CN109463729A - The preparation method of Cordyceps militaris extract, functional food - Google Patents
The preparation method of Cordyceps militaris extract, functional food Download PDFInfo
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- CN109463729A CN109463729A CN201811605698.4A CN201811605698A CN109463729A CN 109463729 A CN109463729 A CN 109463729A CN 201811605698 A CN201811605698 A CN 201811605698A CN 109463729 A CN109463729 A CN 109463729A
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- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 116
- 239000000284 extract Substances 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 235000013376 functional food Nutrition 0.000 title claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 103
- 239000003480 eluent Substances 0.000 claims abstract description 92
- 239000000126 substance Substances 0.000 claims abstract description 62
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 62
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 58
- 238000010828 elution Methods 0.000 claims abstract description 58
- 239000013076 target substance Substances 0.000 claims abstract description 52
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- 238000005406 washing Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 235000019441 ethanol Nutrition 0.000 claims description 46
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 238000000926 separation method Methods 0.000 claims description 28
- 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 22
- 150000004676 glycans Chemical class 0.000 claims description 18
- 229920001282 polysaccharide Polymers 0.000 claims description 18
- 239000005017 polysaccharide Substances 0.000 claims description 18
- 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 claims description 17
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 claims description 17
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
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- 239000007864 aqueous solution Substances 0.000 claims description 13
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- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 11
- 229960005305 adenosine Drugs 0.000 claims description 11
- 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 9
- 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 9
- 238000004108 freeze drying Methods 0.000 claims description 8
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Classifications
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- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/10—General methods of cooking foods, e.g. by roasting or frying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/066—Clavicipitaceae
- A61K36/068—Cordyceps
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Botany (AREA)
- Animal Behavior & Ethology (AREA)
- Alternative & Traditional Medicine (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A kind of preparation method of Cordyceps militaris extract, functional food, belong to field of agricultural products processing.Preparation method includes: to provide object to be extracted, and object to be extracted carries out extraction acquisition by Cordyceps militaris, and extraction includes that water mentions Cordyceps militaris to obtain Aqueous extracts.First elution action, the second elution action after the first elution action are executed to Aqueous extracts using micro-porous resin.Wherein, the first elution action includes washing and collecting the first eluent, carries out the first object substance that alcohol precipitation is precipitated to obtain as sediment to the first eluent.Wherein, the second elution action includes that alcohol is washed and collects the second eluent with the second target substance.Lock out operation is executed to obtain the second target substance to the second eluent 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, in particular to preparation method, the function of a kind of Cordyceps militaris extract
Property food.
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 preparation method of Cordyceps militaris extract, functional food,
Partly or entirely to improve, even solve problem above.
The present invention is implemented as follows:
In a first aspect, example of the invention provides a kind of preparation method of Cordyceps militaris extract.
The targeted extract of preparation method provided in example include by Cordyceps militaris generate first object substance and
Second target substance.In other words, extracting method from Cordyceps militaris for extracting target substance.
Preparation method includes:
Object to be extracted is provided, object to be extracted carries out extraction acquisition by Cordyceps militaris, and extraction includes that water mentions Cordyceps militaris to obtain
Aqueous extracts；
The second elution that first elution action is executed to Aqueous extracts using micro-porous resin, is carried out after the first elution action
Operation；Wherein, the first elution action includes washing and collecting the first eluent, carries out alcohol precipitation to the first eluent and generates to precipitate
The form of object is precipitated and through being separated by solid-liquid separation the first object substance obtained；Wherein, the second elution action includes that alcohol is washed and collects tool
There is the second eluent of the second target substance；
It is optionally right to the second eluent execution lock out operation to obtain the second target substance by way of liquid chromatogram
Second target substance is crystallized or is recrystallized.
By being separated the metabolite in Cordyceps militaris using water extraction, then join in conjunction with micro-porous resin and liquid chromatogram
With being purified, ingredient can have been divided to separate with small the macromoleculars such as polysaccharide protein in metabolite, play for Cordyceps militaris it is secondary
The effect of metabolite removal of impurities.Adopting said method can effectively lock onto target metabolite, directly quickly and effectively separation, improve point
From efficiency, separation costs are reduced, high-purity Cordyceps militaris metabolite sterling is obtained.
