inonotus obliquus D extract as claimed in claim 3, which is characterized in that inonotus obliquus D extract is to use Following method preparation:
(1) prepared by Inonotus obliquus coarse powder: it takes Inonotus obliquus to dry through 40 DEG C, crushes, cross 24 meshes, obtain Inonotus obliquus coarse powder, It is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water is 1g : 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, and is then inoculated with saccharomyces uvarum, and inoculum concentration 15% adds Portugal The xylose of grape juice yeast weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: by saccharomycetes to make fermentation liquid obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2), And pure water is mixed, Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, At 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), the ethyl alcohol of 4 times of amounts 75% is added Solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are under the above conditions Continuous ultrasound adverse current is repeated to extract once, the filtrate of extraction twice merges, the filtrate after merging is concentrated, vacuum drying, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: taking Inonotus obliquus extractive from fermentative obtained by step (4), chloroform is added, adds Concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3It is water-soluble The ratio of liquid three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to above-mentioned side Method, then extract once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation, extraction Object, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is subjected to column chromatography for separation, H-30 type large pore resin absorption column is selected, it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, and flow velocity 1BV/h is washed De- liquid discards, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, flow velocity 1BV/h, collects Eluent, eluent concentration are dry to get inonotus obliquus D extract.
5. a kind of content assaying method of inonotus obliquus D extract, which is characterized in that use high effective liquid chromatography for measuring birch The content of inonotus obliquus D in brown linteus extract；Inonotus obliquus D extract be fermented using biological fermentation process, Extract preparation.
6. the content assaying method of inonotus obliquus D extract as claimed in claim 5, which is characterized in that use efficient liquid Phase chromatography measures the content of inonotus obliquus D in Inonotus obliquus extract, and steps are as follows:
(1) chromatographic condition: chromatographic column: C18Column, mobile phase: -0.15% phosphoric acid solution of acetonitrile-water-isopropyl alcohol mixture；Detection Wavelength: 410~430nm；Flow velocity: 0.5~2.0mLmin-1；20~40 DEG C of column temperature；Sample volume: 5~20 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D reference substance is placed in measuring bottle, the acetonitrile-water mixing added Solution is dissolved to scale, shakes up to get reference substance solution；
(3) preparation of test solution: accurately weighed Inonotus obliquus extract is placed in measuring bottle, adds acetonitrile-water mixed solution, Ultrasound, then plus acetonitrile-water mixed solution to scale, shake up, filter, precision measures subsequent filtrate and is placed in measuring bottle, adds acetonitrile-water mixed Solution is closed to scale, is shaken up to get test solution；
(4) measure: precision draws reference substance solution, test solution injects high performance liquid chromatograph, is measured；
The inonotus obliquus D extract is prepared with the following method:
(1) prepared by Inonotus obliquus coarse powder: it takes Inonotus obliquus to dry, crushes, obtain Inonotus obliquus coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour is dissolved in pure water, is sterilized, it is cooling, and then it is inoculated with grape juice ferment Mother adds xylose, then cultivates, and obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: by saccharomycetes to make fermentation liquid obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2), And pure water is mixed, fermented and cultured, obtains Inonotus obliquus fermentation liquid, it is spare；
