CN102936271A - Method for extracting cordycepin - Google Patents
Method for extracting cordycepin Download PDFInfo
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- CN102936271A CN102936271A CN2012104588210A CN201210458821A CN102936271A CN 102936271 A CN102936271 A CN 102936271A CN 2012104588210 A CN2012104588210 A CN 2012104588210A CN 201210458821 A CN201210458821 A CN 201210458821A CN 102936271 A CN102936271 A CN 102936271A
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- 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 title claims abstract description 127
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 title claims abstract description 127
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 238000000034 method Methods 0.000 title claims abstract description 66
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 238000000605 extraction Methods 0.000 claims abstract description 37
- 238000000746 purification Methods 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 9
- 238000000926 separation method Methods 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 82
- 239000006228 supernatant Substances 0.000 claims description 41
- 229960004756 ethanol Drugs 0.000 claims description 31
- 239000007791 liquid phase Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000011347 resin Substances 0.000 claims description 18
- 229920005989 resin Polymers 0.000 claims description 18
- 239000012535 impurity Substances 0.000 claims description 15
- 230000005855 radiation Effects 0.000 claims description 14
- 238000001556 precipitation Methods 0.000 claims description 13
- 238000010521 absorption reaction Methods 0.000 claims description 11
- 239000012071 phase Substances 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 238000011068 loading method Methods 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 230000000274 adsorptive effect Effects 0.000 claims description 7
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 7
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 6
- 239000012467 final product Substances 0.000 claims description 6
- 230000035614 depigmentation Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 235000020247 cow milk Nutrition 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 235000015099 wheat brans Nutrition 0.000 claims description 3
- 241001248610 Ophiocordyceps sinensis Species 0.000 claims description 2
- 150000001298 alcohols Chemical class 0.000 claims description 2
- 239000003610 charcoal Substances 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 abstract description 9
- 239000000243 solution Substances 0.000 description 27
- 238000004128 high performance liquid chromatography Methods 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 10
- 239000000284 extract Substances 0.000 description 10
- 241001264174 Cordyceps militaris Species 0.000 description 9
- 241000190633 Cordyceps Species 0.000 description 8
- 239000012530 fluid Substances 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 4
- 240000001307 Myosotis scorpioides Species 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000008176 lyophilized powder Substances 0.000 description 3
- 229930014626 natural product Natural products 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 238000007670 refining Methods 0.000 description 3
- 238000005057 refrigeration Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000004308 accommodation Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
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- 238000005342 ion exchange Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 238000005507 spraying Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- CKZWLFLXOQDVEX-UHFFFAOYSA-N [2-(6-aminopurin-9-yl)-5-[[[2-(6-aminopurin-9-yl)-5-(hydroxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]oxolan-3-yl] [5-(6-aminopurin-9-yl)-4-hydroxyoxolan-2-yl]methyl hydrogen phosphate Chemical compound C1=NC2=C(N)N=CN=C2N1C1OC(COP(O)(=O)OC2C(OC(CO)C2)N2C3=NC=NC(N)=C3N=C2)CC1OP(O)(=O)OCC(CC1O)OC1N1C(N=CN=C2N)=C2N=C1 CKZWLFLXOQDVEX-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 102000011759 adducin Human genes 0.000 description 1
- 108010076723 adducin Proteins 0.000 description 1
- 239000002487 adenosine deaminase inhibitor Substances 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003430 antimalarial agent Substances 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
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Abstract
The invention relates to a method for extracting cordycepin. The method includes the following steps of subjecting a cordycepin fermentation liquid to reduced pressure concentration, then performing ultrasonic extraction in combinations of microwaves and light waves respectively, and further subjecting an obtained extraction liquid to separation and purification to obtain the cordycepin. Compared with traditional extraction methods, the method has the advantages that by means of the method, the extraction ratio is improved, extraction time is saved, and purity is increased.
Description
Technical field
The present invention relates to the extracting method of effective constituent, be specifically related to the extracting method of cordycepin.
Background technology
Cordycepin (cordycepin) claims again 3'-Deoxyadenosine, cordycepin, cordycepin, Chinese caterpillar fungus filament, Cordycepin, Cordycepin, Cordycepin.
Cordycepin is (especially ucleosides) main active ingredient in the Cordyceps militaris, also is first nucleoside antibiotics of separating from fungi.
