CN106146685A - The extraction of a kind of Polysaccharide of Brasenia Schreberi and separation purifying technique - Google Patents
The extraction of a kind of Polysaccharide of Brasenia Schreberi and separation purifying technique Download PDFInfo
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- CN106146685A CN106146685A CN201610872170.8A CN201610872170A CN106146685A CN 106146685 A CN106146685 A CN 106146685A CN 201610872170 A CN201610872170 A CN 201610872170A CN 106146685 A CN106146685 A CN 106146685A
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- 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
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- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention discloses extraction and the separation purifying technique of a kind of Polysaccharide of Brasenia Schreberi, the present invention utilizes CO_2 supercritical technology, overcomes Hot water extraction and the relatively low shortcoming of NaOH extraction method extraction ratio, and the most there is not NaOH and extract the safety issue that highly basic uses；When the present invention is to Polysaccharide of Brasenia Schreberi purifying crude, basic protein ferment treatment is used to remove protein；Cetyl trimethylammonium bromide can form quaternary amine complex with Polysaccharide of Brasenia Schreberi, and these complex are insoluble in the aqueous solution of low ionic strength, and when ionic strength is big, complex can dissociate, dissolve, and isolated and purified effect is more preferable；Use 312 weak-type anion exchange resin chromatographies, the impurity of some positively chargeds can be removed, isolated and purified better.
Description
Technical field
The invention belongs to the method and technology field of plant extract polysaccharide, be specifically related to the extraction of a kind of Polysaccharide of Brasenia Schreberi and separate
Purifying process.
Background technology
Folium Braseniae Schreberi (Brasenia schreber J.F.Gme1) is original perianth subclass Nymphaeceae (Nymphaeaceae) Herba braseniaeel.
Belong to perennial fresh water aquatic herbaceous plant, have another name called Herba Calthae Membranaceae, lake dish, water certain herbaceous plants with big flowers, dew certain herbaceous plants with big flowers, originate in China southeast, be grown on
In pond, lake and marsh.Folium Braseniae Schreberi has abundant nutritive value and medical value, to record according to Compendium of Material Medica, Folium Braseniae Schreberi has
Quench one's thirst pyretic arthralgia, control hot subcutaneous ulcer, thick the intestines and stomach, the peace part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels, relieve oedema or abdominal distension through diuresis or purgation, solve the effects such as hundred poison.Research shows, Polysaccharide of Brasenia Schreberi have antitumor,
Antioxidation, blood fat reducing, blood sugar lowering, antiinflammatory is antibacterial, improve 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 in terms of Polysaccharide of Brasenia Schreberi extraction is few at present, and present stage extracting method substantially has ultrasound wave, microwave
After compound enzyme pretreatment, then carry out hot water extraction；Room temperature NaOH removes removing protein with alkaline protease after extracting.But due to Folium Braseniae Schreberi
Polysaccharide viscosity is big, sad filter, difficult concentration, difficult purification, therefore Hot water extraction and NaOH extraction method extraction ratio are relatively low, and NaOH extracts again
There is the safety issue that highly basic uses.And these method extraction yields are low, and utilization rate is low, greatly constrain Folium Braseniae Schreberi industry
Development.
So study the extraction of a kind of efficient Polysaccharide of Brasenia Schreberi and separating and purifying technology for the development of Folium Braseniae Schreberi industry be to
Close important.
Summary of the invention
In view of this, it is an object of the invention to provide extraction and the separation purifying technique of a kind of Polysaccharide of Brasenia Schreberi.It extracts
Rate is high, effective, overcomes Hot water extraction and NaOH extraction method extraction ratio is relatively low, and NaOH extracts and there is again the peace that highly basic uses
Full sex chromosome mosaicism.
For achieving the above object, following technical scheme is specifically provided:
The extraction of a kind of Polysaccharide of Brasenia Schreberi and separation purifying technique, comprise the steps:
(1) extraction of Polysaccharide of Brasenia Schreberi crude product:
(1.1) Folium Braseniae Schreberi being crushed to 100～200 mesh, the Folium Braseniae Schreberi after pulverizing is put to supercritical CO2In extraction kettle, seal still,
Getting rid of air, be passed through and carry agent and carry out extracting to obtain extracting solution, extraction temperature is 44.0-45.5 DEG C, and extracting pressure is 20.5-
21.5MPa, extract carbon dioxide flow is 20-28L/h, and carrying agent is distilled water, and consumption is 400-450ml/kg, extracts 4-
5h；
(1.2) in extracting solution, add the mixed liquor of chloroform and n-butyl alcohol and shake up, being centrifuged 5 3000～5000r/min
～10min, taking supernatant I, in described mixed liquor, chloroform is 4:1 with the volume ratio of n-butyl alcohol, described extracting solution and mixed liquor
Volume ratio be 4～5:1；
(1.3) regulating the supernatant with NaOH is 7.5-8.5 to pH, then adds activated carbon in clear liquid and carries out decolouring also
Sucking filtration obtains filtrate；Filtrate use distilled water dialysis 36-48h obtain dialysis solution further；
(1.4) dialysis solution 3000-5000r/min is centrifuged 5-10min and obtains supernatant II, by supernatant II 75
It is concentrated into the 1/3-1/4 of original volume under DEG C-80 DEG C of water bath condition, adds the 95wt% ethanol of 3-4 times of volume, stir
After, 3000-5000r/min is centrifuged 5-10min must precipitate I, precipitation I is used dehydrated alcohol and washing with acetone respectively, is dried and to obtain Herba braseniaeel.
