AU2020102126A4 - A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof - Google Patents
A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof Download PDFInfo
- Publication number
- AU2020102126A4 AU2020102126A4 AU2020102126A AU2020102126A AU2020102126A4 AU 2020102126 A4 AU2020102126 A4 AU 2020102126A4 AU 2020102126 A AU2020102126 A AU 2020102126A AU 2020102126 A AU2020102126 A AU 2020102126A AU 2020102126 A4 AU2020102126 A4 AU 2020102126A4
- Authority
- AU
- Australia
- Prior art keywords
- cordyceps militaris
- eluent
- extract
- elution
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 113
- 239000000284 extract Substances 0.000 title claims abstract description 62
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000005516 engineering process Methods 0.000 title claims abstract description 23
- 235000013376 functional food Nutrition 0.000 title claims abstract description 22
- 239000003480 eluent Substances 0.000 claims abstract description 83
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 61
- 238000010828 elution Methods 0.000 claims abstract description 56
- 238000000926 separation method Methods 0.000 claims abstract description 51
- 238000000605 extraction Methods 0.000 claims abstract description 43
- 239000011347 resin Substances 0.000 claims abstract description 37
- 229920005989 resin Polymers 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000000047 product Substances 0.000 claims abstract description 26
- 238000001556 precipitation Methods 0.000 claims abstract description 24
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 21
- 230000008569 process Effects 0.000 claims abstract description 13
- 239000006286 aqueous extract Substances 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 9
- 238000002386 leaching Methods 0.000 claims abstract description 8
- 239000013076 target substance Substances 0.000 claims description 73
- 239000007788 liquid Substances 0.000 claims description 71
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 71
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 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 28
- 239000007864 aqueous solution Substances 0.000 claims description 19
- 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 16
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 claims description 16
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 claims description 15
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 14
- 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 14
- 229960005305 adenosine Drugs 0.000 claims description 14
- 239000006185 dispersion Substances 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 12
- 230000002829 reductive effect Effects 0.000 claims description 11
- 238000011068 loading method Methods 0.000 claims description 10
- 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
- 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 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- 230000001376 precipitating effect Effects 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- 238000004821 distillation Methods 0.000 claims description 6
- 241000190633 Cordyceps Species 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 239000002207 metabolite Substances 0.000 abstract description 35
- 238000012545 processing Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 description 23
- 239000013543 active substance Substances 0.000 description 18
- 229930000044 secondary metabolite Natural products 0.000 description 18
- 150000004676 glycans Chemical class 0.000 description 17
- 229920001282 polysaccharide Polymers 0.000 description 17
- 239000005017 polysaccharide Substances 0.000 description 17
- 239000012071 phase Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- 238000000746 purification Methods 0.000 description 13
- 239000007787 solid Substances 0.000 description 12
- 239000002904 solvent Substances 0.000 description 10
- 238000003809 water extraction Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 239000012535 impurity Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000002994 raw material Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 5
- 239000012141 concentrate Substances 0.000 description 5
- 238000002425 crystallisation Methods 0.000 description 5
- 230000008025 crystallization Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000001953 recrystallisation Methods 0.000 description 4
- 238000009835 boiling Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 238000004262 preparative liquid chromatography Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011403 purification operation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000208341 Hedera Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001775 anti-pathogenic effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- -1 ethanol Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000956 solid--liquid extraction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
-
- 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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/12—Preparation by evaporation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A preparation technology of Cordyceps militaris extract and functional food thereof,
belonging to the field of agricultural product processing. The preparation technology
comprises providing an extract to be exacted, obtaining the extract from extracting of
Cordyceps militaris, which includes the process of obtaining aqueous extract by leaching
Cordyceps militaris. Using microporous resin to perform the first elution on obtained
aqueous extract, then perform the second elution in sequence, wherein, the process of first
elution comprises collecting the first eluent, on which performs alcohol precipitation to
generate the precipitate as the first target product, and wherein, the process of second elution
comprises obtaining the second elution with the second target product by alcohol extraction.
Executing liquid chromatography on the second elution for separation to obtain the second
target product. The above extraction technology can completely extract the metabolites from
Cordyceps militaris.
Description
PATENTS ACT 1990
A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof
The invention is described in the following statement:-
A Preparation Technology of Cordyceps militarisExtract and Functional Food thereof
The invention relates to the field of agricultural product processing, in particular to a preparation method of Cordyceps militaris extract and a functional food.
The invention relates to the field of agricultural product processing, in particular to a preparation method of Cordyceps militaris extract and a functional food. It is a medicinal and edible fungus with both nutritious and medicinal value.Modern pharmacological studies have prove that Cordyceps militaris possesses various sorts of pharmacological activities, such as antibacterial, anti-inflammatory, anti-tumor, anti-fatigue, anti-diabetes and immunity improvement, etc. Its medicinal and edible value is paralleled with Cordyceps sinensis, thus, nowadays, it is common to replace natural Cordyceps sinensis with artificially cultivated Cordyceps militaris.
Cordyceps militaris contains multiple sorts of bioactive components, such as adenosine, cordycepin, Cordycepic acid, polysaccharide, etc.
Adenosine has physiological effects on cardiovascular system and other systems and tissues of human body, it is also the index component to examine Cordyceps militaris according to the 2015 edition of Pharmacopoeia of the People's Republic of China.
Cordycepin is also known as 3 '- deoxyadenosine and other Chinese names. Researchers have launched a great deal of studies on Cordycepin in terms of its effect on anti-inflammation, immune adjustment, anti-tumor, hypoglycemic action and lipid- lowering, anti-pathogenic microorganism, anti-aging and neuro-protectio, and have attained a breakthrough from the studies.
Cordycepic acid can inhibit the growth of various germs, and prevent and treat cerebral thrombosis, cerebral hemorrhage, myocardial infarction and long-term failure of organs.
