CN105273024A - Method for separating and purifying cordycepin by using mixed resins - Google Patents
Method for separating and purifying cordycepin by using mixed resins Download PDFInfo
- Publication number
- CN105273024A CN105273024A CN201410322498.3A CN201410322498A CN105273024A CN 105273024 A CN105273024 A CN 105273024A CN 201410322498 A CN201410322498 A CN 201410322498A CN 105273024 A CN105273024 A CN 105273024A
- Authority
- CN
- China
- Prior art keywords
- cordycepin
- resin
- purification
- hybrid resin
- separation
- 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.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a method for separating and purifying cordycepin from cordyceps militaris sporophore (cordyceps flower) or a medium thereof. According to the method, cordycepin is extracted from cordyceps militaris sporophore or the medium thereof by using a microwave, ultrasonic, constant-temperature oscillation or hydro-thermal reflux method, and then a plurality of different resins (macroporous adsorption resins or ion exchange resins) are mixed according to according to a certain manner and are used for separation and purification of cordycepin in crude extract. Under optimized process conditions, cordycepin content in extract of eluate of the mixed resins is increased to 20 to 40% from 0.2 to 0.4% in the cordyceps flower or the medium thereof, and the purity of cordycepin is more than 90% after further refining and purification. According to the invention, solid deserted medium obtained after harvesting of cordyceps militaris can be used as an extraction raw material, so the method has the advantages of changing the waste into the valuable and increasing the resource utilization rate of cordyceps militaris; and produced cordycepin extract products with different purities can be used in the medicine industry or functional food industry.
Description
Technical field
The invention belongs to medicine industry or functional food industrial technical field, relate to a kind of method of extraction purification cordycepin from Cordyccps-militaris-(L.)-link. Sporophore or its substratum.
Background technology
Cordyceps militaris (L.) Link. is called for short Cordyceps militaris, and also claim Cordyceps militaris (L.) Link. or pupa grass, be the medicinal fungi that entomogenous fungi combines, modern Valuable Herbal Medicine, has curative effects such as " Gan Pingbao lung, kidney-nourishing, benefit marrow, hemostasis and phlegms ".In recent years, the demand of domestic and international market to Cordyceps sinensis is increasing, and cause wild cordyceps resource to become very rare due to overexploitation, price also rises steadily.Given this, the concern of the research of the cultivation of artificial Chinese caterpillar fungus, the aspect such as extraction purification and application and development more and more extremely enterprise and scholar.Cordycepin is that Cordyceps militaris (L.) Link. is most representative effectively or one of activeconstituents, and have the effects such as antitumor, antiviral, antifatigue, the main source of natural cs element is Cordyceps militaris (L.) Link..Polymeric adsorbent starts in late 1970s the extraction and isolation being applied to Chemical Constituents of Chinese Traditional And Folk Medicine, China is mainly used in the purification of medicine industry and natural active matter and the separation and purification of Chinese herbal medicine effective ingredients, in the separation and purification of cordycepin, have ion exchange resin absorption, charcoal absorption, alumina column chromatography, silica gel column chromatography and supercritical fluid extraction etc., but the research that application mix resin carries out separation and purification is still rare.Its physicals of different polymeric adsorbents (size in specific surface area and aperture), and resin structure (functional group etc.) aspect may be different, its to target compound as cordycepin the absorption that shows and desorption performance just different.The resin of different performance, type mixes by the present invention, is reached the object of separation and purification cordycepin, improve the content of cordycepin in extracting solution medicinal extract by hybrid resin absorption and wash-out.
Research shows, still containing some activeconstituentss in the solid deserted medium of Cordyceps militaris (L.) Link. after having cultivated, wherein the content of cordycepin is suitable with the content of cordycepin in sporophore, but at present except a small amount of residue of cultivating is used as except feed, is most ofly discarded by the producer.Therefore, research is simple and effective ground extraction purification cordycepin isoreactivity composition from solid deserted medium how, to raising Cordyceps militaris (L.) Link. comprehensive resource utilization rate, promotes that the exploitation of Cordyceps militaris (L.) Link. high added value byproduct are significant.