With reference to first aspect, in some optional examples of the first 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.
In conjunction with the first possible embodiment, the one of second of possible embodiment of the first aspect of the present invention
In a little optional examples, 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 third possible embodiment of the first aspect of the present invention
In, the first elution action, the second elution action for carrying out after the first elution action are executed to Aqueous extracts using micro-porous resin
During, the applied sample amount of Aqueous extracts is 3~6 times of micro-porous resin volume, loading adsorption time 30~120 minutes.
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 applied sample amount and adsorption time, preferably elution effect can be obtained
Rate avoids the loss of target substance.
With reference to first aspect, in some optional examples of the 4th kind of possible embodiment of the first aspect of the present invention
In, the first elution action includes: that the first eluent is concentrated before carrying out alcohol precipitation to the first eluent；
Optionally, the method that the first eluent is concentrated includes: vacuum distillation；
Optionally, distillation under pressure condition includes: 45~65 DEG C of 0.06~0.09MPa of vacuum degree, temperature；
Optionally, first object substance includes Polysaccharides in Cultured Cordyceps militaris.
Optionally, first object substance is trehalose.
After concentration, concentration/content of the first object substance in the first eluent is improved, it is easier to then pass through alcohol precipitation
It is precipitated, and loss can be reduced, reduce alcohol dosage etc..
In conjunction with the 4th kind of possible embodiment, the one of the 5th kind of possible embodiment of the first aspect of the present invention
In a little optional examples, alcohol precipitation is carried out to the first eluent and generates first for being precipitated in the form of sediment and obtaining through separation of solid and liquid
In the step of target substance, the condition of alcohol precipitation includes: to use dosage for 2~4 times of volume of second of the first eluent after concentration
Alcohol is as precipitating reagent, and the sedimentation time 24~48 hours；
Optionally, the method for separation of solid and liquid includes centrifugation；
Optionally, it is separated by solid-liquid separation and obtains first object substance by being dried；
Optionally, be dried includes freeze-drying.
Handled by alcohol precipitation, first object species precipitate and from liquid be precipitated, be layered.The side of liquid separation can so be passed through
Formula obtains first object substance.In order to obtain better yield, avoids losing, the first of precipitation is collected by way of centrifugation
Target substance.
It, can in view of there are the possibility of liquid to dry to it in first object substance, and further by freeze-drying
The damage that may cause to avoid heat drying to target substance.
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, in the second elution action step, the eluant, eluent that alcohol washes use includes methanol aqueous solution；
Optionally, the volumetric concentration of methanol is 60% in methanol aqueous solution, and elution requirement includes: the dosage of methanol aqueous solution
It is 2~7 times of micro-porous resin volume, 1~2BV/h of flow velocity.
It isolates and purifies in operation using micro-porous resin, can be obtained with higher efficiency by elution requirement more than control
The target substance of higher purity is obtained, and reduces loss.
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, before executing lock out operation to the second eluent by way of liquid chromatogram, the second eluent is concentrated；
Optionally, in the first elution step, pass through remaining liquid portion quilt after separation of solid and liquid acquisition first object object
It is merged into the second eluent, and is carried out before the second eluent is concentrated.
Concentration can reduce solvent, improve target substance concentration, to be conducive to subsequent isolate and purify operation.Together
When, in view of being separated by solid-liquid separation in the first elution step in the liquid (supernatant) of generation there may be a certain amount of target substance,
Being incorporated into the second eluent can be improved yield to carry out subsequent liquid chromatogram, reduces loss.
With reference to first aspect, in some optional examples of the 8th kind of possible embodiment of the first aspect of the present invention
In, the condition for executing lock out operation to the second eluent by way of liquid chromatogram includes:
Mobile phase includes acetonitrile solution, and chromatographic column is reverse-phase chromatographic column, preferably C8 reverse-phase chromatographic column；
It is highly preferred that the middle acetonitrile volume content of acetonitrile solution is 2~5%, 30~100mL/min of flow velocity, wave is detected
Long 254nm；
Optionally, the second target substance includes cordycepin, adenosine, cordycepic acid.
Chromatographic isolation is carried out with above-mentioned condition, each target substance can be extracted with higher efficiency and purity.
In second aspect, the embodiment of the invention provides a kind of functional foods.
