CN101229199B - Integrative extract method of multi-active ingredient in cordyceps militaris mycelium - Google Patents
Integrative extract method of multi-active ingredient in cordyceps militaris mycelium Download PDFInfo
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Abstract
The invention provides a comprehensive method of extracting various effective components from cordyceps militaris. The dry and crushed cordyceps militaris is extracted and filtered with organic solvent to get filtrate and leftover; the filtrate is condensed to recycling the solvent, and the condensed liquor after being saponified, extracted and dried can present aweto sterols; the leftover is extracted and filtered, and the sediment and supernatant are obtained after condensing, alcohol sinking and centrifugating the filtrate; cordycepin polysaccharide is obtained through operations such as dissolving the sediment into water and removing the albumen; the supernatant is processed by methods such as ion-exchange resin column and crystallization to obtain cordycepin. The method is material saving, with high purity of the extracts, low cost and simple operation, thus realizing the comprehensive extraction of various effective components from cordyceps militaris.
Description
Technical field
The present invention relates to extraction of effective components in the Chinese herbal medicine, particularly the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium.
Background technology
Cordyceps is the edible and medicinal fungi that a class has remarkable medicinal health care function, and wherein foremost is Cordyceps (Cordyceps sinensis).Because the Cordyceps habitat is special, the host is single-minded, the artificial culture difficulty is very big, and the destruction Of resources that causes is dug in coyoting excessively in addition, makes the price of Cordyceps seriously deviate from value own.That most possible replacement Cordyceps is Cordyceps militaris (Cordyceps militaris) in Cordyceps, claims Cordyceps militaris (L.) Link., Cordyceps militaris (L.) Link. again.Cordyceps militaris is the sibling species of Cordyceps, also is the type sepecies of Cordyceps, and Cordyceps militaris is good medical material and Cordyceps succedaneum.The Cordyceps militaris mycopowder enters clinical as a kind new medicine (Chinese medicine) to obtain National Drug Administration's approval in China, the product sign has invigorating the lung and the kidney, the effect of relieving cough and resolving phlegm, can regulate body's immunity, improve the comprehensive resistance against diseases of body, be mainly used in chronic bronchitis, the treatment of various tumors and other disease of immune system, main cause is to contain great deal of bioactive substances in the Cordyceps militaris, mainly contains Cordyceps sterol (ergosterol, cupreol etc.), Cordyceps polysaccharide, cordycepin and mannitol, adenosine, several amino acids, vitamin and inorganic salt etc.Sterols composition in the Cordyceps has been identified cupreol, ergosterol etc.Sterol can be blocked the absorption of cholesterol, reduces content of cholesterol in body and the blood, prevents the formation of hypercholesterolemia, thereby prevents that atherosclerosis, venothrombotic generation from guaranteeing health; Have the antiinflammatory action that is similar to crovaril or hydrocortisone, can also block the formation that carcinogen brings out cancer, simultaneously good physiological potency is being arranged all aspect skin-nourishing and the protection, have effects such as anti-cancer, treatment, attenuation and nutrition protection in a word.Cordyceps polysaccharide is the highest bioactive substance of content in the Cordyceps; a large amount of medical science confirm; Cordyceps polysaccharide has potentiation to mononuclear phagocyte system; the pair cell immunologic function has regulating action; can strengthen humoral immune function, but activated macrophage stimulates antibody to produce, and improves the human body immunity; and have blood sugar lowering, antitumor and hepatic injury is had protective effect, and can be used as the Radiological Defense agent.Cordycepin claims Cordycepin again, it is first ucleosides antibiotics of separating from fungus, it is a kind of multiple bioactive nucleosides material that has, has antiviral, antibiotic, obviously suppress tumor growth, disturb human body RNA and the synthetic effect of DNA, can be used for clinically treating cancer, leukemia, expansion bronchus being arranged, significantly strengthen adrenal hormone and the anti-ageing effect of waiting for a long time.