Summary of the invention
Utilize the research of macroporous adsorbent resin or ion exchange resin separation and purification cordycepin more at present, but it is less to utilize hybrid resin to carry out the report of separation and purification, the invention provides a kind of method utilizing hybrid resin separation and purification cordycepin.It is characterized in that: can Cordyccps-militaris-(L.)-link. Sporophore be not only raw material, also can Cordyceps militaris (L.) Link. solid medium or cultivation complete after solid deserted medium be raw material, reduce the cost producing cordycepin, improve comprehensive resource utilization rate, improve ecotope.Adopt hybrid resin adsorption-desorption, the process for purification such as binding silica gel column chromatography, can prepare highly purified cordycepin product.
The method utilizing hybrid resin separation and purification cordycepin provided by the invention, concrete steps are as follows:
1. raw materials pretreatment is with Cordyccps-militaris-(L.)-link. Sporophore or Cordyceps militaris (L.) Link. solid medium for raw material, and vacuum-drying at 60 DEG C, grinding and sieving, chooses 60-100 order sample particle, prepares skimming treatment.
2. degreasing is owing to containing the nutritive substances such as albumen, fat or nutrient solution in Cordyccps-militaris-(L.)-link. Sporophore or solid medium, extracts cordycepin again after need carrying out skimming treatment.The sherwood oil adding 10-30 times amount in the substratum particle after drying and crushing carries out backflow degreasing 1-3h, or the degreasing 1-3h that vibrates in constant temperature oscillator at 50 DEG C, and degreasing sample is air-dry rear for subsequent use after filtration.
3. cordycepin extracts and takes a certain amount of Cordyccps-militaris-(L.)-link. Sporophore after pretreatment or solid medium, add the extracting solution of 8 ~ 30 times amount, Extraction solvent is pure water or aqueous ethanolic solution (ethanol content 10-70%, v/v), cordycepin extraction is carried out with microwave, ultrasonic wave, constant temperature oscillation or hydro-thermal circumfluence method.Parallel extraction 2 times, united extraction filtrate, as being Aqua pure extract liquid, then alcohol precipitation removing polysaccharide, decompression recycling ethanol, thin up constant volume, for subsequent use as cordycepin crude extract.
4. hybrid resin purifying takes several macroporous adsorbent resin of filtering out in advance or ion exchange resin respectively, carries out pre-treatment to resin, organic solvent residual in removing production of resins process.Several resins good for pre-treatment are carried out by a certain percentage mix rear filling resin chromatography post, or the difference of the absorption property such as the polarity of pressing resin, resin column is loaded in layering, the upper prop adsorption flow rate of control cordycepin crude extract is 2-10BV/h(BV is resin bed volume), the content of cordycepin in upper prop absorption raffinate is detected with high performance liquid chromatograph (be called for short HPLC, join UV-detector or photodiode array detector).Upon adsorption reach balance after, add 10-70% ethanolic soln (macroporous adsorbent resin) or 0.05-1.0mol/L ammonia ethanolic soln (ion exchange resin, ethanol content 10-70%) carry out wash-out, eluent flow rate 1-3BV/h, every 1-2BV collects a elutriant, the content of cordycepin in elutriant is detected, determined wavelength 259nm with HPLC.
5. concentrate drying presses HPLC detected result, elution fraction higher and close for cordycepin content in hybrid resin bed elutriant is merged, underpressure distillation removing ethanol, be concentrated near dry, vacuum-drying at 60 DEG C, can obtain the medicinal extract that cordycepin content is 20-40%.
6. refining extract medicinal extract after concentrate drying, is further purified refining with methods such as organic solution crystallization such as silica gel column chromatography, polyamide column chromatography or ethanol, and after dry, obtained cordycepin content reaches the extract product of more than 90%.
The cordycepin product of the different purity adopting the present invention to produce, can be used for the suitability for industrialized production of medicine industry or functional food, is especially applicable to Chinese caterpillar fungus manufacturer lengthening manufacturing chain, improves resource utilization, increases added value of product.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates that embodiment 1 rice cultivates that residue extracts stoste, and in collection of illustrative plates, 3.70min goes out the compound at peak is cordycepin.
Fig. 2 is the HPLC collection of illustrative plates of embodiment 1 elutriant after hybrid resin separation and purification, and wherein 3.22min goes out the compound at peak is adenosine (structure is close with cordycepin), and what 3.65min went out peak is cordycepin.