Functional food includes that the preparation method of above-mentioned Cordyceps militaris extract obtains first object substance and/or the second mesh
Mark 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
Functional food, 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 preparation method of the Cordyceps militaris extract of the embodiment of the present invention, functional food:
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 extract includes by produced from Cordyceps militaris L
Raw first object substance and the second target substance.In view of, the complexity of the components/ingredients of the metabolite of Cordyceps militaris, usually
It can according to need and select condition appropriate to control the desired substance obtained.For example, the Cordyceps militaris that will be mentioned later
Polysaccharide, cordycepin, adenosine, cordycepic acid.Wherein, Polysaccharides in Cultured Cordyceps militaris is first object substance.Cordycepin, adenosine, cordycepic acid
Two target substances.
It should be understood that first object 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. -).Second target 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.
Preparation 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 include first object product and
Second target product.
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 carry out extraction acquisition by Cordyceps militaris.
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
(based on the needs that uniform particle sizes are distributed, can be sieved in a manner of being such as to grind, cut by milling machinery progress
Point, wherein the mesh number sieved is 40~120 mesh).Alternatively, passing through low temperature assisted comminution in other examples.Cordyceps militaris is adopted
It is carried out being cooled to frozen state with cryogenic media, then be crushed in the frozen state.It should be noted that in freezing process
Temperature it is unsuitable too low (within 100 degree Celsius such as subzero), otherwise will cause the destruction of the active material configuration of part.
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, ultrasonic wave is specifically selected and is controlled
System, which is one, 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
Easily and it is thoroughly distributed in Aqueous extracts.
Step S102, Aqueous extracts are isolated and purified using micro-porous resin.
Using micro-porous resinThe method isolated and purified to Aqueous extracts includes the first elution action, first
The second elution action carried out after elution action.Substantially, the first elution action and the second elution action are successive progress,
And carry out the second elution action again after the completion of the first elution action.In other words, after micro-porous resin adsorbs Aqueous extracts, column is filled out,
Then elution processing is carried out.After the processing of first time elution action, then carry out the second elution action.Micro-porous resin may be selected to use
Following model: NS4205, DAC-HB50, DAC-HB80, DAC-HB100, DAC-HB150, DAC-HB200.
Due to the degree that Aqueous extracts and micro-porous resin adsorb, subsequent the elution isolated efficiency and yield etc. of carrying out is deposited
It is influencing, therefore, sample loading mode can selected.For example, the applied sample amount of Aqueous extracts is 3~6 times of micro-porous resin volume
(being also possible to 4 times, 5 times etc.) (is also possible to 36 minutes, 42 minutes, 51 minutes, 67 points for loading adsorption time 30~120 minutes
Clock, 79 minutes, 88 minutes etc.).When loading, micro-porous resin is mixed with Aqueous extracts, Aqueous extracts can then be adsorbed by micro-porous resin.
After completion of the sample, carry out elution processing with will it is expected obtain substance separate, so as to collect and optionally into
The subsequent purification process (such as subsequent liquid chromatography process referred to) of row.In example, elution processing is included in following part will be by
The first elution action and the second elution action referred to.
Wherein, using micro-porous resin to Aqueous extracts isolate and purify in the first elution action include washing and collecting the
One eluent is carried out alcohol precipitation generation to first eluent and is precipitated in the form of sediment and through being separated by solid-liquid separation described in acquisition
First object substance.
In the first elution action step, using water as eluent (mobile phase).It can will be a part of as eluent using water
Substance is obtained in a manner of being dissolved in eluent, i.e. the first eluent.First eluent include water and first object substance or
Other a small amount of impurity that can have.Normally, the first eluent can directly carry out subsequent alcohol precipitation processing.But in order to
Efficiency/yield/yield of alcohol precipitation is improved, can carry out carrying out suitable degree of concentration to the first eluent between alcohol precipitation.
Be concentrated the first eluent mode can there are many, in example of the present invention, condensing mode be vacuum distillation.Decompression is steamed
The boiling point that can lower liquid is evaporated, to remove liquid evaporation by heating at relatively lower temperature, and is unlikely to high
Temperature destroys the structure of active material.In example, distillation under pressure condition includes: 0.06~0.09MPa of vacuum degree, temperature 45~65
℃.In other alternative examples, vacuum degree can be 0.07~0.08MPa, temperature is 50~61 DEG C.
