A kind of isolation and purification method of white polysaccharide
Technical field
A kind of the invention belongs to separating and purifying technology field, more particularly to isolation and purification method of white polysaccharide.
Background technology
White Acanthopanax trifoliatus (L.) Merr., also known as:The goose palm, Trifoliate Acanthopanax Root, Radix Acanthopanacis Trifoliati etc.,
Shape shrub is climbed up by holding on to for Araliaceae (Araliaceae).It is the conventional traditional herbal medicine in the area such as China south China, southwest, Central China.This product
Bitter in the mouth, pungent, cool in nature, root, stem, leaf can be used as medicine, and put down with heat-clearing and toxic substances removing, expelling wind and removing dampness, relaxing muscles and tendons to promote blood circulation, reducing swelling and alleviating pain, cough-relieving
The effects such as breathing heavily.Its tender leaf is rich in Vc, chlorophyll, crude protein, soluble sugar, aminoacid and mineral element needed by human, is one
Plant nutrition and health care and be worth all very high wild vegetable, with higher economic worth and extensive DEVELOPMENT PROSPECT.
Research finds that polyoses content is higher compared with other medicinal parts in white stem, in view of Araliaceae polysaccharide has exempting from
The extensive physiologically active such as epidemic disease regulation, antitumor, hematopoietic regulation, antiinflammatory, anti-hypoxia, defying age, resisting fatigue, CNS inhibition and poison
The characteristics of side effect is low, Chinese scholars are in the ascendant to the research of Araliaceae polysaccharide.At present with regard to white Polyose extraction
The research of technique is more superficial, is only limitted to by traditional method for extracting crude polysaccharides, and containing a large amount of nucleic acid, protein and peptides etc.
Impurity, product purity are low, are the mixture of various ingredients.Also not providing one kind isolates each polysaccharide group in white polysaccharide at present
The method that divides, the further structural research of dialogue polysaccharide each component are also almost blank.
The present invention is by dialogue polysaccharide (Acanthopanax trifoliatus polysaccharides, ATP)
Extracting and developing and purification, obtain neutral homogeneous polysaccharide ATP1-1 and two acidic polysaccharose components ATP2, ATP3, and right
Wherein the physicochemical property of ATP1-1 polysaccharide, molecular weight, monosaccharide composition etc. have carried out the Analysis and Identification of system, be in the future food,
Solid foundation has been established in health product, medicine and other fields application.
Content of the invention
The present invention is in order to solve the deficiency of existing white separation of polysaccharides purification process, there is provided a kind of separation of white polysaccharide
Purification process, i.e., white stem through hot water extraction, to filtration after lixiviating solution carry out alcohol precipitation, successively with dehydrated alcohol, ether, third
Ketone washs the alcohol precipitation, obtains final product white crude polysaccharides after spray drying;White crude polysaccharides after Sevage method deproteinization through
DEAE-cellulose column chromatographies, isolated neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3, neutral polysaccharide ATP1 are passed through
Neutral homogeneous Polysaccharide A TP1-1 is obtained after the purification of Sephadex column chromatography for separation.
Further, the isolation and purification method of the white polysaccharide, comprises the steps:
1) extraction of white crude polysaccharides:
1. take dry white stem to crush, cross 20 mesh sieves, after adding distilled water, in 90~100 DEG C of extractions twice, every time
0.5~4h;
2. merge lixiviating solution, after concentrating under reduced pressure, be centrifuged and discard precipitation, in supernatant, add dehydrated alcohol to carry out precipitate with ethanol
Form sediment, be centrifuged and collect alcohol precipitate, and use alcohol precipitate described in dehydrated alcohol, ether, washing with acetone successively, after spray drying i.e.
