CN106397622A - Fritillariae cirrhosae bulbus polysaccharide extraction, separation and purification technology - Google Patents
Fritillariae cirrhosae bulbus polysaccharide extraction, separation and purification technology Download PDFInfo
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- CN106397622A CN106397622A CN201610886229.9A CN201610886229A CN106397622A CN 106397622 A CN106397622 A CN 106397622A CN 201610886229 A CN201610886229 A CN 201610886229A CN 106397622 A CN106397622 A CN 106397622A
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- Prior art keywords
- fritillariae cirrhosae
- polysaccharide
- extraction
- bulbus fritillariae
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention discloses a fritillariae cirrhosae bulbus polysaccharide extraction, separation and purification technology. As a novel supercritical CO2 extraction technology is utilized, the defects that a general method is low in extraction rate and not high in purity are overcome; quaternary ammonium salt cetyl pyridinium chloride monohydrate and fritillariae cirrhosae bulbus polysaccharide can precipitate in water solution with low ionic strength; when the ionic strength is high, precipitate can be dissolved, dissociated and released, and the purification effect is good; DEAE-Sephadex ionic exchange column chromatography is utilized to further remove neutral impurities and impurities with positive charges, and the separation and purification effect is better.
Description
Technical field
The invention belongs to the method and technology field of plant extract polysaccharide and in particular to the extraction of Bulbus Fritillariae Cirrhosae polysaccharide with separate pure
Metallization processes.
Background technology
Bulbus Fritillariae Cirrhosae is Liliaceae Fritillaria Linn, and perennial herb is distributed in the ground such as Sichuan, Yunnan and Sichuan.Bulbus Fritillariae Cirrhosae
There is nourishing the lung to arrest cough, at present, Bulbus Fritillariae Cirrhosae is concentrated mainly on the research of its alkaloid, the extraction to Bulbus Fritillariae Cirrhosae polysaccharide and pharmacology
Research is less.
At present, the extraction of Bulbus Fritillariae Cirrhosae polysaccharide and separation method have hot water extraction, an ethanol extraction, ultrasonic extraction and cable-styled carry
The method such as take.But these conventional method extraction ratios all low (2% about), purity not high (80% about).
So studying the development for Bulbus Fritillariae Cirrhosae industry of a kind of extraction of efficient Bulbus Fritillariae Cirrhosae polysaccharide and separating and purifying technology
It is critical that.
Content of the invention
In view of this, it is an object of the invention to provide a kind of extraction of Bulbus Fritillariae Cirrhosae polysaccharide and separation purifying technique, described
Technology utilization supercritical CO2After abstraction technique extracts Bulbus Fritillariae Cirrhosae polysaccharide, E.C. 3.4.21.64 process removing protein, CPC quaternary salt deposit, then
It is further purified through ion-exchange chromatography.This method extraction ratio and purity are high, can overcome the extraction ratio that conventional method exists low,
The shortcomings of purity is high.
For achieving the above object, specifically provide following technical scheme：
The extraction of Bulbus Fritillariae Cirrhosae polysaccharide and separation purifying technique, comprise the steps：
(1) extraction of Bulbus Fritillariae Cirrhosae polysaccharide crude
(1.1) extract：Take Bulbus Fritillariae Cirrhosae bulb, dry and be crushed to particle diameter and be less than 60 mesh, by the Bulbus Fritillariae Cirrhosae bulb after pulverizing
Put to supercritical CO2In extraction kettle, seal kettle, exclude air, be passed through and carry agent and carry out extracting to obtain extracting solution, extraction temperature is 47.3
DEG C, extracting pressure is 23.7MPa, and extract carbon dioxide flow is 20-28L/h, and carrying agent is distilled water, and consumption is 390ml/
Kg, extracts 2.5h；
(1.2) decolour：Step (1.1) gained extracting solution is adjusted to pH 7.8 with 1mol/L NaOH, and adds kieselguhr to add
Heat boils 5min decolouring, and filtration under diminished pressure obtains filtrate, and added kieselguhr is 1.5% with the mass ratio of extracting solution；
(1.3) concentrate drying：Step (1.2) gained filtrate is just evaporated to original volume in 75 DEG C of water bath condition
30%, add the 95wt% ethanol of 4 times of volumes, stirring, under the conditions of 3000r/min, centrifugation 10min must precipitate I, and precipitation I divides
Do not washed with dehydrated alcohol and ether, be vacuum dried to obtain Bulbus Fritillariae Cirrhosae polysaccharide crude；
(2) purification of Bulbus Fritillariae Cirrhosae polysaccharide crude
(2.1) removing protein：Polysaccharide of Brasenia Schreberi crude product and distilled water mix homogeneously, 3000r/min are taken with mass volume ratio 1%
Centrifugation 10min removes insoluble matter, takes supernatant to add chloroform and the amylalcohol mixed liquor of 1/5 volume, 3000r/min is centrifuged 10min
Obtain supernatant I, in described mixed liquor, the volume ratio of chloroform and amylalcohol is 5:1；
