CN109553647A - The extracting method of metabolite, additive - Google Patents
The extracting method of metabolite, additive Download PDFInfo
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- CN109553647A CN109553647A CN201811605699.9A CN201811605699A CN109553647A CN 109553647 A CN109553647 A CN 109553647A CN 201811605699 A CN201811605699 A CN 201811605699A CN 109553647 A CN109553647 A CN 109553647A
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- substance
- liquid
- metabolite
- cordyceps militaris
- solid
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- 238000000034 method Methods 0.000 title claims abstract description 67
- 239000002207 metabolite Substances 0.000 title claims abstract description 55
- 239000000654 additive Substances 0.000 title claims abstract description 19
- 230000000996 additive effect Effects 0.000 title claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 148
- 239000000126 substance Substances 0.000 claims abstract description 137
- 241001264174 Cordyceps militaris Species 0.000 claims abstract description 102
- 239000007787 solid Substances 0.000 claims abstract description 44
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 68
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 64
- 235000019441 ethanol Nutrition 0.000 claims description 53
- 239000000047 product Substances 0.000 claims description 39
- 239000013076 target substance Substances 0.000 claims description 35
- 238000000605 extraction Methods 0.000 claims description 33
- 239000003480 eluent Substances 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 26
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 16
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- 238000002425 crystallisation Methods 0.000 claims description 13
- 230000008025 crystallization Effects 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 9
- 238000001953 recrystallisation Methods 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 7
- 241000382353 Pupa Species 0.000 claims description 6
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- 238000004108 freeze drying Methods 0.000 claims description 5
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- 239000013618 particulate matter Substances 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
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- -1 and uses eluant Substances 0.000 claims description 3
- 239000002270 dispersing agent Substances 0.000 claims description 3
- 235000013376 functional food Nutrition 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
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- 240000006698 Spigelia anthelmia Species 0.000 claims 1
- 238000012545 processing Methods 0.000 abstract description 8
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 description 17
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 description 17
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 description 16
- 229930000044 secondary metabolite Natural products 0.000 description 14
- 239000011149 active material Substances 0.000 description 13
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- 239000008367 deionised water Substances 0.000 description 8
- 229910021641 deionized water Inorganic materials 0.000 description 8
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 7
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/167—Purine radicals with ribosyl as the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/04—Disaccharides
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A kind of extracting method of metabolite, additive, belong to field of agricultural products processing.Extracting method includes: to provide object to be extracted.The object to be extracted includes the first liquid portion substance with first object product, the solid portion substance with the second target product.First purification process is executed to the first liquid portion substance using micro-porous resin, the second liquid moieties with first object product are obtained, and the second purification process is executed to obtain the third liquid portion substance with first object substance to second liquid moieties by way of liquid chromatogram.The above extracting method may be implemented more completely to extract the metabolite in Cordyceps militaris.
Description
Technical field
The present invention relates to field of agricultural products processing, extracting method, additive in particular to a kind of metabolite.
Background technique
Cordyceps militaris (Cordyceps militaris L.Link) is also known as pupa grass, Cordceps militaris, northern Chinese caterpillar Fungus.Itself and winter worm
Summer grass belongs to xenogenesis, is that a kind of dual-purpose of drug and food fungi has nutritive value abundant and medical value.Modern pharmacology research card
Real: Cordyceps militaris has antibacterial, anti-inflammatory, antitumor, antifatigue, anti-diabetic and improves a variety of pharmacological activity such as immunity of organisms.
Its medicinal and edible value can compare favourably with cordyceps sinensis, therefore it is natural to substitute to generally use the Cordyceps militaris of artificial cultivation now
Cordyceps sinensis.
Contain the various bioactive components such as adenosine, cordycepin, cordycepic acid, polysaccharide in Cordyceps militaris.
Wherein, adenosine has physiological action and 2015 to many other systems and tissue of cardiovascular system and human body
The index ingredient in cordyceps sinensis is examined in version " Chinese Pharmacopoeia ".
Cordycepin be otherwise known as cordycepin, Cordyceps militaris element, 3'-Deoxyadenosine.Researcher is to cordycepin in anti-inflammatory and tune
It saves immune, antitumor, hypoglycemic, resisting pathogenic microbes, anti-aging and neuroprotection etc. and has carried out numerous studies, and take
Obtained breakthrough.
Cordycepic acid can inhibit the growth of various germs, can prevention and treatment cerebral thrombosis, cerebral hemorrhage, myocardial infarction, decline for a long time
It exhausts.
It therefore, is necessary by various substances progress high efficiency extraction from Cordyceps militaris.However, current some extractions
The high extractions emphasized to one-component in method more, and for multi-component in Cordyceps militaris while extracting means and study very
It is few.
Summary of the invention
Based on the deficiencies of the prior art, the present invention provides a kind of extracting method of metabolite, additive, with part or
Fully improve, even solve problem above.
The present invention is implemented as follows:
In a first aspect, example of the invention provides a kind of extracting method of metabolite.
The targeted metabolite of extracting method provided in example includes the first object substance generated by Cordyceps militaris
With the second target substance.In other words, extracting method from Cordyceps militaris for extracting target substance.
Extracting method includes:
Object to be extracted is provided, object to be extracted includes the first liquid portion substance with first object product, has second
The solid portion substance of target product.
Wherein, object to be extracted is successively extracted by Cordyceps militaris, alcohol precipitation, is separated by solid-liquid separation acquisition.And extracting includes water
Cordyceps militaris is mentioned to obtain Aqueous extracts, alcohol precipitation includes alcohol extracting Aqueous extracts to obtain alcohol extract, and alcohol extract obtains the by being separated by solid-liquid separation
One liquid portion substance, solid portion substance.
First purification process is executed to the first liquid portion substance using micro-porous resin, obtaining has first object product
Second liquid moieties, and the second purification process is executed to obtain to second liquid moieties by way of liquid chromatogram
Third liquid portion substance with first object substance.
