CN107266513A - A kind of isolation and purification method of Cordyceps militaris active component - Google Patents
A kind of isolation and purification method of Cordyceps militaris active component Download PDFInfo
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- CN107266513A CN107266513A CN201710554975.2A CN201710554975A CN107266513A CN 107266513 A CN107266513 A CN 107266513A CN 201710554975 A CN201710554975 A CN 201710554975A CN 107266513 A CN107266513 A CN 107266513A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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Abstract
Disclosed by the invention is a kind of isolation and purification method of Cordyceps militaris active component, specifically includes following steps：By Cordyceps militaris dries pulverizing, Cordyceps militaris powder is with 75% 100% ethanolic extractions, the supernatant of extraction is spin-dried for obtaining solid, carried out after solid is dissolved with water with Oligo (dT) affine, heating water bath, remove supernatant, obtain cordycepin solution, the present invention is removed the nucleic acid material in Cordyceps militaris by alcohol, avoid the pollution of nucleic acid in subsequent process, cordycepin is isolated and purified by affinity chromatography, adsorptivity is strong, it is specific high, avoid effective loss, improve the purity of active ingredient, it is with short production cycle, cost is low, it is low for equipment requirements, it is simple to operate, market potential easy to spread is big, the process of isolating and purifying avoids the pollution of hazardous solvent, extract safety non-toxic, environmental protection.
Description
Technical field
The present invention relates to a kind of isolation and purification method, more specifically say, be related to a kind of separation of Cordyceps militaris active component
Purification process.
Background technology
Cordyceps militaris is Ascomycota, meat seat mesh, Clavicipitaceae, the type sepecies of Cordyceps, and scientific name is Cordyceps
Militaris, also known as northern Cordyceps militaris.Contain a variety of chemical compositions in Cordyceps militaris, with a variety of medicinal efficacies, with cordyceps sinensis
Pharmacological function is similar with clinical effectiveness, but price is well below cordyceps sinensis, therefore Cordyceps militaris is increasingly becoming cordyceps sinensis
Substitute, with great potential market.Its main active includes adenosine, cordycepin, Cordyceps sinensis polysaccharide etc., and research at present is most
More most widely used is undoubtedly cordycepin, in view of the antitumor isoreactivity of cordycepin, people are also increasing to the demand of cordycepin.
The mode of currently acquired cordycepin is mainly obtained by separation and Extraction, such as water extraction of cordycepin general extraction methods
Method, water extraction and alcohol precipitation method, alcohol extracting method etc., extraction process is single, it is impossible to the thorough active ingredient extracted in Cordyceps militaris, and assisted extraction
Means generally have microwave, ultrasound, enzyme process etc., and the domestic technological means separated on cordycepin is mainly chromatography, on the whole
For, these extracting methods still suffer from not abundant enough, the active ingredient of extraction in terms of active ingredient, reject invalid components are retained
Loss is big, and process is more, and the cycle is long, the shortcomings of cost is high.
The content of the invention
It is an object of the invention to solve prior art problem, there is provided with the technology such as separation is simple, purity is high, the cycle is short
A kind of isolation and purification method of Cordyceps militaris active component of feature.
The present invention is to be achieved through the following technical solutions：
A kind of isolation and purification method of Cordyceps militaris active component of the present invention, this method uses following steps：
Step 1)：Dried under the conditions of Cordyceps militaris is placed in into 50-70 DEG C to Cordyceps militaris water content below 8%, ultramicro grinding
5-15min, obtains ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 75-100% ethanol solution in, fill
Divide stirring and dissolving, ultrasonic 30-60min stands 1-12h, it is centrifugation acquisition supernatant in 8000-10000rpm centrifuges to be placed in rotating speed
Liquid, is repeated 1-2 times, merges supernatant；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation
Water is dissolved, and is sufficiently stirred for, and acquisition concentration is 0.9-1.1mg/ml solution, then solution is placed in into rotating speed for 8000-10000rpm
Centrifuged in centrifuge, obtain preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 400-600 parts is with 400-600 parts of water washings two
It is secondary, 100-300 parts of preliminary extract solutions are added, magnetic bead is reset, is placed under room temperature condition and reacts 5-10min, entered with magnetic separator
Row Magnetic Isolation, is discarded supernatant with pipettor on magnetic separator；100-300 parts of water are added, magnetic bead is mixed, uses magnetic
Property separator carry out Magnetic Isolation, on magnetic separator pipetting supernatant with pipettor discards, and is repeated twice；Continuously add
50-200 parts of water, mix magnetic bead, are placed in 65-100 DEG C of heating water bath 5-30min, are quickly placed on magnetic separation rack, carry out magnetic
Separation, quickly collects supernatant, obtains cordycepin solution.