Obtain white crude polysaccharides;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained and be configured to the white crude polysaccharides aqueous solution that concentration is 2~6mg/mL,
And n-butyl alcohol-chloroform mixed liquor is added in the solution, acutely vibrate, be centrifuged and collect upper solution, according to above-mentioned deproteinization
Step repeatable operation 2~6 times, merges upper solution, through being spray-dried to obtain white polysaccharide after concentrating under reduced pressure;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to the aqueous solution that mass concentration is 40~60mg/mL, from
The heart takes supernatant DEAE-cellulose ions column chromatography and carries out initial gross separation, is carried out with distilled water, sodium chloride solution successively
Gradient elution, collects eluent, carries out respectively concentrating after merging identical flow point, dialyses, lyophilizing, obtains final product neutral polysaccharide ATP1 and acid
Property Polysaccharide A TP2, ATP3;
2. Sephadex column chromatographies
Take the neutral polysaccharide ATP1 of step 1. middle DEAE-cellulose column chromatographies, be configured to mass concentration for 40~
The aqueous solution of 60mg/mL, with membrane filtration after, gained filtrate carries out purification & isolation through Sephadex chromatographic columns, with distillation washing
Take off and collect eluent, identical flow point is merged according to elution curve, concentration, dialysis, lyophilizing obtain final product neutral homogeneous polysaccharide
ATP1-1.
Further, the isolation and purification method of the white polysaccharide can be used in the range of following separation condition, including such as
Lower step:
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, extract in 90~100 DEG C
Twice, 0.5~4h every time;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 5~15min in 3500~5500r/min, discards precipitation;To supernatant
The dehydrated alcohol of 2 times of volumes is added in liquid, 12~36h is precipitated, 5~15min is centrifuged in 3500~5500r/min, collects precipitate with ethanol
Thing, and use alcohol precipitate described in dehydrated alcohol, ether, washing with acetone successively, white crude polysaccharides are spray-dried to obtain in 60~90 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained and be configured to the white crude polysaccharides aqueous solution that concentration is 2~6mg/mL,
And n-butyl alcohol-chloroform mixed liquor (the volume of n-butyl alcohol-chloroform of 1/4 white crude polysaccharides aqueous solution volume is added in the solution
Than 1:4) 15~25min is acutely vibrated, 5~15min is centrifuged in 3500~5500r/min, collect upper solution, according to above-mentioned
Deproteinization step repeatable operation 2~6 times, merges upper solution, through being spray-dried to obtain white polysaccharide after concentrating under reduced pressure;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to the aqueous solution that mass concentration is 40~60mg/mL, in
6000~10000r/min is centrifuged 5~15min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation,
It is 0.05mol/L with distilled water, concentration successively and the sodium chloride solution of 0.2mol/L carries out gradient elution, flow velocity 1.3mL/min,
Eluent is collected, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, is closed according to elution curve
And identical flow point, concentration, dialysis, lyophilizing, respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
After filtration, gained filtrate carries out purification & isolation through Sephadex chromatographic columns, and with distillation water elution, flow velocity 0.5mL/min, collection are washed
De- liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges according to elution curve identical
Flow point, concentration, dialysis, lyophilizing obtain final product neutral homogeneous Polysaccharide A TP1-1.
Further preferred separation condition, the isolation and purification method of the white polysaccharide comprise the steps:
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, in 100 DEG C of extractions twice,
Each 2h;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 10min in 4500r/min, discards precipitation;2 are added in supernatant
The dehydrated alcohol of times volume, precipitates 24h, in 4500r/min centrifugation 10min centrifugation 10min, collects alcohol hypostasis, and uses nothing successively
Alcohol precipitate described in water-ethanol, ether, washing with acetone, is spray-dried to obtain white crude polysaccharides in 80 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained be configured to concentration be 4mg/mL white crude polysaccharides aqueous solution, and to
N-butyl alcohol-chloroform the mixed liquor of 1/4 white crude polysaccharides aqueous solution volume is added in the solution, acutely vibrates 20min, in 4500r/
Min is centrifuged 10min, collects upper solution, according to above-mentioned deproteinization step repeatable operation 4 times, merges upper solution, concentrating under reduced pressure
Polysaccharide in vain to be obtained by being spray-dried;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to mass concentration be 50mg/mL aqueous solution, in
8000r/min is centrifuged 10min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation, successively with distillation
Water, concentration carry out gradient elution for the sodium chloride solution of 0.05mol/L and 0.2mol/L, and flow velocity 1.3mL/min collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
Filtrate after filtration carries out purification & isolation through Sephadex chromatographic columns, with distillation water elution, flow velocity 0.5mL/min, collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing, respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3 obtain final product neutral homogeneous polysaccharide
ATP1-1.
