CN106349403A - Yantai sweet potato polysaccharide with alpha-1,3-glucan chain and preparation method of Yantai sweet potato polysaccharide - Google Patents
Yantai sweet potato polysaccharide with alpha-1,3-glucan chain and preparation method of Yantai sweet potato polysaccharide Download PDFInfo
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- CN106349403A CN106349403A CN201610694411.4A CN201610694411A CN106349403A CN 106349403 A CN106349403 A CN 106349403A CN 201610694411 A CN201610694411 A CN 201610694411A CN 106349403 A CN106349403 A CN 106349403A
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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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
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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
Abstract
The invention provides Yantai sweet potato polysaccharide with an alpha-1,3-glucan chain and a preparation method of the Yantai sweet potato polysaccharide, relates to Yantai sweet potato polysaccharide and a preparation method thereof, and aims to solve the technical problems that at present, comprehensive utilization ratio of sweet potatoes is low, varieties of the sweet potato polysaccharide are wide, and structural information such as monosaccharide compositions of sweet potato polysaccharide in different varieties, relative molecular mass and a glucosidic bond connection mode differs in thousands of ways. The structural formula of the Yantai sweet potato polysaccharide with the alpha-1,3-glucan chain is as shown in the specification, wherein n is 340-345, and the relative molecular mass is 2.40*105Da-2.46*105Da. The preparation method of the Yantai sweet potato polysaccharide comprises the following steps: firstly, preparing Yantai sweet potato crude polysaccharide; and secondly, purifying. The Yantai sweet potato polysaccharide with the alpha-1,3-glucan chain has the advantages that water-soluble branching type alpha-1,3-glucan is extracted and purified from Yantai sweet potatoes, and the polysaccharide has effect of protecting the liver; and the polysaccharide content of the Yantai sweet potato polysaccharide with the alpha-1,3-glucan chain is 98.47%.
Description
Technical field
The present invention relates to a kind of cigarette potato polysaccharide and preparation method thereof.
Background technology
Polysaccharide is the complicated polymeric carbohydrate of a class, big with the four of protein, nucleic acid and lipid composition life entity
Base substance.Sweet Potato Polysaccharide also has antioxidation and immunoregulatory effect.China is Rhizoma Dioscoreae esculentae big producing country, comprehensive by Grey Multidimensional
Close analysis in the application of food sweet potato variety evaluation, cigarette potato 25 yield and comprehensive assessment all make number one, it is comprehensively commented
Estimating the degree of association is 0.8621, higher by 20.22% than compareing, and illustrates that cigarette potato 25 solves the contradiction of high yield and high-quality.Therefore, to cigarette potato
The extraction of polysaccharide and purification are applied to antitumor research for efficient utilization cigarette potato polysaccharide and provide previous work basis, are also simultaneously
Mid-term scale-up, later stage industrialized production, Polysaccharide Modification and modify after polysaccharide structure activity relationship, modify after polysaccharide in cell
Biology aspect research, sweet potato health care product research and development etc. provide reference.
Content of the invention
The present invention be in order to solve current Rhizoma Dioscoreae esculentae make a low multiple use and Sweet Potato Polysaccharide is various in style, different cultivars
The monosaccharide composition of Sweet Potato Polysaccharide, relative molecular mass, the technical problem that the structural information such as glycosidic bond connected mode varies, and
Offer is a kind of to have cigarette potato polysaccharide of α -1,3- dextran chain and preparation method thereof.
The structural formula of the cigarette potato polysaccharide with α -1,3- dextran chain of the present invention is:
Wherein n is
340～345, relative molecular mass is 2.40 × 105Da～2.46 × 105Da, contains 1 → 3 and 1 → 6 liang in constitutional repeating unit
Plant glycosidic bond, main chain is 1 → 3 glycosidic bond, side chain is made up of 1 → 6 glycosidic bond, saccharide residue is α configuration, degree of branching is 0.33.