(2.2) purification：Add hexadecylpyridinium chloride to precipitating completely to upper strata clear liquid I, stand 5h, 3000r/min from
Heart 10min must precipitate II, precipitates II 70 DEG C of hot washes three times, then to precipitation II addition 2mol/L NaCl solution 60
Dissociate under DEG C temperature conditionss 4h, and obtains supernatant II in 3000r/min centrifugation 10min；
(2.3) ion-exchange chromatography is further purified：Supernatant II is adsorbed by DEAE-Sephadex post, frozen water
It is washed till clarification, uses 0.1mol/LNaCl eluting, flow velocity is 0.2ml/min, collects eluent；
(2.4) dialyse, concentrate, be dried：Step (2.3) gained eluent is used distilled water dialysis 18h, gained is dialysed
Liquid is evaporated to the 30% of original volume under 75 DEG C of water bath condition, adds the 95wt% ethanol of 4 times of volumes, stirring,
3000r/min centrifugation 10min must precipitate III, and precipitation III is washed with dehydrated alcohol and ether respectively, and vacuum drying obtains refined river
Bulbus Fritillariae Uninbracteatae polysaccharide.
Preferably, the described hexadecylpyridinium chloride mass fraction of step (2.2) is 2%.
The beneficial effects of the present invention is：
1) present invention adopts supercritical CO2Novel extraction technology, overcome conventional method presence extraction ratio is low, purity not
High the shortcomings of；
2). quaternary ammonium salt hexadecylpyridinium chloride can be precipitated out in the aqueous solution of low ionic strength with Bulbus Fritillariae Uninbracteatae polysaccharide,
When high ionic strength, precipitation can dissolve, dissociate, discharging again, and purification effect is good；
3) utilize DEAE-Sephadex ion-exchange chromatography, remove some not charged and positively charged impurity further,
Isolate and purify effect more preferable.
Specific embodiment
Below the preferred embodiments of the present invention are described in detail.The experiment side of unreceipted actual conditions in embodiment
Method, generally according to normal condition or according to the condition proposed by manufacturer.
Following examples agents useful for same and equipment：Supercritical extraction system (U.S. STF-100), bag filter (Sigma),
DEAE-Sephadex ion exchange resin (Shanghai Heng Xin chemical reagent company limited), conventional chemical reagent are purchased from Beijing ancient cooking vessel
Bioisystech Co., Ltd of state.
Embodiment 1
(1) extraction of Bulbus Fritillariae Cirrhosae polysaccharide crude
(1.1) extract：Take Bulbus Fritillariae Cirrhosae bulb, dry and be crushed to particle diameter and be less than 60 mesh, by the Bulbus Fritillariae Cirrhosae bulb after pulverizing
Put to supercritical CO2In extraction kettle, seal kettle, exclude air, be passed through and carry agent and carry out extracting to obtain extracting solution, extraction temperature is 47.3
DEG C, extracting pressure is 23.7MPa, and extract carbon dioxide flow is 20-28L/h, and carrying agent is distilled water, and consumption is 390ml/
Kg, extracts 2.5h；
(1.2) decolour：Step (1.1) gained extracting solution is adjusted to pH 7.8 with 1mol/L NaOH, and adds kieselguhr to add
Heat boils 5min decolouring, and filtration under diminished pressure obtains filtrate, and added kieselguhr is 1.5% with the mass ratio of extracting solution；
(1.3) concentrate drying：Step (1.2) gained filtrate is just evaporated to original volume in 75 DEG C of water bath condition
30%, add the 95wt% ethanol of 4 times of volumes, stirring, under the conditions of 3000r/min, centrifugation 10min must precipitate I, and precipitation I divides
Do not washed with dehydrated alcohol and ether, be vacuum dried to obtain Bulbus Fritillariae Cirrhosae polysaccharide crude；
(2) purification of Bulbus Fritillariae Cirrhosae polysaccharide crude
(2.1) removing protein：Polysaccharide of Brasenia Schreberi crude product and distilled water mix homogeneously, 3000r/min are taken with mass volume ratio 1%
Centrifugation 10min removes insoluble matter, takes supernatant to add chloroform and the amylalcohol mixed liquor of 1/5 volume, 3000r/min is centrifuged 10min
Obtain supernatant I, in described mixed liquor, the volume ratio of chloroform and amylalcohol is 5:1；
(2.2) purification：Add the hexadecylpyridinium chloride that mass fraction is 2% to precipitating completely to upper strata clear liquid I, stand
5h, 3000r/min centrifugation 10min must precipitate II, precipitates II 70 DEG C of hot washes three times, then adds 2mol/L to precipitation II
NaCl solution dissociate under 60 DEG C of temperature conditionss 4h, and obtain supernatant II in 3000r/min centrifugation 10min；
(2.3) ion-exchange chromatography is further purified：Supernatant II is adsorbed by DEAE-Sephadex post, frozen water
It is washed till clarification, uses 0.1mol/LNaCl eluting, flow velocity is 0.2ml/min, collects eluent；
(2.4) dialyse, concentrate, be dried：Step (2.3) gained eluent is used distilled water dialysis 18h, gained is dialysed
Liquid is evaporated to the 30% of original volume under 75 DEG C of water bath condition, adds the 95wt% ethanol of 4 times of volumes, stirring,
3000r/min centrifugation 10min must precipitate III, and precipitation III is washed with dehydrated alcohol and ether respectively, and vacuum drying obtains refined river
Bulbus Fritillariae Uninbracteatae polysaccharide.