After concentration, liquid (water) content in the first eluent is reduced, and can promote target substance (first object object
Matter, such as Polysaccharides in Cultured Cordyceps militaris；More specifically, first object substance can be trehalose) precipitation.It then can be more by alcohol precipitation
In large quantities it is precipitated first object substance.Due to dissolution sex differernce of the active material in water, alcohol in different Cordyceps militaris, and water
There is relatively good compatibility with alcohol (such as ethyl alcohol), therefore, when adding alcohol in the first eluent, between different material
The dissolution sex differernce form that causes the active material of part to precipitate be precipitated, and the active material of another part can then continue
In the presence of in water layer.
As a kind of alternative example, alcohol (ethyl alcohol, 95% alcohol) used by the alcohol precipitation carried out after concentration operates
Dosage is 2~4 times of the volume after the first eluent is concentrated, is also possible to 3 times.Correspondingly, the time of alcohol precipitation can be
24~48 hours.As just aforementioned, after alcohol precipitation, the case where being layered substantially is presented in the first eluent in appearance.In this way, point
Not Shou Ji water layer and the beds of precipitation separation of active material may be implemented.Wherein, liquid level is as supernatant (or clear liquid), and its
In there may be the second a small amount of target substances.The beds of precipitation wherein have most first object substance as solid phase.
After layering, the beds of precipitation and liquid level can be separated by way of such as separatory funnel.Further, in example,
Selection is separated by solid-liquid separation by way of centrifugation.Centrifugally operated can solid-liquid separation effect with higher, can be more preferable
Ground reduces the content of solid matter in liquid, while preferably reducing the content of liquid substance in solid.
Further, since first object substance is primarily present in the beds of precipitation (in solid phase), and wherein there may be not
Therefore the liquid of equivalent to obtain purer first object substance, can choose and it is dried, may deposit to remove
Water and alcohol (such as ethyl alcohol).In example, the mode of freeze-drying is selected to remove liquid.Solid phase can be dried or be taken off
Water process (alcohol-that may wherein mix such as ethyl alcohol-can also be removed simultaneously).Optionally, the condition of freeze-drying includes true
Reciprocal of duty cycle is 0.06~0.09MPa, and temperature is 45~65 DEG C.Freeze-drying will be by that will have substance to be dried lower than natural conditions
Heating appropriate is carried out under air pressure to realize the removal of liquid.By decompression, the boiling point of liquid, which reduces, (to be easier to be heated and gas
Change), so as to be removed in relatively lower temperature by heating.In this way, can be steamed to avoid high boiling liquid by heating
Required high temperature when hair, and then avoid active material impaired.
While the above processing obtains purer first object substance, the second elution action can also be carried out.Wherein, it adopts
With micro-porous resin to Aqueous extracts isolate and purify in the second elution action include that alcohol is washed and collected with second target
Second eluent of substance.In this step, the processing mode that alcohol is washed can be (can choose using including methanol aqueous solution
Methanol aqueous solution is that the volumetric concentration of methanol is that eluant, eluent 60%) comes to adsorbing aforementioned Aqueous extracts (and by the first elution behaviour
Separate the first object substance of the overwhelming majority) it is rinsed.In a kind of alternative scheme, elution requirement includes: methanol
The dosage of aqueous solution be micro-porous resin volume 2~7 times (or 3 times, 4 times, 5 times etc.), 1~2BV/h of flow velocity (alternatively,
1.1BV/h, 1.3BV/h, 1.7BV/h, 1.9BV/h).
It is handled by the elution of methanol aqueous solution, the second target substance (combination that can be the substance of a variety of one-components)
It enters in the second eluent.It obviously, include water and methanol and the second target substance in the second eluent, therefore, in order to obtain
The second pure target substance is obtained, separating treatment can be carried out.As previously mentioned, subsequent point can be improved by way of concentration
Efficiency and effect from processing.Therefore, optionally, before executing lock out operation to the second eluent, the second eluent is carried out
Concentration.