Extraction process of effective component in the Cordyceps militaris is more, but nearly all only lay particular emphasis on the one-side extracting method such as cordycepin, Cordyceps polysaccharide and cordycepic acid or simply the compound extraction of cordycepin and Cordyceps polysaccharide is studied, yet to other compositions, as Cordyceps sterol, vitamin, pigment etc., remove or do not consider as impurity and neglected, the utilization of resources is insufficient to cause the huge wasting of resources thereby so just cause.Application number provides a kind of method of continuous extraction effective ingredients from fruitbodies of Cordyceps militaris to relate to the comprehensive extraction of sterols, Cordyceps polysaccharide and cordycepin for 200610023578.4 Chinese patent, but it still has following defective: raw material is a Cordyceps militaris fruiting body, and cost is higher like this; Purification process such as the positive low pressure silica gel chromatography of the supercritical extraction of the sterols that it adopted and cordycepin and preparation type reversed-phase high-performance liquid chromatography, operating procedure is many, complexity etc. too, general medium-sized and small enterprises are difficult to realize; Its sterol for preparing, Cordyceps polysaccharide are not further purified processing, and purity is not high.
Summary of the invention
The combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium of the purpose of this invention is to provide a kind of conservation, extract fully, purity is higher and simple to operate.The technical scheme that adopts is as follows:
(1) extraction of Cordyceps sterol
Pulverize: get fresh cordyceps militaris mycelium and place drying under the environmental condition that is lower than 95 ℃, exsiccant mycelium uses pulverizer to be crushed to Powdered; Described cordyceps militaris mycelium comprises leftover bits and pieces or its mixture after liquid fermentation mycelium, solid culture mycelium, cultivation sporophore and the sporophore results;
Extract: add organic solvent in dry, cordyceps militaris mycelium after pulverizing, add 3～40 milliliters of organic solvents by per 1 gram mycelium, 30～95 ℃ were extracted 0.5～5 hour down, filter, filtrate, repeat to extract 1～4 time; Each filtrate is mixed, and residue is standby; Wherein said organic solvent is a kind of in normal hexane, normal butane, cyclohexane extraction, acetone or the petroleum ether;
Concentrate: filtrate is concentrated into the organic solvent-free flavor under 30～95 ℃ of temperature, normal pressure or decompression, reclaims organic solvent, get concentrate;
Saponification: add 0.2～5 milliliter of alkaline solution in per 1 gram concentrate under the room temperature, mixing left standstill 0.5～24 hour, remove the sub-cloud saponifiable matter, get not saponification liquor, repeat saponification 1～3 time, wherein said alkaline solution is that concentration is 1% to 50% sodium hydroxide or potassium hydroxide solution;
Extraction: add the organic solvent of 0.2～10 times of volume in saponification liquor not, mixing left standstill 0.5～24 hour, divide and get organic solvent layer, extract, re-extract 1～5 time, wherein said organic solvent are a kind of in normal hexane, normal butane, cyclohexane extraction, acetone or the petroleum ether;
Dry: extract at 30～95 ℃ of following normal pressures or be evaporated to the organic solvent-free flavor, is reclaimed organic solvent, and the concentrate drying promptly obtains Cordyceps sterol after handling.
