CN106146685B - A kind of extraction of Polysaccharide of Brasenia Schreberi and separation purifying technique - Google Patents
A kind of extraction of Polysaccharide of Brasenia Schreberi and separation purifying technique Download PDFInfo
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- CN106146685B CN106146685B CN201610872170.8A CN201610872170A CN106146685B CN 106146685 B CN106146685 B CN 106146685B CN 201610872170 A CN201610872170 A CN 201610872170A CN 106146685 B CN106146685 B CN 106146685B
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- 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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- 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
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- 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
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Abstract
The invention discloses a kind of extraction of Polysaccharide of Brasenia Schreberi and separation purifying technique, the present invention utilizes CO_2 supercritical technology, overcomes Hot water extraction and the relatively low disadvantage of NaOH extraction method recovery rates, and the safety issue that NaOH extraction highly basic uses also is not present；When the present invention is to Polysaccharide of Brasenia Schreberi purifying crude, protein is removed using basic protein enzymatic treatment；Cetyl trimethylammonium bromide can form quaternary amine salt complex with Polysaccharide of Brasenia Schreberi, these complex compounds are insoluble in the aqueous solution of low ionic strength, and when ionic strength is big, complex compound can be dissociated, be dissolved, and it is more preferable to isolate and purify effect；It is chromatographed using 312 weak-type anion exchange resin, some positively charged impurity can be removed, isolated and purified better.
Description
Technical field
The invention belongs to the method and technology fields of plant extract polysaccharide, and in particular to a kind of extraction of Polysaccharide of Brasenia Schreberi with detach
Purifying process.
Background technology
Water shield (Brasenia schreber J.F.Gme1) is original perianth subclass Nymphaeceae (Nymphaeaceae) water shield
Belong to perennial fresh water aquatic herbaceous plant also known as membranaceous marshmarigold herb, lake dish, water certain herbaceous plants with big flowers, dew certain herbaceous plants with big flowers, originates in China southeast, be grown on
In pond, lake and marsh.Water shield has abundant nutritive value and medical value, according to《Compendium of Materia Medica》It records, water shield has
Quench one's thirst hot numbness, control hot subcutaneous ulcer, thick stomach, An Xiajiao, relieve oedema or abdominal distension through diuresis or purgation, solve hundred poison and other effects.Studies have shown that Polysaccharide of Brasenia Schreberi have it is antitumor,
Anti-oxidant, reducing blood lipid, hypoglycemic, anti-inflammatory is antibacterial, improves constipation and orectic effect, has higher health value.State
Outer Polysaccharide of Brasenia Schreberi is expensive, and international market price has reached 3000 dollars/kilogram.
But the research at present in terms of Polysaccharide of Brasenia Schreberi extraction is few, and extracting method substantially has ultrasonic wave, microwave at this stage
After compound enzyme pretreatment, then carry out hot water extraction；After room temperature NaOH extractions removing protein is removed with alkali protease.But due to water shield
Polysaccharide viscosity is big, sad filter, difficult concentration, difficult purifying, therefore Hot water extraction and NaOH extraction method recovery rates are relatively low, and NaOH is extracted again
There are the safety issues that highly basic uses.And these method extraction yields are low, and utilization rate is low, greatly constrain water shield industry
Development.
So a kind of extraction of efficient Polysaccharide of Brasenia Schreberi of research and separating and purifying technology for the development of water shield industry be to
It closes important.
Invention content
In view of this, the purpose of the present invention is to provide a kind of extraction of Polysaccharide of Brasenia Schreberi and separation purifying techniques.It is extracted
Rate is high, and effect is good, overcomes Hot water extraction and NaOH extraction method recovery rates are relatively low, and there are the peaces that highly basic uses again for NaOH extractions
Full sex chromosome mosaicism.