In addition, obtaining the first object substance of solid (by consolidating in separation of solid and liquid due in the first elution action
Body is handled and is obtained), still, there may be the second target substances of part for liquid portion during its, right to avoid wasting
It is recycled.In example, in the first elution step, pass through remaining liquid portion after separation of solid and liquid acquisition first object object
It point is incorporated into the second eluent, and is that carry out before the second eluent is concentrated (can certainly be right
Second eluent carries out before being concentrated, but this may may require that and increase the concentration again after being incorporated to).
It could be aware that according to the analysis of Cordyceps militaris metabolite, the second eluent is mainly the second target substance, and main
Including cordycepin, adenosine, cordycepic acid.It is apparent that the property of various components and function exist due in the second target substance
Considerable degree of difference, and be also usually used separately in the time with single component, therefore can choose and distinguished
Extraction.Certainly, relatively purer in order to obtain, and the component that ingredient is more single, it can also be separated, be purified again.
Step S103, the separating treatment purification process of the second eluent.
It holds above-mentioned, when in order to obtain the substance of each single component in the second target substance (when for various ingredients), needs
Separating treatment appropriate is carried out according to its component.To containing multi-component second target substance by way of liquid chromatogram in example
The second eluent carry out component lock out operation.
Due to consideration that the content of the solvent (eluant, eluent) in the second eluent is excessive, the dense of the second target substance will lead to
It spends low, is also unfavorable for carrying out subsequent liquid chromatography process, can choose and first carry out concentration.Pass through the side of liquid chromatogram
Before formula executes lock out operation to the second eluent, the second eluent is concentrated.
The concentration of the second target substance increases in the second eluent (or concentrate) after concentration, and solvent (the
Eluant, eluent in two elution actions) it reduces.The condition packet of lock out operation is executed to the second eluent by way of liquid chromatogram
Include: mobile phase includes acetonitrile solution, and chromatographic column is reverse-phase chromatographic column (such as C8 reverse-phase chromatographic column).C18 reverse-phase chromatographic column is (specific
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 is 254nm, and 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, after liquid chromatography process, the second eluent (multiple liquid mixtures as the aforementioned) obtains the second target
The substance (such as cordycepin, adenosine, cordycepic acid) of the higher one-component of the various purity of product.It is apparent that by liquid chromatogram
Reason obtain aforementioned various one-components substance be also be obtained in a manner of liquid (i.e. arbitrary one-component is dissolved in liquid
The mobile phase of phase chromatography is constituted).In order to which a certain specific substance is obtained from liquid.Optionally, the object for obtaining the expectation
Matter is detached from from liquid.Optionally, the method for being detached from component and liquid includes crystallization and optional recrystallization or evaporation etc..Knot
Crystalline substance can be realized by the selection to temperature, since solubility of the different material in a certain liquid (can be mixture) is
It is associated with temperature.Therefore, by third liquid portion substance be placed in it is different at a temperature of make it through dissolution, crystallization mode can
To obtain the substance of higher purity.
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, micro-porous resin and high performance liquid preparative chromatography combination point
From that ingredient can have been divided to separate with small the macromoleculars such as polysaccharide protein in extracting solution using water extraction and alcohol precipitation method after purification, play as pupa
The effect of cordyceps sinensis secondary metabolite removal of impurities.Adopting said method can effective lock onto target metabolite, directly fast and effective point
From raising separative efficiency reduces separation costs, obtains high-purity Cordyceps militaris secondary metabolite sterling.Product Cordyceps militaris is secondary
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 has a vast market development prospect.
In some alternative specific examples, preparation method is had the advantage that
1. extracting effective metabolite in Cordyceps militaris using water extraction, maintains in Cordyceps militaris be effectively metabolized to the maximum extent
The bioactivity and recovery rate of product, total recovery rate reach 90% or more.
2. separating the polysaccharide and protein ingredient in extract after isolating and purifying using micro-porous resin, while again to water elution
Part is recycled, and the recovery rate of secondary metabolite is improved.After isolating and purifying, Polysaccharides in Cultured Cordyceps militaris yield 46.6% contains
For amount up to 50%, the content of secondary metabolite yield 14.8%, cordycepin, adenosine, cordycepic acid, trehalose can reach 98%
More than.
It, significantly can be with compared with conventional separation techniques 3. high performance liquid preparative chromatography lock onto target metabolite is accurate
Separating difficulty is reduced, separative efficiency is improved, reduces separation costs, saves disengaging time.