Further, described DEAE-cellulose is 52 ion columns of DEAE-cellulose, and described Sephadex is
Sephadex G-50 chromatographic columns.
The invention has the advantages that:
(1) the invention provides a kind of high efficiency separation method for purifying white polysaccharide, through water extract-alcohol precipitation and Sevage methods
Deproteinization, can make the deproteinizing rate of white polysaccharide up to 78.21%, polysaccharide retention rate up to 84.57%, compared to traditional method for extracting
The impurity such as a large amount of nucleic acid, protein and peptides contained by crude polysaccharides, this method significantly improve product purity;
(2) present invention is through 52 ion columns of DEAE-cellulose, Sephadex G-50 chromatographic column column chromatography for separation, purification
Neutral homogeneous polysaccharide ATP1-1 and acidic polysaccharose component ATP2, ATP3, and physics and chemistry to wherein ATP1-1 polysaccharide are obtained first
Property, molecular weight, monosaccharide composition etc. have carried out the Analysis and Identification of system, are in the future in food, health product, medicine and other fields application
Solid foundation is established;
(3) present invention is easy to operate, efficient, it is adaptable to large-scale industrial production, and acquisition polysaccharide properties are single-minded, physiologically active
High.
Description of the drawings
The present invention is further described with reference to the accompanying drawings and examples:
Fig. 1:Elution curve of the crude polysaccharides on 52 posts of DEAE-cellulose in embodiment 1;
Fig. 2:Elution curves of the ATP1 on Sephadex G-50 posts in embodiment 1.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawings and examples:
The present invention can make the deproteinizing rate of ATP up to 78.21% by the process for separation and purification in embodiment, and polysaccharide retains
Rate is up to 84.57%.Neutral homogeneous polysaccharide ATP1-1 and acidic polysaccharose component ATP2, ATP3 are obtained through column chromatography for separation, purification, such as
Shown in Fig. 1 and Fig. 2.Each polysaccharide component (ATP1-1, ATP2, ATP3) is made the solution that mass concentration is 1.0mg/mL, 200
Ultraviolet full wavelength scanner is carried out under~400nm, (figure is omitted) is shown by ultraviolet spectra, without suction at 260nm and 280nm
Receive, show polysaccharide component after purification without impurity such as nucleic acid, protein and peptides.
As shown in table 1, with reference to existing document, the present invention is to ATP1-1 and Vc to DPPH, ABTS+, OH and O2 ˉ·
Free radical scavenging situation is studied.By neutral homogeneous polysaccharide ATP1-1 and positive control ascorbic acid (Vc) respectively with distillation
Water is made into the sample solution of series concentration, carries out antioxidant activity detection.Absorbance parallel assay 3 times, averages.With sample
Product solution concentration is abscissa (X), and free radical scavenging activity (Y) is that vertical coordinate draws curve, and calculates IC50Value.ATP1-1's is anti-
Oxidability is remarkably reinforced with the increase of mass concentration, and in the range of experimental concentration, the linear pass of clearance rate and concentration
System is good, thus calculates the IC to free radical50Value.ATP1-1 is most strong to ABTS Scavenging activities, suitable with Vc abilities.
Half elimination ratio (IC of 1 each sample of table to dissimilar free radical50)
Result of study provides theoretical foundation for the white polysaccharide of reasonable development, while isolating and purifying to acidic polysaccharose, is
The deep of white polysaccharide resource provides valuable reference using with high value added product exploitation.
Embodiment described below, the only preferred version of the present invention, in practical operation, can be selected different as needed
Separation condition.
In embodiment, DEAE-Cellulose 52, Sephadex G-50 derive from the limited public affairs of Shanghai ancient cooking vessel state biotechnology
Department;White stem picks up from Enping City of Guangdong Province, is accredited as Araliaceae through Guangdong pharmaceutical university associate professor Liu Jizhu white
A.trifoliatus (L.) Merr. dries stem.