Fig. 3 is the uv absorption spectra (photodiode array detector obtains at 200-400nm range scans) of cordycepin reference material, showing cordycepin maximum absorption wavelength in figure is 208nm and 259nm, because 208nm is in ultraviolet stub area and the interference of solvent background is large, therefore 259nm is selected to detect cordycepin content.
Embodiment
Below in conjunction with accompanying drawing and preferred case study on implementation, the present invention is set forth further, its object is to understand content of the present invention better, but not limit the scope of the invention.
embodiment 1
1. degreasing: the sherwood oil adding 15 times amount in the Cordyceps militaris (L.) Link. rice medium after sieving to drying and crushing, vibrate at 50 DEG C degreasing 2h, for subsequent use after reclaiming solid drying.
2. extract: the water adding 25 times of volumes in the substratum after skimming treatment, hydro-thermal refluxing extraction 2h, filter, filter residue repeats to extract once, merges extracted twice liquid, discards solid filter residue.
3. alcohol precipitation: add ethanol in extracting solution and carry out alcohol precipitation, removing polysaccharide, with water dilution constant volume after decompression recycling ethanol, makes crude extract.
4. hybrid resin purifying: two kinds of macroporous adsorbent resin WYU-005 and WYU-006 filtered out in advance are carried out pre-treatment, then entirely mix in the ratio of resin dry weight 1:1, the resin mixed loads in the glass chromatography column of internal diameter 10mm in a wet process, with the extracting solution of 5BV/h flow velocity absorption (2) step, 25% aqueous ethanolic solution wash-out is added after having adsorbed, elution flow rate 2BV/h, the content of cordycepin in absorption raffinate and elutriant is detected with HPLC, the part that in merging elutriant, cordycepin content is higher also makes medicinal extract, in medicinal extract, the content of cordycepin is 27%.
5. refining: the extract medicinal extract after hybrid resin purifying, dissolved with 95% ethanol, be placed in refrigerator and place one week at 5 DEG C, adularescent crystal is separated out, and suction filtration, at 60 DEG C, vacuum-drying obtains the extract product that cordycepin content is 95%.
embodiment 2
1. degreasing: the sherwood oil adding 30 times amount in the Cordyceps militaris (L.) Link. rice/corn mixed culture medium after sieving to drying and crushing, vibrate at 50 DEG C degreasing 1.5h, for subsequent use after reclaiming solid drying.
2. extract: take the solid medium after a certain amount of degreasing, add 40% ethanolic soln of 20 times amount, microwave extraction 1.5min under 240W power, filter, filter residue repeats extraction 1 time, merges extracted twice liquid, with water dilution constant volume after decompression recycling ethanol, make crude extract.
3. hybrid resin purifying: by the three kinds of macroporous adsorbent resin WYU-005 filtered out in advance, WYU-004, after WYU-006 carries out pre-treatment, layering filling is carried out by the resin dry ratio of 1:2:1, be followed successively by WYU-005 from top to bottom, WYU-004, WYU-006, wet method loads in the glass chromatography column of internal diameter 20mm, by this layering hybrid resin post on (2) step microwave extraction liquid, elutriant is the ethanolic soln of 35%, absorption and elution flow rate are 3BV/h, the content of cordycepin in absorption raffinate and elutriant is detected with HPLC, the part that in merging elutriant, cordycepin content is higher also makes medicinal extract, in medicinal extract, the content of cordycepin is 33%.
4. refining: the extract medicinal extract after hybrid resin purifying, refine with silica gel column chromatography again, collect the cordycepin elutriant after silica gel adsorption, the content of cordycepin in elutriant is detected with HPLC, merge the part that in elutriant, cordycepin content is higher, decompression removing eluting solvent, vacuum-drying at 60 DEG C, obtains the extract product that cordycepin content is 92%.
embodiment 3
1. degreasing: the sherwood oil adding 25 times amount in the Cordyccps-militaris-(L.)-link. Sporophore (cordyceps flower) after sieving to drying and crushing, reflux degreasing 2h, filter, solid is air-dry rear for subsequent use.
2. extract: take the Cordyceps sporophore after a certain amount of degreasing, add 50% ethanolic soln of 15 times amount, supersound extraction 30min under ultrasonic frequency 45kHz, temperature 60 C, filter, filter residue repeats extraction 1 time, merges extracted twice liquid, with water dilution constant volume after decompression recycling ethanol, make crude extract.