By being separated metabolite from Cordyceps militaris using water extraction and alcohol precipitation method, then in conjunction with micro-porous resin and liquid phase color
Spectrum combination is purified, and ingredient can have been divided to separate with small the macromoleculars such as polysaccharide protein in metabolite, played as Cordyceps militaris
The effect of secondary metabolite removal of impurities.Adopting said method can be mentioned effectively in lock onto target metabolite, directly fast and effective separation
High separating efficiency reduces separation costs, obtains high-purity Cordyceps militaris metabolite sterling.
With reference to first aspect, in some optional examples of the first possible embodiment of the first aspect of the present invention
In, the second target product is Polysaccharides in Cultured Cordyceps militaris, includes: that freezing is dry by the method that solid portion substance obtains the second target product
It is dry.
Optionally, the second target product is trehalose.
Optionally, it is 0.06~0.09MPa that the condition of freeze-drying, which includes vacuum degree, and temperature is 45~65 DEG C.
Due to being related to water extract-alcohol precipitation, the liquid such as water that solid portion substance contains part mention in water and alcohol precipitation in
Alcohol.Therefore, the liquid in solid portion substance is removed by being separated by solid-liquid separation, is conducive to the content for improving the second target product,
Also it is conducive to save or be used as other processing.
With reference to first aspect, in some optional examples of second of possible embodiment of the first aspect of the present invention
In, first object substance includes cordycepin, adenosine, cordycepic acid, and the side of first object product is obtained by third liquid portion substance
Method includes: that first object substance is precipitated from third liquid portion substance.
Optionally, the method that first object substance is precipitated from third liquid portion substance is including crystallization and optionally
Recrystallization.
Being crystallized from liquid to obtain solid matter is a kind of means easy to implement, and is easy to carry out scale behaviour
Make.Meanwhile the energy consumption of crystallization treatment is small, the purity is high of crystalline solid.Further, weight is carried out by the crystal generated to crystallization
Crystallization can further increase purity, reduce impurity.
With reference to first aspect, in some optional examples of the third possible embodiment of the first aspect of the present invention
In, water mentions Cordyceps militaris and includes: in the method for obtaining Aqueous extracts
To being crushed the Cordyceps militaris for particulate matter and the dispersion liquid being dispersed in water heats.
Water, which mentions, to be extracted by extraction lock out operation a certain or certain constituents in solvent extraction solid material or solid-liquid
It takes.Wherein, solvent is selected to be adopted as water.Water as solvent is used, by medicinal material (Cordyceps militaris) heating regular hour to extract it
Ingredient.Water proposes, the advantage of raw material environmental protection controllable with solvent Costco Wholesale.
By crushing Cordyceps militaris for particulate matter, it is easier to which it is impregnated with Yu Shuizhong, so that the substance in target be made to be easier to
Into in water, is conducive to the utilization rate for improving Cordyceps militaris, reduces the loss of target substance, improve the yield of target substance.
The third possible embodiment with reference to first aspect, in the 4th kind of possible reality of the first aspect of the present invention
It applies in some optional examples of mode, the solid-liquid ratio of dispersion liquid is 1 kilogram/5~50 liters, and the temperature by heating dispersion liquid reaches
50~85 DEG C.
Optionally, each extraction time is 1~3 hour, and extraction time is 1~3 time.
Due to being related to solid-liquid mass transport process in leaching process, it is directed primarily to Cordyceps militaris transfer of the target substance from solid
The mass transport process in liquid phase water is moved to, and it includes the stages such as moistening, permeating, desorb, dissolve and spread.And in this example, temperature
Degree and solid-liquid ratio can play comparable influence to the leaching of target substance in relatively bigger degree.
With reference to first aspect, in some optional examples of the 5th kind of possible embodiment of the first aspect of the present invention
In, alcohol extracting Aqueous extracts include: to mix extractant with Aqueous extracts to generate precipitating in the method for obtaining alcohol extract.
Optionally, in the step of alcohol extracting Aqueous extracts are to obtain alcohol extract, extractant includes ethyl alcohol, and the dosage of extractant
It is 2~4 times of the volume of Aqueous extracts.
Optionally, the dosage of extractant is 3 times of the volume of Aqueous extracts；
Optionally, before the second target product being made by solid portion substance, solid portion substance is carried out at least once
It separates again, then isolated method includes using water to disperse solid portion substance as dispersing agent, and then carry out alcohol extracting, then pass through
Centrifugation obtains the solid matter that the content of the second target product improves；
Optionally, when carrying out lock out operation again twice or more to solid portion substance, in lock out operation again every time
In centrifugation step caused by supernatant be incorporated into the first liquid portion substance.
It has been observed that obtaining target substance by way of water extract-alcohol precipitation in example of the present invention.Wherein, target substance is water
Dissolubility can preferably dissolve in water.Then by water mention also in be added alcohol, it is contemplated that between target substance and alcohol with
And the dissolution sex differernce between water and alcohol, target substance can be made to be precipitated in a manner of precipitating in competitive course of dissolution,
It is separated to reach with solids and liquid.
With reference to first aspect, in some optional examples of the 6th kind of possible embodiment of the first aspect of the present invention
In, include: using the method that micro-porous resin executes the first purification process to the first liquid portion substance
The first liquid portion substance is adsorbed using micro-porous resin, and uses eluant, eluent to be eluted to collect second liquid portion
Divide substance.
Optionally, the dosage of the first liquid portion substance is the 1~5% of the weight of micro-porous resin, and the first liquid portion
Substance mixes the time to be adsorbed with micro-porous resin as 30~120 minutes；
Optionally, the method for using eluant, eluent to be eluted includes: successively in the method for collecting second liquid moieties
The first elution step and the second elution step carried out.
Optionally, eluent is concentrated, the eluant, eluent in the first elution step is water；In the second elution step
Eluant, eluent be methanol aqueous solution.
Optionally, in the second elution step, the flow velocity of eluant, eluent is the collecting amount of 1~2BV/h and the eluent of collection
It is 2~7 times of the volume of micro-porous resin.