Therefore, it is imperative to extract the various substances from Cordyceps militaris with high efficiency. However, the prior art only emphasizes only on extraction rate of single component, there is little research done on simultaneous extraction of multiple component from Cordyceps militaris.
On the basis of defect of the prior art, the present invention provides an extracting technology of Cordyceps militaris extract and a functional food to partially or completely improve or even solve the issues described above.
The present invention is realized as follows
In a first aspect, an embodiment of the present invention provides a preparation technology of Cordyceps militaris extract.
Cordyceps militaris extract is aimed by the preparation technology is to obtain the first target product and the second target product,in other words, the technology is to extract target product from Cordyceps militaris.
The preparation technology comprises:
Providing an extract to be obtained by leaching Cordyceps militaris, wherein the leaching comprises extracting Cordyceps militariswith water to obtain an aqueous extract;
Using microporous resin to perform the first elution operation and a second elution operation afterwards, wherein, the first elution operation includes washing with water and collecting the first eluent, precipitating the first eluent with alcohol to generate a first target substance which is precipitated in the form of precipitate and obtained by solid liquid separation;Wherein the second elution operation comprises alcohol washing and collecting a second eluent with a second target substance;
Separating the second eluent by liquid chromatography to obtain a second target substance, and optionally crystallizing or recrystallizing the obtained target substance.
The metabolites in Cordyceps militaris were separated by water extraction, and then purified by combination of microporous resin and liquid chromatography. Macromolecules such as polysaccharideand protein in metabolites were separated from small components, which played a role in removing impurities from the secondary metabolites of Cordyceps militaris.Theapplication of this technology can effectively lock the target metabolite, and separating it directly, quickly and effectively, improving the separation efficiency, reducing the separation cost, and obtaining high-purity Cordyceps militaris metabolite.
In combination with the first aspect, in some optional examples of the first possible embodiment of the first aspect of the present invention, the method for extracting Cordyceps militaris with water to obtain an aqueous extract comprises:
A dispersion obtained by pulverizing Cordyceps militaris into particles and dispersing in water is heated.
Water extraction is an extraction and separation operation, or solid-liquid extraction, in which one or some components in solid raw materials are extracted by solvent.Among them, the solvent is selected as water.That is to say, water is used as solvent, and the medicinal material (Cordyceps militaris) is heated for a certain time to extract its components.Water extraction has the advantages of controllable solvent price and cost and environmental protection of raw materials.
By crushing Cordyceps militaris into particles, it is easier to soak in water, so that the target substances can easily enter into water, which is beneficial to improve the utilization rate of Cordyceps militaris, reduce the loss of target substances and improve the yield of target substances.
Combined with the first possible embodiment, in some optional examples of the second possible embodiment of the first aspect of the present invention, the material liquid ratio of the dispersion liquid is 1 kg/5-50 liters, and the temperature of the dispersion liquid reaches 50-85°C by heating.
Alternatively, each extraction time is 1-3 hours and the extraction times are 1-3 times.
As the leaching process involves solid-liquid mass transfer process, it mainly involves the mass transfer process of target substances from solid Cordyceps militaristo liquid water, and it includes wetting, permeation, desorption, dissolution and diffusion.In an example, temperature and the ratio of material to liquid can have considerable influence on the leaching of target substances to a relatively greater extent.
Combined with the first aspect, in some optional examples of the third possible embodiment of the first aspect of the present invention, in the process of performing the first elution operation and the second elution operation after the first elution operation with microporous resin, the loading amount of the aqueous extract is 3-6 times the volume of the microporous resin, and the loading adsorption time is 30-120 minutes.
Because Cordyceps militaris contains a variety of metabolites, solid impurities can be removed better (more thoroughly) by microporous resin.In addition, in order to eliminate other impurities, the target substance can be obtained by eluting step by step and collecting the corresponding eluent correspondingly.Furthermore, by controlling the loading amount and adsorption time, we can obtain better elution efficiency and avoid the loss of target substances.
In combination with the first aspect, in some alternative examples of the fourth possible embodiment of the first aspect of the present invention, the first elution operation includes: concentrating the first eluent before alcohol precipitation of the first eluent;
Optionally, the method for concentrating the first eluent comprises: distillation under reduced pressure;
Optionally, the pressure distillation conditions include: vacuum degree 0.06-0.09 MPa, temperature 45 ~ 65°C;
Optionally, the first target substance comprises Cordyceps militaris polysaccharide, and the Cordyceps militaris polysaccharide comprises trehalose.
After concentration, the concentration/content of the first target substance in the first eluent is increased, which is easier to precipitate by alcohol precipitation, and the loss and alcohol consumption can be reduced.
Combined with the fourth possible embodiment, in some optional examples of the fifth possible embodiment of the first aspect of the present invention, in the step of performing alcohol precipitation on the first eluent to generate the first target substance precipitated in the form of precipitate and obtained by solid-liquid separation, the alcohol precipitation conditions include: using ethanol with the amount of 2-4 times the volume of the concentrated first eluent as precipitant, and the precipitation time is 24-48 hours;
Optionally, the method of solid-liquid separation includes centrifugation;
Optionally, that first target substance obtain by solid-liquid separation is dried;
Optionally, the drying treatment includes freeze drying.
By alcohol precipitation treatment, the first target substance is precipitated and separated from the liquid and layered.In this way, the first target substance can be obtained by liquid separation.In order to obtain better yield and avoid loss, the precipitated first target substance was collected by centrifugation.
Considering the possibility of liquid in the first target substance being dried, and further freeze drying, the possible damage to the target substance caused by heat drying can be avoided.
Combined with the first aspect, in some optional examples of the sixth possible embodiment of the first aspect of the present invention, in the second elution operation step, the eluent used for alcohol washing includes methanol aqueous solution;
Optionally, the volume concentration of methanol in methanol aqueous solution is %, and the elution conditions include: the amount of methanol aqueous solution is 2-7 times of the volume of microporous resin, and the flow rate is 1-2 BV/h.