Measurement of the polysaccharide content
Polyoses content is detected using sulfuric acid-phynol method.Polysaccharide in the presence of concentrated sulphuric acid, dehydration generate alditol and its
Derivant, is combined rear generation yellow substance with phenol, and this material has maximum light absorption value, the size of light absorption value and polysaccharide in 490nm
Content is proportional.
(1) making of glucose standard curve：
Weigh after a certain amount of glucose dries to constant weight in 105 DEG C, accurately weigh 0.100g, pure water is settled to 100mL,
Obtain final product Glucose standards storing solution.Accurately draw 0,2.0,4.0,6.0,8.0,10.0mL storing solutions respectively, be respectively placed in 100mL
Volumetric flask, with pure water constant volume, is configured to 0,20,40,60,80,100 μ g/mL prepare liquids.Accurately draw 1.0mL prepare liquid in test tube
In, sequentially add 1mL 6% phenol, 5mL concentrated sulphuric acid, mix, room temperature stands 30min.Separately take a test tube, accurately draw
1.0mL pure water, sequentially adds 1mL6% phenol, 5mL concentrated sulphuric acid, mixes, room temperature stands 30min, as blank.490nm
Place surveys light absorption value, with absorbance, concentration of glucose is returned, obtains regression equation y=0.0111x-0.0098, R2=
0.9661.In formula：Y is the light absorption value at wavelength 490nm, and x is the concentration of glucose. (μ g/mL).
Polysaccharide yield is evaluated with total sugar leaching rate, total sugar content calculates as follows：
Total sugar content %=(m ' × V × N)/(Vs × m × 106) × 100%, in formula：
m’：The glucose content checking on standard curve, μ g；
V：Sample liquid volume, mL；
N：Extension rate；
Vs：The volume of sample liquid, mL is drawn during mensure；
m：The quality of sample, g.
Bulbus Fritillariae Cirrhosae polysaccharide extraction liquid is settled to 250mL volumetric flask, accurately draws 1.0mL in 10mL volumetric flask, with pure
Water constant volume.Accurately draw 1.0mL prepare liquid after mixing in test tube, sequentially add freshly prepared 6% phenol of 1mL, the dense sulfur of 5mL
Acid mixes, and room temperature stands 30min.Separately take a test tube, accurately draw 1.0mL pure water, sequentially add 1mL6% phenol, 5ml is dense
Sulphuric acid, mixes, and room temperature stands 30min, as blank, measures light absorption value at 490nm.
Through assay, Bulbus Fritillariae Cirrhosae polysaccharide extract rate reaches 80%, and purity reaches 90.8%.
The present invention adopts supercritical CO2Novel extraction technology, overcome conventional method presence extraction ratio is low, purity is not high
The shortcomings of；Quaternary ammonium salt hexadecylpyridinium chloride can be precipitated out in the aqueous solution of low ionic strength with Bulbus Fritillariae Uninbracteatae polysaccharide, works as height
During ionic strength, precipitation can dissolve, dissociate, discharging again, and purification effect is good；Using DEAE-Sephadex ion-exchange chromatography,
Remove some not charged and positively charged impurity further, isolate and purify effect more preferable.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and various changes are made to it, without departing from claims of the present invention limited range in details.