(4) prepared by Inonotus obliquus extractive from fermentative: Inonotus obliquus fermentation liquid obtained by step (3) is taken, ethanol solution is added, it is continuous super Sound adverse current is extracted, filtration, collects filtrate, and the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract primary, mentioning twice The filtrate taken merges, and the filtrate after merging is concentrated, and vacuum drying obtains Inonotus obliquus extractive from fermentative, spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: taking Inonotus obliquus extractive from fermentative obtained by step (4), chloroform is added, adds NaHCO3Aqueous solution after being sufficiently mixed immersion, collects chloroform extract liquor, and surplus materials according to the method described above, then extracts once, will Extract liquor twice merges, and filtration, filtrate volatilizes chloroform, dry, obtains Inonotus obliquus fermentation, extraction object, spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is subjected to column chromatography for separation, Large pore resin absorption column is selected, washing, eluent discards, with acetone -0.25moLL-1Phosphoric acid solution mixed solution is eluant, eluent It is eluted, collects eluent, eluent concentration is dry to get inonotus obliquus D extract.
7. the content assaying method of inonotus obliquus D extract as claimed in claim 6, which is characterized in that use efficient liquid Phase chromatography measures the content of inonotus obliquus D in Inonotus obliquus extract, and steps are as follows:
(1) chromatographic condition: chromatographic column: C18Column, specification: 4.6mm × 250mm, 5 μm；Mobile phase: volume ratio 10: 38~42: 28 ~32: 18~22-0.15% phosphoric acid solution of acetonitrile-water-isopropanol；Detection wavelength: 418~426nm；Flow velocity: 0.5~ 1.5mL·min-1；25~30 DEG C of column temperature；Sample volume: 5~20 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D 5~20mg of reference substance is placed in 10~20mL measuring bottle, Adding volume ratio is that 75~85: 25~15 acetonitrile-aqueous solution is dissolved to scale, is shaken up to get reference substance solution；
(3) preparation of test solution: 90~110mg of accurately weighed Inonotus obliquus extract is placed in 10~20mL measuring bottle, adds The acetonitrile-aqueous solution that volume ratio is 75~85: 25~15, ultrasound 10~20min, then plus volume ratio be 75~85: 25~15 Acetonitrile-aqueous solution shakes up to scale, filters, and precision measures 1~2mL of subsequent filtrate and is placed in 10~20mL measuring bottle, adds the volume ratio to be 75~85: 25~15 acetonitrile-aqueous solution shakes up to scale to get test solution；
(4) measure: precision draws reference substance solution, each 5~20 μ L of test solution, injects high performance liquid chromatograph, is surveyed It is fixed；
The inonotus obliquus D extract is prepared with the following method:
(1) prepared by Inonotus obliquus coarse powder: taking Inonotus obliquus to dry through 35~45 DEG C, crushes, crosses 20~30 meshes, obtain the brown hole of birch Bacterium coarse powder, it is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water is 1g : 8~12mL, sterilize 20~30min at 110~130 DEG C, is cooled to room temperature, is then inoculated with saccharomyces uvarum, inoculum concentration 13 ~17%, the xylose of saccharomyces uvarum weight 0.03%~0.05% is added, then cultivates 25 under conditions of 36~38 DEG C ~35h obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: by saccharomycetes to make fermentation liquid obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2), And pure water is mixed, Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 0.5~1.5mL: 25~35mL, at 36~38 DEG C, 32~40h of fermented and cultured obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: take Inonotus obliquus fermentation liquid obtained by step (3), be added 3~5 times of amounts 70%~ 80% ethanol solution, continuous ultrasound adverse current extracts 40~50min under conditions of 35~45 DEG C, 25~35KHz, filters, and receives Collect filtrate, the dregs of a decoction repeat continuous ultrasound adverse current under the above conditions and extract once, and the filtrate of extraction twice merges, and will merge Filtrate concentration afterwards, vacuum drying obtains Inonotus obliquus extractive from fermentative, spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: taking Inonotus obliquus extractive from fermentative obtained by step (4), chloroform is added, adds Concentration is 0.4~0.6moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1's NaHCO3The ratio of aqueous solution three is 1g: 10~20mL: 0.5~1.5mL, after being sufficiently mixed 20~30h of immersion, collects chloroform Extract liquor, surplus materials according to the method described above, then extract once, extract liquor twice are merged, and filtration, filtrate volatilizes chloroform, It is dry, Inonotus obliquus fermentation, extraction object is obtained, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is subjected to column chromatography for separation, H-30 type large pore resin absorption column is selected, it is 1: 7~9 that resin column diameter height, which compares, 1~2BV of loading volume, washes 3~7BV, flow velocity For 0.5~1.5BV/h, eluent is discarded, with acetone: 0.25moLL-1Phosphoric acid solution=1: 0.5~1.5 eluant, eluent carries out 7~9BV is eluted, flow velocity is 0.5~1.5BV/h, collects eluent, and eluent concentration is dry to get inonotus obliquus D extraction Object.