(2) extraction of Cordyceps polysaccharide
Extract: the residue that step (1) is extracted the back gained adds water, and press per 1 gram residue and add 3～20 ml waters, mixing, 50～100 ℃ were extracted 0.5～4 hour, filtered, and got filtrate and water and carried residue, repeated to extract 1～4 time; It is capable of using as feed that water is carried residue;
Concentrate: filtrate is concentrated into 1/3～1/8 of original volume at 50～100 ℃, decompression or normal pressure, concentrated solution;
Precipitate with ethanol for the first time: in concentrated solution, add 2～6 times of volume of ethanol, place-15～40 ℃ 0.5～48 hour down;
Filter: precipitate with ethanol solution is filtered, can get filtrate and filter cake, filter cake is thick Cordyceps polysaccharide;
Remove albumen: after thick Cordyceps polysaccharide was dissolved in 0.1～10 times of volume water, the Sevage reagent that adds 0.5～3 times of volume again removed albumen, repeats 1～6 time;
Precipitate with ethanol for the second time: add 2～5 times of volume of ethanol through removing in the proteic thick Cordyceps polysaccharide liquid, place-15～40 ℃ 0.5～24 hour down;
Filter: precipitate with ethanol solution is filtered, get filtrate and filter cake;
Dry: filter cake places 25～100 ℃ of oven dry down, can get highly purified Cordyceps polysaccharide;
(3) extraction of cordycepin
Concentrate: get filtrate behind step (2) ethanol precipitation twice and be blended in 40～80 ℃, normal pressure or decompression and be concentrated into down and do not have the alcohol flavor, get concentrated solution;
Transfer pH: get concentrated solution and transfer pH to 1.5～6.0 with acid solution, wherein said acid solution is that concentration is 1% to 50% hydrochloric acid or sulfuric acid solution;
Column chromatography: choose cation exchange resin, dress post, by specification pretreatment resin; Cation exchange column on the concentrated solution that above-mentioned pH is mixed up is used the alkaline solution eluting then, collects the alkaline solution eluent, and wherein said alkaline solution is the ammonia spirit of 0.05～5.0mol/L; The cation exchange resin of choosing can be polystyrene macropore strong acid cation exchanger resin, polystyrene gel storng-acid cation exchange resin etc.;
Crystallization: eluent is concentrated into dried under 50～100 ℃, normal pressure or decompression, the heating dissolve with ethanol places-15～25 ℃ of crystallizations, collects crystal, places 25～100 ℃ of oven dry, the high-purity cordycepin crystal.
The preferred reflux extraction of extracting method of the described Cordyceps sterol of step (1) also can be taked extraction, ultrasonic or microwave extraction method etc.
The extracting method of the described Cordyceps polysaccharide of step (2) can the water heat reflow method, hot water extraction, ultrasonic or microwave extraction method etc., preferred water heat reflow method of the present invention.
The method of described separating filtrate of step (2) and filter cake can be with Filtration or centrifuging, the preferred Filtration of the present invention.
Realized the comprehensive extraction of main three major types effective ingredient such as Cordyceps sterol, Cordyceps polysaccharide, cordycepin in the cordyceps militaris mycelium by the employing technical solution of the present invention, and the final residue thing can be used as fine animal feed.Accompanying drawing 1 has clearly shown the comprehensive method flow that extracts of this three effective constituents.
For confirming extract obtained purity, we analyze the purity of cordycepin: carry out purification process by technology such as 732 cation exchange resiies, crystallizations; Wherein 732 cation exchange resin treatment steps are, will contain cordycepin solution is 2 upper props with HCl accent pH, with 0.15mol/L ammonia eluting, collects the ammonia eluent, and the liquid phase purity that detects cordycepin under 205nm reaches 74.0% (seeing accompanying drawing 2); To collect liquid again and obtain crystal through steps such as concentrated, dissolve with ethanol, crystallizations, get the crystal efficient liquid phase chromatographic analysis that carries out soluble in water, under 205nm, detect the liquid phase purity (seeing accompanying drawing 3) of cordycepin, as seen from the figure, nearly all impurity all is removed, and this moment, the purity of cordycepin reached more than 98%.