Embodiment 1
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, in 100 DEG C of extractions twice,
Each 2h;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 10min in 4500r/min, discards precipitation;2 are added in supernatant
The dehydrated alcohol of times volume, precipitates 24h, in 4500r/min centrifugation 10min centrifugation 10min, collects alcohol hypostasis, and uses nothing successively
Alcohol precipitate described in water-ethanol, ether, washing with acetone, is spray-dried to obtain white crude polysaccharides in 80 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained be configured to concentration be 4mg/mL white crude polysaccharides aqueous solution, and to
N-butyl alcohol-chloroform the mixed liquor of 1/4 white crude polysaccharides aqueous solution volume is added in the solution, acutely vibrates 20min, in 4500r/
Min is centrifuged 10min, collects upper solution, according to above-mentioned deproteinization step repeatable operation 4 times, merges upper solution, concentrating under reduced pressure
Polysaccharide in vain to be obtained by being spray-dried;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to mass concentration be 50mg/mL aqueous solution, in
8000r/min is centrifuged 10min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation, successively with distillation
Water, concentration carry out gradient elution for the sodium chloride solution of 0.05mol/L and 0.2mol/L, and flow velocity 1.3mL/min collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
Filtrate after filtration carries out purification & isolation through Sephadex chromatographic columns, with distillation water elution, flow velocity 0.5mL/min, collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing, respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3 obtain final product neutral homogeneous polysaccharide
ATP1-1.
Embodiment 2
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, in 90 DEG C of extractions twice,
Each 0.5h;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 5min in 3500r/min, discards precipitation;2 are added in supernatant
The dehydrated alcohol of times volume, precipitates 12h, is centrifuged 5min in 3500r/min, collects alcohol hypostasis, and uses dehydrated alcohol, second successively
Alcohol precipitate described in ether, washing with acetone, is spray-dried to obtain white crude polysaccharides in 60 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained be configured to concentration be 2mg/mL white crude polysaccharides aqueous solution, and to
N-butyl alcohol-chloroform mixed liquor (volume ratio 1 of n-butyl alcohol-chloroform of 1/4 white crude polysaccharides aqueous solution volume is added in the solution:
4) 15min is acutely vibrated, 5min is centrifuged in 3500r/min, collect upper solution, according to above-mentioned deproteinization step repeatable operation 2
Secondary, merge upper solution, through being spray-dried to obtain white polysaccharide after concentrating under reduced pressure;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to mass concentration be 40mg/mL aqueous solution, in
6000r/min is centrifuged 5min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation, successively with distillation
Water, concentration carry out gradient elution for the sodium chloride solution of 0.05mol/L and 0.2mol/L, and flow velocity 1.3mL/min collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
After filtration, gained filtrate carries out purification & isolation through Sephadex chromatographic columns, and with distillation water elution, flow velocity 0.5mL/min, collection are washed
De- liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges according to elution curve identical
Flow point, concentration, dialysis, lyophilizing obtain final product neutral homogeneous Polysaccharide A TP1-1.
Embodiment 3
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, in 100 DEG C of extractions twice,
Each 4h;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 15min in 5500r/min, discards precipitation;2 are added in supernatant
The dehydrated alcohol of times volume, precipitates 36h, is centrifuged 15min in 5500r/min, collects alcohol hypostasis, and uses dehydrated alcohol, second successively
Alcohol precipitate described in ether, washing with acetone, is spray-dried to obtain white crude polysaccharides in 90 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained be configured to concentration be 6mg/mL white crude polysaccharides aqueous solution, and to
N-butyl alcohol-chloroform mixed liquor (volume ratio 1 of n-butyl alcohol-chloroform of 1/4 white crude polysaccharides aqueous solution volume is added in the solution:
4) 25min is acutely vibrated, 15min is centrifuged in 5500r/min, collect upper solution, according to above-mentioned deproteinization step repeatable operation
6 times, merge upper solution, through being spray-dried to obtain white polysaccharide after concentrating under reduced pressure;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to mass concentration be 60mg/mL aqueous solution, in
10000r/min is centrifuged 15min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation, successively with distillation
Water, concentration carry out gradient elution for the sodium chloride solution of 0.05mol/L and 0.2mol/L, and flow velocity 1.3mL/min collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
After filtration, gained filtrate carries out purification & isolation through Sephadex chromatographic columns, and with distillation water elution, flow velocity 0.5mL/min, collection are washed
De- liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges according to elution curve identical
Flow point, concentration, dialysis, lyophilizing obtain final product neutral homogeneous Polysaccharide A TP1-1.