Due to containing in Cordyceps militaris there are many metabolite, it is miscellaneous that solid preferably (more thoroughly) can be removed by micro-porous resin
Matter.In addition, in order to exclude other impurity, by way of being eluted step by step, and the corresponding corresponding eluent of collection, so as to
Target substance can be obtained.Further, by the control to elution requirement, better elution efficiency can be obtained.
With reference to first aspect, in some optional examples of the 7th kind of possible embodiment of the first aspect of the present invention
In, the second purification process is executed to obtain with first object substance to second liquid moieties by way of liquid chromatogram
Third liquid portion substance the step of in, liquid chromatogram is using reverse-phase chromatographic column, preferably C18 reverse-phase chromatographic column.
Reverse chromatograms column is that the polarity of stationary phase is less than the polarity of mobile phase.Its liquid conduct that polarity can be used bigger
Nonpolar medium correspondingly can also be used as stationary phase in mobile phase.C18 column is a kind of common versatility chromatographic column.Instead
Have performance stabilization, separative efficiency high to chromatographic column, separates the advantage of substance classes multiplicity.
The 7th kind of possible embodiment with reference to first aspect, in the 8th kind of possible reality of the first aspect of the present invention
It applies in some optional examples of mode, second liquid moieties is executed in such a way that reverse chromatograms are lived through liquid chromatogram
In the step of second purification process is to obtain the third liquid portion substance with first object substance, the operation item of liquid chromatogram
Part includes: that the flow velocity of mobile phase is 30~100mL/min, and Detection wavelength 254nm, mobile phase is acetonitrile solution, and acetonitrile
Volume content be 95~98%.
It can by such as flowing phase composition, flowing phase velocity and the Detection wavelength-of the selection-to liquid chromatogram operating condition
To realize better separative efficiency, separation accuracy, to improve purity, the yield etc. that separation obtains substance.
In second aspect, example of the invention provides a kind of additive.
The additive is used for health care product and functional food.Additive includes being obtained according to the extracting method of metabolite above-mentioned
Obtain first object substance and/or the second target substance.
The utility model has the advantages that
Extracting method provided in an embodiment of the present invention can extract together a greater variety of metabolites in Cordyceps militaris,
And recovery rate can be improved preferably.It, can be more acurrate and efficiently by pupa by the way that micro-porous resin and liquid chromatogram to be used in conjunction
Polysaccharide, albumen in cordyceps sinensis are separated with other impurity, improve the purity and yield of the product of separation.Also, due to pupa worm
Careless metabolite activity is high, can be applied to the related fieldss such as medicine food, can also be directly as health care product and functional food
Additive, have a vast market development prospect.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
It is specifically described below for the extracting method of the metabolite of the embodiment of the present invention, additive:
Due to Cordyceps militaris numerous pharmacy and medicine in terms of application potential, about Cordyceps militaris research report it is more.And
Its key is to obtain the various active materials in Cordyceps militaris.In the related technology, mentioning to the various active materials in Cordyceps militaris
It takes and is mainly obtained from its metabolite.
Inventor firmly believes: current, the extraction research of Cordyceps militaris active material, which focuses primarily upon, obtains a certain specific components
?.Due to active matter qualitative diversities various in Cordyceps militaris, obtained under the conditions of more preferably single group with high purity, high income
Expectation is divided to reach.However, be based on actual needs, it is therefore desirable to be able in a better way to Cordyceps militaris active material into
Row extracts.One of them main task and difficult point are, the synchronous of various active substance in Cordyceps militaris is extracted and separated.
Currently, some means known for inventor can not preferably realize the above task and expectation.In view of this, this hair
Express and gives the extracting method of various active substance in a kind of pair of Cordyceps militaris in example.
The extracting method object to be operated is the metabolite of Cordyceps militaris.Also, the metabolite includes by Cordyceps militaris
The first object substance and the second target substance of generation.In view of the complexity of the components/ingredients of the metabolite of Cordyceps militaris is led to
It often can according to need and select condition appropriate to control the desired substance obtained.For example, the pupa worm that will be mentioned later
Grass polysaccharide, cordycepin, adenosine, cordycepic acid.Wherein, Polysaccharides in Cultured Cordyceps militaris is the second target substance.Cordycepin, adenosine, cordycepic acid are
First object substance.
It should be understood that the second target substance above-mentioned can be single substance (such as trehalose), it is also possible to a variety of
(such as trehalose and pentosan, many kinds of substance usually can be such as water-soluble with same or similar performance-, oily molten substance
The substance of property, polarity etc. -).First object substance can be single substance (such as cordycepin), be also possible to many kinds of substance (such as gland
Glycosides and cordycepic acid).In other words, first object substance and the second target substance may each be the substance of one pack system, can be multiple groups
The substance of the composition divided.
Extracting method includes:
Step S101, object to be extracted is provided.
Object to be extracted is a kind of crude product, and is obtained from Cordyceps militaris.The object to be extracted includes that there is first object to produce
The first liquid portion substance, the solid portion substance with the second target product of object.It should be pointed out that the first liquid above-mentioned
Its overwhelming majority that body portion substance refers to is liquid, but is also not excluded for wherein being contaminated with the solid of part or indissoluble object.Phase
Ying Di, its overwhelming majority that solid portion substance refers to is solid, but is also not excluded for wherein being contaminated with the liquid of part.Example
Such as, when carrying out coagulation operation in the solution to be separated by solid-liquid separation, after liquid and solids stratification, there may be part in liquid
Sediment correspondingly may also can have the liquid of part in solid.
In addition, object to be extracted above-mentioned can be pre-production, it is also possible to live directly production and obtains.In other words,
Object to be extracted can directly be bought or as prefabricated raw material as raw material.
It is considered that the stability, and the difficulty and complexity saved etc. of various active materials consider in Cordyceps militaris,
The usage mode that can need to select to treat extract according to actual production.
In example, object to be extracted can successively be extracted by Cordyceps militaris, alcohol precipitation, be separated by solid-liquid separation acquisition.