For achieving the above object, the following technical solution is specifically provided：
A kind of extraction of Polysaccharide of Brasenia Schreberi and separation purifying technique, include the following steps：
(1) extraction of Polysaccharide of Brasenia Schreberi crude product：
(1.1) water shield is crushed to 100~200 mesh, the water shield after crushing is put to supercritical CO2In extraction kettle, kettle is sealed,
Air is excluded, carrying agent is passed through and carries out extracting to obtain extracting solution, extraction temperature is 44.0-45.5 DEG C, extracting pressure 20.5-
21.5MPa, extract carbon dioxide flow are 20-28L/h, and carrying agent is distilled water, and dosage 400-450ml/kg extracts 4-
5h；
(1.2) mixed liquor of chloroform and n-butanol is added into extracting solution and shakes up, 5 are centrifuged in 3000~5000r/min
~10min, takes supernatant liquor I, and the volume ratio of chloroform and n-butanol is 4 in the mixed liquor:1, the extracting solution and mixed liquor
Volume ratio be 4~5:1；
(1.3) it is 7.5-8.5 to adjust supernatant liquor to pH with NaOH, and activated carbon is then added into clear liquid is decolourized simultaneously
Filter to obtain filtrate；Filtrate is further used into distilled water dialysis 36-48h and obtains dialyzate；
(1.4) dialyzate 3000-5000r/min centrifugations 5-10min is obtained into supernatant liquor II, by supernatant liquor II 75
It is concentrated into the 1/3-1/4 of original volume under DEG C -80 DEG C of water bath conditions, adds the 95wt% ethyl alcohol of 3-4 times of volume, stirs evenly
Afterwards, 3000-5000r/min, which centrifuges 5-10min, must precipitate I, precipitation I be washed with absolute ethyl alcohol and acetone respectively, dry water shield
Dish polysaccharide crude；
(2) purifying of Polysaccharide of Brasenia Schreberi crude product
(2.1) Polysaccharide of Brasenia Schreberi crude product and distilled water are taken with mass volume ratio 1%-2% and are uniformly mixed, 3000-5000r/
Min centrifuges 5-10min and removes insoluble matter, takes supernatant 1mol/L NaOH tune pH to 8.6-9.2, the alkalinity of 0.1wt% is added
Protease, 46 DEG C of -50 DEG C of enzymolysis 4-5h removing proteins, 8000-10000r/min centrifugations 8-10min obtain supernatant liquor III；
(2.2) add cetyl trimethylammonium bromide complete to precipitating to upper layer clear liquid I II, stand 5h, 3000-5000r/
Min centrifuges the precipitation II of 5-10min, and precipitation II is washed three times with ice water, adds 52-57 DEG C of dissociation 3- of NaCl solution to precipitation II
4h, and obtain supernatant liquor IV in 3000-5000r/min centrifugations 5-10min；
(2.3) supernatant liquor IV is adsorbed by the 312 weak-type anion-exchange resin columns handled well, then by resin
It moves into beaker, ice water, which is washed till clarification, to be stopped, then upper prop, with 0.5mol/L Na2HPO4-0.3mol/L Na2SO4Elution,
Flow velocity 1ml/min, obtains eluent；
(2.4) eluent adds 95% ethyl alcohol of three times volume using distilled water dialysis 12h, 80 DEG C of water-baths concentrations of dialyzate,
After stirring evenly, 3000r/min centrifuge 10min, precipitation washed respectively with absolute ethyl alcohol and acetone again, be dried in vacuo water shield is more
Sugar.
Preferably, 45.2 DEG C, extracting pressure 21.3MPa of step (1.1) extraction temperature, carrying agent are distilled water, are used
Amount is 420.0ml/kg, extracts 4.0h.
The beneficial effects of the present invention are：
1, the present invention utilizes CO_2 supercritical technology, overcomes Hot water extraction and NaOH extraction method recovery rates are relatively low
The shortcomings that, and the safety issue that NaOH extraction highly basic uses also is not present.
2, when the present invention is to Polysaccharide of Brasenia Schreberi purifying crude, protein is removed using basic protein enzymatic treatment；Cetyl three
Methyl bromide ammonium can form quaternary amine salt complex with Polysaccharide of Brasenia Schreberi, these complex compounds are insoluble in the aqueous solution of low ionic strength,
When ionic strength is big, complex compound can be dissociated, be dissolved, and it is more preferable to isolate and purify effect.
3, it is chromatographed using 312 weak-type anion exchange resin, some positively charged impurity can be removed, isolate and purify effect
Fruit is more preferably.
Specific implementation mode
The preferred embodiment of the present invention is described in detail below.The experiment side of actual conditions is not specified in embodiment
Method, usually according to conventional conditions or according to the manufacturer's recommendations.
Following embodiment agents useful for same and equipment：Bag filter (Sigma), 312 weak-type anion exchange resin (Luo Menha
This), alkali protease (the green skies), conventional chemical reagent are purchased from Beijing Ding Guo Bioisystech Co., Ltd.