Claims (2)
1. the extraction of Bulbus Fritillariae Cirrhosae polysaccharide with separation purifying technique it is characterised in that comprising the steps：
(1) extraction of Bulbus Fritillariae Cirrhosae polysaccharide crude
(1.1) extract：Take Bulbus Fritillariae Cirrhosae bulb, dry and be crushed to particle diameter be less than 60 mesh, by pulverize after Bulbus Fritillariae Cirrhosae bulb put to
Supercritical CO2In extraction kettle, seal kettle, exclude air, be passed through and carry agent and carry out extracting to obtain extracting solution, extraction temperature is 47.3 DEG C,
Extracting pressure is 23.7MPa, and extract carbon dioxide flow is 20-28L/h, and carrying agent is distilled water, and consumption is 390ml/kg, extraction
Take 2.5h；
(1.2) decolour：Step (1.1) gained extracting solution is adjusted to pH 7.8 with 1mol/L NaOH, and adds kieselguhr to boil
Boiling 5min decolouring, filtration under diminished pressure obtains filtrate, and added kieselguhr is 1.5% with the mass ratio of extracting solution；
(1.3) concentrate drying：Step (1.2) gained filtrate is just evaporated to the 30% of original volume in 75 DEG C of water bath condition,
Add the 95wt% ethanol of 4 times of volumes, stirring, under the conditions of 3000r/min, centrifugation 10min must precipitate I, and precipitation I uses respectively
Dehydrated alcohol and ether washing, are vacuum dried to obtain Bulbus Fritillariae Cirrhosae polysaccharide crude；
(2) purification of Bulbus Fritillariae Cirrhosae polysaccharide crude
(2.1) removing protein：Polysaccharide of Brasenia Schreberi crude product and distilled water mix homogeneously are taken with mass volume ratio 1%, 3000r/min is centrifuged
10min removes insoluble matter, takes supernatant to add chloroform and the amylalcohol mixed liquor of 1/5 volume, 3000r/min centrifugation 10min must go up
Layer clear liquid I, in described mixed liquor, the volume ratio of chloroform and amylalcohol is 5:1；
(2.2) purification：Add hexadecylpyridinium chloride to precipitating completely to upper strata clear liquid I, stand 5h, 3000r/min is centrifuged
10min must precipitate II, precipitates II 70 DEG C of hot washes three times, then adds the NaCl solution of 2mol/L at 60 DEG C to precipitation II
Dissociate under temperature conditionss 4h, and obtains supernatant II in 3000r/min centrifugation 10min；
(2.3) ion-exchange chromatography is further purified：Supernatant II is adsorbed by DEAE-Sephadex post, frozen water is washed till
Clarification, uses 0.1mol/LNaCl eluting, and flow velocity is 0.2ml/min, collects eluent；
(2.4) dialyse, concentrate, be dried：Step (2.3) gained eluent is used distilled water dialysis 18h, gained dialysis solution is existed
It is evaporated to the 30% of original volume under 75 DEG C of water bath condition, add the 95wt% ethanol of 4 times of volumes, stirring, 3000r/min
Centrifugation 10min must precipitate III, and precipitation III is washed with dehydrated alcohol and ether respectively, and vacuum drying obtains refined Bulbus Fritillariae Cirrhosae polysaccharide.
2. according to claim 1 the extraction of Bulbus Fritillariae Cirrhosae polysaccharide and separation purifying technique it is characterised in that step (2.2) institute
Stating hexadecylpyridinium chloride mass fraction is 2%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108047343A (en) * | 2017-12-06 | 2018-05-18 | 中国科学院新疆理化技术研究所 | The preparation method and applications of Siberian fritillary bulb total starches |
| CN108530551A (en) * | 2018-04-25 | 2018-09-14 | 河北化工医药职业技术学院 | The preparation of fritillaria polysaccharide and application in preparation of anti-tumor drugs |
| CN111040040A (en) * | 2019-11-11 | 2020-04-21 | 佳木斯大学 | Preparation method and application of fritillaria ussuriensis polysaccharide zinc complex |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108047343A (en) * | 2017-12-06 | 2018-05-18 | 中国科学院新疆理化技术研究所 | The preparation method and applications of Siberian fritillary bulb total starches |
| CN108530551A (en) * | 2018-04-25 | 2018-09-14 | 河北化工医药职业技术学院 | The preparation of fritillaria polysaccharide and application in preparation of anti-tumor drugs |
| CN108530551B (en) * | 2018-04-25 | 2020-07-24 | 河北化工医药职业技术学院 | Preparation of fritillaria polysaccharide and application of fritillaria polysaccharide in preparation of antitumor drugs |
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| CN111040040B (en) * | 2019-11-11 | 2021-10-22 | 佳木斯大学 | Preparation method and application of fritillaria ussuriensis polysaccharide zinc complex |
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