In the separation and purification operation with microporous resin, the target substance with higher purity can be obtained with higher efficiency and the loss can be reduced by controlling the above elution conditions.
Combined with the first aspect, in some optional examples of the seventh possible embodiment of the first aspect of the present invention, the second eluent is concentrated before the separation operation is performed on the second eluent by liquid chromatography;
Optionally, in the first elution step, the remaining liquid part after the first target is obtained by solid-liquid separation is combined into the second eluent, and is performed before the second eluent is concentrated.
Concentration treatment can reduce the solvent and increase the concentration of target substances, which is beneficial to subsequent separation and purification operations.At the same time, in view of the fact that there may be a certain amount of target substances in the supernatant produced by solid-liquid separation in thefirst elution step, it can be combined into the second eluent for subsequent liquid chromatography to improve the yield and reduce the loss.
Combined with the first aspect, in some optional examples of the eighth possible embodiment of the first aspect of the present invention, the conditions for performing the separation operation on the second eluent by liquid chromatography include:
The mobile phase includes acetonitrile aqueous solution, and the chromatographic column is reversed-phase chromatographic column, preferably C8 reversed-phase chromatographic column;
More preferably, the volume content of acetonitrile in the acetonitrile aqueous solution is 2-5%, the flow rate is 30-100 ml/min, and the detection wavelength is 254nm;
Optionally, the second target substance comprises cordycepin, adenosine and cordycepic acid.
By chromatographic separation under the above conditions, each target substance can be extracted with higher efficiency and purity.
In a second aspect, an embodiment of the present invention provides a functional food.
The functional food comprises the first target substance and/or the second target substance obtained by the preparation method of the Cordyceps militaris extract.
Beneficial effects are described as below:
According to the extraction method provided by the embodiment of the invention, more kinds of metabolites in Cordyceps militaris can be extracted together, and the extraction rate can be better improved.By using microporous resin and liquid chromatography, polysaccharides and proteins in Cordyceps militaris can be separated from other impurities more accurately and efficiently, and the purity and yield of separated products can be improved.Moreover, due to the high activity of Cordyceps militaris metabolites, it can be applied to related fields such as medical food, and can also be directly used as functional food of health care products and functional foods, which has broad market development prospects.
The embodiments of the present invention will be described in detail with examples below, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention.If the specific conditions are not indicated in the embodiment, the conventional conditions or the conditions suggested by the manufacturer shall be followed.The reagents or instruments used are conventional products that can be obtained through commercial purchase without indicating the manufacturer.
The preparation method and functional food of Cordyceps militaris extract according to the embodiment of the present invention are described in detail below.
There are many reports about Cordyceps militaris due to its potential applications in pharmacy and medicine.The key is to obtain various active substances in Cordyceps militaris.In related technologies, the extraction of various active substances from Cordyceps militaris is mainly obtained from its metabolites.
The inventor is convinced that at present, the extraction of active substances from Cordyceps militaris mainly focuses on obtaining a specific component.Due to the diversity of various active substances in Cordyceps militaris, it is expected to obtain a single component with high purity and high yield under more ideal conditions.However, based on the actual needs, we hope to extract the active substances from Cordyceps militaris in a better way.One of the main tasks and difficulties lies in the simultaneous extraction and separation of various active substances from Cordyceps militaris.
At present, some methods known by the inventors can not achieve the above tasks and expectations well.In view of this, an example of the present invention provides a method for extracting various active substances from Cordyceps militaris.
The extraction method operates on the metabolites of Cordyceps militaris.Furthermore, the extract includes a first target substance and a second target substance produced by Cordyceps militaris.In view of the complexity of the components/ingredients of the metabolites of Cordyceps militaris, it is usually possible to select appropriate conditions to control the desired substances as needed.For example, Cordyceps militaris polysaccharide, cordycepin, adenosine and cordycepic acid which will be mentioned later.Among them, Cordyceps militaris polysaccharide is the first target substance.Cordycepin, adenosine and cordycepic acid are the second target substances.
It should be understood that the aforementioned first target substance can be a single substance (such as trehalose) or a plurality of substances (such as trehalose and pentosan, which are usually substances with the same or similar properties (such as water solubility, oil solubility, polarity, etc.).The second target substance can be a single substance (such as cordycepin) or multiple substances (such as adenosine and cordycepic acid).In other words, both the first target substance and the second target substance may be single component substances or multi-component compositions.
The preparation method comprises:
S101: provide the extract to be extracted.
The extract to be extracted is a crude product and is obtained from Cordyceps militaris.The extract to be extracted comprises a first target product and a second target product.
In addition, the extract to be extracted can be prepared in advance or directly prepared on site.In other words, the extract to be extracted can be purchased directly as raw material or as prefabricated raw material.
Considering the stability of various active substances in Cordyceps militaris, as well as the difficulty and complexity of preservation, we can choose the use mode of the extract according to the actual production needs.
In an example, the extract to be extracted can be obtained by leaching Cordyceps militaries, Wherein, the extraction comprises water extraction of Cordyceps militaris to
obtain an aqueous extract.The method for extracting Cordyceps militaris with water to obtain an aqueous extract comprises: heating a dispersion obtained by pulverizing Cordyceps militaris into particles and dispersing the particles in water.
For the convenience of processing, Cordyceps militaris can be newly collected.Of course, dried Cordyceps militaris can also be collected.Furthermore, based on the need of subsequent treatment, good water extraction effect can be obtained by crushing Cordyceps militaris.The crushing method can be carried out by crushing machinery, such as grinding, cutting, etc. (based on the requirement of uniform particle size distribution, screening can be carried out, wherein the mesh number of the screen is 40-120 meshes).Or, in other examples, by low temperature assisted pulverization.That is, Cordyceps militaris is cooled to a frozen state by using a low-temperature medium, and then crushed in the frozen state.It should be noted that the temperature in the freezing process should not be too low (for example, within-100 degrees Celsius), otherwise, the structure of some active substances will be destroyed.