The technology of the present invention and technology mainly show following some: 1. the present invention utilizes organic solvent that the sterols extracts active ingredients of low pole is come out, the raw materials used leftover bits and pieces that comprises after liquid fermentation mycelium, solid culture mycelium, cultivation sporophore and sporophore are gathered in the crops, and existing other technology all has this part composition is removed as impurity, basically be raw material with Cordyceps Stroma (sporophore), cost an arm and a leg the cost height; The present invention has made full use of resource, avoids waste; This is a bright spot of the present invention.2. extracting aspect Cordyceps polysaccharide or the cordycepin, prior art adopt mostly earlier ethanol extraction, again water carry, the reuse ethanol precipitation, to waste a large amount of ethanol like this, production cost is higher; Then to be that first water is carried, concentrated, reuse is pure analyse in the present invention, saves the ethanol consumption like this and disposablely just can obtain Cordyceps polysaccharide and cordycepin, compares production cost with it and reduce greatly.3. the content that existing technology is extracted Cordyceps polysaccharide is lower, and the Cordyceps polysaccharide content that the present invention extracts is greater than 90%.4. most of existing processes are single or two kinds of composition simple composite are extracted, and extract the back and are carrying out a series of purification work, complex operation; 5. the product purity that most of prior arts obtain is not high, and three series products purity such as the Cordyceps sterol that the present invention obtains, Cordyceps polysaccharide and cordycepin are all higher, and wherein the purity of cordycepin is up to 98%, is respectively colourless, canescence and white crystal.
The invention has the beneficial effects as follows raw materials used go up and leaching process in saved cost, plurality of active ingredients in the Cordyceps militaris is extracted fully, make extract obtained purity higher through extracting, remove impurity repeatedly, and simple to operate, can realize the comprehensive extraction of plurality of active ingredients.
Description of drawings
Fig. 1 is the process chart of the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium
Fig. 2 is that cation exchange resin is handled eluent high performance liquid chromatogram collection of illustrative plates
Fig. 3 is the pure product high performance liquid chromatogram of a crystallization collection of illustrative plates
The specific embodiment (totally 3 examples)
Embodiment 1:
(1) extraction of Cordyceps sterol
Pulverize: get fresh Cordyceps militaris (L.) Link. solid culture mycelium 1000 grams and place 50 ℃ of oven for drying to constant weight, exsiccant mycelium uses pulverizer to pulverize;
Extract: restrain 1000 milliliters of normal butanes of adding in the mycelium in 100 of pulverizing, 40 ℃ were extracted 2 hours down, and filtration gets filtrate, repeats to extract 3 times, and residue is standby;
Concentrate: filtrate is concentrated into no normal butane flavor at 40 ℃ of following normal pressures, gets concentrate, reclaim normal butane;
Saponification: add 10% sodium hydroxide solution of its 2 times of volumes at normal temperatures to concentrate, mixing left standstill 2 hours, removed the sub-cloud saponifiable matter, saponification liquor, do not repeat 2 times;
Extraction: add the not normal butane of 3 times of volumes of saponification liquor, mixing left standstill 2 hours, and normal butane liquid is got in extraction, repeats 3 times;
Dry: as above-mentioned extract to be concentrated into no normal butane flavor at 40 ℃ of following normal pressures, to reclaim normal butane, after the concentrate drying is handled, get 0.56 gram Cordyceps sterol.
(2) extraction of Cordyceps polysaccharide
Extract: the residue after step (1) is extracted adds water, residue and water w/v 1: 20, and mixing, 95 ℃ were extracted 3 hours, and repeated to extract 2 times;
Filter: water is carried residue filter, obtain filtrate and filter cake, filter cake is capable of using as feed, and filtrate is evaporated to about 1/4 of original volume at 85 ℃;
Precipitate with ethanol for the first time: add 3 times of volume of ethanol in the filtrate after concentrating, place-4 ℃ following 24 hours;
Filter: precipitate with ethanol solution is filtered, can contain the filtrate and the filter cake that contains Cordyceps polysaccharide of cordycepin, filter cake is thick Cordyceps polysaccharide, filtrate for later use;
Remove albumen: after thick Cordyceps polysaccharide precipitation was dissolved in 2 times of volume water, the Sevage reagent (volume ratio of chloroform and n-butyl alcohol is 4: 1) that adds 3 times of volumes of amount of water again removed albumen, repeats 2 times;
Precipitate with ethanol for the second time: will remove 3 times of volume of ethanol of adding in the proteic thick Cordyceps polysaccharide liquid, place-4 ℃ following 24 hours;
Filter: precipitate with ethanol solution is filtered, and filter cake places 65 ℃ of oven dry, gets 3.4 gram Cordyceps polysaccharides.