4. present invention process is simple, reproducible, stable and controllable for quality.
It, can also be directly as health care product 5. Cordyceps militaris metabolite can be applied to the related fieldss such as medical scientific research food
With the additive of functional food, development prospect is had a vast market.
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
Supplied mainly include based on the above-mentioned extract for extracting from Cordyceps militaris metabolite functional food.Such functional food
The ingredient of product can adjust as needed.In other words, functional food includes first object substance.Alternatively, functional food includes
Second target substance.Alternatively, functional food includes first object substance and the second target substance.
Functional food can be used as the modes such as pulvis or granule and be produced and use.
The preparation method of Cordyceps militaris extract of the invention, functional food are made with reference to embodiments further detailed
Thin description.
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:
By MCI micro-porous resin column on Cordyceps militaris metabolite extracting solution, loading liquid measure is 3 times of resin column volumes；Absorption 30
Minute, de-, collection eluent is then washed with deionized water.Eluent is concentrated under reduced pressure under temperature 60 C with rotary evaporator and obtains water
Concentrate is mentioned, is the ethyl alcohol that its 3 times amounts 95% are added in eluant, eluent concentrating part by water, for 24 hours, supernatant is collected in centrifugation to precipitating.It will
Eccentric part freeze-drying, obtains Polysaccharides in Cultured Cordyceps militaris extract.
It is again eluent resin column with 60% methanol aqueous solution, eluent is 2 times of resin column volumes, flow velocity 1BV/h,
Collect eluent.Eluent merges with the supernatant after water-wash section alcohol precipitation, and concentration obtains secondary metabolite concentrate.Decompression
Drying condition 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 46.6%, content 50.40%, secondary metabolite yield 14.80%, cordycepin, adenosine,
The content of 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:
By MCI micro-porous resin column on Cordyceps militaris metabolite extracting solution, loading liquid measure is 6 times of resin column volumes；Adsorb 30-
120 minutes, de-, collection eluent is then washed with deionized water.Eluent is concentrated under reduced pressure under temperature 60 C with rotary evaporator
Water extracting liquid is obtained, is the ethyl alcohol that its 3 times amounts 95% are added in eluant, eluent concentrating part by water, precipitates 48h, centrifugation, collection supernatant
Liquid.Eccentric part is freeze-dried, Polysaccharides in Cultured Cordyceps militaris extract is obtained.
It is again eluent resin column with 60% methanol aqueous solution, eluent is 7 times of resin column volumes, flow velocity 2BV/h,
Collect eluent.Eluent merges with the supernatant after water-wash section alcohol precipitation, and concentration obtains secondary metabolite concentrate.Decompression
Drying condition each means that vacuum degree is 0.09MPa, and temperature is 45~65 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 45.9%, content 51.82%, secondary metabolite yield 15.39%, 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:
By MCI micro-porous resin column on Cordyceps militaris metabolite extracting solution, loading liquid measure is 5 times of resin column volumes；Absorption 60
Minute, de-, collection eluent is then washed with deionized water.Eluent is concentrated under reduced pressure under temperature 60 C with rotary evaporator and obtains water
Concentrate is mentioned, is the ethyl alcohol that its 3 times amounts 95% are added in eluant, eluent concentrating part by water, supernatant is collected in precipitating 36h centrifugation.It will
Eccentric part freeze-drying, obtains Polysaccharides in Cultured Cordyceps militaris extract.
It is again eluent resin column with 60% methanol aqueous solution, eluent is 5 times of resin column volumes, flow velocity 2BV/h,
Collect eluent.Eluent merges with the supernatant after water-wash section alcohol precipitation, and concentration obtains secondary metabolite concentrate.Decompression
Drying condition each means that vacuum degree is 0.09MPa, and temperature is 55 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.
