CN102633900A - Method for extracting and purifying polysaccharide from Cordyceps militaris - Google Patents
Method for extracting and purifying polysaccharide from Cordyceps militaris Download PDFInfo
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- CN102633900A CN102633900A CN2012101341964A CN201210134196A CN102633900A CN 102633900 A CN102633900 A CN 102633900A CN 2012101341964 A CN2012101341964 A CN 2012101341964A CN 201210134196 A CN201210134196 A CN 201210134196A CN 102633900 A CN102633900 A CN 102633900A
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Abstract
The invention relates to a method for extracting and purifying polysaccharide from Cordyceps militaris, which belongs to the technical field of farm produce deep processing. The method comprises three major steps: extracting the polysaccharide from Cordyceps militaris, preparing crude polysaccharide from Cordyceps militaris and refining and purifying Cordyceps polysaccharide. The method has the characteristics of high efficiency, mildness and simple process, so as to be suitable for large scale popularization and application. According to the invention, the polysaccharide in a Cordyceps militaris material can be extracted at a lower temperature, so as to keep the bioactivity of the polysaccharide to the largest extent; and polysaccharide and protein components in the extract can be removed after separation through macroporous resin and purification through an ultrafiltration membrane, so that the polysaccharide content in the contract reaches 68%, and yield of refined polysaccharide is 6.7%. With the adoption of the technology, the method provided by the invention has the advantages of simple process, high efficiency, moderate conditions, no pollution and the like and can effectively reduce the cost for separation. According to the invention, the polysaccharide extracted from Cordyceps militaris not only can be used as a material for producing a Cordyceps polysaccharide monomer and but also can be directly used as additive of health care products and functional foods.
Description
Technical field
The invention provides the extracting and purifying method of polysaccharide in a kind of Cordyceps militaris (L.) Link., belong to deep-processing technical field of agricultural products.
Background technology
Cordyceps militaris (L.) Link. (Cordyceps militaris) has another name called Cordyceps militaris (L.) Link.; With China rare traditional Chinese medicine entomophyte (Cordyeeps sinensis) for belonging to xenogenesis together; Cordyceps polysaccharide (CMPS) is the important active substance in the Cordyceps militaris (L.) Link.; The monosaccharide component of Cordyceps polysaccharide is semi-lactosi: seminose=6:5, and it is a trunk with the seminose that β (1 → 2) connects, and side chain is made up of more a large amount of β (1 → 6) semi-lactosi and more a large amount of β (1 → 2) galactofuranoses; Being connected on the 0-4 and 0-6 of trunk, is to contain the mixed polysaccharide that a small amount of albumen has hyperbranched structure.Cordyceps polysaccharide has been proved to have antitumor, hypoglycemic, anti hepatic fibrosis and to the enhancement of the huge macrophage system of monokaryon, can also improve the viral infection of immunologic function and treatment liver etc., has the wide development prospect.
Ultra-filtration membrane is that what to hold back the technology of separating different substances according to different molecular weight sizes is the stripping technique of new development both at home and abroad in recent years; Its ultimate principle is at normal temperatures with certain pressure and flow, utilizes asymmetric microvoid structure and semi-permeable membranes medium, and the pressure difference that relies on the film both sides is as impellent; Filter with the cross-flow mode; Solvent and small-molecule substance are passed through, and macromolecular substance and micropartical such as protein, water soluble polymer, bacterium etc. are by the filter membrane detention, thereby reach separation, classification, purifying, concentrate a kind of new membrane stripping technique of purpose; And in field of medicaments (particularly natural drug is refining), widely use, be fit to suitability for industrialized production.
Summary of the invention
The object of the present invention is to provide a kind of method of from Cordyceps militaris (L.) Link., extracting Cordyceps polysaccharide.
The present invention includes following steps:
1) extraction of polysaccharide in the Cordyceps militaris (L.) Link.: Cordyceps militaris (L.) Link. raw material after will pulverizing and water extract suction filtration behind the 2h with the weight ratio continuous backflow of 1:5～8; Filter residue behind the suction filtration and water are extracted suction filtration behind the 2h with the weight ratio continuous backflow of 1:3～5, merge suction filtration liquid;
2) preparation of Crude polysaccharides in the Cordyceps militaris (L.) Link.: the suction filtration liquid after will merging uses zero(ppm) water to be dispersed to the Chinese caterpillar fungus water dispersion solution that Cordyceps militaris (L.) Link. crude drug amount is 0.3～0.5g/ml; The Chinese caterpillar fungus aqueous dispersions is splined on the macroporous resin behind the dress post, collects effluent with sodium hydroxide adjust pH to 9～10 back circulations;
3) the refining purifying of Cordyceps polysaccharide: the effluent pH value of collecting is transferred to neutrality,, collect and see through liquid with the ultrafiltration membrane permeate of 10KDa; Again with the said liquid that sees through of the ultrafiltration membrance filter of 200KDa; Obtain Cordyceps polysaccharide solution, will concentrate Cordyceps polysaccharide solution again, obtain Cordyceps polysaccharide.