(3) extraction of cordycepin
Concentrate: the filtrate of getting behind step (2) ethanol precipitation twice is mixed, and being evaporated under 65 ℃ does not have the alcohol flavor;
Transfer pH: get concentrated solution and transfer pH to 2.0 with 10% hydrochloric acid solution, pH meter is followed the tracks of and is detected;
Column chromatography: 732 cation exchange resin by specification pretreatment; 732 cation exchange columns on the concentrated solution that above-mentioned pH is mixed up are used 0.1mol/L ammonia spirit eluting then then, collect eluent;
Crystallization: eluent is concentrated, and the heating dissolve with ethanol places-4 ℃ of crystallizations, collects crystal, places 45 ℃ of oven dry, gets 0.2 gram cordycepin.
Embodiment 2:
(1) extraction of Cordyceps sterol
Pulverize: mycelium 12000 grams of getting fresh Cordyceps militaris (L.) Link. liquid fermentation and culture place 65 ℃ of oven for drying to constant weight, use pulverizer to pulverize;
Extract: the mycelium 1000 that accurately takes by weighing pulverizing restrains and adds 8 liters of normal hexane, and 65 ℃ were extracted 3 hours down, filtered, and got filtrate, repeated to extract 2 times, and residue is standby;
Concentrate: will extract filtrate and be evaporated to no normal hexane flavor, and get concentrated solution at 50 ℃;
Saponification: above-mentioned concentrated solution adds 10% sodium hydroxide solution of 3 times of volumes of concentrated solution at normal temperatures, and mixing left standstill 4 hours, removes the sub-cloud saponifiable matter, saponification liquor, repeat saponification 2 times;
Extraction: add not that the normal hexane of 3 times of volumes of saponification liquor extracts, mixing left standstill 2 hours, re-extract 3 times;
Dry: as above-mentioned organic solvent extraction liquid to be evaporated to no normal hexane flavor under 50 ℃, to reclaim normal hexane, after the concentrate drying is handled, promptly obtain 4.7 gram Cordyceps sterol.
(2) extraction of Cordyceps polysaccharide
Extract: the residue after step (1) is extracted adds water, residue and water w/v 1: 8, and mixing, 90 ℃ were extracted 3 hours, and repeated to extract 2 times;
Filter: water is carried residue filter, obtain filtrate and filter cake, filter cake is evaporated to 1/5 of original volume with filtrate at 85 ℃ as feedstuff;
Precipitate with ethanol for the first time: add 3 times of volume of ethanol in the filtrate after concentrating, place-4 ℃ following 12 hours;
Filter: precipitate with ethanol solution is filtered, can contain the filtrate and the filter cake that contains Cordyceps polysaccharide of cordycepin, filter cake is thick Cordyceps polysaccharide;
Remove albumen: after thick Cordyceps polysaccharide precipitation was dissolved in 2 times of volume water, the Sevage reagent (volume ratio of chloroform and n-butyl alcohol is 5: 1) that adds 1 times of volume of amount of water again removed albumen, repeats 3 times;
Precipitate with ethanol for the second time: will remove 3 times of volume of ethanol of adding in the proteic thick Cordyceps polysaccharide liquid, place-4 ℃ following 12 hours;
Filter: precipitate with ethanol solution is filtered, and filter cake places 60 ℃ of oven dry, gets Cordyceps polysaccharide 28.3 grams.