8. the content assaying method of inonotus obliquus D extract as claimed in claim 7, which is characterized in that use efficient liquid Phase chromatography measures the content of inonotus obliquus D in Inonotus obliquus extract, and steps are as follows:
(1) chromatographic condition: chromatographic column: C18Column, specification: 4.6mm × 250mm, 5 μm；Mobile phase: volume ratio 10: 40: 30: 20 - 0.15% phosphoric acid solution of acetonitrile-water-isopropanol；Detection wavelength: 422nm；Flow velocity: 1.0mLmin-1；28 DEG C of column temperature；Sample introduction Amount: 10 μ L；
(2) preparation of reference substance solution: accurately weighed inonotus obliquus D reference substance 10mg is placed in 10mL measuring bottle, adds volume ratio It is dissolved to scale for 80: 20 acetonitrile-aqueous solution, shaking up to get concentration is 1.0mgmL-1Reference substance stock solution；
(3) preparation of test solution: accurately weighed Inonotus obliquus extract 100mg is placed in 10mL measuring bottle, adds the volume ratio to be 80: 20 acetonitrile-aqueous solution, ultrasonic 15min, then plus volume ratio be 80: 20 acetonitrile-aqueous solution to scale, shake up, filter, Precision measures subsequent filtrate 1mL and is placed in 10mL measuring bottle, adds the acetonitrile-aqueous solution that volume ratio is 80: 20 to scale, shake up to get Test solution；
(4) measure: precision draws reference substance solution, each 10 μ L of test solution, injects high performance liquid chromatograph, is measured；
The inonotus obliquus D extract is prepared with the following method:
(1) prepared by Inonotus obliquus coarse powder: it takes Inonotus obliquus to dry through 40 DEG C, crushes, cross 24 meshes, obtain Inonotus obliquus coarse powder, It is spare；
(2) saccharomycetes to make fermentation liquid is prepared: defatted soy flour being dissolved in pure water, the ratio of defatted soy flour and pure water is 1g : 10mL, sterilize 25min at 120 DEG C, is cooled to room temperature, and is then inoculated with saccharomyces uvarum, and inoculum concentration 15% adds Portugal The xylose of grape juice yeast weight 0.04%, then cultivates 30h under conditions of 37 DEG C, obtains saccharomycetes to make fermentation liquid, spare；
(3) prepared by Inonotus obliquus fermentation liquid: by saccharomycetes to make fermentation liquid obtained by Inonotus obliquus coarse powder obtained by step (1) and step (2), And pure water is mixed, Inonotus obliquus coarse powder, saccharomycetes to make fermentation liquid, the ratio of pure water three are 1g: 1mL: 30mL, At 37 DEG C, fermented and cultured 36h obtains Inonotus obliquus fermentation liquid, spare；
(4) prepared by Inonotus obliquus extractive from fermentative: taking Inonotus obliquus fermentation liquid obtained by step (3), the ethyl alcohol of 4 times of amounts 75% is added Solution, continuous ultrasound adverse current extracts 45min under conditions of 40 DEG C, 30KHz, and filtrate is collected in filtration, and the dregs of a decoction are under the above conditions Continuous ultrasound adverse current is repeated to extract once, the filtrate of extraction twice merges, the filtrate after merging is concentrated, vacuum drying, Inonotus obliquus extractive from fermentative is obtained, it is spare；
(5) prepared by Inonotus obliquus fermentation, extraction object: taking Inonotus obliquus extractive from fermentative obtained by step (4), chloroform is added, adds Concentration is 0.5moLL-1NaHCO3Aqueous solution, Inonotus obliquus extractive from fermentative, chloroform, 0.5moLL-1NaHCO3It is water-soluble The ratio of liquid three is 1g: 15mL: 1mL, after being sufficiently mixed immersion for 24 hours, collects chloroform extract liquor, surplus materials is according to above-mentioned side Method, then extract once, extract liquor twice is merged, filtration, filtrate volatilizes chloroform, and it is dry, obtain Inonotus obliquus fermentation, extraction Object, it is spare；
(6) prepared by inonotus obliquus D extract: the resulting Inonotus obliquus fermentation, extraction object of step (5) is subjected to column chromatography for separation, H-30 type large pore resin absorption column is selected, it is 1: 8 that resin column diameter height, which compares, loading volume 1BV, washes 5BV, and flow velocity 1BV/h is washed De- liquid discards, with acetone: 0.25moLL-1Phosphoric acid solution=1: 1 eluant, eluent carries out elution 8BV, flow velocity 1BV/h, collects Eluent, eluent concentration are dry to get inonotus obliquus D extract.
CN201811292267.7A 2018-11-01 2018-11-01 Inonotus obliquus D extract and preparation method and detection method thereof Active CN109337953B (en)