In addition, Cordyceps militaris includes insect body part (fruiting body) and grass body part (mycelium).However, the components and distribution of metabolites of Cordyceps militaris may be different, so it is possible to obtain better purity and yield of desired products by treating them separately.In addition, because they are extracted independently, the interference of more impurities can be reduced, which is beneficial to the acquisition of target substances.
Considering the possible adverse effects of impurities in water on separation and purification, water is used with higher purity.In some examples, deionized water, purified water, distilled water, reverse osmosis water and ultrapure water can be selected for water extraction.
Furthermore, proper extraction effect can be achieved by selecting the water consumption and temperature of water extraction.The material-liquid ratio of the dispersion liquid is lkg/5 ~ 501, and the temperature of the dispersion liquid reaches -85°C by heating.The ratio of material to liquid refers to the ratio between Cordyceps militaris particles and water.That is, 5-50 liters of water is used for 1 kg of Cordyceps militaris particles.
In other examples, the material-liquid ratio of the dispersion liquid can also be lkg/12L, lkg/16L, lkg/21L, lkg/25L, lkg/30L, lkg/37L or 1kg/46L.Similarly, the temperature of the dispersion may be 53°C, 56°C, 62°C, 67°C, 72°C, 79°C, or 83°C.The material-liquid ratio and temperature of the dispersion liquid can be freely selected and matched, which is not specifically limited by the present invention.For example, the dispersion liquid may have a material-liquid ratio of 1 kg /21 liter and a temperature of 82°C;Or, the ratio of material to liquid is 1kg/101 and the temperature is 51°C.The dispersion liquid can also have a material-liquid ratio of 1 kg /48 L and a temperature of °C;Or, the ratio of material to liquid is lkg/49L and the temperature is 50°C.In some studies, ultrasonic-assisted extraction is attempted, but ultrasonic extraction often requires specific ultrasonic frequency and intensity (such as power), and can only be used for relatively single component substances.When multi-components need to be extracted, the specific selection and control of ultrasonic wave is a problem to be considered.
Generally, the increase of temperature can be beneficial to improve the solubility of the target substance in water.However, too high a temperature may also lead to irreversible and undesirable transformation of the target substance.Similarly, by adjusting the ratio of material to liquid (such as increasing the ratio of water), the dispersion can dissolve more target substances.When the water ratio is too high, it may bring disadvantages to the subsequent purification steps.E.g., water, can be easily removed under mild conditions that are relatively simple and easy to implement.
Optionally, in other examples of the present invention, the extraction time may be controlled to balance the extraction efficiency and the thoroughness of the royalty.For example, each extraction time is 1-3 hours, and the extraction times are 1-3 times.
After the above water extraction treatment, the desired target substances (the first target substance and the second target substance) are relatively easily and thoroughly dispersed in the water extract.
S102, separating and purifying the water extract with microporous resin.
The method for separating and purifying the water extract by using microporous resin (MCI) includes a first elution operation and a second elution operation after the first elution operation.Essentially, the first elution operation and the second elution operation are carried out successively, and the second elution operation is carried out after the first elution operation is completed.In other words, after the microporous resin absorbs the water extract, it is packed in a column and then eluted.After the first elution operation, the second elution operation is carried out.The following types of microporous resins can be selected: NS4205, DAC-HB50, DAC-HB80, DAC-HB100, DAC-HB150 and DAC-HB200.
As the adsorption degree of water extract and microporous resin has influence on the efficiency and yield of subsequent elution separation, the loading method can be selected.For example, the loading amount of water extract is 3-6 times (4 times, 5 times, etc.) of the volume of microporous resin, and the loading adsorption time is 30-120 minutes (36 minutes, 42 minutes, 51 minutes, 67 minutes, 79 minutes, 88 minutes, etc.).When loading samples, the microporous resin is mixed with the water extract, and the water extract will be adsorbed by the microporous resin.
After the loading is completed, elution treatment is performed to separate the desired substances for collection and optionally subsequent purification treatment (such as liquid chromatography treatment mentioned later).In an example, the elution treatment includes a first elution operation and a second elution operation which will be mentioned in the following section.
The first elution operation in the separation and purification of the water extract with microporous resin includes washing with water and collecting the first eluent, precipitating the first eluent with alcohol to generate the first target substance which is precipitated in the form of precipitate and obtained by solid-liquid separation.
In this first elution step, water is used as eluent (mobile phase).With water as eluent, a part of substances can be obtained by dissolving in the eluent, that is, the first eluent.The first eluent comprises water and a first target substance or other small amount of impurities which can exist.Generally, the first eluent can be directly subjected to subsequent alcohol precipitation treatment.However, in order to improve the efficiency/yield/yield of alcohol precipitation, the first eluent can be concentrated to an appropriate degree before alcohol precipitation.
There are many ways to concentrate the first eluent. In the example of the present invention, the concentration way is reduced pressure distillation.Vacuum distillation can reduce the boiling point of the liquid, so that the liquid can be evaporated and removed by heating at a relatively lower temperature, without damaging the structure of active substances at high temperature.In the example, the pressure distillation conditions include: vacuum degree 0.06-0.09 MPa, temperature 45-65°C.In other alternative examples, the vacuum degree may be 0.07-0.08 MPa and the temperature may be -61 0C.