Cordycepin (Cordycepin) is the analogue of adenosine, molecular formula: C
10H
13N
5O
3, molecular weight: 251.24, alkalescence, needle-like or plate crystal, 230 ℃-231 ℃ of fusing points, maximum absorption wavelength is 259.0nm.
Nineteen fifty-one, the Cunningham of Germany etc. separate the discovery cordycepin from the culturing filtrate of Cordyceps militaris.
The eighties of last century sixties rise, the synthetic and extraction of cordycepin, and at home and abroad scientific and technological circle just attract wide attention and pay attention to.Although once there was the report of synthetic cordycepin in China in 1964, do not seen the report that continues further investigation.
Just be found the inhibition tumour at 20 century 70 cordycepins, the effect of antimalarial protozoon and inhibition mRNA translation; The nineties research invention, the interpolation adenosine deaminase inhibitors plays an important role to the expression of its anti-tumor activity, thus the research of cordycepin obtains breakthrough; The U.S. began cordycepin is used for the first phase clinical experiment in 1997, treated acute front B and pre-T lymphocyte leukaemic; Cordycepin also shows extremely strong antimycotic, and anti-HTV-I C-type virus C and selectivity suppress the fusobacterium bacterial activity.
The mechanism of action of cordycepin: cordycepin can disturb synthesizing of cell RNA and DNA; suppress the division of undesired cell (cancer cells); and can be as the instrument of RNA polymerase different in the discriminate between cells; has the reparation gene; the special efficacy of protection life entity genetic code; now cordycepin in the U.S. as anticancer, new antiviral drug, it is clinical to enter for three phases.Because its effect is more outstanding, it is in great demand to market, at present its prices are rather stiff, reach ten thousand yuan of every grams.
The domestic and international disclosed patent of analysis-by-synthesis and document are as can be known, the main method that obtains at present cordycepin has organic synthesis, from the Cordyccps-militaris-(L.)-link. Sporophore of artificial culture, extract, from the artificial liquid Cordyceps sinensis of cultivating or cordyceps mycelium, extract, after sporophore is gathered, cover with and extract the mycelial solid culture medium residue of cordyceps militaris etc.
At present, the method for cordycepin extraction mainly contains:
Alcohol immersion, alcohol reflux: CN00130376.7 discloses the method for extracting cordycepin from Chinese caterpillar fungus, comprises drying and crushing, soak refluxing extraction, Recycled ethanol, aqueous precipitation filters, and upper ion changes post, collect elutriant, concentrated, refrigeration, crystallization are filtered, butanol crystal, the Chinese caterpillar fungus crystallization.
Water soaking, extraction using alcohol or water extract-alcohol precipitation: CN02109887.5 disclose a kind of extracting method and Chinese medicine preparation of compound cordycepin, and Cordyceps fungus powder was soaked in water 8-14 hour, the ethanol that adds 50-70% extracts, or extract filtering extracting liquid, concentrating return-flow ethanol with decoction alcohol precipitation method, upper ion column wash-out is removed impurity, collect washing lotion and carry out 30%, 0 ℃ of refrigeration 24 hours that reconcentration is original volume, crystallization, use the propyl carbinol recrystallization, get compound cordycepin.
Water soaking, cross macroporous adsorptive resins: CN200710170485.9 discloses a kind of separation purification method of cordycepin, it is raw material that the method adopts Cordyceps militaris (L.) Link. solid drying substratum, carries out purifying with NKA-II type macroporous resin column and C18 post, carries out repeatedly recrystallization with distilled water at last.
Alcohol coldly oozes, pure ultrasonic extraction, upper ion exchange column: CN200710122409.0 relates to a kind of method of extracting refining cordycepin and Cordyceps polysaccharide from Cordyceps militaris (L.) Link., the method may further comprise the steps: the Cordyceps militaris (L.) Link. powder is extracted with the cold method of oozing filter, filter residue mixes with ethanol, ultrasonic-assisted extraction, centrifugal, supernatant concentration, alcohol are analysed.Alcohol is analysed supernatant liquor concentrating under reduced pressure, wash-out, and the elutriant condensing crystal is used the propyl carbinol recrystallization, obtains refining cordycepin; Alcohol is analysed precipitation and is carried out enzymolysis deproteinated processing, centrifugal, supernatant concentration, wash-out, and effluent liquid is concentrated, alcohol is analysed, and obtains refining polysaccharide after the lyophilize.