3. hybrid resin purifying: by the two kinds of macroporous adsorbent resin WYU-005 filtered out in advance, after WYU-004 and a kind of ion exchange resin WYU-002 carries out pre-treatment respectively, layering filling is carried out by the resin dry ratio of 1:1:2, be followed successively by WYU-005 from top to bottom, WYU-004, WYU-002, wet method loads in the glass chromatography column of internal diameter 18mm, by this layering hybrid resin post on (2) step supersound extraction liquid, successively with 40% ethanolic soln and 0.2mol/L ammonia ethanolic soln (ethanol content 70%) carry out wash-out, adsorption liquid flow velocity is 6BV/h, eluent flow rate 2BV/h, the content of cordycepin in absorption raffinate and elutriant is detected with HPLC, the part that in merging elutriant, cordycepin content is higher also makes medicinal extract, in medicinal extract, the content of cordycepin is 35%.
4. refining: the extract medicinal extract after hybrid resin purifying, refine with Thin-layer chromatography again, collect the cordycepin elutriant after adsorption cleaning, the content of cordycepin in elutriant is detected with HPLC, merge the part that in elutriant, cordycepin content is higher, decompression removing eluting solvent, vacuum-drying at 60 DEG C, obtains the extract product that cordycepin content is 94%.
Claims (9)
1. one kind utilizes the method for hybrid resin technology separation purification of cordycepin, it is characterized in that: with Cordyccps-militaris-(L.)-link. Sporophore or its solid medium for raw material, after drying, pulverizing, degreasing, extraction, hybrid resin purifying and drying, obtain the extract medicinal extract that cordycepin content is 20-40%, be further purified refining rear cordycepin purity and reach more than 90%.
2. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, is characterized in that, described raw material is Cordyccps-militaris-(L.)-link. Sporophore or Cordyceps militaris (L.) Link. solid medium, or sporophore gather after remain discarded solid deserted medium.
3. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, it is characterized in that, described solid medium is using food crop such as rice, wheat or corns as substratum major ingredient, or wherein two or more by different ratios mixing after carry out Artificial culture as major ingredient.
4. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, it is characterized in that, described extracting method is the methods such as microwave extraction, ultrasonic extraction, constant temperature oscillation extraction or hydro-thermal refluxing extraction, and extracting solid-liquid ratio is 1:8 ~ 1:30(raw materials quality/extracting liquid volume).
5. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, is characterized in that, described Extraction solvent is water or aqueous ethanolic solution, its ethanol content 10-70%.
6. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, it is characterized in that, described hybrid resin is that two or more macroporous adsorbent resin or ion exchange resin mix by different ratios (1:1 ~ 1:10), or macroporous adsorbent resin and ion exchange resin press the hybrid resin of different ratios (1:1 ~ 1:10).
7. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, it is characterized in that, the hybrid mode of described hybrid resin is layering mixing (namely different resins is filled into resin bed in certain sequence successively), or full mixing (namely each resin all mixes rear entirety loading resin bed).
8. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, it is characterized in that, with 10-70% ethanolic soln (macroporous adsorbent resin) or 0.05-1.0mol/L ammonia ethanolic soln (ion exchange resin after described hybrid resin has adsorbed, alcohol concn 10-70%) carry out wash-out as elutriant, the dried medicinal extract cordycepin content of elutriant is 20-40%.