After concentration, the liquid (water) content in the first eluent is reduced, which can promote the precipitation of the target substance (first target substance, such as Cordyceps militaris polysaccharide, more specifically trehalose).Subsequently, the first target substance can be precipitated in a larger amount by alcohol precipitation.Because the solubility of active substances in different Cordyceps militaris is different in water and alcohol, and the compatibility between water and alcohol (such as ethanol) is relatively good, when alcohol is added to the first eluent, the solubility difference between different substances leads to the precipitation of some active substances, while the other active substances can continue to exist in the water layer
As an alternative example, the alcohol (ethanol, 95% alcohol) used in the alcohol precipitation operation after concentration is 2-4 times or 3 times the volume of the first eluent after concentration.Correspondingly, the alcohol precipitation time can be 24-48 hours.As mentioned above, after alcohol precipitation, the first eluent is approximately layered in appearance.In this way, the separation of active substances can be realized by collecting the water layer and the precipitation layer respectively.Among them, the liquid layer serves as supernatant (or clear liquid), and there may be a small amount of second target substance in it.The precipitation layer serves as a solid phase and has most of the first target substance therein.
After layering, the precipitation layer and the liquid layer can be separated by means such as a separatory funnel.Further, in the example, centrifugation is selected for solid liquid separation.Centrifugal operation can have a high solid-liquid separation effect, which can better reduce the content of solid matter in the liquid, and at the same time better reduce the content of liquid matter in the solid.
Furthermore, because the first target substance mainly exists in the precipitation layer (solid phase), and there may be different amounts of liquid in it, in order to obtain a more pure first target substance, it can be dried to remove water and alcohol (such as ethanol) that may exist.In the example, freeze drying is selected to remove the liquid.The solid phase can be dried or dehydrated (in which possibly mixed alcohols, such as ethanol, can also be removed at the same time).Optionally, freeze-drying conditions include vacuum degree of 0.06-0.09 MPa and temperature of 45-65°C.Freeze-drying realizes the removal of liquid by heating the substance to be dried at a pressure lower than natural conditions.By depressurizing, the boiling point of the liquid is lowered (it is easier to be heated and gasified), so that it can be removed by heating at a relatively lower temperature.In this way, the high temperature required by evaporation of high boiling point liquid by heating can be avoided, and thus the damage of active substances can be avoided.
While obtaining the purer first target substance by the above treatment, the second elution operation can also be performed.Wherein, the second elution operation in the separation and purification of the water extract by using microporous resin comprises alcohol washing and collecting the second eluent with the second target substance.In this step, the treatment mode of alcohol washing may be to wash the adsorbed water extract (and most of the first target substance is separated by the first elution operation) with an eluent including methanol aqueous solution (the optional methanol aqueous solution is methanol with a volume concentration of 60%).In an alternative scheme, the elution conditions include: the amount of methanol aqueous solution is 2-7 times (or 3 times, 4 times, 5 times, etc.) of the volume of microporous resin, and the flow rate is 1-2 BV/h (or 1.1 BV/h, 1.3 BV/h, 1.7 BV/h, 1.9 BV/h).
Through the elution treatment of methanol aqueous solution, the second target substance (which can be a combination of multiple single-component substances) enters the second eluent.Obviously, the second eluent includes water, methanol and the second target substance, so in order to obtain the pure second target substance, separation treatment can be carried out.As mentioned above, the efficiency and effect of subsequent separation treatment can be improved by concentration.Therefore, optionally, the second eluent is concentrated before the separation operation is performed on the second eluent.
In addition, in the first elution operation, a solid first target substance (obtained by solid treatment in solid-liquid separation) is obtained, but a part of the second target substance may exist in the liquid part in the process, so it is reused to avoid waste.In an example, in the first elution step, the remaining liquid part after the first target is obtained by solid-liquid separation is merged into the second eluent, and it is carried out before concentrating the second eluent (of course, it can also be carried out before concentrating the second eluent, but it may need to increase the re-concentration treatment after merging).
According to the analysis of metabolites of Cordyceps militaris, the second eluent is mainly the second target substance, and mainly includes cordycepin, adenosine and cordycepic acid.Obviously, because the properties and functions of various components in the second target substance are quite different, and they are usually used as single components in time, so they can be extracted separately.Of course, in order to obtain relatively pure and single components, re-separation and purification can also be carried out.
S103: separation and purification of the second eluent.
Therefore, in order to obtain each single component of the second target substance (when it is a plurality of components), it is necessary to carry out appropriate separation treatment according to its components.In the example, the second eluent containing multi-component second target substances is subjected to component separation operation by means of liquid chromatography.
Considering that the excessive content of solvent (eluent) in the second eluent will lead to the low concentration of the second target substance, which is not conducive to the subsequent liquid chromatography treatment, it is possible to choose to concentrate first.That is, before separating the second eluent by liquid chromatography, the second eluent is concentrated.
After concentration, the concentration of the second target substance in the second eluent (or concentrated solution) increases, and the solvent (eluent in the second elution operation) decreases.Conditions for separating the second eluent by liquid chromatography include that the mobile phase includes acetonitrile aqueous solution, and the chromatographic column is reversed-phase chromatographic column (such as C8 reversed-phase chromatographic column).C18 reversed-phase chromatographic columns (specific models such as dubhe C18, hedera C18, megres C18, Benetnach C18, phecda C18, Xmide C18, XCharge C18, ODS C18, etc.).The operating conditions of liquid chromatography can be as follows: the flow rate of mobile phase is 30-100 mL/min, the detection wavelength is 254 nm, the mobile phase is acetonitrile aqueous solution, and the volume content of acetonitrile is 95~98%.
Through liquid chromatography, the active substances can be clearly and definitely distinguished, so as to obtain various substances with high purity and relatively single components (high content of target substances to be extracted).Material obtained in liquid chromatography are mixture of different liquids.The liquid mixture comprises a mobile phase and corresponding one or more substances dissolved in the mobile phase.
Among them, after liquid chromatography treatment, the second eluent (such as the above-mentioned multiple liquid mixtures) can obtain various high-purity single- component substances (such as cordycepin, adenosine and cordycepic acid) as the second target product.Obviously, all kinds of single-component substances obtained by liquid chromatography are also obtained in liquid form (that is, any single component is dissolved in the mobile phase of liquid chromatography).For a particular substance to be obtained from a liquid.Optionally, the desired substance is separated from the liquid.Optionally, methods for separating components and liquids include crystallization and optional recrystallization or evaporation, etc.Crystallization can be realized by choosing temperature, because the solubility of different substances in a certain liquid (which can be a mixture) is related to temperature.Therefore, higher purity substances can be obtained by dissolving and crystallizing the substances in the third liquid part at different temperatures.