Water soaking, pure supersound extraction: CN200910037874.3 relate to a kind of from Cordyccps-militaris-(L.)-link. Sporophore the method for extracting effective components cordycepin, may further comprise the steps: Cordyccps-militaris-(L.)-link. Sporophore is dry, soak after pulverizing; In thick solution, add 95% ethanol, utilize ultrasonic-assisted extraction, extract finish after, filter, collect filtrate, filter cake is carried out alcohol precipitation and supersound process second time, repeatedly filtrate is collected in merging; Concentrating under reduced pressure is distinguished the flavor of without ethanol under certain condition, gets concentrated solution; Getting concentrated solution transfers pH to acid with hydrochloric acid or sulphuric acid soln; Upper ion exchange column is removed impurity with the ammoniacal liquor wash-out, collects elutriant and carries out reconcentration, and the refrigeration crystallize out is used the propyl carbinol recrystallization, obtains cordycepin.
Provide extraction and purification process for cordycepin in a kind of Cordyceps militaris (L.) Link. with water with ultrasonic extraction, upper macroporous adsorptive resins: CN200910026113.8, the method may further comprise the steps: mix adding water with 1: 20 ratio of material-water ratio after the Cordyceps militaris (L.) Link. raw material pulverizing, carry out ultrasonic extraction, with macroporous resin column in the extracting solution centrifuging, absorption flow velocity 1.8BV/hr; Take 15% methanol aqueous solution as eluent wash-out resin column, with rotatory evaporator concentrating under reduced pressure elutriant under temperature 60 C, make the cordycepin enriched material; Other has CN201110004151.0 also to disclose the method that adopts the ultrasonic extraction cordycepin.
Prior art prepare the cordycepin time cycle long, extracts active ingredients is incomplete, the cordycepin content of preparation is lower, cost is high especially, the technique circulation ratio is poor.
The microwave/light wave combined method be in the natural product extraction a kind of new technique that development potentiality arranged very much to have equipment simple, applied widely, extraction efficiency is high, favorable reproducibility saves time, and saves reagent, pollutes the characteristics such as little.
At present, the microwave/light wave combined method also is used for biochemistry, food, the fields such as technical analysis and natural product extraction except being mainly used in the environmental sample pre-treatment.
But at home, microwave/light wave combined method extractive technique is also fewer for the research report that natural drug extracts.The principle that the microwave/light wave combined method is extracted is in microwave field, the difference of the absorbing microwave ability of different substances, so that some zone in the handled object or some component are by the heating of selectivity, thereby so that extract mass-energy is separated from processed object enough fast, it is less to enter specific inductivity, in the extraction agent of microwave absorption capacity relative mistake.When the microwave/light wave combined method penetrates processed object simultaneously, can make processed object inner, the outside almost is heated intensification simultaneously, and inside and outside homogeneous heating is consistent, forms the heat source body state, has greatly shortened the heat conduction time in the conventional heating.
Up to this point, not yet see the report of microwave/light wave aspect the cordycepin extraction.
Summary of the invention
The objective of the invention is to overcome the problem of prior art, and a kind of new extracting method of cordycepin is provided.
The extracting method of a kind of cordycepin provided by the invention, the method may further comprise the steps: cordycepin fermented liquid concentrating under reduced pressure, supersound extraction under the combination of microwave and light wave respectively then, the extracting solution separation and purification that obtains, and get final product.
In the extracting method of described cordycepin:
Described cordycepin fermented liquid adopts existing method that cordycepin is fermented and obtains, also can adopt the following methods preparation: in fermentor tank, add substratum, access bacterial classification after the sterilization, inoculum size 10%, fermentation condition is: 22 ℃, 150r/min, pH6-7 makes bacterial classification suspension growth in liquid, and incubation time is 6d, cordycepin content reaches more than 0.5%, namely gets the cordycepin fermented liquid;
Described fermention medium is liquid nutrient medium, and the weight percent of each composition is in the substratum: sucrose 4.0%, corn juice 3.0%, wheat bran 3.0%, full-fat cow milk 1.0%, VB 0.01%, KH
2P0
41%, MgS0
40.05%.
At present the kind of Cordyceps is a lot, for so that in the fermentation culture cordycepin content increase, must select suitable Cordyceps, condition be purity high, without miscellaneous bacteria, without aging, infectivity is strong, the bacterial classification of wide accommodation.