Wherein, extraction includes that water mentions Cordyceps militaris to obtain Aqueous extracts.Water mentions Cordyceps militaris and includes: in the method for obtaining Aqueous extracts
To being crushed the Cordyceps militaris for particulate matter and the dispersion liquid being dispersed in water heats.
For the ease of being handled, Cordyceps militaris, which can choose, is newly collected.Certainly, Cordyceps militaris can also acquire dry product.
Further, it is needed based on subsequent processing, the water that Cordyceps militaris progress pulverization process can obtain is proposed into effect.The method of crushing can
It is carried out in a manner of being such as to grind, cut by milling machinery progress.Alternatively, assisting powder by low temperature in other examples
It is broken.Cordyceps militaris is carried out being cooled to frozen state using cryogenic media, then be crushed in the frozen state.It should be noted that
, the temperature in freezing process is unsuitable too low (within 100 degree Celsius such as subzero), otherwise will cause the activity of part
The destruction of the structure of matter.
In addition, since Cordyceps militaris includes polypide part (fructification) and cursive script part (mycelium).And the metabolism of Cordyceps militaris
There may be differences for ingredient of the product in the two and distribution, therefore, may obtain the better phase by handling the two respectively
Hope product purity and yield.Also, since the two is carried out extraction operation independently, it is possible to reduce more kinds of impurity
Interference, to be conducive to the acquisition of target substance.
In view of the adverse effect to separation, purifying that impurities in water may cause, water is used with higher purity.One
In a little examples, water, which mentions, may be selected to use deionized water, pure water, distilled water, reverse osmosis water, ultrapure water.
Further, extraction effect appropriate may be implemented by the selection of the water consumption and temperature that mention to water.Dispersion liquid
Solid-liquid ratio be 1 kilogram/5~50 liters, by heat dispersion liquid temperature reach 50~85 DEG C.Solid-liquid ratio is the Cordyceps militaris referred to
Ratio between particulate matter and water.I.e. 1 kilogram of cordyceps militaris particle object uses 5~50 liters of water.
In other some examples, the solid-liquid ratio of dispersion liquid is also possible to 1 kilogram/12 liters or 1 kilogram/16 liters or 1
Kilogram/21 liters or 1 kilogram/25 liters or 1 kilogram/30 liters or 1 kilogram/37 liters or 1 kilogram/46 liters.Similarly, dispersion liquid
Temperature is also possible to 53 DEG C or 56 DEG C or 62 DEG C or 67 DEG C or 72 DEG C or 79 DEG C or 83 DEG C.The solid-liquid ratio and temperature of dispersion liquid
Degree can carry out freely selecting cooperation, and the present invention is not specifically limited it.For example, it is 1,000 that dispersion liquid, which can be solid-liquid ratio,
Gram/21 liters, temperature be 82 DEG C；Alternatively, solid-liquid ratio is 1 kilogram/10 liters, temperature is 51 DEG C.It is 1 that dispersion liquid, which is also possible to solid-liquid ratio,
Kilogram/48 liters, temperature be 80 DEG C；Alternatively, solid-liquid ratio is 1 kilogram/49 liters, temperature is 50 DEG C.In some researchs, attempt by super
Sound wave assisted extraction, still, since ultrasonic extraction usually may require that specific ultrasonic frequency and intensity (such as power), and it is past
Toward the substance that can only be directed to opposite one-component.When for needing to extract multicomponent, the selection and control of ultrasonic wave are
One has considered problem.
Generally, it is beneficial that temperature increase can obtain the solubility raising of target substance in water to desired extraction
's.However, the too high irreversible unexpected transformation of generation that may also lead to target substance of temperature.Similarly, by adjusting material
Liquor ratio (as increased water ratio) can enable dispersion liquid dissolve more target substances.It may be right when too high with water ratio
It is unfavorable that subsequent purification step is brought.Such as shipwreck is easily removed under the temperate condition being relatively simplistic, easy to implement.
Optionally, in other examples of the invention, extraction time can be controlled so as to balance extraction efficiency and
The thorough degree of deduction.For example, each extraction time is 1~3 hour, extraction time is 1~3 time.
After proposing processing by the above water, it is expected that obtaining target substance (first object substance and the second target substance) relatively
It easily and is thoroughly distributed in Aqueous extracts, then by alcohol extracting Aqueous extracts to obtain alcohol extract.Alcohol extracting Aqueous extracts are to obtain alcohol extracting
The method of liquid includes: to mix extractant with Aqueous extracts to generate precipitating.
Due to dissolution sex differernce of the active material in water, alcohol in different Cordyceps militaris, and water has with alcohol (such as ethyl alcohol)
Relatively good compatibility, therefore, when adding alcohol in Aqueous extracts, the dissolution sex differernce between different material leads to part
Active material be precipitated in the form that precipitates, and the active material of another part can then exist in water layer.
Optionally, in the step of alcohol extracting Aqueous extracts are to obtain alcohol extract, extractant (can be ethyl alcohol using ethyl alcohol
Refer to that content is 95% alcohol), and the dosage of extractant is 2~4 times of the volume of Aqueous extracts, is also possible to 3 times.
As just aforementioned, after alcohol precipitation, the case where being layered substantially is presented in Aqueous extracts in appearance.In this way, collecting respectively
The separation of active material may be implemented in water layer and the beds of precipitation.Wherein, water layer is as the first liquid portion substance, and wherein has the
One target substance；The beds of precipitation wherein have the second target substance as solid portion substance.
It could be aware that according to the analysis of Cordyceps militaris metabolite, wherein the first object substance in water layer mainly includes cordyceps sinensis
Element, adenosine, cordycepic acid.The second target substance in the beds of precipitation is mainly Polysaccharides in Cultured Cordyceps militaris；For example, the second target product can also be with
It is trehalose.
It is apparent that since the second target substance in the beds of precipitation is mainly Polysaccharides in Cultured Cordyceps militaris, and various physical properties and function
It can be close, therefore can be used as independent substance and separated together, and can choose direct extraction.Certainly, in order to obtain phase
To purer, and the component that ingredient is more single, Polysaccharides in Cultured Cordyceps militaris can also be separated again, be purified.
