CN103772460B - The extraction process of cordycepin in silkworm Chinese caterpillar fungus - Google Patents
The extraction process of cordycepin in silkworm Chinese caterpillar fungus Download PDFInfo
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- CN103772460B CN103772460B CN201410051001.9A CN201410051001A CN103772460B CN 103772460 B CN103772460 B CN 103772460B CN 201410051001 A CN201410051001 A CN 201410051001A CN 103772460 B CN103772460 B CN 103772460B
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Abstract
The invention discloses the extraction process of active substance cordycepin in silkworm Chinese caterpillar fungus.A kind of natural nucleus glucoside new drug that Chinese caterpillar fungus prime system extracts from Cordyceps sinensis, has the activity of Tumor suppression, antiplasmodial, antimycotic, anti-H IV-I C-type virus C and Selective depression clostridium, causes the great attention of medicine sector.Though but it can synthetic, also can extract from Cordyceps sinensis, output is very low, expensive, at present its extract, purification process not only troublesome poeration, and toxic organic solvents can bring pollution, not easily produces in enormous quantities.The present invention adopts aqueous two-phase system to extract silkworm Chinese caterpillar fungus cordycepin, and not only extractive content is high, and extraction yield is high, and saves time, laborsaving, and applicable industrialization is extracted on a large scale.
Description
Technical field
The present invention relates to the field of Chinese medicines, relate to a kind of extraction process of active Chinese drug component material.
Background technology
Cordyceps militaris (L.) Link. (Cordycepsmilitaris) is also known as Cordyceps militaris or Cordyceps militaris (L.) Link., and being a kind of not only edible but also pharmaceutically useful fungi of generally acknowledging both at home and abroad, is the another kind of Chinese caterpillar fungus being different from Cordyceps sinensis parasitized on the pupal cells such as Noctuidae.Current Cordyceps militaris generally adopts silkworm chrysalis to make host or rice etc. and cultivates as substratum.It is new resource food that health ministry ratifies Cordyceps militaris (L.) Link. in March, 2009.Silkworm Chinese caterpillar fungus also claims silkworm Chinese caterpillar fungus or silkworm Chinese caterpillar fungus, is the entomogenous fungi combination with artificial culture on wild Natural C.militaris (Cordycepsmilitaris) bacterial strain spore inoculating to 5 age silkworms.
Research confirm, silkworm Chinese caterpillar fungus is compared with silkworm chrysalis grass, and cordycepic acid is close, and Cordyceps polysaccharide is 2.5 times of silkworm chrysalis grass, and cordycepin is then its 4 times, only have adenosine be its 1/2; Silkworm Chinese caterpillar fungus is compared with Cordyceps sinensis, and cordycepin is hundreds of times of Cordyceps sinensis, and adenosine is also its 4 times, and Cordyceps polysaccharide and adenosine are then its about 1/2.Above comparative result illustrates, the active component content of silkworm Chinese caterpillar fungus is better than silkworm chrysalis grass, is also better than Cordyceps sinensis simultaneously, especially the content of cordycepin far above silkworm chrysalis careless and Cordyceps sinensis.Visible silkworm Chinese caterpillar fungus is the optimum feed stock that cordycepin extracts.
Cordycepin formal name used at school is cordycepin, and foreign language name is called 3 '-deoxyadenosine, is a kind of natural nucleus glucoside new drug extracted from Cordyceps sinensis, and 20 century 70s are found to have the effect of Tumor suppression, antiplasmodial and suppression mRNA translation; The nineties in 20th century, research found, added the expression of adenosine deaminase inhibitors to its anti-tumor activity and played an important role.Cordycepin can disturb the synthesis of gene cell RNA and DNA, the division of the abnormal cell such as anticancer, and can as the instrument of RNA polymerase different in discriminate between cells; Meanwhile, cordycepin also shows the activity of extremely strong antimycotic, AntiHIV1 RT activity-I C-type virus C and Selective depression clostridium, causes the great attention of medicine sector, cordycepin the U.S. as anticancer, that new antiviral drug entered for three phases is clinical.
Only report that both at home and abroad cordycepin can synthetic, also can extract from Cordyceps sinensis, but output is very low, expensive.Current cordycepin extracts, purification process mainly contains: the methods such as solvent extraction-column chromatography purification, ultrasonic enhanced extraction-column chromatography purification, supercritical fluid extraction-ultrasonic wave purifying.But solvent extraction-column chromatography purification, ultrasonic enhanced extraction-column chromatography purification method needs upper prop, also need decolouring, not only troublesome poeration, and toxic organic solvents can bring pollution; Although supercritical fluid extraction-ultrasonic wave purifying does not carry out chemical reaction, do not generate objectionable impurities, required equipment is special, not easily produces in enormous quantities.