As a preferred embodiment, step 2) described in micro- Cordyceps militaris powder and concentration be that 70-100% ethanol mass ratioes are 1:5-
20。
A kind of isolation and purification method of Cordyceps militaris active component of the present invention, this method uses following steps：
Step 1)：Cordyceps militaris is placed under the conditions of 60 DEG C and dried to Cordyceps militaris water content below 8%, ultramicro grinding
8min, obtains ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 85% ethanol solution in, fully stir
Dissolving is mixed, ultrasonic 45min stands 8h, be placed in rotating speed and obtain supernatant for centrifugation in 9000rpm centrifuges, be repeated 1 times, merge
Supernatant；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation
Water dissolves, and is sufficiently stirred for, and acquisitions concentration is 1.0mg/ml solution, then by solution be placed in rotating speed be in 9000rpm centrifuges from
The heart, obtains preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 500 parts twice, is added with 500 parts of water washings
200 parts of preliminary extract solutions, reset magnetic bead, are placed under room temperature condition and react 8min, Magnetic Isolation is carried out with magnetic separator, in magnetic
Property separator on supernatant is discarded with pipettor, add 200 parts of water, mix magnetic bead, with is carried out on magnetic separator magnetic divide
From on magnetic separator pipetting supernatant with pipettor discards, and is repeated twice, and adds 125 parts of water, mixes magnetic bead, is placed in 80 DEG C
Heating water bath 18min, is quickly placed on magnetic separation rack, carries out Magnetic Isolation, quickly collects supernatant, obtains cordycepin molten
Liquid.
As a preferred embodiment, step 2) described in micro- Cordyceps militaris powder and concentration be that 85% ethanol mass ratio is 1:8.
Beneficial effects of the present invention：The present invention begins through alcohol and removes the nucleic acid material in Cordyceps militaris, it is to avoid
Pollution of nucleic acid in subsequent process；Pure cordycepin is separated by affinity chromatography, adsorptivity is strong, and specificity is high.Avoid effectively
Loss, improves the purity of active ingredient；With short production cycle, cost is low, low for equipment requirements, simple to operate, it is easy to promote city
Field potentiality are big；The process of isolating and purifying avoids the pollution of hazardous solvent, makes the extract safety non-toxic finally obtained, green ring
Protect；It is domestic using reagent instrument, cost is low.
Embodiment
Below by way of specific embodiment, the present invention is further illustrated, but embodiments of the present invention not by
The limitation of following examples.
A kind of isolation and purification method of Cordyceps militaris active component of the invention of embodiment 1, this method uses following steps：
Step 1)：Cordyceps militaris is placed under the conditions of 50 DEG C and dried to Cordyceps militaris water content below 8%, ultramicro grinding
5min, obtains ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 75% ethanol solution in, fully stir
Dissolving is mixed, ultrasonic 30min stands 1h, be placed in rotating speed and obtain supernatant for centrifugation in 8000rpm centrifuges, be repeated 1 times, merge
Supernatant, micro- Cordyceps militaris powder and concentration are that 70% ethanol mass ratio is 1:5；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation
Water dissolves, and is sufficiently stirred for, and acquisitions concentration is 0.9mg/ml solution, then by solution be placed in rotating speed be in 8000rpm centrifuges from
The heart, obtains preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 400 parts twice, is added with 400 parts of water washings
100 parts of steps 3) obtain preliminary extract solution, reset magnetic bead, be placed under room temperature condition and react 5min, carried out with magnetic separator
Magnetic Isolation so that magnetic bead is precipitated, separation of solid and liquid is discarded supernatant with pipettor on magnetic separator；Add 100 parts
Water, mixes magnetic bead, carries out Magnetic Isolation with magnetic separator, pipetting supernatant with pipettor on magnetic separator discards, weight
Again twice；50 parts of water are continuously added, magnetic bead is mixed, is placed in 65 DEG C of heating water bath 5min, is quickly placed on magnetic separation rack, are carried out
Magnetic Isolation, quickly pipettes supernatant with pipettor and discards, and obtains cordycepin solution, is evaporated solution, can obtain powdered height
Purity cordycepin.