Embodiment 1
(1) extraction of Polysaccharide of Brasenia Schreberi crude product：
(1.1) water shield is crushed to 100~200 mesh, the water shield after crushing is put to supercritical CO2In extraction kettle, kettle is sealed,
Air is excluded, carrying agent is passed through and carries out extracting to obtain extracting solution, extraction temperature is 45.2 DEG C, extracting pressure 21.3MPa, extraction two
Oxidation carbon flow is 25L/h, and carrying agent is distilled water, and dosage 420ml/kg extracts 4h；
(1.2) mixed liquor of chloroform and n-butanol is added into extracting solution and shakes up, centrifuges 10min in 3000r/min, takes
Supernatant liquor I, the volume ratio of chloroform and n-butanol is 4 in the mixed liquor:1, the volume ratio of the extracting solution and mixed liquor
It is 4:1；
(1.3) it is 8.0 to adjust supernatant liquor to pH with NaOH, and activated carbon is then added into clear liquid is decolourized and filtered
Obtain filtrate；Filtrate is further used into distilled water dialysis 48h and obtains dialyzate；
(1.4) dialyzate 3000r/min centrifugations 10min is obtained into supernatant liquor II, by supernatant liquor II in 80 DEG C of water-bath items
It is concentrated into the 1/3 of original volume under part, adds the 95wt% ethyl alcohol of 3 times of volumes, after stirring evenly, 3000r/min centrifuges 10min
I must be precipitated, precipitation I is washed with absolute ethyl alcohol and acetone respectively, dry Polysaccharide of Brasenia Schreberi crude product；
(2) purifying of Polysaccharide of Brasenia Schreberi crude product
(2.1) Polysaccharide of Brasenia Schreberi crude product and distilled water are taken with mass volume ratio 1% and are uniformly mixed, 3000r/min centrifugations
10min removes insoluble matter, takes supernatant 1mol/L NaOH tune pH to 9.0, the alkali protease of 0.1wt%, 50 DEG C of enzymes are added
5h removing proteins are solved, 8000r/min centrifugations 10min obtains supernatant liquor III；
(2.2) add cetyl trimethylammonium bromide complete to precipitating to upper layer clear liquid I II, stand 5h, 3000r/min from
Precipitation II is washed three times with ice water, adds 55 DEG C of solutions of 100ml 2mol/L NaCl solutions to precipitation II by the precipitation II of heart 10min
From 4h, 3000r/min centrifugations 10min obtains supernatant liquor IV；
(2.3) supernatant liquor IV is adsorbed by the 312 weak-type anion-exchange resin columns handled well, then by resin
It moves into beaker, ice water, which is washed till clarification, to be stopped, then upper prop, with 0.5mol/L Na2HPO4-0.3mol/L Na2SO4Elution,
Flow velocity 1ml/min, obtains eluent；
(2.4) eluent adds 95% ethyl alcohol of three times volume using distilled water dialysis 12h, 80 DEG C of water-baths concentrations of dialyzate,
After stirring evenly, 3000r/min centrifuge 10min, precipitation washed respectively with absolute ethyl alcohol and acetone again, be dried in vacuo water shield is more
Sugar.
Measurement of the polysaccharide content
Polyoses content is detected using sulfuric acid-phynol method.Polysaccharide under the action of the concentrated sulfuric acid, dehydration generate alditol and its
Derivative generates yellow substance after being combined with phenol, which has maximum light absorption value, the size and polysaccharide of light absorption value in 490nm
Content is proportional.
(1) making of glucose standard curve：
After weighing a certain amount of glucose drying to constant weight in 105 DEG C, 0.100g is accurately weighed, pure water is settled to 100mL,
Up to Glucose standards storing solution.It is accurate respectively to draw 0,2.0,4.0,6.0,8.0,10.0mL storing solutions, it is respectively placed in 100mL
Volumetric flask is configured to 0,20,40,60,80,100 μ g/mL prepare liquids with pure water constant volume.Accurate 1.0mL prepare liquids of drawing are in test tube
In, 6% phenol of 1mL, the 5mL concentrated sulfuric acids are sequentially added, mixing is stored at room temperature 30min.A test tube separately is taken, it is accurate to draw
1.0mL pure water sequentially adds 1mL6% phenol, and the 5mL concentrated sulfuric acids, mixing is stored at room temperature 30min, as blank control.490nm
Place surveys light absorption value, is returned to concentration of glucose with absorbance, obtains regression equation y=0.0101x-0.0191, R2=
0.9841.In formula：Y is the light absorption value at wavelength 490nm, and x is the concentration (μ g/mL) of glucose.