The present invention adopts the macroporous resin technology that coupling separates the Cordyceps militaris (L.) Link. polysaccharide with ultra-filtration membrane, has efficient, gentle, technology characteristic of simple, is fit to large-scale promotion and uses.The present invention can keep the biological activity of polysaccharide to greatest extent with the polysaccharide extract at low temperature in the Cordyceps militaris (L.) Link. raw material; Can remove polysaccharide and the protein ingredient in the extract after utilizing macroporous resin separation and ultra-filtration membrane purifying, make the polysaccharide content in the extract reach 68%, smart polysaccharide yield is 6.7%.The polysaccharide thing impurity that contracts is few in the product Cordyceps militaris (L.) Link., and active high, it is simple to adopt this technology to have technology, and efficient is high, and mild condition, and advantage such as pollution-free can effectively reduce separation costs.Active polysaccharide is high in the product Cordyceps militaris (L.) Link., both can be used as to produce the monomeric raw material of Cordyceps polysaccharide, also can be directly as the additive of healthcare products and functional food.
Major advantage of the present invention and positively effect specify as follows:
1, the present invention adopts hydro-thermal backflow formulation to extract polysaccharide, has kept the biological activity of polysaccharide to greatest extent, Crude polysaccharides extraction rate reached to 88%.
2, utilize macroporous resin and ultra-filtration membrane coupling separation and purification polysaccharide; Can be according to the molecular weight size of active polysaccharide; Carry out specific aim and separate, utilize macroporous resin effectively to remove most of polysaccharide and protein ingredient in the extract, utilize ultra-filtration membrane can be directed against the polysaccharide molecular weight size; Specific aim is collected active polysaccharide, and the biological activity, the purity that obtain product are higher.
3, in addition, it is to carry out at normal temperatures that the present invention adopts hyperfiltration process to have ultra-filtration process, and mild condition does not have composition and destroys; Filter not undergoing phase transition of journey, need not heating, energy consumption is low, need not to add chemical reagent, and is pollution-free, is a kind of stripping technique of energy-conserving and environment-protective; The ultra-filtration technique separation efficiency is high, and is all very effective to concentrating of the recovery of the trace ingredients in the dilute solution, low concentration solution; Tripping device is simple, flow process is short, easy and simple to handle, be easy to control and safeguard.
In addition, step 2 according to the invention) the Chinese caterpillar fungus water dispersion solution is 1:1.5～2 with the weight ratio of macroporous resin in, adsorbs fully with glycocalix in the assurance Chinese caterpillar fungus aqueous dispersions.
Said step 2) take-off rate of effluent is 30～50ml/min in, and this speed flows out and can guarantee that polysaccharide is not flowed out influence and the desorption of power by effluent.
Description of drawings
Fig. 1 is the polysaccharide determination canonical plotting.
Embodiment
One, extraction process:
1, the extraction of Cordyceps polysaccharide:
Cordyccps-militaris-(L.)-link. Sporophore 3kg pulverizes (meal is advisable), drops in the multi-function extractor, and the water continuous backflow that adds 15～24 kg is extracted 2h, suction filtration.
The filter residue of getting behind the suction filtration continues with the water refluxing extraction that adds 3 to 5 times of weight, and continuous backflow is extracted 2h, suction filtration.
Merge two filtratings.
2, the preparation of Crude polysaccharides in the Cordyceps militaris (L.) Link.:
(1) the suction filtration liquid after will merging uses zero(ppm) water to be dispersed to the Chinese caterpillar fungus water dispersion solution 6L that Cordyceps militaris (L.) Link. crude drug amount is 0.3～0.5g/ml, and is with sodium hydroxide solution regulator solution pH to 9～10, subsequent use.
(2) preliminary purification of macroporous resin:
Get HPD-D macroporous resin (the precious grace chemical industry in Cangzhou ltd produces) the 3kg φ 800 ㎜ * 1000 ㎜ stainless steel chromatography posts of packing into; It is for use (earlier with two column volumes of hydrochloric acid wash-out of 3% to handle the back; Be washed till neutrality with tap water then; Use two column volumes of sodium hydroxide wash-out of 3% then, be washed till neutrality with tap water again, use behind two column volumes of zero(ppm) water wash-out subsequent use at last).