A kind of isolation and purification method of Cordyceps militaris active component of the invention of embodiment 2, this method uses following steps：
Step 1)：Cordyceps militaris is placed under the conditions of 60 DEG C and dried to Cordyceps militaris water content below 8%, ultramicro grinding
10min, obtains ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 85% ethanol solution in, fully stir
Dissolving is mixed, ultrasonic 45min stands 8h, be placed in rotating speed and obtain supernatant for centrifugation in 9000rpm centrifuges, be repeated 1 times, merge
Supernatant, micro- Cordyceps militaris powder and concentration are that 85% ethanol mass ratio is 1:8；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation
Water dissolves, and is sufficiently stirred for, and acquisitions concentration is 1.0mg/ml solution, then by solution be placed in rotating speed be in 9000rpm centrifuges from
The heart, obtains preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 500 parts twice, is added with 500 parts of water washings
200 parts of steps 3) obtain preliminary extract solution, reset magnetic bead, be placed under room temperature condition and react 8min, carried out with magnetic separator
Magnetic Isolation so that magnetic bead is precipitated, separation of solid and liquid is discarded supernatant with pipettor on magnetic separator；Add 200 parts
Water, mixes magnetic bead, carries out Magnetic Isolation with magnetic separator, pipetting supernatant with pipettor on magnetic separator discards, weight
Again twice；125 parts of water are continuously added, magnetic bead is mixed, is placed in 80 DEG C of heating water bath 18min, is quickly placed on magnetic separation rack, enters
Row Magnetic Isolation, quickly pipettes supernatant with pipettor and discards, and obtains cordycepin solution, is evaporated solution, can obtain powdered
High-purity cordycepin.
A kind of isolation and purification method of Cordyceps militaris active component of the invention of embodiment 3, this method uses following steps：
Step 1)：Cordyceps militaris is placed under the conditions of 70 DEG C and dried to Cordyceps militaris water content below 8%, ultramicro grinding
15min, obtains ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 100% ethanol solution in, fully stir
Dissolving is mixed, ultrasonic 60min stands 12h, be placed in rotating speed and obtain supernatant for centrifugation in 10000rpm centrifuges, be repeated 2 times, close
And supernatant, micro- Cordyceps militaris powder and concentration are that 100% ethanol mass ratio is 1:20；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation
Water dissolves, and is sufficiently stirred for, and acquisitions concentration is 1.1mg/ml solution, then by solution be placed in rotating speed be in 10000rpm centrifuges from
The heart, obtains preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 600 parts twice, is added with 600 parts of water washings
300 parts of steps 3) obtain preliminary extract solution, reset magnetic bead, be placed under room temperature condition and react 10min, carried out with magnetic separator
Magnetic Isolation so that magnetic bead is precipitated, separation of solid and liquid is discarded supernatant with pipettor on magnetic separator；Add 300 parts
Water, mixes magnetic bead, carries out Magnetic Isolation with magnetic separator, pipetting supernatant with pipettor on magnetic separator discards, weight
Again twice；200 parts of water are continuously added, magnetic bead is mixed, is placed in 100 DEG C of heating water bath 30min, is quickly placed on magnetic separation rack,
Magnetic Isolation is carried out, quickly supernatant is pipetted with pipettor and discards, cordycepin solution is obtained, is evaporated solution, powder can be obtained
Shape high-purity cordycepin.
Finally it should be noted that the invention is not restricted to above example, can also there is many variations.This area it is general
All deformations that logical technical staff directly can export or associate from present disclosure, are considered as the present invention's
Protection domain.
Claims (4)
1. a kind of isolation and purification method of Cordyceps militaris active component, it is characterised in that this method uses following steps：
Step 1)：Dried under the conditions of Cordyceps militaris is placed in into 50-70 DEG C to Cordyceps militaris water content below 8%, ultramicro grinding 5-
15min, obtains ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 75-100% ethanol solution in, fully stir
Dissolving is mixed, ultrasonic 30-60min stands 1-12h, is placed in rotating speed and obtains supernatant for centrifugation in 8000-10000rpm centrifuges,
Repeat 1-2 times, merge supernatant；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation it is water-soluble
Solution, and be sufficiently stirred for, acquisition concentration is 0.9-1.1mg/ml solution, then it is 8000-10000rpm centrifugations that solution is placed in into rotating speed
Centrifuged in machine, obtain preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 400-600 parts with 400-600 parts of water washings twice,
100-300 parts of preliminary extract solutions are added, magnetic bead is reset, is placed under room temperature condition and reacts 5-10min, magnetic is carried out with magnetic separator
Property separation, supernatant is discarded with pipettor on magnetic separator；100-300 parts of water are added, magnetic bead is mixed, with magnetic point
Magnetic Isolation is carried out from device, pipetting supernatant with pipettor on magnetic separator discards, and is repeated twice；Continuously add 50-200
Part water, mixes magnetic bead, is placed in 65-100 DEG C of heating water bath 5-30min, is quickly placed on magnetic separation rack, carries out Magnetic Isolation,
Quickly supernatant is pipetted with pipettor to discard, obtain cordycepin solution.