Polysaccharide yield is evaluated with total reducing sugar leaching rate, total sugar content calculates as follows：
/ (Vs × m × 10 total sugar content %=(m ' × V × N)6) × 100%, in formula：
m’：The glucose content checked on standard curve, μ g；
V：Sample liquid volume, mL；
N：Extension rate；
Vs：The volume of sample liquid, mL are drawn when measurement；
m：The quality of sample, g.
Polysaccharide of Brasenia Schreberi extracting solution is settled to 250mL volumetric flasks, the accurate 1.0mL that draws is in 10mL volumetric flasks, with pure water
Constant volume.1.0mL prepare liquids are accurately drawn after mixing in test tube, sequentially add 6% phenol of 1mL Fresh, the 5mL concentrated sulfuric acids
Mixing is stored at room temperature 30min.A test tube separately is taken, it is accurate to draw 1.0mL pure water, sequentially add 1mL6% phenol, the dense sulphur of 5ml
Acid, mixing are stored at room temperature 30min, and as blank control, light absorption value is measured at 490nm.
Through assay, for the Polysaccharide of Brasenia Schreberi extraction rate reached in water shield to 85.2%, purity reaches 80.5%.
The present invention utilizes supercritical CO2Abstraction technique, overcomes Hot water extraction and NaOH extraction method recovery rates are lower
Disadvantage, and the safety issue that NaOH extraction highly basic uses also is not present.When the present invention is to Polysaccharide of Brasenia Schreberi purifying crude, use
Basic protein enzymatic treatment removes protein；Cetyl trimethylammonium bromide can form quaternary amine salt complex with Polysaccharide of Brasenia Schreberi, this
A little complex compounds are insoluble in the aqueous solution of low ionic strength, and when ionic strength is big, complex compound can be dissociated, be dissolved, and separation is pure
It is more preferable to change effect.It is chromatographed using 312 weak-type anion exchange resin, some positively charged impurity can be removed, isolated and purified
It is better.And Polysaccharide of Brasenia Schreberi its extraction efficiency height extracted using the method for the invention, purity are good.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (2)
1. extraction and the separation purifying technique of a kind of Polysaccharide of Brasenia Schreberi, which is characterized in that include the following steps：
（1）The extraction of Polysaccharide of Brasenia Schreberi crude product：
（1.1）Water shield is crushed to 100 ~ 200 mesh, the water shield after crushing is put to supercritical CO2In extraction kettle, kettle is sealed, is excluded empty
Gas is passed through carrying agent and carries out extracting to obtain extracting solution, and extraction temperature is 44.0-45.5 DEG C, extracting pressure 20.5-21.5MPa, extraction
It is 20-28L/h to take carbon dioxide flow, and carrying agent is distilled water, and dosage 400-450ml/kg extracts 4-5h；
（1.2）The mixed liquor of chloroform and n-butanol is added into extracting solution and shakes up, 5 are centrifuged in 3000 ~ 5000r/min ~
10min, takes supernatant liquor I, and the volume ratio of chloroform and n-butanol is 4 in the mixed liquor:1, the extracting solution and mixed liquor
Volume ratio is 4 ~ 5:1；
（1.3）It is 7.5-8.5 to adjust supernatant liquor to pH with NaOH, and activated carbon is then added into clear liquid is decolourized and filtered
Obtain filtrate；Filtrate is further used into distilled water dialysis 36-48h and obtains dialyzate；
（1.4）Dialyzate 3000-5000r/min centrifugations 5-10min is obtained into supernatant liquor II, by supernatant liquor II at 75 DEG C -80
It is concentrated into the 1/3-1/4 of original volume under DEG C water bath condition, adds the 95wt% ethyl alcohol of 3-4 times of volume, after stirring evenly, 3000-
5000r/min centrifugations 5-10min must precipitate I, and precipitation I is washed with absolute ethyl alcohol and acetone respectively, dry that Polysaccharide of Brasenia Schreberi is thick
Product；
（2）The purifying of Polysaccharide of Brasenia Schreberi crude product