Explain: with the used resin of the inventive method is that Cangzhou precious grace chemical industry ltd produces, and model is HPD-D.
With mix up the pH value the Chinese caterpillar fungus water dispersion solution on the HPD-D macroporous resin; Appearance in the circulation; Controlling flow fluid speed is about 30～50ml/min, collects effluent, handles macroporous resin column (ethanol of the recyclable high density of elutriant continues to use) with the ethanol elution of higher concentration again after wash-out is intact; Alcohol is washed the back and is eluted to nothing alcohol flavor with tap water, with carrying out going up appearance next time behind two column volumes of water elution).
(3) make with extra care purifying: collect effluent and also transfer pH to neutral, with the ultrafiltration membrane permeate of 10KDa, behind the removal small molecular weight impurity with sulphuric acid soln; Collect to see through liquid again with the ultrafiltration membrance filter of 200KDa; Get Cordyceps polysaccharide solution, reduced vacuum concentrates, the Cordyceps polysaccharide 200g that obtains; Yield is 6.7%, and purity is 66%.
Two, product analysis
Measuring method: utilize the sulfuric acid anthrone method to carry out measurement of the polysaccharide content, measuring method is following:
1, experiment material
Electric-heated thermostatic water bath (production of Beijing Medical Equipment Plant), Bu Shi suction funnel, 95% ethanol, acetone, the vitriol oil are analyzes alcohol; Tianjin, island UV-2501PC ultraviolet spectrophotometer; Tianjin, 1200SM island electronic analytical balance.
2, experimental technique
2.1 measure the selection of wavelength
(Chinese pharmaceutical biological product is identified institute with the D-dextrose anhydrous to get assay; Lot number 110833-200302) be dried to constant weight in 105 ℃, precision takes by weighing dried D-dextrose anhydrous and is dissolved in water in right amount and processes solution that every 1ml contains 0.2mg as reference substance solution.In the 500-700nm scope, do spectral scan, confirm that its maximum absorption is at the 624nm place
2.2 typical curve
Precision take by weighing 105% be dried to constant weight anhydrous D-dextrose anhydrous reference substance 10mg, put in the 50ml volumetric flask, adding distil water is diluted to scale; Shake up, respectively accurate draw above-mentioned reference substance each 0.5,1,1.5,2 and 2.5,3ml is in the 10ml volumetric flask; Thin up shakes up to scale.Respectively accurately again draw 1ml in the 10ml nessler colorimetric tube, add anthrone test solution 0.5ml and vitriol oil 3ml, put be chilled to room temperature after, the mensuration optical density value in the 624nm place.Add reagent colour development as blank with water with method.Be respectively X-coordinate with concentration, the optical density value is that ordinate zou is done typical curve, and the result is as shown in Figure 1.
2.3 measuring method:
Precision takes by weighing polysaccharide 1g water constant volume in the 50ml volumetric flask, and thin up is to scale, and accurate again absorption 2ml places the 10ml volumetric flask to use the zero(ppm) water constant volume; Shake up; The accurate again 1ml that draws adds anthrone test solution 0.5ml and vitriol oil 3ml in nessler colorimetric tube, put be chilled to room temperature after; Measure the optical density value in the 624nm place, calculate and promptly get polysaccharide content.
Claims (3)
1. polysaccharide extracting and purifying method in the Cordyceps militaris (L.) Link. is characterized in that may further comprise the steps:
1) extraction of polysaccharide in the Cordyceps militaris (L.) Link.: Cordyceps militaris (L.) Link. raw material after will pulverizing and water extract suction filtration behind the 2h with the weight ratio continuous backflow of 1:5～8; Filter residue behind the suction filtration and water are extracted suction filtration behind the 2h with the weight ratio continuous backflow of 1:3～5, merge suction filtration liquid;
2) preparation of Crude polysaccharides in the Cordyceps militaris (L.) Link.: the suction filtration liquid after will merging uses zero(ppm) water to be dispersed to the Chinese caterpillar fungus water dispersion solution that Cordyceps militaris (L.) Link. crude drug amount is 0.3～0.5g/ml; The Chinese caterpillar fungus aqueous dispersions is splined on the macroporous resin behind the dress post, collects effluent with sodium hydroxide adjust pH to 9～10 back circulations;
3) the refining purifying of Cordyceps polysaccharide: the effluent pH value of collecting is transferred to neutrality,, collect and see through liquid with the ultrafiltration membrane permeate of 10KDa; Again with the said liquid that sees through of the ultrafiltration membrance filter of 200KDa; Obtain Cordyceps polysaccharide solution, will concentrate Cordyceps polysaccharide solution again, obtain Cordyceps polysaccharide.