Supersound extraction refers to that in the microwave oven combination of 55% microwave and 45% light wave, radiation power are supersound extraction 4-10min under the condition of 800W under described microwave and the light wave;
The extracting solution separation and purification may further comprise the steps: centrifugal, alcohol precipitation, and centrifugal, cross macroporous adsorptive resins, high-efficient liquid phase chromatogram purification.Wherein:
In described two centrifugation step: the speed of two times centrifugal can be the same or different: centrifugal rotating speed is 4000-6000r/min, and the centrifugal time is 10-30min;
The described macroporous adsorptive resins step of crossing is: with the absorption of 0.5-3BV/h flow velocity loading, with the distilled water wash-out impurity of 3-5 times of column volume, again with concentration be the ethanol of 20-30% with the flow wash-out of 2-6BV/h 1-5 hour, the collection ethanol eluate gets final product;
Described high-efficient liquid phase chromatogram purification condition: chromatographic column is Kromasil 100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85 flow velocity 1mL/min; The detection wavelength is 260nm.
Preferably, the extracting method of described cordycepin may further comprise the steps:
1) gets the cordycepin fermented liquid, be evaporated to the 1/3-1/5 of stoste doubly, supersound extraction 4-10min under the condition that 55% microwave and 45% light wave make up in microwave oven, radiation power is 800W, the extracting solution that obtains is centrifugal, centrifugal speed is 4000-6000r/min, centrifugation time is 10-30min, collects supernatant liquor;
2) supernatant liquor that obtains in the step 1) is evaporated to the 1/3-1/6 of original volume, with concentrated solution volume 3-5 dehydrated alcohol precipitation doubly, spends the night, and centrifugal speed is 4000-6000r/min, and centrifugation time is 10-30min, collects supernatant liquor;
3) with step 2) supernatant liquor collected crosses macroporous resin column, adsorb with 0.5-3BV/h flow velocity loading, with the distilled water wash-out impurity of 3-5 times of column volume, again with concentration be the ethanol of 20-30% with the flow wash-out of 2-6BV/h 1.5-3.5 hour, collect ethanol eluate;
4) the ethanol eluate high-efficient liquid phase chromatogram purification that step 3) is obtained, then that the refined solution of collecting is concentrated, use again high-efficient liquid phase chromatogram purification, repeat again above-mentioned concentrated, purification step 2-5 time, the refined solution of collecting is concentrated into dried, namely gets cordycepin.
Further preferred, the extracting method of described cordycepin may further comprise the steps:
1) the cordycepin fermented liquid is evaporated to 1/3 times of stoste volume, in microwave oven 55% and the combination of microwave 45% light wave, radiation power be radiation supersound extraction 4min under the condition of 800W, centrifugal, collect supernatant liquor;
2) the supernatant liquor concentrating under reduced pressure that step 1) is obtained is to 1/5 of original volume, and concentrated solution precipitates with 3 times of volume dehydrated alcohols, spends the night, and then collects supernatant liquor, and the centrifugal 15min of 5000r/min obtains the cordycepin supernatant liquor;
3) with step 2) the cordycepin supernatant liquor that obtains crosses AB-8 type macroporous resin column, with the absorption of 1BV/h flow velocity loading, with the distilled water wash-out impurity of 4 times of column volumes, uses 20% ethanol with the flow wash-out of 4BV/h 2 hours, collection ethanol eluate again;
4) then ethanol eluate is used the high-efficient liquid phase chromatogram purification elutriant, wherein high-efficient liquid phase chromatogram condition is: chromatographic column is Kromasil100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85 flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃, collects cordycepin peak effluent liquid;
5) cordycepin peak effluent liquid is evaporated to 1/3 of its volume, then adopts the high-efficient liquid phase chromatogram purification of step 3); And then concentrating under reduced pressure, high-efficient liquid phase chromatogram purification, namely repeat above-mentioned steps 3 times, the refined solution that finally obtains is evaporated to dried, and get final product.
In the described method:
Described concentrating under reduced pressure adopts Rotary Evaporators concentrated;
In step 2) can also comprise and be specially the process of charcoal absorption impurity: with the gac mixing of equivalent, shaking table jolting 12h filters depigmentation.
Described macroporous adsorptive resins is selected from AB-8 type macroporous resin column.