Hold it is above-mentioned, in order to obtain the second target substance, solid portion substance can be dried or dehydration (wherein
The alcohol that may mix can also be removed simultaneously).For example, including: cold by the method that solid portion substance obtains the second target product
It is lyophilized dry.Optionally, it is 0.06~0.09MPa that the condition of freeze-drying, which includes vacuum degree, and temperature is 45~65 DEG C.Freeze-drying
By will there is substance to be dried carrying out the removal appropriate heated to realize liquid under air pressure lower than natural conditions.It is logical
Decompression is crossed, the boiling point of liquid reduces and (is easier to heated and gasifies), so as to remove in relatively lower temperature by heating.
In this way, high temperature required when heating evaporation can be passed through to avoid high boiling liquid, and then avoid active material impaired.
In addition, in order to avoid the damage of other active components (such as first object substance) that may be present in solid portion substance
It loses, which can be recycled.For example, before the second target product is made by solid portion substance,
Solid portion substance is separated again at least once.The method separated again includes using water as dispersing agent by solid portion substance
Dispersion, and alcohol extracting is then carried out, then the solid matter improved by the content that centrifugation obtains the second target product.Further, when
When carrying out lock out operation again twice or more to solid portion substance, every time again produced by the centrifugation step in lock out operation
Supernatant be incorporated into the first liquid portion substance.
As above-mentioned, Cordyceps militaris mentions by a water, alcohol precipitation, separates the first liquid portion substance of acquisition and solid portion object
Matter.However, it is understood that being likely present only by the first liquid portion substance successively operated a certain amount of
First object substance is also likely to be present a certain amount of second target substance in solid portion substance.Therefore, pass through multiple water
It mentions, alcohol precipitation, lock out operation, and each liquid portion is mixed, solid portion mixing can make first object substance and second
Target substance is more thoroughly separated from each other.
Step S102, treating to obtain in extract, there is the first liquid portion substance of first object substance to be purified.
As mentioned in step S01, first object substance is usually the substance of a variety of properties and structure notable difference, such as
(on the other side to be, the second target substance is usually substance similar in a variety of properties and structure, pupa for cordycepin, adenosine, cordycepic acid
Cordyceps sinensis polysaccharide, trehalose).Therefore, it is necessary to be separated to the various substances in first object substance.
In example, the first liquid portion substance obtains one of the various active materials by two purification process processing
Obtained relatively good separation.
For example, the first liquid portion substance includes: using micropore tree the first step in processing by two purification process
Rouge executes the first purification process to the first liquid portion substance, obtains the second liquid moieties with first object product.
It include: to utilize using the method that micro-porous resin executes the first purification process to the first liquid portion substance in example
Micro-porous resin adsorbs the first liquid portion substance, and uses eluant, eluent to be eluted to collect second liquid moieties.
As a kind of optional example, the dosage of the first liquid portion substance is micro-porous resinWeight 1~
5% (being also possible to 1.3%, 2%, 2.7%, 3%, 4% etc.), and the first liquid portion substance is mixed with micro-porous resin to carry out
The time of absorption is 30~120 minutes (being also possible to 33 minutes, 41 minutes, 58 minutes, 62 minutes, 75 minutes, 89 minutes etc.).
The adsorption time of micro-porous resin be not easy it is too long, avoid its desorb difficulty increase.Micro-porous resin may be selected to use following model:
NS4205, DAC-HB50, DAC-HB80, DAC-HB100, DAC-HB150, DAC-HB200.
The method for using eluant, eluent to be eluted includes: successively carried out in the method for collecting second liquid moieties
One elution step and the second elution step, and collect the eluent of the second elution step and optionally eluent is carried out dense
Contracting.
Eluant, eluent in the first elution step is water.First elution step can go out the impurity of part, avoid then
Desorption target substance when bring pollution and drying.
Eluant, eluent in the second elution step is methanol aqueous solution.Further, in the second elution step, eluant, eluent
Flow velocity is chosen as 1~2BV/h and the collecting amount of eluent collected is 2~7 times of volume of micro-porous resin.
Brought forward is stated, and the first liquid portion substance includes: to pass through liquid phase by the second step in two purification process processing
The mode of chromatography executes the second purification process to second liquid moieties to obtain the third liquid with first object substance
Moieties.
Optionally, the second purification process is executed to be had to second liquid moieties by way of liquid chromatogram
In the step of third liquid portion substance of first object substance, liquid chromatogram is anti-using reverse-phase chromatographic column, preferably C18
Phase chromatographic column (concrete model such as dubhe C18, hedera C18, megres C18, Benetnach C18, phecda
C18, Xmide C18, XCharge C18, ODS C18 etc.).The operating condition of liquid chromatogram may is that the flow velocity of mobile phase is
30~100mL/min, Detection wavelength 254nm, mobile phase is acetonitrile solution, and the volume content of acetonitrile is 95~98%.
Each middle active material can be subjected to more visible and specific differentiation by liquid chromatogram, to obtain various purity
The object of higher and component relatively single (content for the target substance that expectation is extracted is higher) is extremely.Substance is obtained in liquid chromatogram is
Different liquid mixtures.Liquid mixture includes mobile phase and the one or more substances of correspondence that are dissolved in mobile phase.
Wherein, the method for first object product being obtained by third liquid portion substance (multiple liquid mixtures as the aforementioned)
It include: that first object substance is precipitated from third liquid portion substance.Optionally, make first object substance from third liquid portion
Dividing the method being precipitated in substance includes crystallization and optional recrystallization.Crystallizing can be realized by the selection to temperature, by
In solubility of the different material in a certain liquid (can be mixture) be associated with temperature.Therefore, by third liquid
Moieties be placed in it is different at a temperature of make it through dissolution, crystallization mode can obtain the substance of higher purity.
For example, when cordycepin, adenosine, cordycepic acid three are respectively present in different liquid mixtures, in order to obtain respectively
When obtaining the higher cordycepin of purity, adenosine, cordycepic acid, by way of making corresponding liquid mixture be crystallized and be recrystallized
It is set respectively to be precipitated and obtain from liquid in a manner of solid.