The active substances mentioned above mainly refer to various biomedical active substances in Cordyceps militaris, such as adenosine, cordycepin, cordycepic acid, polysaccharide, etc.
Experiments show that the extraction method provided above, the separation and purification of microporous resin combined with high performance liquid chromatography, and the water extraction and alcohol precipitation method can separate macromolecular and small components such as polysaccharide and protein in the extraction solution, which plays a role in removing impurities from the secondary metabolites of Cordyceps militaris.The application of this method can effectively lock the target metabolite, separate it directly, quickly and effectively, improve the separation efficiency, reduce the separation cost, and obtain the pure secondary metabolite of Cordyceps militaris with high purity.The secondary metabolite of Cordyceps militaris has high activity, which can be used in related fields such as medicine and food, and can also be directly used as an additive of health care products and functional foods, thus having broad market development prospects.
In some alternative specific examples, the preparation method has the following advantages:
1. The effective metabolites in Cordyceps militaris were extracted by water extraction, which kept the biological activity and extraction rate of effective metabolites in Cordyceps militaris to the maximum extent, and the total extraction rate reached over
2. The polysaccharide and protein components in the extract can be separated by microporous resin separation and purification, and the water-eluting part is recovered at the same time, which improves the extraction rate of secondary metabolites.After separation and purification, the yield of Cordyceps militaris polysaccharide is 46.6%, the content can reach 50%, the yield of secondary metabolites is 14.8%, and the contents of cordycepin, adenosine, cordycepic acid and trehalose can all reach above 98%.
3. The target metabolite is accurately locked by HPLC. Compared with the traditional separation technology, it can greatly reduce the separation difficulty, improve the separation efficiency, reduce the separation cost and save the separation time.
4. The invention has simple process, good repeatability and stable and controllable quality.
5. Metabolites of Cordyceps militaris can be used in related fields such as medicine, scientific research and food, and can also be directly used as additives of health care products and functional foods, which has broad market development prospects.
As far as the inventors know, the metabolites of Cordyceps militaris have many pharmacological activities, and have potential and great medicinal and edible values.In view of this, the existing Cordyceps militaris extract is used by direct administration. In an example of the present invention, a functional food based on the extract extracted from Cordyceps militaris mainly includes metabolites is provided.The ingredients of such functional foods can be adjusted as required.In other words, the functional food includes the first target substance.Or, the functional food includes a second target substance.Or, the functional food includes a first target substance and a second target substance.
Functional food can be prepared and used as powder or granule.
The preparation method and functional food of Cordyceps militaris extract of the present invention will be further described in detail with examples.
Example 1
(1) Extraction of metabolites from Cordyceps militaris: 2kg of Cordyceps militaris raw material is crushed, sieved with 40 mesh sieve, then deionized water is added for extraction, and then the extract is filtered to prepare Cordyceps militaris metabolite extract;The extraction conditions are as follows: the ratio of solid to liquid is 1kg: 51, the temperature is 50°C, the extraction time is 1, and the extraction times are 1.
(2) Separation of metabolites from Cordyceps militaris:
The extract of cordyceps militaris metabolite is put on MCI microporous resin column, and the sample volume is 3 times the volume of resin column;Adsorbing for 30 minutes, eluting with deionized water, and collecting eluate.Concentrate the eluent under reduced pressure at 60°C with a rotary evaporator to obtain water extract concentrated solution, add 3 times of 95% ethanol to the concentrated part of the eluent, precipitate for 24h, centrifuge and collect supernatant.Freeze-drying the centrifuged part to obtain Cordyceps militaris polysaccharide extract.
Eluting the resin column with 60% methanol aqueous solution as eluent, wherein the eluent is twice the volume of the resin column and the flow rate is1BV/h, and collecting the eluent.The eluent is combined with the supernatant after washing with water and precipitating with alcohol, and concentrated to obtain the concentrated solution of secondary metabolites.The vacuum drying conditions are 0.06MPa and 45°C.
(3) Purification of metabolites from Cordyceps militaris:
Put the concentrated solution of secondary metabolites on preparative liquid chromatography (DAC-HB50), collect fractions of secondary metabolites, and crystallize and recrystallize.Chromatographic conditions for preparing hue were acetonitrile: water (2%: 98%), flow rate 30mL/min, detection wavelength 254nm, and C18 reversed-phase chromatographic column (megres C18).Solvents for crystallization and recrystallization are ethanol and water.
The yield of Cordyceps militaris polysaccharide is 46.6%, the content is 50.40%, the yield of secondary metabolites is 14.80%, and the contents of cordycepin, adenosine, cordycepic acid and trehalose can all reach above 98%.
Example 2
(1) extraction of metabolites from cordyceps militaris: 4kg of cordyceps militaris raw material is crushed, sieved with 120 meshes, then deionized water is added for extraction, and then the extraction solution is filtered to prepare an extraction solution of cordyceps militaris metabolites;The extraction conditions were as follows: the ratio of solid to liquid was 1kg : 50L, the temperature was 85°C, the extraction time was 3h, and
the extraction times were three times.
(2) Separation of metabolites from Cordyceps militaris:
The extract of cordyceps militaris metabolite is put on MCI microporous resin column, and the sample volume is 6 times the volume of resin column;Adsorb for 30 120min, eluting with deionize water, and collecting eluate.Concentrate the eluent under reduced pressure at 60°C with a rotary evaporator to obtain water extract concentrated solution, add 3 times of 95% ethanol to the concentrated part of the eluent, precipitate for 48 hours, centrifuge and collect supematant.Freeze-drying the centrifuged part to obtain Cordyceps militaris polysaccharide extract.