Dish polysaccharide crude；
(2) purification of Polysaccharide of Brasenia Schreberi crude product
(2.1) Polysaccharide of Brasenia Schreberi crude product and distilled water mix homogeneously, 3000-5000r/ are taken with mass volume ratio 1%-2%
Min is centrifuged 5-10min and removes insoluble matter, takes supernatant 1mol/L NaOH and adjusts pH to 8.6-9.2, adds the alkalescence of 0.1wt%
Protease, 46 DEG C-50 DEG C enzymolysis 4-5h removing protein, 8000-10000r/min is centrifuged 8-10min and obtains supernatant III；
(2.2) add cetyl trimethylammonium bromide to precipitation completely to upper strata clear liquid I II, stand 5h, 3000-5000r/
Min is centrifuged precipitation II of 5-10min, is washed three times by precipitation II frozen water, adds NaCl solution 52-57 DEG C to precipitation II and dissociates 3-
4h, and be centrifuged 5-10min at 3000-5000r/min and obtain supernatant IV；
(2.3) supernatant IV is adsorbed, then by resin by the 312 weak-type anion-exchange resin columns handled well
Moving in beaker, frozen water is only washed till clarification, then upper prop, uses 0.5mol/L Na2HPO4-0.3mol/L Na2SO4Elution,
Flow velocity 1ml/min, obtains eluent；
(2.4) eluent uses distilled water dialysis 12h, and 80 DEG C of water-baths of dialysis solution concentrate, and add 95% ethanol that triploid is long-pending,
After stirring, 3000r/min is centrifuged 10min, precipitation the most respectively with dehydrated alcohol and washing with acetone, be vacuum dried Folium Braseniae Schreberi is many
Sugar.
Preferably, described step (1.1) extraction temperature 45.2 DEG C, extracting pressure 21.3MPa, carrying agent is distilled water, uses
Amount is 420.0ml/kg, extracts 4.0h.
The beneficial effects of the present invention is:
1, the present invention utilizes CO_2 supercritical technology, overcomes Hot water extraction and NaOH extraction method extraction ratio is relatively low
Shortcoming, and the most do not exist NaOH extract highly basic use safety issue.
2, when the present invention is to Polysaccharide of Brasenia Schreberi purifying crude, basic protein ferment treatment is used to remove protein；Cetyl three
Methyl bromide ammonium can form quaternary amine complex with Polysaccharide of Brasenia Schreberi, and these complex are insoluble in the aqueous solution of low ionic strength,
When ionic strength is big, complex can dissociate, dissolve, and isolated and purified effect is more preferable.
3, use 312 weak-type anion exchange resin chromatographies, the impurity of some positively chargeds, isolated and purified effect can be removed
Fruit is more preferably.