Active material mentioned above is primarily referred to as various with the active substance of biologic pharmacological science, such as gland in Cordyceps militaris
Glycosides, cordycepin, cordycepic acid, polysaccharide etc..
By experimental verification, using the extracting method of above-mentioned offer, in some specific examples, in the product of extraction
For Polysaccharides in Cultured Cordyceps militaris content up to 66%, the content of cordycepin, adenosine, cordycepic acid, trehalose can reach 98% or more.By micro-
Hole resin and high performance preparative liquid chromatography joint technology substantially increase the recovery rate of metabolite in Cordyceps militaris, greatly improve
The purity of effective secondary metabolite.It is low with production cost, the features such as technology maturation, the period is short.
According to the inventors knowledge, Cordyceps militaris metabolite has a variety of pharmacological activity, has potential and huge medicinal and food
With value.In view of this, existing Cordyceps militaris extract is by way of directly taking come using mentioning in example of the invention
The additive based on the above-mentioned metabolite for extracting from Cordyceps militaris is supplied.Such additive can be used for health care product and function
Food.The ingredient of additive can adjust as needed.In other words, additive includes first object substance.Alternatively, additive includes
Second target substance.Alternatively, additive includes first object substance and the second target substance.
Additive can be used as the modes such as pulvis or granule and be produced and use.
The extracting method of metabolite of the invention, additive are described in further detail with reference to embodiments.
Embodiment 1
(1) in Cordyceps militaris metabolite extraction: Cordyceps militaris raw material 2kg crush, cross 40 meshes, deionized water is then added
It extracts, then extracting solution is filtered, Cordyceps militaris metabolite extracting solution is made；Extraction conditions be solid-liquid ratio be 1kg:5L, temperature is
50 DEG C, extraction time 1, extraction time be 1 time.
(2) in Cordyceps militaris metabolite separation:
Cordyceps militaris metabolite extracting solution is concentrated, Cordyceps militaris metabolite is obtained and extracts concentrated liquor, its 3 times amounts are added
95% ethyl alcohol, precipitating for 24 hours, collect the beds of precipitation and collect supernatant by centrifugation.Eccentric part is re-dissolved in deionized water, weight
Multiple aforesaid operations 1 time, the beds of precipitation and supernatant are collected, merges supernatant.Eccentric part is freeze-dried, Polysaccharides in Cultured Cordyceps militaris is obtained and mentions
Take object.
Supernatant is concentrated with Rotary Evaporators, by MCI micro-porous resin on Cordyceps militaris secondary metabolite concentrating part, on
Sample amount is the 1% of amount of filler；Absorption 30 minutes, is washed with deionized water de-, then with 60% methanol aqueous solution is eluent tree
Rouge column, eluent are 2 times of resin column volumes, and flow velocity 1BV/h collects 60% meoh eluate.Concentration, obtains Cordyceps militaris secondary metabolism
Product concentrate.The condition of being dried under reduced pressure each means that vacuum degree is 0.06MPa, and temperature is 45 DEG C.
(3) in Cordyceps militaris metabolite purifying:
By above-mentioned secondary metabolite concentrate, upper preparative liquid chromatography (DAC-HB50) collects each secondary metabolite
Fraction, crystallization and recrystallization.Preparing form and aspect chromatographic condition is acetonitrile: water (2%:98%), flow velocity 30mL/min, Detection wavelength
254nm, chromatographic column are C18 reverse-phase chromatographic column (megres C18).Crystallization is ethyl alcohol, water with recrystallization solvent.
Polysaccharides in Cultured Cordyceps militaris yield 43.98%, content 65.40%, secondary metabolite yield 14.80%, cordycepin, gland
The content of glycosides, cordycepic acid, trehalose can reach 98% or more.
Embodiment 2
(1) in Cordyceps militaris metabolite extraction: Cordyceps militaris raw material 4kg crush, cross 120 meshes, deionization is then added
Water extracts, then extracting solution is filtered, and Cordyceps militaris metabolite extracting solution is made；Extraction conditions be solid-liquid ratio be 1kg:50L, temperature
It is 3 times for 85 DEG C, extraction time 3h, extraction time.
(2) in Cordyceps militaris metabolite separation:
Cordyceps militaris metabolite extracting solution is concentrated, Cordyceps militaris metabolite is obtained and extracts concentrated liquor, its 3 times amounts are added
95% ethyl alcohol, precipitates 48h, and centrifugation collects the beds of precipitation and collects supernatant.Eccentric part is re-dissolved in deionized water, weight
Multiple aforesaid operations 3 times, the beds of precipitation and supernatant are collected, merges supernatant.Eccentric part is freeze-dried, Polysaccharides in Cultured Cordyceps militaris is obtained and mentions
Take object.
Supernatant is concentrated with Rotary Evaporators, by MCI micro-porous resin on Cordyceps militaris secondary metabolite concentrating part, on
Sample amount is the 5% of amount of filler；Absorption 120 minutes, is washed with deionized water de-, then with 60% methanol aqueous solution is eluent tree
Rouge column, eluent are 7 times of resin column volumes, and flow velocity 2BV/h collects 60% meoh eluate.Concentration, obtains Cordyceps militaris secondary metabolism
Product concentrate.The condition of being dried under reduced pressure each means that vacuum degree is 0.06MPa, and temperature is 45 DEG C.
(3) in Cordyceps militaris metabolite purifying:
By above-mentioned secondary metabolite concentrate, upper preparative liquid chromatography (DAC-HB80) collects each secondary metabolite
Fraction, crystallization and recrystallization.Preparing form and aspect chromatographic condition is acetonitrile: water (5%:95%), flow velocity 100mL/min, Detection wavelength
254nm, chromatographic column are C18 reverse-phase chromatographic column (dubhe C18).Crystallization is ethyl alcohol, methanol, water with recrystallization solvent.
Polysaccharides in Cultured Cordyceps militaris yield 47.3%, content 65.82%, secondary metabolite yield 17.39%, cordycepin, adenosine,
The content of cordycepic acid, trehalose can reach 98% or more.