（2.1）Polysaccharide of Brasenia Schreberi crude product and distilled water are taken with mass volume ratio 1%-2% and are uniformly mixed, 3000-5000r/min centrifugations
5-10min removes insoluble matter, takes supernatant 1mol/L NaOH tune pH to 8.6-9.2, the alkali protease of 0.1wt% is added,
46 DEG C of -50 DEG C of enzymolysis 4-5h removing proteins, 8000-10000r/min centrifugations 8-10min obtain supernatant liquor III；
（2.2）Add cetyl trimethylammonium bromide complete to precipitating to upper layer clear liquid I II, stands 5h, 3000-5000r/min
Precipitation II is washed three times with ice water, adds 100ml 2mol/L NaCl solutions 52- to precipitation II by the precipitation II for centrifuging 5-10min
57 DEG C of dissociation 3-4h, and obtain supernatant liquor IV in 3000-5000r/min centrifugations 5-10min；
（2.3）Supernatant liquor IV is adsorbed by the 312 weak-type anion-exchange resin columns handled well, then moves into resin
In beaker, ice water, which is washed till clarification, to be stopped, then upper prop, with 0.5mol/L Na2HPO4-0.3mol/L Na2SO4Elution, flow velocity
1ml/min obtains eluent；
（2.4）Eluent is added 95% ethyl alcohol of three times volume, is stirred using distilled water dialysis 12h, 80 DEG C of water-baths concentrations of dialyzate
After uniformly, 3000r/min centrifuges 10min, and precipitation is washed with absolute ethyl alcohol and acetone respectively again, is dried in vacuo to obtain Polysaccharide of Brasenia Schreberi.
2. a kind of extraction of Polysaccharide of Brasenia Schreberi and separation purifying technique according to claim 1, which is characterized in that the step
（1.1）45.2 DEG C, extracting pressure 21.3MPa of extraction temperature, carrying agent are distilled water, and dosage 420.0ml/kg extracts 4.0h.
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| CN108484784B (en) * | 2018-01-11 | 2020-12-11 | 浙江工业大学 | Physical separation method of in-vitro polysaccharide gum of water shield |
| CN110194810A (en) * | 2019-07-19 | 2019-09-03 | 浙江工业大学 | A kind of grading purification method for the external glue polysaccharide of water shield improving hypoglycemic activity |
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|---|---|---|---|---|
| CN102060933A (en) * | 2010-12-06 | 2011-05-18 | 湖北民族学院 | Method for extracting and separating in-vivo water-soluble polysaccharide from old water shield leaves |
| CN102964460A (en) * | 2012-11-16 | 2013-03-13 | 北京石油化工学院 | Method for continuously extracting and separating polysaccharides and 1-deoxynojirimycin from mulberry leaves |
| CN103755822A (en) * | 2013-12-06 | 2014-04-30 | 山东好当家海洋发展股份有限公司 | Brasenia schreberi polysaccharide preparation method |
| CN104341533A (en) * | 2014-10-31 | 2015-02-11 | 西南大学 | Brasenia schreberi polysaccharide extraction method |
| CN104367853A (en) * | 2014-10-22 | 2015-02-25 | 苏州市博力生物科技有限公司 | Aquatic plant compound, extraction method of polysaccharide in aquatic plant compound, and novel use of polysaccharide in aquatic plant compound |
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| KR100544052B1 (en) * | 2003-10-31 | 2006-01-23 | 주식회사 엠디바이오알파 | Polysaccharides Of Plants Belonging To Panax Having Effect On Treatment And Prevention Of Obesity |
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- 2016-09-30 CN CN201610872170.8A patent/CN106146685B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102060933A (en) * | 2010-12-06 | 2011-05-18 | 湖北民族学院 | Method for extracting and separating in-vivo water-soluble polysaccharide from old water shield leaves |
| CN102964460A (en) * | 2012-11-16 | 2013-03-13 | 北京石油化工学院 | Method for continuously extracting and separating polysaccharides and 1-deoxynojirimycin from mulberry leaves |
| CN103755822A (en) * | 2013-12-06 | 2014-04-30 | 山东好当家海洋发展股份有限公司 | Brasenia schreberi polysaccharide preparation method |
| CN104367853A (en) * | 2014-10-22 | 2015-02-25 | 苏州市博力生物科技有限公司 | Aquatic plant compound, extraction method of polysaccharide in aquatic plant compound, and novel use of polysaccharide in aquatic plant compound |
| CN104341533A (en) * | 2014-10-31 | 2015-02-11 | 西南大学 | Brasenia schreberi polysaccharide extraction method |
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