2. polysaccharide extracting and purifying method in the said a kind of Cordyceps militaris (L.) Link. of claim 1 is characterized in that said step 2) in the weight ratio of Chinese caterpillar fungus water dispersion solution and macroporous resin be 1:1.5～2.
3. polysaccharide extracting and purifying method in claim 1 or the 2 said a kind of Cordyceps militaris (L.) Link. is characterized in that said step 2) in the take-off rate of effluent be 30～50ml/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2012101341964A CN102633900A (en) | 2012-05-03 | 2012-05-03 | Method for extracting and purifying polysaccharide from Cordyceps militaris |
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| CN2012101341964A CN102633900A (en) | 2012-05-03 | 2012-05-03 | Method for extracting and purifying polysaccharide from Cordyceps militaris |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103271952A (en) * | 2013-06-07 | 2013-09-04 | 陈玉龙 | Preparation method and preparation of cordyceps sinensis active concentration powder |
| CN104650255A (en) * | 2015-03-20 | 2015-05-27 | 海安县泓寿生物技术有限责任公司 | Method for separating and purifying cordyceps polysaccharide contained in cordyceps militaris solid culture medium |
| CN106749742A (en) * | 2017-01-18 | 2017-05-31 | 河南大学 | Polysaccharides in Cultured Cordyceps militaris, extracting method and its application in terms for the treatment of functional consitipation medicine is prepared |
| CN107114779A (en) * | 2017-05-26 | 2017-09-01 | 吉林省华惠生物科技有限公司 | The chewable tablets and preparation method of the extract of Cordyceps militaris containing ginseng stem and leave general saponin |
| CN109021132A (en) * | 2018-07-23 | 2018-12-18 | 广东省微生物研究所（广东省微生物分析检测中心） | Guangdong Cordyceps militaris polysaccharide CCG-PSW and CCG-PSS and preparation method and application |
| CN109400731A (en) * | 2018-09-04 | 2019-03-01 | 庆云堂生物科技（北京）有限公司 | A kind of cold-water-soluble astragalus polyose and preparation method thereof and its extracorporeal anti-tumor application |
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| CN101124988A (en) * | 2007-09-25 | 2008-02-20 | 江苏瑞迪生科技有限公司 | Method for extracting refined cordycepin and cordycepin polysaccharide from cordyceps mititaris |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101124988A (en) * | 2007-09-25 | 2008-02-20 | 江苏瑞迪生科技有限公司 | Method for extracting refined cordycepin and cordycepin polysaccharide from cordyceps mititaris |
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| 《食用菌学报》 20111215 朱丽娜 等 "不同分子量段蛹虫草多糖的超滤分离及其对巨噬细胞释放NO的影响" 47-50 1-3 第18卷, 第4期 * |
| 朱丽娜 等: ""不同分子量段蛹虫草多糖的超滤分离及其对巨噬细胞释放NO的影响"", 《食用菌学报》, vol. 18, no. 4, 15 December 2011 (2011-12-15), pages 47 - 50 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103271952A (en) * | 2013-06-07 | 2013-09-04 | 陈玉龙 | Preparation method and preparation of cordyceps sinensis active concentration powder |
| CN104650255A (en) * | 2015-03-20 | 2015-05-27 | 海安县泓寿生物技术有限责任公司 | Method for separating and purifying cordyceps polysaccharide contained in cordyceps militaris solid culture medium |
| CN106749742A (en) * | 2017-01-18 | 2017-05-31 | 河南大学 | Polysaccharides in Cultured Cordyceps militaris, extracting method and its application in terms for the treatment of functional consitipation medicine is prepared |
| CN107114779A (en) * | 2017-05-26 | 2017-09-01 | 吉林省华惠生物科技有限公司 | The chewable tablets and preparation method of the extract of Cordyceps militaris containing ginseng stem and leave general saponin |
| CN109021132A (en) * | 2018-07-23 | 2018-12-18 | 广东省微生物研究所（广东省微生物分析检测中心） | Guangdong Cordyceps militaris polysaccharide CCG-PSW and CCG-PSS and preparation method and application |
| CN109021132B (en) * | 2018-07-23 | 2021-01-29 | 广东省微生物研究所（广东省微生物分析检测中心） | Cordyceps guangdongensis polysaccharide CCG-PSW and CCG-PSS as well as preparation method and application thereof |
| CN109400731A (en) * | 2018-09-04 | 2019-03-01 | 庆云堂生物科技（北京）有限公司 | A kind of cold-water-soluble astragalus polyose and preparation method thereof and its extracorporeal anti-tumor application |
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Application publication date: 20120815 |