2. a kind of isolation and purification method of Cordyceps militaris active component according to claim 1, it is characterised in that：Step 2) in
Micro- Cordyceps militaris powder and concentration are that 70-100% ethanol mass ratioes are 1:5-20.
3. a kind of isolation and purification method of Cordyceps militaris active component, it is characterised in that this method uses following steps：
Step 1)：Cordyceps militaris is placed under the conditions of 60 DEG C and dried to Cordyceps militaris water content below 8%, ultramicro grinding 10min is obtained
Obtain ultra micro Cordyceps militaris powder；
Step 2)：By step 1) obtain ultra micro Cordyceps militaris powder be added to concentration be 85% ethanol solution in, be sufficiently stirred for molten
Solution, ultrasonic 45min stands 8h, is placed in rotating speed and obtains supernatant for centrifugation in 9000rpm centrifuges, is repeated 1 times, merges supernatant
Liquid；
Step 3)：By step 2) merge supernatant be spin-dried for obtain solid, solid is placed in beaker, be slowly added to distillation it is water-soluble
Solution, and be sufficiently stirred for, acquisition concentration is 1.0mg/ml solution, then solution is placed in into rotating speed to be centrifuged in 9000rpm centrifuges, is obtained
Obtain preliminary extract solution；
Step 4)：By mass fraction, the magnetic bead for being coupled Oligo (dT) by 500 parts twice, adds 200 parts with 500 parts of water washings
Preliminary extract solution, resets magnetic bead, is placed under room temperature condition and reacts 8min, Magnetic Isolation is carried out with magnetic separator, in magnetic point
From being discarded supernatant with pipettor on device, 200 parts of water are added, magnetic bead is mixed, with carrying out Magnetic Isolation on magnetic separator,
Supernatant is pipetted with pipettor on magnetic separator to discard, be repeated twice, add 125 parts of water, mix magnetic bead, be placed in 80 DEG C of water-baths and add
Hot 18min, is quickly placed on magnetic separation rack, carries out Magnetic Isolation, quickly pipettes supernatant with pipettor and discard, and obtains cordyceps sinensis
Plain solution.
4. a kind of isolation and purification method of Cordyceps militaris active component according to claim 3, it is characterised in that：Step 2) in
Micro- Cordyceps militaris powder and concentration are that 85% ethanol mass ratio is 1:8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109619192A (en) * | 2018-11-06 | 2019-04-16 | 广州健康玖号健康管理科技有限公司 | A kind of fine lucky health solid beverage and preparation method thereof |
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| CN101508715A (en) * | 2009-04-01 | 2009-08-19 | 江苏省农业科学院 | Extraction and purification process for cordycepin in cordyceps militaris link |
| CN101830954A (en) * | 2009-03-13 | 2010-09-15 | 五邑大学 | Method for extracting active ingredient of cordycepin from fruiting bodies of cordyceps militaris |
| CN103800385A (en) * | 2014-01-27 | 2014-05-21 | 正源堂（天津）生物科技有限公司 | Method for extracting cordycepin component from Cordyceps militaris and application of cordycepin component |
| CN106924298A (en) * | 2017-04-06 | 2017-07-07 | 广州市嘛咪食品有限公司 | A kind of Cordyceps militaris extract and its preparation method and application |
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2017
- 2017-07-10 CN CN201710554975.2A patent/CN107266513B/en active Active
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| CN101830954A (en) * | 2009-03-13 | 2010-09-15 | 五邑大学 | Method for extracting active ingredient of cordycepin from fruiting bodies of cordyceps militaris |
| CN101508715A (en) * | 2009-04-01 | 2009-08-19 | 江苏省农业科学院 | Extraction and purification process for cordycepin in cordyceps militaris link |
| CN103800385A (en) * | 2014-01-27 | 2014-05-21 | 正源堂（天津）生物科技有限公司 | Method for extracting cordycepin component from Cordyceps militaris and application of cordycepin component |
| CN106924298A (en) * | 2017-04-06 | 2017-07-07 | 广州市嘛咪食品有限公司 | A kind of Cordyceps militaris extract and its preparation method and application |
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| CN109619192A (en) * | 2018-11-06 | 2019-04-16 | 广州健康玖号健康管理科技有限公司 | A kind of fine lucky health solid beverage and preparation method thereof |
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