The extracting method of cordycepin provided by the invention has the following advantages:
1, the Cordyceps infectivity of selecting is strong, and bacterial classification has stronger vitality, can infect rapidly the dead kind of falling ill to insect; Wide accommodation particularly changes ambient moisture and other miscellaneous bacteria infects the kind that certain resistivity will be arranged.
2, the fermentation culture with Cordyceps concentrate, microwave extraction, reconcentration, alcohol precipitation, centrifugal,
Wherein centrifugal purpose is to remove the insoluble impurity of the alcohol such as Cordyceps polysaccharide;
By the different purifying of high performance liquid chromatography, with the methyl alcohol in the cordycepin extracting solution, water and other Impurity removals, finally obtain the sterling of cordycepin.
2, adopt conventional ultrasonic extraction, extraction time is long, and the extraction yield of cordycepin is low, only is 69%;
The present invention adopts the cordycepin of microwave/light wave assisted extraction Cordyceps militaris.The contrast of different microwave/light wave array modes draws, the combination of 55% microwave and 45% light wave, radiation power are that the ultrasonic effect of radiation is better under the condition of 800W, and the extraction yield of cordycepin is 81%, more simple extraction extraction yield has increased by 12%, and has greatly shortened time loss.Compare with traditional extracting method, the microwave/light wave combination has improved extraction yield, has saved extraction time, and very wide prospect is being arranged aspect the active ingredient of natural product extraction.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The preparation of cordycepin fermented liquid:
Described cordycepin fermented liquid can obtain in commercially available purchase, and the present invention adopts the following methods preparation in order to guarantee the controllability of its raw material:
Fermention medium is liquid nutrient medium, and the weight percent of each composition is in the substratum: sucrose 4.0%, corn juice 3.0%, wheat bran 3.0%, full-fat cow milk 1.0%, VB 0.01%, KH
2P0
41%, MgS0
40.05%.
Concrete fermentation process is: in fermentor tank, add substratum, and access bacterial classification after the sterilization, inoculum size 10%, fermentation condition is: 22 ℃, 150r/min, pH6-7 makes bacterial classification suspension growth in liquid, incubation time is 6d, and cordycepin content reaches more than 0.5%, namely gets the cordycepin fermented liquid.
The quality control index of the content of cordycepin adopts liquid chromatography for measuring in the fermented liquid, and concrete chromatographic condition is:
Chromatographic column is Kromasil100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85, flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃.
Embodiment 1: the extracting method of cordycepin
1, will be evaporated to 1/3 times of stoste volume with Rotary Evaporators at the cordycepin fermented liquid, extract 8min under the condition that 55% microwave and 45% light wave make up in microwave oven, radiation power is 800W, extracting solution is centrifugal, and 5000 leave heart speed and time 10min, collects supernatant liquor;
2, step 1 is obtained Rotary Evaporators on the supernatant liquor, concentrating under reduced pressure is to 1/5 of original volume, then with the dehydrated alcohol precipitation of 3 times of volumes of concentrated solution, spend the night, then collect supernatant liquor, with the centrifugal 15min of 5000r/min, collect supernatant liquor, then use the gac mixing of equivalent, shaking table jolting 12h, filter depigmentation, obtain the cordycepin supernatant liquor;
3, the cordycepin supernatant liquor that step 2 is obtained is crossed AB-8 type macroporous resin column, with 1BV/h flow velocity loading absorption, with the distilled water wash-out impurity of 4 times of column volumes, uses 20% ethanol with the flow wash-out of 4BV/h 2 hours, collection ethanol eluate again;
4, then with the ethanol eluate high-efficient liquid phase chromatogram purification, wherein high-efficient liquid phase chromatogram condition is: chromatographic column is Kromasil 100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85 flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃, collects cordycepin peak effluent liquid, and it is 83.5 that analysis mode HPLC detects purity, and purity reaches export standard;
5, Rotary Evaporators on the cordycepin peak effluent liquid that step 4 is obtained, be evaporated to 1/3rd of cordycepin peak effluent volume, gained cordycepin peak concentrated solution adopts the high performance liquid chromatography in the step 3 to be further purified, chromatographic condition is the same, collect cordycepin peak secondary stream fluid, it is 99.84% that analysis mode HPLC detects purity, and purity reaches the reference substance standard.