Embodiment 3
(1) in Cordyceps militaris metabolite extraction: Cordyceps militaris raw material 10kg crush, cross 60 meshes, deionization is then added
Water extracts, then extracting solution is filtered, and Cordyceps militaris metabolite extracting solution is made；Extraction conditions be solid-liquid ratio be 1kg:20L, temperature
It is 2 times for 75 DEG C, extraction time 2h, extraction time.
(2) in Cordyceps militaris metabolite separation:
Cordyceps militaris metabolite extracting solution is concentrated, Cordyceps militaris metabolite is obtained and extracts concentrated liquor, its 3 times amounts are added
95% ethyl alcohol, precipitates 36h, and centrifugation collects the beds of precipitation and collects supernatant.Eccentric part is re-dissolved in deionized water, weight
Multiple aforesaid operations 2 times, the beds of precipitation and supernatant are collected, merges supernatant.Eccentric part is freeze-dried, Polysaccharides in Cultured Cordyceps militaris is obtained and mentions
Take object.
Supernatant is concentrated with Rotary Evaporators, by MCI micro-porous resin on Cordyceps militaris secondary metabolite concentrating part, on
Sample amount is the 2% of amount of filler；Absorption 60 minutes, is washed with deionized water de-, then with 60% methanol aqueous solution is eluent tree
Rouge column, eluent are 4 times of resin column volumes, and flow velocity 1BV/h collects 60% meoh eluate.Concentration, obtains Cordyceps militaris secondary metabolism
Product concentrate.The condition of being dried under reduced pressure each means that vacuum degree is 0.06MPa, and temperature is 45 DEG C.
(3) in Cordyceps militaris metabolite purifying:
By above-mentioned secondary metabolite concentrate, upper preparative liquid chromatography (DAC-HB80) collects each secondary metabolite
Fraction, crystallization and recrystallization.Preparing form and aspect chromatographic condition is acetonitrile: water (3%:97%), flow velocity 100mL/min, Detection wavelength
254nm, chromatographic column are C18 reverse-phase chromatographic column (dubhe C18).Crystallization is ethyl alcohol, methanol, water with recrystallization solvent.Cordyceps militaris
Polysaccharide yield 47.34%, content 64.13%, secondary metabolite yield 15.22%, cordycepin, adenosine, cordycepic acid, trehalose
Content can reach 98% or more.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of extracting method of metabolite, the metabolite includes the first object substance and second generated by Cordyceps militaris
Target substance, which is characterized in that the extracting method includes:
Object to be extracted is provided, the object to be extracted includes the first liquid portion substance with first object product, has second
The solid portion substance of target product；
Wherein, the object to be extracted is successively extracted by Cordyceps militaris, alcohol precipitation, is separated by solid-liquid separation acquisition, and the extraction includes water
The Cordyceps militaris is mentioned to obtain Aqueous extracts, the alcohol precipitation includes Aqueous extracts described in alcohol extracting to obtain alcohol extract, the alcohol extract warp
It crosses separation of solid and liquid and obtains the first liquid portion substance, the solid portion substance；
First purification process is executed to the first liquid portion substance using micro-porous resin, obtaining, there is the first object to produce
The second liquid moieties of object, and the second purifying is executed to the second liquid moieties by way of liquid chromatogram and is grasped
Make to obtain the third liquid portion substance with first object substance.
2. the extracting method of metabolite according to claim 1, which is characterized in that second target product is pupa worm
Grass polysaccharide includes: freeze-drying by the method that the solid portion substance obtains second target product, it is preferable that described
Second target product is trehalose；It is further preferred that it is 0.06~0.09MPa, temperature that the condition of freeze-drying, which includes vacuum degree,
Degree is 45~65 DEG C.
3. the extracting method of metabolite according to claim 1, which is characterized in that the first object substance includes worm
Careless element, adenosine, cordycepic acid, by the method that the third liquid portion substance obtains the first object product include: make described in
First object substance is precipitated from the third liquid portion substance；Preferably, make the first object substance from the third
The method being precipitated in liquid portion substance includes crystallization and optional recrystallization.
4. the extracting method of metabolite according to claim 1, which is characterized in that water mentions the Cordyceps militaris to obtain water
The method of extract includes:
To being crushed the Cordyceps militaris for particulate matter and the dispersion liquid being dispersed in water heats.
5. the extracting method of metabolite according to claim 4, which is characterized in that the solid-liquid ratio of the dispersion liquid is 1
Kilogram/5~50 liters, the temperature by heating the dispersion liquid reaches 50~85 DEG C；Preferably, each extraction time is 1~3 small
When, extraction time is 1~3 time.
6. the extracting method of metabolite according to claim 1, which is characterized in that Aqueous extracts described in alcohol extracting are to obtain alcohol
The method of extract includes: to mix extractant with the Aqueous extracts to generate precipitating；
Preferably, in the step of Aqueous extracts described in alcohol extracting are to obtain alcohol extract, extractant includes ethyl alcohol, and the dosage of extractant
For 2~4 times of the volume of the Aqueous extracts, preferably 3 times；
It is highly preferred that before second target product is made by the solid portion substance, to the solid portion substance into
Row separates again at least once, and the method separated again includes that water is used to disperse solid portion substance as dispersing agent, and subsequent
Carry out alcohol extracting, then the solid matter improved by the content that centrifugation obtains second target product；
It is further preferred that dividing again every time when carrying out lock out operation again twice or more to the solid portion substance
The first liquid portion substance is incorporated into from supernatant caused by the centrifugation step in operation.