Polysaccharides in Cultured Cordyceps militaris yield 47.34%, content 50.67%, secondary metabolite yield 17.22%, cordycepin, gland
The content of glycosides, cordycepic acid, trehalose 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 preparation method of Cordyceps militaris extract, the extract includes the first object substance and the generated by Cordyceps militaris
Two target substances, which is characterized in that the preparation method includes:
Object to be extracted is provided, the object to be extracted carries out extraction acquisition by Cordyceps militaris, and the extraction includes that water mentions the pupa worm
Grass is to obtain Aqueous extracts；
First elution action is executed to the Aqueous extracts using micro-porous resin, is carried out after first elution action second
Elution action；Wherein, the first elution action includes washing and collecting the first eluent, carries out alcohol precipitation production to first eluent
It is raw to be precipitated in the form of sediment and through being separated by solid-liquid separation the first object substance obtained；Wherein, the second elution action includes
Alcohol is washed and collects the second eluent with second target substance；
It is optionally right to second eluent execution lock out operation to obtain the second target substance by way of liquid chromatogram
Second target substance is crystallized or is recrystallized.
2. the preparation method of Cordyceps militaris extract according to claim 1, which is characterized in that water mentions the Cordyceps militaris to obtain
The methods of Aqueous extracts includes:
To being crushed the Cordyceps militaris for particulate matter and the dispersion liquid being dispersed in water heats.
3. the preparation method of Cordyceps militaris extract according to claim 2, which is characterized in that the solid-liquid ratio of the dispersion liquid
It is 1 kilogram/5~50 liters, the temperature by heating the dispersion liquid reaches 50~85 DEG C；Preferably, each extraction time be 1~
3 hours, extraction time was 1~3 time.
4. the preparation method of Cordyceps militaris extract according to claim 1, which is characterized in that using micro-porous resin to described
Aqueous extracts execute the first elution action, during the second elution action carried out after first elution action, and water mentions
The applied sample amount of liquid is 3~6 times of micro-porous resin volume, loading adsorption time 30~120 minutes.
5. the preparation method of Cordyceps militaris extract according to claim 1, which is characterized in that the first elution action includes:
Before carrying out alcohol precipitation to first eluent, first eluent is concentrated；
Preferably, the method that first eluent is concentrated includes: vacuum distillation；
It is highly preferred that distillation under pressure condition includes: 45~65 DEG C of 0.06~0.09MPa of vacuum degree, temperature；
It is further preferred that the first object substance includes Polysaccharides in Cultured Cordyceps militaris or the first object substance is trehalose.
6. the preparation method of Cordyceps militaris extract according to claim 5, which is characterized in that first eluent into
Row alcohol precipitation generate in the form of sediment be precipitated and through be separated by solid-liquid separation obtain the first object substance the step of in, alcohol precipitation
Condition include: use dosage be concentration after the first eluent 2~4 times of volume of ethyl alcohol as precipitating reagent, sedimentation time 24
~48 hours；
Preferably, the method for separation of solid and liquid includes centrifugation；
The first object substance is obtained by being dried it is highly preferred that being separated by solid-liquid separation；
It is further preferred that described be dried includes freeze-drying.
7. the preparation method of Cordyceps militaris extract according to claim 1, which is characterized in that the second elution action step
In rapid, the eluant, eluent that alcohol washes use includes methanol aqueous solution；
Preferably, the volumetric concentration of methanol is 60% in methanol aqueous solution, and elution requirement includes: that the dosage of methanol aqueous solution is micro-
2~7 times of hole resin volume, 1~2BV/h of flow velocity.
8. the preparation method of Cordyceps militaris extract according to claim 1, which is characterized in that by way of liquid chromatogram
Before executing lock out operation to second eluent, second eluent is concentrated；
Preferably, in first elution step, pass through remaining liquid portion quilt after separation of solid and liquid acquisition first object object
It is merged into second eluent, and is carried out before second eluent is concentrated.
9. the preparation method of Cordyceps militaris extract according to claim 1, which is characterized in that by way of liquid chromatogram
To second eluent execute lock out operation condition include:
Mobile phase includes acetonitrile solution, and chromatographic column is reverse-phase chromatographic column, preferably C8 reverse-phase chromatographic column；
It is highly preferred that the middle acetonitrile volume content of acetonitrile solution is 2~5%, and 30~100mL/min of flow velocity, Detection wavelength
254nm；
It is further preferred that second target substance includes cordycepin, adenosine, cordycepic acid.
10. a kind of functional food, which is characterized in that the functional food includes according to claim 1~any one of 9
The preparation method of the Cordyceps militaris extract obtains the first object substance and/or second target substance.
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