Detailed description of the invention
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 example agents useful for same and equipment: bag filter (Sigma), 312 weak-type anion exchange resin (Luo Menha
This), alkaline 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) Folium Braseniae Schreberi being crushed to 100～200 mesh, the Folium Braseniae Schreberi after pulverizing is put to supercritical CO2In extraction kettle, seal still,
Getting rid of air, be passed through and carry agent and carry out extracting to obtain extracting solution, extraction temperature is 45.2 DEG C, and extracting pressure is 21.3MPa, extracts two
Carbonoxide flow is 25L/h, and carrying agent is distilled water, and consumption is 420ml/kg, extracts 4h；
(1.2) in extracting solution, add the mixed liquor of chloroform and n-butyl alcohol and shake up, being centrifuged 10min at 3000r/min, take
Supernatant I, in described mixed liquor, the volume ratio of chloroform and n-butyl alcohol is 4:1, described extracting solution and mixed liquor volume ratio
For 4:1；
(1.3) regulating the supernatant with NaOH is 8.0 to pH, then adds activated carbon in clear liquid and carries out decolouring sucking filtration
Obtain filtrate；Filtrate use distilled water dialysis 48h obtain dialysis solution further；
(1.4) dialysis solution 3000r/min is centrifuged 10min and obtains supernatant II, by supernatant II at 80 DEG C of water-bath bars
Being concentrated into the 1/3 of original volume under part, add the 95wt% ethanol of 3 times of volumes, after stirring, 3000r/min is centrifuged 10min
I must be precipitated, precipitation I is used dehydrated alcohol and washing with acetone respectively, is dried and to obtain Polysaccharide of Brasenia Schreberi crude product；
(2) purification of Polysaccharide of Brasenia Schreberi crude product
(2.1) taking Polysaccharide of Brasenia Schreberi crude product and distilled water mix homogeneously with mass volume ratio 1%, 3000r/min is centrifuged
10min removes insoluble matter, takes supernatant 1mol/L NaOH and adjusts pH to 9.0, adds the alkaline protease of 0.1wt%, 50 DEG C of enzymes
Solving 5h removing protein, 8000r/min is centrifuged 10min and obtains supernatant III；
(2.2) add cetyl trimethylammonium bromide to precipitation completely to upper strata clear liquid I II, stand 5h, 3000r/min from
Precipitation II of heart 10min, washs precipitation II frozen water three times, adds 100ml 2mol/L NaCl solution 55 DEG C solution to precipitation II
It is centrifuged 10min from 4h, 3000r/min and obtains supernatant IV；
(2.3) supernatant IV is adsorbed, then by resin by the 312 weak-type anion-exchange resin columns handled well
Moving in beaker, frozen water is only washed till clarification, then upper prop, uses 0.5mol/L Na2HPO4-0.3mol/L Na2SO4Elution,
Flow velocity 1ml/min, obtains eluent；
(2.4) eluent uses distilled water dialysis 12h, and 80 DEG C of water-baths of dialysis solution concentrate, and add 95% ethanol that triploid is long-pending,
After stirring, 3000r/min is centrifuged 10min, precipitation the most respectively with dehydrated alcohol and washing with acetone, be vacuum dried Folium Braseniae Schreberi is many
Sugar.
Measurement of the polysaccharide content
Polyoses content uses sulfuric acid-phynol method to detect.Polysaccharide under the effect of concentrated sulphuric acid, dehydration generate alditol and
Derivant, produces yellow substance with phenol after being combined, this material has maximum light absorption value at 490nm, the size of light absorption value and polysaccharide
Content is proportional.
(1) making of glucose standard curve:
Weighing a certain amount of glucose to dry to constant weight in 105 DEG C, accurately weigh 0.100g, pure water is settled to 100mL,
Obtain Glucose standards storing solution.The most accurately draw 0,2.0,4.0,6.0,8.0,10.0mL storing solutions, be respectively placed in 100mL
Volumetric flask, with pure water constant volume, is configured to 0,20,40,60,80,100 μ g/mL liquid to be measured.Accurately draw 1.0mL liquid to be measured in test tube
In, it being sequentially added into 1mL 6% phenol, 5mL concentrated sulphuric acid, mixing, room temperature stands 30min.Separately take a test tube, accurately draw
1.0mL pure water, is sequentially added into 1mL6% phenol, 5mL concentrated sulphuric acid, mixing, and room temperature stands 30min, as blank.490nm
Place surveys light absorption value, returns 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, x is the concentration of glucose. (μ g/mL).
Evaluating polysaccharide yield 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 checked on standard curve, μ g；
V: sample liquid amasss, mL；
N: extension rate；
Vs: draw the volume of sample liquid, mL during mensuration；
The quality of m: sample, g.
Polysaccharide of Brasenia Schreberi extracting solution is settled to 250mL volumetric flask, and accurately absorption 1.0mL is in 10mL volumetric flask, with pure water
Constant volume.After mixing, accurately absorption 1.0mL liquid to be measured, in test tube, is sequentially added into freshly prepared 6% phenol of 1mL, 5mL concentrated sulphuric acid
Mixing, room temperature stands 30min.Separately take a test tube, accurately draw 1.0mL pure water, be sequentially added into 1mL6% phenol, the dense sulfur of 5ml
Acid, mixing, room temperature stands 30min, as blank, measures light absorption value at 490nm.
Polysaccharide of Brasenia Schreberi extraction rate reached in assay, Folium Braseniae Schreberi is to 85.2%, and purity reaches 80.5%.
The present invention utilizes supercritical CO2Abstraction technique, overcomes Hot water extraction and NaOH extraction method extraction ratio is relatively low
Shortcoming, and the most there is not the safety issue that NaOH extraction highly basic uses.When the present invention is to Polysaccharide of Brasenia Schreberi purifying crude, use
Basic protein ferment treatment removes protein；Cetyl trimethylammonium bromide can form quaternary amine complex with Polysaccharide of Brasenia Schreberi, this
A little complex are insoluble in the aqueous solution of low ionic strength, and when ionic strength is big, complex can dissociate, dissolve, and separates pure
Change effect more preferable.Use 312 weak-type anion exchange resin chromatographies, the impurity of some positively chargeds can be removed, isolated and purified
Better.And its extraction efficiency of Polysaccharide of Brasenia Schreberi using the method for the invention to extract is high, and purity is good.