6, Rotary Evaporators on the cordycepin peak secondary stream fluid that step 5 is obtained, be evaporated to 1/3rd of cordycepin peak secondary effluent volume, the described high performance liquid chromatography of step 3 on the cordycepin peak secondary concentration liquid of gained, chromatographic condition is the same, collect three effluent liquid in cordycepin peak, it is 99.99% that analysis mode HPLC detects purity;
7, Rotary Evaporators on three effluent liquid in the cordycepin peak that step 6 is obtained is evaporated to 1/3rd of three effluent volumes in cordycepin peak, and becoming cordycepin lyophilized powder sterling 10g(yield after the gained concentrated solution vacuum lyophilization is 87.7%).
Embodiment 2: the extracting method of cordycepin
1, with the cordycepin fermented liquid in concentrated ratio be decompressed to 1/5 times of stoste 55% microwave and the combination of 45% light wave in microwave oven, radiation power is the condition supersound extraction 5min of 800W, extracting solution is centrifugal, centrifugal speed 5000r/min and time are 10min, collect supernatant liquor;
2, Rotary Evaporators on the supernatant liquor that step 1 is obtained, concentrating under reduced pressure is to 1/5 of original volume, then the dehydrated alcohol with 3 times of volumes of concentrated solution precipitates, spend the night, then collect supernatant liquor, with the centrifugal 15min of supernatant liquor 5000r/min, then use the activated carbon mixing of equivalent, shaking table jolting 12h filters depigmentation, gets the cordycepin supernatant liquor;
3, the cordycepin supernatant liquor that step 2 is obtained is crossed AB-8 type macroporous resin column, with 0.5BV/h flow velocity loading absorption, with the distilled water wash-out impurity of 5 times of column volumes, uses 30% ethanol with the flow wash-out of 3BV/h 3.5 hours, collection ethanol eluate again;
4, with the ethanol eluate high-efficient liquid phase chromatogram purification, high-efficient liquid phase chromatogram condition is: chromatographic column is Kromasil100-25, C18,250mm * 4.6mm; Moving phase is methyl alcohol: water=15:85; Flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃.Collect cordycepin peak effluent liquid, it is 84.5% that analysis mode HPLC detects purity, and purity reaches export standard;
5, Rotary Evaporators on the cordycepin peak effluent liquid that step 4 is obtained, be evaporated to 1/3rd of cordycepin peak effluent volume, the gained concentrated solution adopts the high performance liquid chromatography in the step 4 to be further purified, chromatographic condition is the same, collect cordycepin peak secondary stream fluid, it is 95.6% that analysis mode HPLC detects purity, and purity reaches the reference substance standard;
6, Rotary Evaporators on the cordycepin peak secondary stream fluid that step 5 is obtained, be evaporated to 1/3rd of cordycepin peak secondary effluent volume, the gained concentrated solution is gone up the described high performance liquid chromatography of step 3 for the third time, chromatographic condition is the same, collect three effluent liquid in cordycepin peak, it is 99.9% that analysis mode HPLC detects purity, purity accurate product standard up to standard;
7, Rotary Evaporators on three effluent liquid in the cordycepin peak that step 6 is obtained is evaporated to 1/3rd of three effluent volumes in cordycepin peak, and becoming cordycepin lyophilized powder sterling 9.5g(yield after the gained concentrated solution spraying drying is 83.32%).