Summary of the invention
The object of the invention is to provide the extraction process of cordycepin in a kind of silkworm Chinese caterpillar fungus, this operational path is simple to operate, efficiency is high, both decreased upper prop in old technology, decolour bring troublesome poeration, turn avoid the toxic organic solvents that brings in upper prop process and pollute, and can carry out producing in enormous quantities, improving yield, thus reduce cost, belong to green technology and new cleanproduction process, solve Problems existing described in background technology.
The present invention seeks to be realized by technical scheme below:
The extraction process of cordycepin in silkworm Chinese caterpillar fungus, its technique is:
1) silkworm Chinese caterpillar fungus through 65 DEG C dry after, be placed in totally-enclosed powder equipment, adopt at a certain temperature mechanical presses, clash into the mode of solids component emulsification is pulverized;
2) by the silkworm Chinese caterpillar fungus powder after pulverizing with get 6-10 doubly after the 10-50% polyethylene glycol 6000 aqueous solution of bulk drug weight fully dissolves, be salt in mass ratio: polyethylene glycol 6000=0.5-3:1 adds salt and fully vibrates, static phase-splitting, centrifugation, goes its throw out to obtain supernatant liquid;
3) by step 2) after the supernatant liquid that obtains uses ethyl acetate and petroleum ether successively, vacuum-drying, obtains cordycepin.
Technical solution of the present invention adopts aqueous two-phase system to extract silkworm Chinese caterpillar fungus cordycepin, be different from the extracting method of cordycepin routine, Chinese patent 2012510084508.5 discloses a kind of method of Hydrolysis kinetics cordycepin from Cordyccps-militaris-(L.)-link. Sporophore, the technical scheme adopted is: add alcohol reflux after being pulverized by Cordyccps-militaris-(L.)-link. Sporophore, filtrate is upper silicagel column elution after n-butanol extraction, elutriant crystallizing at room temperature after concentrated cordycepin, disclosed in this method and Chinese patent 00130376.7, cordycepin extracting method is similar.Existing document " Productionofcordycepinbysurfacecultureusingthemedicinalm ushroomCordycepsmilitaris " (MinaMasuda, ErikoUrabe, " EnzymeandMicrobialTechnology " the 01st phase in 2006) adopt Dowex-I-OH-1 resin column to be separated from the culturing filtrate of Cordyceps militaris (L.) Link. to obtain cordycepin.US Patent No. 8008060 discloses a kind of method of Isolation and purification cordycepin from solid culture medium residue of cordyceps militaris and prepares the method for cordycepin dry powder, and adopt microwave method to extract, what drastically increase cordycepin slightly raises rate, reaches more than 75%.In above-mentioned document, ordinary method is to the extraction and purification process of cordycepin, cumbersome, and extraction yield is not high.
The present invention uses aqueous two-phase system, the wetting ability superpolymer utilizing two kinds of chemical structures different or a kind of wetting ability superpolymer and inorganic salt in water with two immiscible aqueous phase systems that suitable concentration is formed, utilize the difference of polysaccharide in system, protein and other biomolecules partition ratio between two aqueous phases, and make respective different solubility, thus be separated.Present invention process mild condition, the treatment time is short, aftertreatment is easy, inactivation and the sex change of bio-active substance can not be caused, eliminate again suck tissue and partial invalidity composition, extend the shelf lives, improve purity and extraction yield, approach is extracted in the new industrialization providing a kind of activeconstituents, this method saves time, laborsaving, extractive content is high, and extraction yield is high.
Embodiment
Be further elaborated the present invention below with reference to specific embodiment, these examples only for illustration of object, and are not used in the restriction scope of the invention.
Embodiment 1
Taking a certain amount of polyethylene glycol 6000 is dissolved in deionized water, according to table 1 obtain solution, for subsequent use.After silkworm Chinese caterpillar fungus is dried through 65 DEG C, be placed in totally-enclosed powder equipment, adopt at 25 DEG C mechanical presses, clash into the mode of solids component emulsification is pulverized, silkworm Chinese caterpillar fungus after pulverizing is taken according to proportional in table 1, after fully dissolving with the polyethylene glycol 6000 aqueous solution, add ammonium citrate in the ratio in table 1 and fully vibrate, static phase-splitting, centrifugation, goes its throw out to obtain supernatant liquid; After gained supernatant liquid ethyl acetate and sherwood oil wash respectively, vacuum-drying, obtains cordycepin.
Embodiment 2 extraction process compares
Silkworm Chinese caterpillar fungus through 65 DEG C dry after, be placed in totally-enclosed powder equipment, adopt at 25 DEG C mechanical presses, clash into the mode of solids component emulsification is pulverized, by pulverize after silkworm Chinese caterpillar fungus prepare cordycepin according to following different methods extraction purification.