(3) extraction of cordycepin
Concentrate: getting being evaporated to filtrate mixing behind step (2) ethanol precipitation twice does not have the alcohol flavor;
Transfer pH: get concentrated solution and transfer pH to 2.5 with 10% hydrochloric acid solution;
Column chromatography: 732 cation exchange resin by specification pretreatment; With this cation exchange column on the above-mentioned concentrated solution that mixes up pH, use 0.1mol/L ammonia eluting then, collect eluent;
Crystallization: eluent is concentrated, and the heating dissolve with ethanol places-4 ℃ of crystallizations, collects crystal, places 45 ℃ of oven dry, gets 1.6 gram high-purity cordycepins.
Embodiment 3:
(1) extraction of Cordyceps sterol
Pulverize: leftover bits and pieces 9000 grams of getting after the cultivation cordyceps militaris fruit body is gathered in the crops place 65 ℃ of oven for drying to constant weight, use pulverizer to pulverize;
Extract: the leftover bits and pieces 1000 that takes by weighing pulverizing restrains and adds 8 liters of petroleum ether, and 65 ℃ were extracted 3 hours down, filtered, and got filtrate, repeated to extract 2 times, and residue is standby;
Concentrate: will extract filtrate and be evaporated to no petroleum ether flavor, and get concentrated solution at 50 ℃;
Saponification: above-mentioned concentrated solution adds 10% sodium hydroxide solution of 3 times of volumes of concentrated solution at normal temperatures, and mixing left standstill 4 hours, removes the sub-cloud saponifiable matter, saponification liquor, repeat saponification 2 times;
Extraction: add not that the petroleum ether of 3 times of volumes of saponification liquor extracts, mixing left standstill 2 hours, re-extract 3 times;
Dry: as above-mentioned organic solvent extraction liquid to be evaporated to no petroleum ether flavor under 50 ℃, to reclaim petroleum ether, after the concentrate drying is handled, promptly obtain 5.7 gram Cordyceps sterol.
(2) extraction of Cordyceps polysaccharide
Extract: the residue after step (1) is extracted adds water, residue and water w/v 1: 8, and mixing, 98 ℃ were extracted 2 hours, and repeated to extract 2 times;
Filter: water is carried residue filter, obtain filtrate and filter cake, filter cake is evaporated to 1/5 of original volume with filtrate at 85 ℃ as feedstuff;
Precipitate with ethanol for the first time: add 3 times of volume of ethanol in the filtrate after concentrating, place-4 ℃ following 12 hours;
Filter: precipitate with ethanol solution is filtered, can contain the filtrate and the filter cake that contains Cordyceps polysaccharide of cordycepin, filter cake is thick Cordyceps polysaccharide;
Remove albumen: after thick Cordyceps polysaccharide precipitation was dissolved in 2 times of volume water, the Sevage reagent (volume ratio of chloroform and n-butyl alcohol is 3: 1) that adds 1 times of volume of amount of water again removed albumen, repeats 3 times;
Precipitate with ethanol for the second time: will remove 3 times of volume of ethanol of adding in the proteic thick Cordyceps polysaccharide liquid, place-4 ℃ following 12 hours;
Filter: precipitate with ethanol solution is filtered, and filter cake places 60 ℃ of oven dry, gets Cordyceps polysaccharide 42.5 grams.
(3) extraction of cordycepin
Concentrate: the filtrate of getting behind step (2) ethanol precipitation twice is mixed, and being evaporated to does not have the alcohol flavor;
Transfer pH: get concentrated solution and transfer pH to 3.0 with 10% hydrochloric acid solution;
Column chromatography: 732 cation exchange resin by specification pretreatment; With this cation exchange column on the above-mentioned concentrated solution that mixes up pH, use 0.1mol/L ammonia eluting then, collect eluent;
Crystallization: eluent is concentrated, and the heating dissolve with ethanol places-4 ℃ of crystallizations, collects crystal, places 45 ℃ of oven dry, gets 3.2 gram high-purity cordycepins.