Embodiment 3: the extracting method of cordycepin
1, Rotary Evaporators on the cordycepin fermented liquid is evaporated to 1/5 times of stoste volume, radiation supersound extraction 10min under the condition that 55% microwave and 45% light wave make up in microwave oven, radiation power is 800W, extracting solution is centrifugal, centrifugal speed 4000r/min, centrifugation time is 20min, collects supernatant;
2, Rotary Evaporators on the supernatant liquor that step 1 is obtained, concentrating under reduced pressure is to 1/5 of original volume, then with the dehydrated alcohol precipitation of 3 times of volumes of concentrated solution, spend the night, then collect supernatant liquor, with the centrifugal 10min of supernatant liquor 6000r/min, collect supernatant liquor, then use the activated carbon mixing of equivalent, shaking table jolting 12h, filter depigmentation, get the cordycepin supernatant liquor;
3, the cordycepin supernatant liquor that step 2 is obtained is crossed AB-8 type macroporous resin column, with 0.5BV/h flow velocity loading absorption, with the distilled water wash-out impurity of 5 times of column volumes, uses 25% ethanol with the flow wash-out of 5BV/h 1.5 hours, collection ethanol eluate again;
4, step 3 is obtained the ethanol eluate high-efficient liquid phase chromatogram purification, middle high-efficient liquid phase chromatogram condition is: chromatographic column is Kromasil100-25, C18,250mm * 4.6mm; Moving phase is methyl alcohol: water=15:85; Flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃.Collect cordycepin peak effluent liquid, it is 84.5% that analysis mode HPLC detects purity, and purity reaches export standard;
5, Rotary Evaporators on the cordycepin peak effluent liquid that step 4 is obtained, be evaporated to 1/3rd of cordycepin peak effluent volume, the gained concentrated solution adopts the high performance liquid chromatography in the step 4 to be further purified, chromatographic condition is the same, collect cordycepin peak secondary stream fluid, it is 95.6% that analysis mode HPLC detects purity, and purity reaches the reference substance standard;
6, Rotary Evaporators on the cordycepin peak secondary stream fluid that step 5 is obtained, be evaporated to 1/3rd of cordycepin peak secondary effluent volume, the gained concentrated solution is gone up the described high performance liquid chromatography of step 3 for the third time, chromatographic condition is the same, collect three effluent liquid in cordycepin peak, it is 99.9% that analysis mode HPLC detects purity, purity accurate product standard up to standard;
7, Rotary Evaporators on three effluent liquid in the cordycepin peak that step 6 is obtained is evaporated to 1/3rd of three effluent volumes in cordycepin peak, and becoming cordycepin lyophilized powder sterling 9.7g(yield after the gained concentrated solution spraying drying is to be 85.07%).
Comparative Examples 1: the extracting method of cordycepin
Cordycepin extracting method with reference to CN200910026113.8, method adopts embodiments of the invention 1, and ultrasonic extracting method wherein adopts the method for CN200910026113.8, and namely ultrasonic frequency 90khz carries out ultrasonic extraction, extracting temperature is 60 ℃, and extraction time is 30 minutes.
Experimental example: yield and purity are relatively
According to the weight comparing embodiment 1-3 of the finished product that finally makes and the yield in the Comparative Examples, then adopt HPLC to detect the purity of the cordycepin product that obtains, the results are shown in Table 1, wherein the HPLC detection method is specially:
Chromatographic column is Kromasil 100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85 flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃.
Table 1: the yield of embodiment 1-3 and Comparative Examples and content are relatively
| ? | Yield (%) | Purity (%) |
| Embodiment 1 | 87.7 | 99.99 |
| Embodiment 2 | 85.07 | 99.99 |
| Embodiment 3 | 83.32 | 99.99 |
| Comparative Examples | 69 | 98..8 |
Table 1 result shows: the yield of Comparative Examples only is 69%, and purity is 98.8%, and yield is higher than 83% in the embodiments of the invention, and purity all can reach 99.99%.
The result shows: cordycepin extracting method yield provided by the invention is high, and purity is high, and effect is better than prior art.
Although, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the extracting method of a cordycepin is characterized in that, the method may further comprise the steps: cordycepin fermented liquid concentrating under reduced pressure, and supersound extraction under the combination of microwave and light wave respectively then, the extracting solution separation and purification that obtains, and get final product.
2. extracting method according to claim 1 is characterized in that, supersound extraction refers to that in the microwave oven combination of 55% microwave and 45% light wave, radiation power are supersound extraction 4-10min under the condition of 800W under described microwave and the light wave.
3. extracting method according to claim 1 is characterized in that, the extracting solution separation and purification may further comprise the steps: centrifugal, alcohol precipitation, and centrifugal, cross macroporous adsorptive resins, high-efficient liquid phase chromatogram purification.
4. extracting method according to claim 3 is characterized in that, in described two centrifugation step: centrifugal rotating speed is 4000-6000r/min, and the centrifugal time is 10-30min.
5. extracting method according to claim 3, it is characterized in that, the described macroporous adsorptive resins step of crossing is: adsorb with 0.5-3BV/h flow velocity loading, distilled water wash-out impurity with 3-5 times of column volume, be that the ethanol of 20-30% was with the flow wash-out of 2-6BV/h 1-5 hour with concentration again, collect ethanol eluate, get final product.
6. extracting method according to claim 3 is characterized in that, described high-efficient liquid phase chromatogram purification condition: chromatographic column is Kromasil 100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85 flow velocity 1mL/min; The detection wavelength is 260nm.