Sample 1 (refluxing extraction-column chromatography purification) (method is with reference to Chinese patent 00130376.7): get silkworm Chinese caterpillar fungus 400 parts, add 5 times amount 30% ethanol (temperature 70 C), soak 48 hours, by reflux on the suspension after immersion, reflux 2 hours, reclaim ethanol, aqueous precipitation, adjust pH3.5, filter after 24 hours, liquid after recovery is dissolved in water and carries out refrigeration precipitation, temperature 4 DEG C, 24 hours time.Carrying out the liquid millipore filtration of cold postpone filtering (0.85 μm) filtrate adjusts 4.0 for subsequent use with 0.1mol/L, upper ion changes post, collect elutriant, elutriant is carried out be evaporated to thick, by the liquid of thickness in 4 DEG C, cold putting makes it crystallization in 24 hours, filter, above-mentioned filtrate adds appropriate propyl carbinol, put 4 DEG C, cold putting makes it crystallization in 24 hours, filter, filtrate processes 2-3 time so repeatedly, the crystallization of filtration gained is put in freeze drier and carries out drying, after measuring, determine that cordycepin extraction yield is 5.387% after drying, the cordycepin purity obtained is 94%.
Sample 2 (ultrasonic extraction-column chromatography purification): get silkworm Chinese caterpillar fungus 400 parts, add 5 times amount 30% ethanol (temperature 70 C) mixing, and stir in agitator, then slowly container is flowed into through pipeline, the tube wall of this pipeline is provided with ultrasonic generator, he carries out sonic oscillation break process to the mixture of sluggish flow in pipeline, treatment time is 30 minutes, channel separator centrifugal (16000r/min), get supernatant liquor, be evaporated to extracting solution 1ml containing 1g crude drug, upper ion changes post, collect elutriant, elutriant is carried out be evaporated to thick, by the liquid of thickness in 4 DEG C, cold putting makes it crystallization in 24 hours, the crystallization of separating out is put in freeze drier and is carried out drying, after measuring, determine that cordycepin extraction yield is 62.18% after drying, the cordycepin purity obtained is 36%.
Sample 3 (supercritical fluid extraction-organic solvent purifying) (method is with reference to Chinese patent 01126910.3): get silkworm Chinese caterpillar fungus 0.75kg, load in extraction kettle, adopt supercritical carbon dioxide extraction method, condition: 50 DEG C, 35Mpa, 20L/h, balances flowing extraction after 15 minutes.Be separated after extraction, enter separating still, separating still pressure 6Mpa, 30 DEG C, sample watery thing is about 40ml (lower floor) after 2 hours: upper strata is oily matter.Point adding each 200ml of 10% ethanol for three times makes entrainment agent, and each extraction 1-2 hour, extraction liquid about 540ml, merge and add remove impurity with active carbon, vacuum concentration, obtain faint yellow granular substance 235mg, cordycepin content 75.05%, yield about 16.79%.Cordycepin content can be brought up to 98% with after organic solvent purifying, yield reduces to 8.09%.
Sample 4 (polyvidone-ammonium citrate two-phase water system): take polyvidone 6000 and be dissolved in deionized water, be formulated as the solution that concentration is 20%, for subsequent use.Getting silkworm Chinese caterpillar fungus 400 parts, add after 6 times of volume polyvidone water liquid fully dissolve, is that 2:1 adds ammonium citrate and fully vibrates by ammonium citrate/polyvidone 6000 mass ratio, static phase-splitting, and centrifugation, goes its throw out to obtain supernatant liquid; After gained supernatant liquid ethyl acetate and sherwood oil wash respectively, vacuum-drying, obtains precipitation, determines that cordycepin extraction yield is 57.29% after measured afterwards, and the cordycepin purity obtained is 78.31%.
Sample 5 (polyoxyethylene glycol-ammonium sulfate two-phase water system): take polyethylene glycol 6000 and be dissolved in deionized water, be formulated as the solution that concentration is 20%, for subsequent use.Getting silkworm Chinese caterpillar fungus 400 parts, add after 6 times of volume polyoxyethylene glycol water liquid fully dissolve, is that 2:1 adds ammonium sulfate and fully vibrates by ammonium sulfate/polyethylene glycol 6000 mass ratio, static phase-splitting, and centrifugation, goes its throw out to obtain supernatant liquid; After gained supernatant liquid ethyl acetate and sherwood oil wash respectively, vacuum-drying, obtains precipitation, determines that cordycepin extraction yield is 53.29% after measured afterwards, and the cordycepin purity obtained is 88.31%.
Result obviously visible the inventive method extraction saves time most, and laborsaving, the potency ratio of extraction yield and purity is the highest, and the method is that industrialization extraction cordycepin provides new way.