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 it is made various change, without departing from claims of the present invention limited range in details.
Claims (2)
1. the extraction of a Polysaccharide of Brasenia Schreberi and separation purifying technique, it is characterised in that comprise the steps:
(1) extraction of Polysaccharide of Brasenia Schreberi crude product:
(1.1) Folium Braseniae Schreberi being crushed to 100～200 mesh, the Folium Braseniae Schreberi after pulverizing is put to supercritical CO2In extraction kettle, seal still, get rid of
Air, is passed through and carries agent and carry out extracting to obtain extracting solution, and extraction temperature is 44.0-45.5 DEG C, and extracting pressure is 20.5-21.5MPa,
Extract carbon dioxide flow is 20-28L/h, and carrying agent is distilled water, and consumption is 400-450ml/kg, extracts 4-5h；
(1.2) in extracting solution, add the mixed liquor of chloroform and n-butyl alcohol and shake up, 3000～5000r/min be centrifuged 5～
10min, takes supernatant I, and in described mixed liquor, chloroform is 4:1 with the volume ratio of n-butyl alcohol, described extracting solution and mixed liquor
Volume ratio be 4～5:1；
(1.3) regulating the supernatant with NaOH is 7.5-8.5 to pH, then adds activated carbon in clear liquid and carries out decolouring sucking filtration
Obtain filtrate；Filtrate use distilled water dialysis 36-48h obtain dialysis solution further；
(1.4) dialysis solution 3000-5000r/min is centrifuged 5-10min and obtains supernatant II, by supernatant 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% ethanol of 3-4 times of volume, after stirring,
3000-5000r/min is centrifuged 5-10min must precipitate I, and precipitation I is used dehydrated alcohol and washing with acetone respectively, dry that Folium Braseniae Schreberi is many
Sugar crude product；
(2) purification of Polysaccharide of Brasenia Schreberi crude product
(2.1) take Polysaccharide of Brasenia Schreberi crude product and distilled water mix homogeneously with mass volume ratio 1%-2%, 3000-5000r/min from
Heart 5-10min removes insoluble matter, takes supernatant 1mol/L NaOH and adjusts pH to 8.6-9.2, adds the basic protein of 0.1wt%
Enzyme, 46 DEG C-50 DEG C enzymolysis 4-5h removing protein, 8000-10000r/min is centrifuged 8-10min and obtains supernatant III；
(2.2) add cetyl trimethylammonium bromide to precipitation completely to upper strata clear liquid I II, stand 5h, 3000-5000r/min
Precipitation II of centrifugal 5-10min, washs precipitation II frozen water three times, adds NaCl solution 52-57 DEG C to precipitation II and dissociates 3-4h,
And be centrifuged 5-10min at 3000-5000r/min and obtain supernatant IV；
(2.3) supernatant IV is adsorbed by the 312 weak-type anion-exchange resin columns handled well, then resin is moved into
In beaker, frozen water is only washed till clarification, then upper prop, uses 0.5mol/L Na2HPO4-0.3mol/L Na2SO4Elution, flow velocity
1ml/min, obtains eluent；
(2.4) eluent uses distilled water dialysis 12h, and 80 DEG C of water-baths of dialysis solution concentrate, and add 95% ethanol that triploid is long-pending, stirring
After Jun Yun, 3000r/min is centrifuged 10min, and precipitation with dehydrated alcohol and washing with acetone, is vacuum dried to obtain Polysaccharide of Brasenia Schreberi the most respectively.
The extraction of a kind of Polysaccharide of Brasenia Schreberi and separation purifying technique, it is characterised in that described step
(1.1) extraction temperature 45.2 DEG C, extracting pressure 21.3MPa, carrying agent is distilled water, and consumption is 420.0ml/kg, extracts 4.0h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108484784A (en) * | 2018-01-11 | 2018-09-04 | 浙江工业大学 | A kind of physical separation method of the external 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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| 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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| WO2005041985A1 (en) * | 2003-10-31 | 2005-05-12 | Md Bioalpha Co., Ltd. | Polysaccharides of plants belonging to panax having effect on treatment and prevention of obesity |
| 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 |
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| CN108484784A (en) * | 2018-01-11 | 2018-09-04 | 浙江工业大学 | A kind of physical separation method of the external polysaccharide gum of water shield |
| 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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| CN106146685B (en) | 2018-07-27 |
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