Claims (5)
1. the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium is characterized in that carrying out according to the following steps:
(1) extraction of Cordyceps sterol
Extract: add organic solvent in dry, cordyceps militaris mycelium after pulverizing, add 3～40 milliliters of organic solvents by per 1 gram mycelium, 30～95 ℃ were extracted 0.5～5 hour down, filter, repeat to extract 1～4 time, the filtrate of each gained is mixed, residue is standby; Wherein said organic solvent is a kind of in normal hexane, normal butane, cyclohexane extraction, acetone, the petroleum ether;
Concentrate: filtrate is concentrated into the organic solvent-free flavor under 30～95 ℃ of temperature, normal pressure or decompression, reclaims organic solvent, get concentrate;
Saponification: add 0.2～5 milliliter of alkaline solution in per 1 gram concentrate under the room temperature, mixing left standstill 0.5～24 hour, remove the sub-cloud saponifiable matter, get not saponification liquor, repeat saponification 1～3 time, wherein said alkaline solution is that concentration is 1% to 50% sodium hydroxide or potassium hydroxide solution;
Extraction: add the organic solvent of 0.2～10 times of volume in saponification liquor not, mixing left standstill 0.5～24 hour, divide and get organic solvent layer, extract, re-extract 1～5 time, wherein said organic solvent are a kind of in normal hexane, normal butane, cyclohexane extraction, acetone, the petroleum ether;
Dry: extract at 30～95 ℃ of following normal pressures or be evaporated to the organic solvent-free flavor, is reclaimed organic solvent, after the concentrate drying is handled, Cordyceps sterol;
(2) extraction of Cordyceps polysaccharide
Extract: the residue behind step (1) the extraction Cordyceps sterol is added water, press per 1 gram residue and add 3～20 ml waters, mixing, 50～100 ℃ were extracted 0.5～4 hour, and filtration gets filtrate and water and carries residue, repeats to extract 1～4 time;
Concentrate: filtrate is concentrated into 1/3～1/8 of original volume under 50～100 ℃, decompression or normal pressure, concentrated solution;
Precipitate with ethanol for the first time: in concentrated solution, add 2～6 times of volume of ethanol, place-15～40 ℃ 0.5～48 hour down;
Filter: precipitate with ethanol solution is filtered, can get filtrate and filter cake, filter cake is thick Cordyceps polysaccharide;
Remove albumen: after thick Cordyceps polysaccharide was dissolved in 0.1～10 times of volume water, the Sevage reagent that adds 0.5～3 times of volume again removed albumen, repeats 1～6 time;
Precipitate with ethanol for the second time: add 2～5 times of volume of ethanol through removing in the proteic thick Cordyceps polysaccharide liquid, place-15～40 ℃ 0.5～24 hour down;
Filter: precipitate with ethanol solution is filtered, get filtrate and filter cake;
Dry: filter cake is placed 25～100 ℃, decompression or normal pressure oven dry down, the high-purity Cordyceps polysaccharide;
(3) extraction of cordycepin
Concentrate: the filtrate of getting behind step (2) ethanol precipitation twice is mixed, and is concentrated into no ethanol flavor under 40～80 ℃, normal pressure or decompression, gets concentrated solution;
Transfer pH: get concentrated solution and transfer pH to 1.5～6.0 with acid solution, wherein said acid solution is that concentration is 1% to 50% hydrochloric acid or sulfuric acid solution;
Column chromatography: choose cation exchange resin, dress post, by specification pretreatment resin; Cation exchange column on the concentrated solution that above-mentioned pH is mixed up is used the alkaline solution eluting then, collects the alkaline solution eluent, and wherein said alkaline solution is the ammonia of 0.05～5.0mol/L;
Crystallization: eluent is concentrated into dried under 50～100 ℃, normal pressure or decompression, the heating dissolve with ethanol places-15～25 ℃ of crystallizations, collects crystal, places 25～100 ℃ of oven dry, high-purity cordycepin.