Embodiment 3 sets up the method measuring cordycepin content
Chromatographic condition chromatographic column: KnauerEuropher – 100C18 (5 μm, 4.6 × 250mm); Moving phase: Yi Jing – 0.05mol/LKH
2pO
4damping fluid (7.5:92.5); Flow velocity: 1mL/min; Determined wavelength: 260nm; Column temperature: 25 DEG C; Sample size: 10 μ L.
The selection of determined wavelength gets cordycepin reference substance in right amount, adds methyl alcohol and makes the solution of every 1mL containing cordycepin 0.2mg, measure by above-mentioned chromatographic condition.Extract reference substance ultraviolet spectrogram visible, cordycepin has maximum absorption at 259.3nm place, selects wavelength 260nm to measure cordycepin so final.
It is appropriate, accurately weighed that the preparation of reference substance solution is taken at the cordycepin reference substance being dried to constant weight at 105 DEG C, puts in 10mL volumetric flask, add dissolve with methanol and be diluted to scale, shaking up.Precision measures 1mL, puts in 50mL volumetric flask, adds methanol dilution to scale, shakes up, and makes the solution of every 1mL containing cordycepin 30 μ g, product solution in contrast.
Linear relationship investigates accurate absorption reference substance solution 1 respectively, 5,10,15,20 μ L inject high performance liquid chromatograph, measure, with the sample size of reference substance solution (ng) for X-coordinate according to above-mentioned chromatographic condition, peak area (A) is ordinate zou drawing standard curve, calculates regression equation.
The extract prepared is got in the preparation of test liquid, every part of about 0.3g, accurately weighed, puts and is dried in the tool plug Erlenmeyer flask of constant weight, adds 20% methyl alcohol 30 times, supersound extraction 10min.Supersound extraction, takes out, is cooled to room temperature, shakes up, centrifugal, gets supernatant liquor 0.45 μm of millipore filtration and filters, discard just filtrate, get subsequent filtrate, to obtain final product.Accurate absorption reference substance solution and each 10 μ L of need testing solution measure, and calculate the content of cordycepin in extract with external standard method (peak area).
The discriminating of embodiment 4 extract and confirmation
Get this product powder 0.5g, accurately weighed, put in tool plug Erlenmeyer flask, precision adds 20% ethanol 10mL, close plug, and supersound extraction 30min, shakes up, and leaves standstill, and filters, gets subsequent filtrate, as need testing solution.Separately get adenosine, VITAMIN B4 and cordycepin reference substance, add 20% ethanol and make the solution of every 1mL containing adenosine 0.284mg, VITAMIN B4 0.1mg, cordycepin 0.39mg, product solution in contrast.Test according to tlc (China's coastal port annex VIB), draw need testing solution, each 10 μ L of reference substance solution, put respectively in the same carboxymethylcellulose sodium solution containing 4% Sodium phosphate dibasic be on the silica GF254 thin layer plate of tamanori, with Yi Suan Yi Zhi – Bing Tong – strong aqua (4:4:1) for developping agent, launch, take out, dry, inspect under putting ultraviolet lamp (254nm).In trial-product chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
Claims (2)
1. the extraction process of cordycepin in silkworm Chinese caterpillar fungus, its technique is:
1) silkworm Chinese caterpillar fungus through 65 DEG C dry after, be placed in totally-enclosed powder equipment, adopt at a certain temperature mechanical presses, clash into the mode of solids component emulsification is pulverized;
2) by the silkworm Chinese caterpillar fungus powder after pulverizing with get 6-10 doubly after the 10-50% polyethylene glycol 6000 aqueous solution of bulk drug weight fully dissolves, be salt in mass ratio: polyethylene glycol 6000=0.5-3:1 adds salt and fully vibrates, static phase-splitting, centrifugation, goes its throw out to obtain supernatant liquid;
3) by step 2) after the supernatant liquid that obtains uses ethyl acetate and petroleum ether successively, vacuum-drying, obtains cordycepin;
The above salt is (NH
4)
2sO
4or ammonium citrate.
2. the extraction process of cordycepin in silkworm Chinese caterpillar fungus as claimed in claim 1, is characterized in that described processing step 1) in certain temperature be-50 DEG C ~ 30 DEG C.
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| CN104327139B (en) * | 2014-09-28 | 2017-02-22 | 西北农林科技大学 | Preparation method of cordycepin crystal |
| CN104311614B (en) * | 2014-11-05 | 2016-07-20 | 桂林益元素生物科技有限公司 | A kind of method extracting Mengiferin from Folium mangiferae |
| CN106279455B (en) * | 2015-06-07 | 2019-02-26 | 山东建筑大学 | A kind of comprehensive method for extracting cordycepin and Cordyceps sinensis polysaccharide in Cordyceps militaris of aqueous two-phase |
| CN114990183B (en) * | 2022-06-11 | 2023-11-21 | 江苏科技大学 | Method for preparing cordycepin and antihypertensive peptide based on rice cordyceps militaris fruiting body |
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