Embodiment 4
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, in 95 DEG C of extractions twice,
Each 1h;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 8min in 4000r/min, discards precipitation;2 are added in supernatant
The dehydrated alcohol of times volume, precipitates 20h, is centrifuged 8min in 4000r/min, collects alcohol hypostasis, and uses dehydrated alcohol, second successively
Alcohol precipitate described in ether, washing with acetone, is spray-dried to obtain white crude polysaccharides in 70 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained be configured to concentration be 3mg/mL white crude polysaccharides aqueous solution, and to
N-butyl alcohol-chloroform mixed liquor (volume ratio 1 of n-butyl alcohol-chloroform of 1/4 white crude polysaccharides aqueous solution volume is added in the solution:
4) 20min is acutely vibrated, 8min is centrifuged in 4000r/min, collect upper solution, according to above-mentioned deproteinization step repeatable operation 3
Secondary, merge upper solution, through being spray-dried to obtain white polysaccharide after concentrating under reduced pressure;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to mass concentration be 45mg/mL aqueous solution, in
7000r/min is centrifuged 8min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation, successively with distillation
Water, concentration carry out gradient elution for the sodium chloride solution of 0.05mol/L and 0.2mol/L, and flow velocity 1.3mL/min collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
After filtration, gained filtrate carries out purification & isolation through Sephadex chromatographic columns, and with distillation water elution, flow velocity 0.5mL/min, collection are washed
De- liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges according to elution curve identical
Flow point, concentration, dialysis, lyophilizing obtain final product neutral homogeneous Polysaccharide A TP1-1.
Embodiment 5
1) extraction of total polysaccharidess:
1. take dry white stem to crush, cross 20 mesh sieves, after adding the distilled water of 10 times of quality, in 100 DEG C of extractions twice,
Each 3h;
2. united extraction liquid, after concentrating under reduced pressure, is centrifuged 12min in 5000r/min, discards precipitation;2 are added in supernatant
The dehydrated alcohol of times volume, precipitates 30h, is centrifuged 12min in 5000r/min, collects alcohol hypostasis, and uses dehydrated alcohol, second successively
Alcohol precipitate described in ether, washing with acetone, is spray-dried to obtain white crude polysaccharides in 80 DEG C;
2) Sevage methods deproteinization:
By step 1) in dry the white crude polysaccharides of gained be configured to concentration be 5mg/mL white crude polysaccharides aqueous solution, and to
N-butyl alcohol-chloroform mixed liquor (volume ratio 1 of n-butyl alcohol-chloroform of 1/4 white crude polysaccharides aqueous solution volume is added in the solution:
4) 15~25min is acutely vibrated, 12min is centrifuged in 5000r/min, collect upper solution, according to above-mentioned deproteinization step repeatedly
Operation 5 times, merges upper solution, through being spray-dried to obtain white polysaccharide after concentrating under reduced pressure;
3) separation of polysaccharide:
1. DEAE-cellulose column chromatographies
Take step 2) deproteinization process after white polysaccharide, be configured to mass concentration be 55mg/mL aqueous solution, in
9000r/min is centrifuged 12min, and taking supernatant DEAE-cellulose ions column chromatography carries out initial gross separation, successively with distillation
Water, concentration carry out gradient elution for the sodium chloride solution of 0.05mol/L and 0.2mol/L, and flow velocity 1.3mL/min collects eluting
Liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges same stream according to elution curve
Point, concentration, dialysis, lyophilizing respectively obtain neutral polysaccharide ATP1 and acidic polysaccharose ATP2, ATP3;
2. Sephadex column chromatographies
100mg neutral polysaccharide ATP1 through DEAE-cellulose after purification are weighed, is dissolved in 2mL distilled water, is used filter membrane
After filtration, gained filtrate carries out purification & isolation through Sephadex chromatographic columns, and with distillation water elution, flow velocity 0.5mL/min, collection are washed
De- liquid, with phend-sulphuric acid detection eluent in the absorbance that Detection wavelength is 490nm, merges according to elution curve identical
Flow point, concentration, dialysis, lyophilizing obtain final product neutral homogeneous Polysaccharide A TP1-1.
Above example is not limited only to protection scope of the present invention, all basic thoughts based on the present invention and carry out
Modification or variation belong to protection scope of the present invention.