Eluting the resin column with 60% methanol aqueous solution as eluent, wherein the eluent is 7 times the volume of the resin column and the flow rate is 2BV/h, and collecting the eluent.The eluent is combined with the supernatant after washing with water and precipitating with alcohol, and concentrated to obtain the concentrated solution of secondary metabolites.The drying conditions under reduced pressure refer to the vacuum degree of 0.09MPa and the temperature of 45-65°C.
(3) Purification of metabolites from Cordyceps militaris:
Put the concentrated solution of secondary metabolites on preparative liquid chromatography (DAC-HB80), collect fractions of secondary metabolites, and crystallize and recrystallize.Chromatographic conditions for preparing hue were acetonitrile: water (5%: 95%), flow rate 100mL/min, detection wavelength 254nm, and C18 reversed-phase chromatographic column (dubhe C18).Solvents for crystallization and recrystallization are ethanol, methanol and water.
The yield of polysaccharide from Cordyceps militaris is 45.9%, the content is 51.82%, the yield of secondary metabolites is 15.39%, and the contents of cordycepin, adenosine, cordycepic acid and trehalose can all reach above 98%.
Example 3
(1) extraction of metabolites from cordyceps militaris: 10kg of cordyceps militaris raw material is crushed, sieved with 60 meshes, then deionized water is added for extraction, and then the extracting solution is filtered to prepare an extracting solution of cordyceps militaris metabolites;The extraction conditions are as follows: the ratio of solid to liquid is 1kg : 20L, the temperature is 75°C, the extraction time is 2h, and the
extraction times are twice.
(2) Separation of metabolites from Cordyceps militaris:
The extract of cordyceps militaris metabolite is put on MCI microporous resin column, and the sample volume is 5 times the volume of resin column;Adsorbing for 60 minutes, eluting with deionized water, and collecting eluate.Concentrate the eluent under reduced pressure at 60°C with a rotary evaporator to obtain water extract concentrated solution, add 3 times of 95% ethanol to the concentrated part of the eluent, precipitate for 36h, centrifuge, and collect supernatant.Freeze-drying the centrifuged part to obtain Cordyceps militaris polysaccharide extract.
Eluting the resin column with 60% methanol aqueous solution as eluent, wherein the eluent is 5 times the volume of the resin column and the flow rate is 2BV/h, and collecting the eluent.The eluent is combined with the supernatant after washing with water and precipitating with alcohol, and concentrated to obtain the concentrated solution of secondary metabolites.The vacuum drying conditions are 0.09MPa and 55°C.
(3) Purification of metabolites from Cordyceps militaris:
Put the concentrated solution of secondary metabolites on preparative liquid chromatography (DAC-HB80), collect fractions of secondary metabolites, and crystallize and recrystallize.Chromatographic conditions for preparing hue were acetonitrile: water (3%: 97%), flow rate 1OOmL/min, detection wavelength 254nm, and C18 reversed-phase chromatographic column (dubhe C18).Solvents for crystallization and recrystallization are ethanol, methanol and water.
The yield of Cordyceps militaris polysaccharide is 47.34%, the content is 50.67%, the yield of secondary metabolites is 17.22%, and the contents of cordycepin, adenosine, cordycepic acid and trehalose can all reach above 98%.
Although the invention has been illustrated and described with specific embodiments, it should be appreciated that many other changes and modifications can be made without departing from the spirit and scope of the invention.Therefore, it is meant that all such changes and modifications as fall within the scope of the present invention are included in the appended claims.
Claims (10)
1. A preparation technology of obtaining the first target product and the second target product by extracting Cordyceps militaris is characterized in comprising the following process:
Providing an extract to be extracted, wherein the extract to be extracted is obtained by leaching Cordyceps militaris, and the leaching comprises extracting Cordyceps militaris with water to obtain an aqueous extract;
Performing the first elution operation and the second elution in sequence on the water extract by adopting microporous resin;The first elution operation comprises collecting the first eluent by washing the extract with water, and executing alcohol precipitation of the first eluent to separate liquid and precipitate to obtain the first target product, wherein, the process of second elution comprises washing the extract with alcohol and collecting the second elution with the second target product.
Separating the second eluent by liquid chromatography to obtain a second target substance, and optionally crystallizing or recrystallizing the obtained target substance.
2. According to the preparation technology of Cordyceps militaris extract in Claim 1, wherein the method for extracting Cordyceps militaris with water to obtain an aqueous extract comprises:Heating the dispersion obtained by pulverizing Cordyceps militaris into particles and dispersing in water.
3. The preparation technology of Cordyceps militaris extract according to Claim 1 is characterized in the feed-liquid ratio is 1 kg/5-50 liters, and the temperature of the dispersion liquid is heated to 50-85°C; Preferably, each extraction time is 1-3 hours, and the extraction times are 1-3 times.
4. The preparation technology of Cordyceps militaris extract according to Claim 1 is characterized in that during the first elution operation and the second elution operation after, the loading amount of the water extract is 3-6 times the volume of the microporous resin, and the loading adsorption time is 30-120 minutes.
5. The preparation technology of Cordyceps militaris extract according to Claim 1 is characterized in that the first elution process comprises: Concentrating the first eluent before performing alcohol precipitation on the first eluent; Preferably, the method for concentrating the first eluent comprises: distillation under reduced pressure; More preferably, the pressure distillation conditions include: vacuum degree 0.06-0.09 MPa, temperature 45 ~ 65°C; Further preferably, the first target product comprises Cordyceps militarispolysaccharide,whichcomprises trehalose.