2. the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium as claimed in claim 1, wherein the described scarlet caterpiller fungus mycelia of step (1) comprises leftover bits and pieces or its mixture after liquid fermentation mycelium, solid culture mycelium, cultivation sporophore and sporophore are gathered in the crops.
3. the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium as claimed in claim 1, wherein step (2) is removed the component chloroform of the used Sevage reagent of albumen and the volume ratio of n-butyl alcohol is 2～8: 1.
4. the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium as claimed in claim 3, wherein step (2) is removed the component chloroform of the used Sevage reagent of albumen and the volume ratio of n-butyl alcohol is 4: 1.
5. the combined extraction method of plurality of active ingredients in the cordyceps militaris mycelium as claimed in claim 1, wherein the described cation exchange resin of step (3) is a polystyrene highly acid gel type cation exchanger resin.
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| CN101407556B (en) * | 2008-11-26 | 2011-10-05 | 吉林大学 | Preparation method of Cordyceps polysaccharide injection and its use in cancer |
| CN101928352B (en) * | 2010-08-13 | 2012-06-06 | 内蒙古昆明卷烟有限责任公司 | Extraction for active ingredients of cordyceps sinensis and application thereof in cigarette filter |
| CN102499435B (en) * | 2011-11-02 | 2014-01-01 | 深圳市大百汇技术有限公司 | Cigarette containing cordyceps militaris extract |
| CN103288909B (en) * | 2012-02-28 | 2015-12-16 | 中国科学院沈阳应用生态研究所 | The extracting method of cordycepin in the fresh substratum of a kind of Chinese caterpillar fungus |
| CN102643318B (en) * | 2012-03-28 | 2014-10-22 | 辽宁大学 | Method for extracting refined cordycepin from Cordyceps militaris fruit body |
| CN103446189A (en) * | 2013-07-03 | 2013-12-18 | 广州市浩立生物科技有限公司 | Medicinal liquor of cordyceps sinensis fungi powder and preparation method of medicinal liquor |
| CN103766545A (en) * | 2014-01-02 | 2014-05-07 | 隋新 | Preparation method of cordyceps militaris extractive-rich cordyceps militaris tea |
| CN104403012A (en) * | 2014-11-10 | 2015-03-11 | 苏州蔻美新材料有限公司 | Extraction technology of cordyceps militaris polysaccharide and polypeptide |
| CN104909972A (en) * | 2015-05-30 | 2015-09-16 | 吴爱群 | Method for extracting amino acids in worm grass |
| CN105039452A (en) * | 2015-06-23 | 2015-11-11 | 中山鼎晟生物科技有限公司 | A kind of extraction method of polysaccharide in Cordyceps sinensis solid fermentation broth |
| CN105294395A (en) * | 2015-09-23 | 2016-02-03 | 安徽阳光药业有限公司 | Method for preparing cordycepic acid and cordycepin by simultaneous extraction-combination with column chromatography-crystallization purification |
| CN106309466A (en) * | 2016-08-18 | 2017-01-11 | 湖州新驰医药科技有限公司 | Cordyceps cicadae cordycepin preparation and extraction process |
| CN108551973A (en) * | 2018-03-08 | 2018-09-21 | 贵州安顺金黔虫草有限公司 | A kind of cordyceps culturing method and cordycepin extracting method |
| CN109651524A (en) * | 2018-12-06 | 2019-04-19 | 桐乡市博奥生物科技有限公司 | A kind of northern Chinese caterpillar Fungus Polyose extraction purifying process based on solid fermentation |
| CN111171169A (en) * | 2020-01-10 | 2020-05-19 | 江苏神华药业有限公司 | Method for efficiently and simultaneously extracting polysaccharide and adenosine from fermented cordyceps sinensis powder |
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| CN115684404B (en) * | 2022-10-27 | 2024-12-13 | 广东省科学院动物研究所 | A method for constructing a fingerprint of Cordyceps sinensis and its application |
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