9. a kind of method utilizing hybrid resin separation and purification cordycepin according to claim 1, it is characterized in that, extract medicinal extract after described hybrid resin purifying, the method of further polishing purification is silica gel column chromatography, polyamide column chromatography or ethanolic soln recrystallization etc., and after refining, cordycepin purity reaches more than 90%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410322498.3A CN105273024A (en) | 2014-07-08 | 2014-07-08 | Method for separating and purifying cordycepin by using mixed resins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410322498.3A CN105273024A (en) | 2014-07-08 | 2014-07-08 | Method for separating and purifying cordycepin by using mixed resins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105273024A true CN105273024A (en) | 2016-01-27 |
Family
ID=55142917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410322498.3A Pending CN105273024A (en) | 2014-07-08 | 2014-07-08 | Method for separating and purifying cordycepin by using mixed resins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105273024A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317148A (en) * | 2016-07-29 | 2017-01-11 | 河北省科学院生物研究所 | Method for extracting cordycepin from cordyceps militaris |
| WO2020177420A1 (en) * | 2019-03-01 | 2020-09-10 | 江南大学 | Ultrasonic composite acidic water extraction method for cordyceps polysaccharide and cordycepin in cordyceps militaris |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008280271A (en) * | 2007-05-09 | 2008-11-20 | Oriza Yuka Kk | Agent for preventing photoaging of skin |
| 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 |
| CN101906127A (en) * | 2010-07-23 | 2010-12-08 | 上海国宝企业发展中心 | Method for extracting cordycepin and cordyceps polysaccharides from residue of cordyceps militaris culture medium |
-
2014
- 2014-07-08 CN CN201410322498.3A patent/CN105273024A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008280271A (en) * | 2007-05-09 | 2008-11-20 | Oriza Yuka Kk | Agent for preventing photoaging of skin |
| 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 |
| CN101906127A (en) * | 2010-07-23 | 2010-12-08 | 上海国宝企业发展中心 | Method for extracting cordycepin and cordyceps polysaccharides from residue of cordyceps militaris culture medium |
Non-Patent Citations (2)
| Title |
|---|
| 曾昱，等: "大孔树脂柱层析法提取蛹虫草废弃培养基中的虫草素", 《湖南师范大学自然科学学报》 * |
| 李辰，等: "蛹虫草废弃培养残基中虫草素的提取工艺研究", 《五邑大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317148A (en) * | 2016-07-29 | 2017-01-11 | 河北省科学院生物研究所 | Method for extracting cordycepin from cordyceps militaris |
| CN106317148B (en) * | 2016-07-29 | 2019-02-19 | 河北省科学院生物研究所 | A method of extracting cordycepin from Cordyceps militaris |
| WO2020177420A1 (en) * | 2019-03-01 | 2020-09-10 | 江南大学 | Ultrasonic composite acidic water extraction method for cordyceps polysaccharide and cordycepin in cordyceps militaris |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103333067B (en) | A kind of extracting method of high-purity chlorogenic acid | |
| CN103087128B (en) | A kind of method extracting peoniflorin from the peony seeds dregs of rice | |
| CN101961371B (en) | Method for extracting and separating ginsenoside, flavone and polysaccharide from sweet gynostemma pentaphylla | |
| CN101747196A (en) | Method for preparing chlorogenic acid by using Jerusalem artichoke | |
| CN101979400A (en) | Method for comprehensively extracting tea saponin and tea polysaccharide from camellia oil seed cake or tea seed cake | |
| CN104177370B (en) | A kind of method preparing high-load sesamin from sesame seed meal | |
| Sato et al. | Improvement in the productivity of xylooligosaccharides from waste medium after mushroom cultivation by hydrothermal treatment with suitable pretreatment | |
| CN103239489B (en) | Process for extracting and purifying punicosides | |
| CN102898341A (en) | Extracting and purifying method of high-purity sulforaphane | |
| CN105693781A (en) | Technology for extracting tea polyphenol in tea leaves | |
| CN102199177A (en) | Manufacturing method of pure natural high-purity stevioside | |
| CN102212144A (en) | Method for preparing pure polysaccharose from alfalfa hay | |
| CN107098942B (en) | Method for subcritical water extraction of kaempferitrin in radish leaves | |
| CN101856427B (en) | Method for extracting solanine from potato peels | |
| CN101961445A (en) | Method for simultaneously extracting various bioactive components from Chinese yam by using subcritical water as medium | |
| CN105273024A (en) | Method for separating and purifying cordycepin by using mixed resins | |
| CN1927857A (en) | Extraction and purification process of buckwheat sprout total flavone | |
| CN111205300B (en) | Method for extracting sesamin from sesame meal | |
| CN102276455B (en) | Method for extracting effective ingredients from dry cinnamomum burmannii fruits | |
| CN108997359B (en) | Method for extracting chlorophyll from stevioside production waste residues | |
| CN107988280B (en) | Method for extracting high-purity isothiocyanate from cruciferous vegetable seeds | |
| CN106701319A (en) | Method for extracting taxus chinensis essential oil | |
| CN108148108B (en) | Method for extracting limonin from lemon peel residues by guanidine salt ionic liquid through two aqueous phases | |
| CN102499377A (en) | Method for preparing apple polyphenol by using sparse dropped apples | |
| CN102936271A (en) | Method for extracting cordycepin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160127 |