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 true CN109337953A (en) 2019-02-15
CN109337953B 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)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109825540A (en) * 2019-03-28 2019-05-31 青岛农业大学 A kind of preparation method and applications of the non-carbohydrate extract of Inonotus obliquus
CN113069484A (en) * 2021-03-12 2021-07-06 黑龙江中医药大学 Anti-aging medicine and preparation method of microcapsule preparation thereof
CN116120480A (en) * 2023-01-06 2023-05-16 湖南杰萃生物技术有限公司 Method for simultaneously extracting inonotus obliquus polysaccharide and inonotus obliquus element

Citations (4)

* Cited by examiner, † Cited by third party
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
US20130287829A1 (en) * 2000-10-04 2013-10-31 Paul Edward Stamets Controlling insects and arthropods using preconidial mycelium and extracts of preconidial mycelium from entomopathogenic fungi
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130287829A1 (en) * 2000-10-04 2013-10-31 Paul Edward Stamets Controlling insects and arthropods using preconidial mycelium and extracts of preconidial mycelium from entomopathogenic fungi
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 (3)

* Cited by examiner, † Cited by third party
Title
张仕瑾: "桦褐孔菌三萜类化学成分研究", 《中草药》 *
范柳萍: "桦褐孔菌多糖的分离纯化及其组成分析", 《食品工业科技》 *
郑勤琴: "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", 《上海畜牧兽医通讯》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109825540A (en) * 2019-03-28 2019-05-31 青岛农业大学 A kind of preparation method and applications of the non-carbohydrate extract of Inonotus obliquus
CN109825540B (en) * 2019-03-28 2020-05-08 青岛农业大学 Preparation method and application of inonotus obliquus non-saccharide extract
CN113069484A (en) * 2021-03-12 2021-07-06 黑龙江中医药大学 Anti-aging medicine and preparation method of microcapsule preparation thereof
CN116120480A (en) * 2023-01-06 2023-05-16 湖南杰萃生物技术有限公司 Method for simultaneously extracting inonotus obliquus polysaccharide and inonotus obliquus element
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

Also Published As

Publication number Publication date
CN109337953B (en) 2021-11-26

Similar Documents

Publication Publication Date Title
CN109337953A (en) A kind of inonotus obliquus D extract and preparation method thereof and detection method
Van Le et al. Ginseng saponins in different parts of Panax vietnamensis
CN100335505C (en) Process for preparing antrodia camphorata polysaccharide and antrodia camphorata triterpene with micro-prorous adsorptive resin and its product
CN105777692B (en) Isoflavones component is extracted and assay method
US20170360802A1 (en) Enrichment Method of Ergosterol Peroxide from Sporoderm-Broken Ganoderma Lucidum Spore Powder
CN102928526A (en) Method for analyzing content of adenosine and cordycepin in cordyceps militaris by virtue of high performance liquid chromatography (HPLC)
CN107823237A (en) A kind of extracting method of burdock root total flavone
CN109942649A (en) A kind of indoles glycosides compound and its purification methods and uses
CN101810696B (en) Total triterpenoids of apples and method for preparing same
CN102967670A (en) Method for measuring cordycepin, adenosine and mannitol in cordyceps sinensis mycelium powder
CN103494844A (en) Yellow mushroom standardized component manufacturing method and application of components to treatment of liver cancer
CN103494843A (en) Yellow mushroom standardized component manufacturing method and application of components to treatment of lung cancer
CN102552476B (en) Quality control method for Rosa laevigata root
Meena et al. ESTIMATION OF CORDYCEPIN BY IMPROVED HPLC METHOD IN THE NATURAL AND CULTURED MYCELIA OF HIGH MEDICINAL VALUE HIMALAYAN ENTOMOGENOUS FUNGUS CORDYCEPS SINENSIS.
Chac et al. Quantification of quercetin, isorhamnetin and ferulic acid in dry extract of Anoectochilus setaceus Blume from Vietnam
CN103512975A (en) Method for analyzing contents of effective substances in Cordyceps martialis fruiting body and residue by HPLC
CN104725455B (en) Preparation method of ganoderic acid T
CN107823235B (en) Processing method for solid fermentation of ginseng, American ginseng and pseudo-ginseng
CN101481379B (en) Compound separated from acetic acid ethyl ester extract of ginko endogenetic fungal bacterial strain fermentation liquor
CN112076237B (en) Extraction process, optimization method and application of triterpenoids in Eyeichhornia crassipes
CN107721857A (en) A kind of method that high-purity chlorogenic acid is prepared from Gynura procumbens (Lour.) Merr
CN102419350B (en) Method for carrying out simultaneous quantitative analysis on four lignan components in Chinese magnoliavine raw material and Chinese magnoliavine extract
CN107576736B (en) Method for simultaneously measuring contents of 4 sterols in fresh cordyceps sinensis by HPLC-ELSD and application
CN110274980A (en) A kind of ginseng under forest and the new differentiation discrimination method of garden ginsent
CN105687367B (en) A kind of jujube leaf standardized extract and its preparation and application

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