6. The preparation technology of Cordyceps militaris extract according to Claim 5 is characterized in obtaining the first target product by performing solid-liquid separation on the precipitate generated from alcohol precipitating the first eluent, the conditions of alcohol precipitation include: using ethanol which is 2-4 times the volume of the concentrated first eluent as precipitant, and precipitating for 24-48 hours;
Preferably, the solid-liquid separation method comprises centrifugation;
More preferably, the first target substance obtained by solid-liquid separation is dried;
Further preferably, the drying treatment comprises freeze drying.
7. The preparation technology of Cordyceps militaris extract according to Claim 1 is characterized in that in the second elution operation step, the eluent used for alcohol washing comprises methanol aqueous solution;
Preferably, the volume concentration of methanol in methanol aqueous solution is %, and the elution conditions are: the amount of methanol aqueous solution is 2-7 times of the volume of microporous resin, and the flow rate is 1-2 BV/h.
8. The preparation technology of Cordyceps militaris extract according to Claim 1 is characterized in concentrating the second elution before using liquid chromatography to perform separation on it.
Preferably, in the first elution step, before concentrating the second eluent, the remaining liquid part after obtaining the first target by solid-liquid separation is combined into the second eluent.
9. The preparation technology of Cordyceps militaris extract according to Claim 1 is characterized in that the conditions for separating the second eluent by liquid chromatography include:
The mobile phase includes acetonitrile aqueous solution, and the chromatographic column is reversed-phase chromatographic column, preferably C8 reversed-phase chromatographic column;
More preferably, the volume content of acetonitrile in the acetonitrile aqueous solution is 2-5%, the flow rate is 30-100 ml/min, and the detection wavelength is 254nm;
Further preferably, the second target product comprises cordycepin, adenosine and cordycepic acid.
10. A functional food is characterized in that it is made from the first and/or the second target product described obtained through the technology described in any claims from the number 1 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020102126A AU2020102126A4 (en) | 2020-09-03 | 2020-09-03 | A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020102126A AU2020102126A4 (en) | 2020-09-03 | 2020-09-03 | A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020102126A4 true AU2020102126A4 (en) | 2020-10-15 |
Family
ID=72750359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020102126A Ceased AU2020102126A4 (en) | 2020-09-03 | 2020-09-03 | A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2020102126A4 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113155999A (en) * | 2021-03-30 | 2021-07-23 | 浙江工业大学 | Method for detecting cordycepin content in cordyceps militaris |
| CN113893385A (en) * | 2021-10-09 | 2022-01-07 | 南京师范大学 | Composition for preparing hydrogel, hydrogel and preparation method and application thereof |
| CN114456278A (en) * | 2022-03-14 | 2022-05-10 | 泸州品创科技有限公司 | Method for continuously extracting cordyceps militaris intracellular polysaccharide |
-
2020
- 2020-09-03 AU AU2020102126A patent/AU2020102126A4/en not_active Ceased
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113155999A (en) * | 2021-03-30 | 2021-07-23 | 浙江工业大学 | Method for detecting cordycepin content in cordyceps militaris |
| CN113893385A (en) * | 2021-10-09 | 2022-01-07 | 南京师范大学 | Composition for preparing hydrogel, hydrogel and preparation method and application thereof |
| CN113893385B (en) * | 2021-10-09 | 2023-05-12 | 南京师范大学 | Composition for preparing hydrogel, preparation method and application thereof |
| CN114456278A (en) * | 2022-03-14 | 2022-05-10 | 泸州品创科技有限公司 | Method for continuously extracting cordyceps militaris intracellular polysaccharide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020102126A4 (en) | A Preparation Technology of Cordyceps militaris Extract and Functional Food thereof | |
| CN109810201A (en) | The ultrasonic wave combination of acidic water extracting method of Cordyceps sinensis polysaccharide and cordycepin in a kind of Cordyceps militaris | |
| CN108752231B (en) | Method for extracting theanine from sweet tea and simultaneously extracting rubusoside and tea polyphenol | |
| CN102675398B (en) | A kind of method extracting momordica grosvenori glycoside V and farnesol from Grosvenor Momordica | |
| CN105713061A (en) | Preparation method of fructus momordicae extract with mogroside V content larger than or equal to 95% | |
| CN108911985B (en) | Method for separating and purifying ethyl p-hydroxycinnamate from camellia pollen and application of ethyl p-hydroxycinnamate | |
| CN104098713B (en) | A kind of method simultaneously preparing garlic polysaccharide and alliin | |
| CN110981921B (en) | Continuous method for synchronously extracting multiple effective components from figs | |
| CN111039914A (en) | Extraction and separation method of cannabinol | |
| CN104744601B (en) | Method for extracting and purifying fleurotus ferulae polysaccharide | |
| CN109021046B (en) | Method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori | |
| CN110878010A (en) | Extraction and separation method of cannabigerol | |
| WO2022052394A1 (en) | Method for preparing delphinidin acylated anthocyanin | |
| CN112266399B (en) | High-purity separation and extraction method of epimedium extract | |
| CN111303236B (en) | Method for simultaneously extracting and separating maslinic acid, oleuropein and oleanolic acid from olive leaves | |
| CN105732741A (en) | Method for extracting anthocyanin and ursolic acid from perilla leaves | |
| CN105440095A (en) | Beta-ecdysterone-rich Cyanotis arachnoids extraction and purification method | |
| CN107988280B (en) | Method for extracting high-purity isothiocyanate from cruciferous vegetable seeds | |
| CN110917240A (en) | Continuous method for separating multiple effective components from cyclocarya paliurus | |
| AU2020102054A4 (en) | An extracting technology and an additives of metabolite | |
| CN113913029B (en) | Method for preparing effective components of gardenia jasminoides ellis | |
| CN109929002A (en) | Preparation method of salidroside | |
| CN114098089B (en) | Composite peptide composition and composite peptide seasoning tea | |
| JP3806693B2 (en) | Method for recovering pinitol from soybean processing by-products in high yield | |
| CN115010618A (en) | Separation and purification method of aureoyl amide alcohol ester capable of reducing uric acid and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |