CN107793489A - A kind of method that polysaccharide grifolin is extracted from grifola frondosus - Google Patents
A kind of method that polysaccharide grifolin is extracted from grifola frondosus Download PDFInfo
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- CN107793489A CN107793489A CN201610802778.3A CN201610802778A CN107793489A CN 107793489 A CN107793489 A CN 107793489A CN 201610802778 A CN201610802778 A CN 201610802778A CN 107793489 A CN107793489 A CN 107793489A
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses a kind of method that polysaccharide grifolin is extracted from grifola frondosus, comprise the following steps：1) water extract-alcohol precipitation：Grifola frondosus raw material is extracted using deionized water, separation of solid and liquid and concentrated extracting solution, make polysaccharide precipitation using ethanol precipitation after extraction；2) alkali lye extracts：Grifolan precipitation is extracted again using sodium hydroxide solution；3) dialysis is neutralized：Using sodium hydroxide is neutralized, dialysis is removed and desalted；4) gel chromatography 1：The component in dialyzate is separated using anionite, is eluted with water；5) ferment treatment：Separation of solid and liquid and dialysed using ferment treatment eluent, after reaction terminating；6) gel chromatography 2：The component in dialyzate is separated using cation-exchanger, is eluted with water；7) it is freeze-dried：Eluent is freeze-dried, that is, obtains grifolin.Grifolin is the beta glucan that a kind of molecular weight is about 5,000,000 dalton, mainly by glucose group into, have highly significant antitumor and immunoregulatory activity.
Description
Technical field
The present invention relates to food processing, medicines and health protection field, and in particular to a kind of extraction of grifolan grifolin.
Technical background
Grifola frondosus (Grifola frondosa) is Basidiomycotina (Basidiomycotina), Hymenomycetes
(Hgmenomycetes), Holobasidiomycetidae (Holobasidiomyceidae), Aphyllophorales (Aphyllophrales),
Polyporaceae (polyporaceae), tree flower Pseudomonas (Grigola) fungi, also known as polyporus frondosus, thousand Buddhist bacterium, chestnut mushroom, cloud
Tan, lotus flower bacterium etc., Japan are referred to as to wave fine and soft (Maitake).1709, Japanese Bei Yuanyi pavilions《Big and book on Chinese herbal medicine》In recorded ash
Tree flower.Its pharmacologic action is recorded in right ((the bacterium spectrum of Japanese slope earliest》In, " sweet, flat, nontoxic, hemorrhoid can be controlled ".It is born in Oak Tree
Or around other broad leaf tree stumps, form is graceful, it is laminated like chrysanthemum.Fructification meat, is overlapped into clump：Handle white, more cauliflower-shapeds point
Branch, top differentiation shellfish shape taupe blade；Hymenium is porous, white：It is colourless to embrace son.Fructification has unique fragranced, mouth
Delicious, it is that one kind that China and Japan are promoting in recent years is precious rich in multivitamin, mineral matter and bioactive substance
Your food, medicine dual-purpose fungi.In Japan, oneself has the history of more than 20 years for the cultivation of grifola frondosus, turn into that Japan gives priority to six
One of big edible mushroom, its sales volume is only second to mushroom and asparagus.Although the domesticating and cultivating of China's grifola frondosus is started late, in recent years
Very fast to develop, current production rate occupies second place of the world, is distributed mainly on the ground such as Hebei, Jilin, Guangxi, Sichuan.
From ohno in 1984.Reported Deng river since grifolan has antitumor activity, the change to grifolan
The research for learning structure and healthcare function progressively deploys, and its physiological function adjustment effect good to human body is also gradually recognized by people
Know.Substantial amounts of pharmacology analysis confirms with clinical trial, grifolan have it is obvious it is antitumor, improve function of immune system,
Anti- HW viruses, regulation blood glucose, blood fat and cholesterol levels, generation and transfer, hypertension, obesity, sugar to preventing cancer cell
The disorder of urine disease, AIDS and function of immune system has the effect of certain.
Japanese scholars carry out the research of grifolan earliest.Ohno etc. is extracted from Grifola Frondosa sporophore with hot water and obtained
A kind of polysaccharide grifolin Grioflan (GRN) with antitumor activity, confirmed through methylation analysis and NMR spectrum,
Its structure is the C every 3 glycosyls6Upper β -1 with a side chain, 3- glucans, with lentinan, Schizophyllum commune Fr polysaccharides, obtain
The structure of an aromatic plant metioned in ancient books polysaccharide is similar, and molecular weight is about 5,000,000 dalton.They also demonstrate that extraction obtains more from maitake mushroom mycelia
Sugared structure is similar to fruitbody polysaccharide structure.Then ohno etc. utilizes CP-MAS13C NMR have studied the conformation of this polysaccharide, hair
Existing its shows as two kinds of conformations, i.e. natural type and screw type in the solid state.Either fructification or mycelium polysaccharides, temperature
The polysaccharide that (such as hot water) extraction obtains with the conditions of is natural type, and the polysaccharide that (cold alkali or thermokalite) extracts under drastic conditions is spiral shell
Rotation type, the polysaccharide of two kinds of conformations are respectively provided with significant antitumor activity.
The content of the invention
It is an object of the invention to provide a kind of extracting method for grifolan grifolin, in order to improve grifolin
Leaching rate and remove the different polysaccharide of structure, employ boiling water bath just extract and alkali lye extract the method being combined for the second time.
The preferable extraction process of grifolan grifolin concretely comprises the following steps：
First, drying, the crushing of Grifola Frondosa sporophore
Before being crushed to Grifola Frondosa sporophore, it should be made fully to dry.Preferable drying condition is in decompression bar
The drying 1.5~2 hours of lower 60 DEG C~70 DEG C of part.After Grifola Frondosa sporophore is fully dried, it is put into mechanical crusher and is crushed to
Fine powder.
2nd, extracting in boiling water
Take the grifola frondosus powder of appropriate quality, add the deionized water of 18~22 times of volumes, as extraction 2 in boiling water bath~
3 hours, temperature can not be less than 95 DEG C.Centrifuged after extraction, discard precipitation, supernatant is grifola frondosus extract solution.
3rd, alcohol precipitation is concentrated
The volume concentration of grifola frondosus extract solution is original volume as in Rotary Evaporators or drier by grifola frondosus extract solution
1/8~1/12.Grifola frondosus extract solution after concentration is poured into progress polysaccharide precipitation precipitation in the ethanol of 3.5~4.5 times of volumes,
It is statically placed in 4 DEG C of environment 10~12 hours.Centrifuged after alcohol precipitation, abandoning supernatant, precipitation is grifola frondosus Thick many candies.
4th, alkali lye extracts
Grifola frondosus Thick many candies are extracted again using the aqueous slkali containing 5%~10% sodium hydroxide, alkali lye quality
For 8~10 times of grifola frondosus Thick many candies quality, extraction time is 12~24 hours.
5th, alkali lye extraction post processing
After alkali extracts, pH to 7.0 is adjusted, solution is dialysed 48 hours as flowing water in bag filter.Will be saturating after dialysis
Analysis liquid is freeze-dried after being concentrated into suitable volume, obtains alkali process grifolan.
6th, DEAE-Sephadex A-25 gel chromatographies
A small amount of alkali process grifolan is taken, adds deionized water dissolving, centrifuges, discards precipitation.Supernatant uses prepackage
DEAE-SephadexA-25 chromatographic column is purified, and is eluted using deionized water, collects eluent main peak.
7th, enzyme reaction
The eluent of collection uses starch ferment treatment.50 mMs of every liter of PBSs are prepared, are mixed in eluent, body
Product, amylase is added, be 1: 100 with solution quality ratio.60 DEG C~70 DEG C are reacted 12~24 hours.
8th, enzyme reaction post-processes
After enzyme reaction carries out the sufficiently long time, solution is transferred to 100 DEG C of water-baths and terminates enzyme reaction within 5~10 minutes.From
The heart, precipitation is discarded, supernatant loads 2000D bag filters, is dialysed 1 day using deionized water.Dialyzate centrifuges 10 minutes, and it is heavy to discard
Form sediment, be freeze-dried after supernatant concentration to suitable volume, obtain decolouring grifolan.
9th, SP-Sephadex C-25 gel chromatographies
A small amount of decolouring grifolan is taken, adds deionized water dissolving, centrifuges, discards precipitation.Supernatant uses prepackage SP-
Sephadex C-25 chromatographic column is purified, and is eluted using deionized water, collects eluent main peak.
Tenth, it is freeze-dried
The eluent of collection is freeze-dried after being concentrated into suitable volume, produces grifolin.
The features of the present invention one is to be that boiling waterbath is combined with alkali lye extraction, and boiling waterbath ensure that strange fruit bacterium
The recovery rate of element, alkali lye extraction then eliminate and remaining incompatible polysaccharide of grifolin soda acid；Second, anionite
With being used in combination for cation-exchanger, the active set of anionite removes depigmentation, molecular sieve and removes negative electrical charge polysaccharide
In one, cation-exchanger then can effectively remove positive charge polysaccharide and retain neutral polysaccharide grifolin；Third, use starch
Enzyme removes the polysaccharide different from grifolin glucosides bond structure.Grifola frondosus Thick many candies are by alkali extraction, anion exchange, amylase
Reaction, the purifying of cation exchange level Four, finally can obtain the polysaccharide component grifolin of single structure, purity more than 95%.
Brief description of the drawings
The preferable extraction process specific steps of Fig. 1 grifolan grifolins illustrate accompanying drawing.
Embodiment
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
The content shown understands the flow and operation skill of the present invention.The present invention can also pass through specific embodiment parties different in addition
Formula is embodied or practiced, and the various details in this specification may be based on different viewpoints and application, without departing from this hair
Modified or changed on the premise of bright spirit.
With reference to embodiment, the present invention is described further, but protection scope of the present invention and not only limits
In this：
Embodiment 1
100 grams of Grifola Frondosa sporophore dry product, it is placed in vacuum drying chamber 60 DEG C and is dried under reduced pressure 2 hours, use mechanical crushing
It is fine powder that machine, which crushes dried Grifola Frondosa sporophore, crosses 80 mesh sieves, obtains 98 grams of Grifola Frondosa sporophore powder.By grifola frondosus
Powder is added in 2L deionized waters, is placed in after powder fully soaks in 100 DEG C of water-baths, is extracted 3 hours, centrifuge the extract,
Rotating speed 5000rpm, 10 minutes time.Grifola Frondosa sporophore residue is precipitated as, is discarded, supernatant liquor uses Rotary Evaporators
200mL is concentrated into, the extract solution after concentration is poured into 800mL ethanol, is statically placed in 4 DEG C of environment and precipitates 12 hours.Centrifugation, turn
Fast 5000rpm, 10 minutes time, abandoning supernatant, precipitation is collected, obtains 7.8 grams of grifola frondosus Thick many candies.By grifola frondosus Thick many candies
It is dissolved in 70mL5% sodium hydroxide solutions, does not stop stirring to dissolving completely, be subsequently placed in 4 DEG C of environment and deposit 24 hours.Adjust molten
Liquid pH to 7.0, polysaccharide solution is transferred in 5000D bag filters, flowing water is dialysed 48 hours.Use Rotary Evaporators concentrate dialysate
To 50mL, freeze-drying removes moisture, obtains 3.6 grams of alkali process grifolan.3.6 grams of alkali process grifolans are dissolved in
In 72mL deionized waters, centrifuge after abundant dissolving, supernatant is carried out using the chromatographic column equipped with DEAE-SephdexA-25
Purifying, is washed with deionized water de-.Eluent main peak 40mL is collected, is added in 100mL PBS (pH=7.4), then is added
Enter 1.4 grams of amylase, be placed in 60 DEG C of water-baths and carry out enzyme reaction 24 hours.Enzyme reaction solution is taken out to be placed in 100 DEG C of water and boiled
Water-bath 10 minutes, 8000rpm are centrifuged 10 minutes, discard precipitation, and supernatant is loaded 5000D bag filters, dialysed using deionized water
24 hours.Dialyzate is concentrated into 50mL using Rotary Evaporators, freeze-drying removes moisture, obtains decolouring grifolan
1.6 gram.1.6 grams of alkali process grifolans are dissolved in 32mL deionized waters, are centrifuged after abundant dissolving, using equipped with SP-
Sephdex C-25 chromatographic column purifies to supernatant, is washed with deionized water de-.Eluent main peak is collected, is freeze-dried,
Obtain 0.7 gram of grifolin.
Claims (7)
1. a kind of method that grifolan grifolin is separated from grifola frondosus, comprises the following steps：
1) water extract-alcohol precipitation：Grifola frondosus raw material is extracted using deionized water, separation of solid and liquid and concentration extraction after extraction
Liquid, make polysaccharide precipitation using ethanol precipitation；
2) alkali lye extracts：Grifolan precipitation is extracted again using sodium hydroxide solution；
3) dialysis is neutralized：Neutralize sodium hydroxide, dialysis is except desalting and urea；
4) gel chromatography 1：The component in dialyzate is separated using anionite, is eluted with water；
5) ferment treatment：Separation of solid and liquid and dialysed using ferment treatment eluent, after reaction terminating；
6) gel chromatography 2：The component in dialyzate is separated using cation-exchanger, is eluted with water；
7) it is freeze-dried：Eluent is freeze-dried, that is, obtains grifolan grifolin.
A kind of 2. grifolan grifolin extracting method as claimed in claim 1, it is characterised in that in the step 1,
The mass ratio of deionized water and ash tree floral material is 18: 1~22: 1, and the volume ratio of ethanol and extract solution is 3.5: 1~4.5: 1.
A kind of 3. grifolan grifolin extracting method as claimed in claim 1, it is characterised in that in the step 1,
Extraction temperature is 95 DEG C~100 DEG C, and extraction time is 2~3 hours.
A kind of 4. grifolan grifolin extracting method as claimed in claim 1, it is characterised in that in the step 2,
Naoh concentration is 5%~10% in alkali lye, and alkali lye quality is 8~10 times of Thick many candies quality.
A kind of 5. grifolan grifolin extracting method as claimed in claim 1, it is characterised in that in the step 4,
Used cation-exchanger is strong cation exchanger.
A kind of 6. grifolan grifolin extracting method as claimed in claim 1, it is characterised in that in the step 5,
Used enzyme is amylase, and reaction temperature is 60 DEG C~70 DEG C, and the reaction time is 12~24 hours.
A kind of 7. grifolan grifolin extracting method as claimed in claim 1, it is characterised in that in the step 6,
Used anionite is strong anion exchanger.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610802778.3A CN107793489A (en) | 2016-09-05 | 2016-09-05 | A kind of method that polysaccharide grifolin is extracted from grifola frondosus |
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| CN201610802778.3A CN107793489A (en) | 2016-09-05 | 2016-09-05 | A kind of method that polysaccharide grifolin is extracted from grifola frondosus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102022408B1 (en) * | 2019-06-18 | 2019-11-25 | 주식회사 라비오 | Anti-aging composition comprising fermented albatrellus confluens products |
| CN116283786A (en) * | 2023-03-03 | 2023-06-23 | 深圳摩尔雾化健康医疗科技有限公司 | Preparation method of Grifolin derivative in Geotrichum candidum and application of Grifolin derivative in anti-aging skin care product |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736053A (en) * | 2010-02-01 | 2010-06-16 | 南京泽朗医药科技有限公司 | Technique for extracting Grifola frondosa water-soluble polysaccharide |
-
2016
- 2016-09-05 CN CN201610802778.3A patent/CN107793489A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736053A (en) * | 2010-02-01 | 2010-06-16 | 南京泽朗医药科技有限公司 | Technique for extracting Grifola frondosa water-soluble polysaccharide |
Non-Patent Citations (2)
| Title |
|---|
| TOSHIO GOTO等: "The structure of grifolin, an antibiotic from a basidiomycete", 《TETRAHEDRON》 * |
| Y. HIRATA等: "grifolin, an antibiotic from a basidiomycete", 《J. BIOL. CHEM.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102022408B1 (en) * | 2019-06-18 | 2019-11-25 | 주식회사 라비오 | Anti-aging composition comprising fermented albatrellus confluens products |
| CN116283786A (en) * | 2023-03-03 | 2023-06-23 | 深圳摩尔雾化健康医疗科技有限公司 | Preparation method of Grifolin derivative in Geotrichum candidum and application of Grifolin derivative in anti-aging skin care product |
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Application publication date: 20180313 |