7. the extracting method of metabolite according to claim 1, which is characterized in that using micro-porous resin to described first
Liquid portion substance execute the first purification process method include:
The first liquid portion substance is adsorbed using micro-porous resin, and uses eluant, eluent to be eluted to collect second liquid
Body portion substance；
Preferably, the dosage of the first liquid portion substance is the 1~5% of the weight of micro-porous resin, and the first liquid portion substance
The time to be adsorbed is mixed with micro-porous resin as 30~120 minutes；
It is highly preferred that the method for using eluant, eluent to be eluted with the method for collecting the second liquid moieties include: according to
The first elution step and the second elution step of secondary progress, and collect the eluent and optionally of second elution step
The eluent is concentrated, the eluant, eluent in first elution step is water；In second elution step
Eluant, eluent is methanol aqueous solution；
It is further preferred that the flow velocity of eluant, eluent is 1~2BV/h and the eluent of collection in second elution step
Collecting amount is 2~7 times of the volume of micro-porous resin.
8. the extracting method of metabolite according to claim 1, which is characterized in that institute by way of liquid chromatogram
It states second liquid moieties and executes the second purification process to obtain the third liquid portion substance with first object substance
In step, liquid chromatogram is using reverse-phase chromatographic column, preferably C18 reverse-phase chromatographic column.
9. the extracting method of metabolite according to claim 8, which is characterized in that lived using reverse chromatograms and pass through liquid phase
The mode of chromatography executes the second purification process to the second liquid moieties to obtain the third with first object substance
In the step of liquid portion substance, the operating condition of liquid chromatogram includes: that the flow velocity of mobile phase is 30~100mL/min, detection
Wavelength is 254nm, and the mobile phase is acetonitrile solution, and the volume content of acetonitrile is 95~98%.
10. a kind of additive is used for health care product and functional food, which is characterized in that the additive includes according to claim 1
The extracting method of metabolite described in any one of~9 obtains the first object substance and/or second object
Matter.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101007025A (en) * | 2006-01-24 | 2007-08-01 | 上海市农业科学院 | A continuous extraction method of effective ingredients from fruitbodies of Cordyceps militaris |
| CN101124988A (en) * | 2007-09-25 | 2008-02-20 | 江苏瑞迪生科技有限公司 | Method for extracting refined cordycepin and cordyceps polysaccharide from Cordyceps militaris |
| WO2008038973A1 (en) * | 2006-09-26 | 2008-04-03 | Konkuk University Industrial Cooperation Corp. | A pharmaceutical composition comprising cordycepin for the treatment and prevention of obesity |
| CN101580755A (en) * | 2009-06-11 | 2009-11-18 | 湖南农业大学 | Process for continuously extracting cordyceps militaris essential oil, cordycepin, cordycepic acid and cordyceps militaris polysaccharide from cordyceps militaris |
| CN102070690A (en) * | 2010-11-30 | 2011-05-25 | 中国科学院大连化学物理研究所 | Method for preparing adenosine, cordycepin and N6-(2-hydroxyethyl)adenosine simultaneously used as chemical reference substances |
| CN102558264A (en) * | 2012-01-18 | 2012-07-11 | 辽宁仙榆湾北冬虫夏草（集团）有限公司 | Method for extracting cordycepin and cordyceps polysaccharide from cordyceps militaris |
| CN104473977A (en) * | 2014-11-18 | 2015-04-01 | 广东省微生物研究所 | Cordyceps militaris water extract as well as preparation method and application thereof |
| CN107556358A (en) * | 2017-09-27 | 2018-01-09 | 黄河科技学院 | Promote blood coagulation Cordyceps militaris active ingredient and its purification methods and uses |
| TW201803571A (en) * | 2016-07-20 | 2018-02-01 | 錢佑 | Cordyceps militaris extract for anti-inflammation and anti-proliferation against human liver cancer cell lines, and its preparation method |
-
2018
- 2018-12-26 CN CN201811605699.9A patent/CN109553647A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101007025A (en) * | 2006-01-24 | 2007-08-01 | 上海市农业科学院 | A continuous extraction method of effective ingredients from fruitbodies of Cordyceps militaris |
| WO2008038973A1 (en) * | 2006-09-26 | 2008-04-03 | Konkuk University Industrial Cooperation Corp. | A pharmaceutical composition comprising cordycepin for the treatment and prevention of obesity |
| CN101124988A (en) * | 2007-09-25 | 2008-02-20 | 江苏瑞迪生科技有限公司 | Method for extracting refined cordycepin and cordyceps polysaccharide from Cordyceps militaris |
| CN101580755A (en) * | 2009-06-11 | 2009-11-18 | 湖南农业大学 | Process for continuously extracting cordyceps militaris essential oil, cordycepin, cordycepic acid and cordyceps militaris polysaccharide from cordyceps militaris |
| CN102070690A (en) * | 2010-11-30 | 2011-05-25 | 中国科学院大连化学物理研究所 | Method for preparing adenosine, cordycepin and N6-(2-hydroxyethyl)adenosine simultaneously used as chemical reference substances |
| CN102558264A (en) * | 2012-01-18 | 2012-07-11 | 辽宁仙榆湾北冬虫夏草（集团）有限公司 | Method for extracting cordycepin and cordyceps polysaccharide from cordyceps militaris |
| CN104473977A (en) * | 2014-11-18 | 2015-04-01 | 广东省微生物研究所 | Cordyceps militaris water extract as well as preparation method and application thereof |
| TW201803571A (en) * | 2016-07-20 | 2018-02-01 | 錢佑 | Cordyceps militaris extract for anti-inflammation and anti-proliferation against human liver cancer cell lines, and its preparation method |
| CN107556358A (en) * | 2017-09-27 | 2018-01-09 | 黄河科技学院 | Promote blood coagulation Cordyceps militaris active ingredient and its purification methods and uses |
Non-Patent Citations (5)
| Title |
|---|
| 张晓峰 等: "《中国虫草：历史·资源·科研》", 31 January 2008, 陕西科学技术出版社 * |
| 惠永正 著: "《中药天然产物大全 10 下 中药》", 31 January 2011, 上海科学技术出版社 * |
| 朱丽娜 等: "不同来源的蛹虫草子实体活性成分的比较", 《菌物学报》 * |
| 薛俊杰: "北冬虫夏草主要有效成分的含量测定研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 陈方圆 等: "蛹虫草活性物质提取技术研究进展", 《江苏农业科学》 * |
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