7. extracting method according to claim 1, it is characterized in that the fermentation process of described cordyceps sinensis fermentation liquor is: in fermentor tank, add substratum, access bacterial classification after the sterilization, inoculum size 10%, fermentation condition is: 22 ℃, and 150r/min, pH6-7, make bacterial classification suspension growth in liquid, incubation time is 6d, and cordycepin content reaches more than 0.5%, namely gets the cordycepin fermented liquid; Described fermention medium is liquid nutrient medium, and the weight percent of each composition is in the substratum: sucrose 4.0%, corn juice 3.0%, wheat bran 3.0%, full-fat cow milk 1.0%, VB 0.01%, KH
2P0
41%, MgS0
40.05%.
8. each described extracting method is characterized in that according to claim 1-7, and described extracting method may further comprise the steps:
1) gets the cordycepin fermented liquid, be evaporated to the 1/3-1/5 of stoste doubly, supersound extraction 4-10min under the condition that 55% microwave and 45% light wave make up in microwave oven, radiation power is 800W, the extracting solution that obtains is centrifugal, centrifugal speed is 4000-6000r/min, centrifugation time is 10-30min, collects supernatant liquor;
2) supernatant liquor that obtains in the step 1) is evaporated to the 1/3-1/6 of original volume, with concentrated solution volume 3-5 dehydrated alcohol precipitation doubly, spends the night, and centrifugal speed is 4000-6000r/min, and centrifugation time is 10-30min, collects supernatant liquor;
3) with step 2) supernatant liquor collected crosses macroporous resin column, adsorb with 0.5-3BV/h flow velocity loading, with the distilled water wash-out impurity of 3-5 times of column volume, again with concentration be the ethanol of 20-30% with the flow wash-out of 2-6BV/h 1.5-3.5 hour, collect ethanol eluate;
4) the ethanol eluate high-efficient liquid phase chromatogram purification that step 3) is obtained, then that the refined solution of collecting is concentrated, use again high-efficient liquid phase chromatogram purification, repeat again above-mentioned concentrated, purification step 2-5 time, the refined solution of collecting is concentrated into dried, namely gets cordycepin.
9. extracting method according to claim 8 is characterized in that, extracting method may further comprise the steps:
1) the cordycepin fermented liquid is evaporated to 1/3 times of stoste volume, in microwave oven 55% and the combination of microwave 45% light wave, radiation power be radiation supersound extraction 4min under the condition of 800W, centrifugal, collect supernatant liquor;
2) the supernatant liquor concentrating under reduced pressure that step 1) is obtained is to 1/5 of original volume, and concentrated solution precipitates with 3 times of volume dehydrated alcohols, spends the night, and then collects supernatant liquor, and the centrifugal 15min of 5000r/min obtains the cordycepin supernatant liquor;
3) with step 2) the cordycepin supernatant liquor that obtains crosses AB-8 type macroporous resin column, with the absorption of 1BV/h flow velocity loading, with the distilled water wash-out impurity of 4 times of column volumes, uses 20% ethanol with the flow wash-out of 4BV/h 2 hours, collection ethanol eluate again;
4) then ethanol eluate is used the high-efficient liquid phase chromatogram purification elutriant, wherein high-efficient liquid phase chromatogram condition is: chromatographic column is Kromasil100-25, C18,4.6mm * 250mm; Moving phase is that the methanol-water volume ratio is 15:85 flow velocity 1mL/min; The detection wavelength is 260nm; Detected temperatures is 30 ℃, collects cordycepin peak effluent liquid;
5) cordycepin peak effluent liquid is evaporated to 1/3 of its volume, then adopts the high-efficient liquid phase chromatogram purification of step 3); Concentrating under reduced pressure, high-efficient liquid phase chromatogram purification namely repeat above-mentioned steps 3 times again, the refined solution that finally obtains are evaporated to dried, and get final product.
10. extracting method according to claim 8, it is characterized in that, in step 2) also comprise the process of charcoal absorption impurity, be specially: the supernatant liquor that obtains in the step 1) is evaporated to the 1/3-1/6 of original volume, with concentrated solution volume 3-5 dehydrated alcohol precipitation doubly, spend the night, centrifugal speed is 4000-6000r/min, and centrifugation time is 10-30min, with the gac mixing of equivalent, shaking table jolting 12h filters depigmentation.
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