The preparation method of the cigarette potato polysaccharide with α -1,3- dextran chain of the present invention is as follows:
First, cigarette potato crude polysaccharides are prepared: cigarette potato 25 is cleaned up, peeling, stripping and slicing, naturally dry, pulverize, cross 50 mesh sieves,
Obtain cigarette potato starch end, immersion 24h～48h in the ethanol that mass fraction is 95% is put at cigarette potato starch end and carries out defat, after taking-up certainly
So dry, then carry out hot water extraction, sucking filtration obtains extracting solution, and extracting solution is carried out concentrating under reduced pressure, obtains concentrated solution i, will concentrate
Liquid i puts into immersion 12h～24h in the ethanol solution that mass fraction is 95%, and sucking filtration must precipitate i, obtains after precipitating i vacuum drying
To precipitation powder, precipitation powder is put in distilled water, obtain the crude polysaccharides solutions i that concentration is 2mg/ml, then use sevag method
Deproteinization, takes supernatant, and supernatant is carried out concentrating under reduced pressure, obtains concentrated solution ii, concentrated solution ii is put into mass fraction and is
Soak 12h～24h in 95% ethanol solution, sucking filtration must precipitate ii, then precipitation ii is vacuum dried, obtains cigarette potato thick
Polysaccharide；The volume ratio of the ethanol that the quality at described cigarette potato starch end is 95% with mass fraction is 1g:(5ml～10ml)；Described
Concentrated solution i is put in the ethanol solution that mass fraction is 95% soak 12h～24h in concentrated solution i with mass fraction be
The volume ratio of 95% ethanol solution is 1:(3～4)；Described puts into the ethanol solution that mass fraction is 95% by concentrated solution ii
Concentrated solution ii in middle immersion 12h～24h and mass fraction are the volume ratio of 95% ethanol solution is 1:(3～4)；
2nd, purification: the cigarette potato crude polysaccharides that step one is obtained are put in distilled water, with 0.45 μm of filtering with microporous membrane,
Obtain crude polysaccharides solutions i i that concentration is 2mg/ml, carry out initial gross separation with deae-52 cellulose chromatographic column, after being separated
Eluent i, the eluent i after separation is carried out flowing water successively and dialyses 1 day, distilled water is dialysed 2 days, then carries out concentrating under reduced pressure and true
Vacuum freecing-dry, obtains cigarette potato polysaccharide component, and cigarette potato polysaccharide component is put in distilled water, with 0.45 μm of microporous filter membrane mistake
Filter, obtains the cigarette potato polysaccharide component solution that concentration is 2mg/ml, then uses sephadexg-200 chromatographic column to cigarette potato polysaccharide component
Solution is isolated and purified, the eluent ii after being separated, and the eluent ii after separation is carried out concentrating under reduced pressure and vacuum is cold
Lyophilizing is dry, obtains with α -1, the cigarette potato polysaccharide of 3- dextran chain；
What the flowing water described in step 2 was dialysed concretely comprises the following steps: it is 8000 dongle that eluent i is loaded molecular cut off
Pause in the bag filter of～11000 dalton, bag filter is placed in the beaker of 1000ml, room temperature down-flow water persistently soaks dialysis 1
My god；
What the distilled water described in step 2 was dialysed concretely comprises the following steps: the bag filter after flowing water dialysis treatment is placed in and fills
In the beaker of 1000ml distilled water, distilled water immersion is dialysed 2 days, and wherein every 6h～12h changes No. 1 distilled water；
The condition that described deae-52 cellulose chromatographic column carries out initial gross separation is: carry out sodium chloride solution gradient elution,
Described concentration of sodium chloride solution is followed successively by 0.5mol/l and 1.0mol/l, and the volume of sodium chloride solution used is deae- every time
52 celluloses chromatograph 1.5 times～2 times of column volume, and it is 1ml/min that constant flow pump controls elution flow rate, the loading of crude polysaccharides solutions i i
Measure as 2ml, collect 1 pipe with the every 3ml of automatic fraction collector, the eluent collecting the 20th pipe to the 60th pipe is eluting after separating
Liquid i；Described deae-52 cellulose chromatography column internal diameter is 2.6cm, and length is 55cm；
The condition that described sephadexg-200 chromatographic column is isolated and purified to cigarette potato crude polysaccharides component solution is: washes
De- liquid is distilled water, and the volume of distilled water used chromatographs 1.5 times～2 times of column volume for sephadexg-200, and constant flow pump controls
The flow velocity of eluting is 0.5ml/min, and the applied sample amount of cigarette potato crude polysaccharides component solution is 1ml, with the every 3ml of automatic fraction collector
Collect 1 pipe, the eluent of collection the 25th pipe to the 50th pipe is the eluent ii after separating；Described sephadexg-200 chromatography
Column internal diameter is 1.6cm, and length is 80cm.
The cigarette potato polysaccharide with α -1,3- dextran chain of the present invention is used for the medicine that preparation has liver protection and antioxidation
Thing.
Advantages of the present invention:
Extraction purification from cigarette potato goes out water solublity branching type α -1,3- glucosan to the present invention first, and this polysaccharide has liver protection
Effect.
The polyoses content of the cigarette potato polysaccharide with α -1,3- dextran chain of the present invention is 98.47%.
It is illustrated the cigarette potato polysaccharide of the α -1,3- dextran chain of the present invention from vitro tests and two parts of in vivo test
The method causing hepatic injury for treatment three-(2,3- dibromopropyl) isocyanates:
In vitro tests adopts wistar rat feeding test.In vivo test with hepg2 cell as target cell, with tbc[tri--
(2,3- dibromopropyl) isocyanates (tris- (2,3-dibromopropyl) isocyanurate, tbc] induction hepg2 cell
After the dosage of apoptosis processes cell, then observe α -1 of the present invention with various dose and different disposal time, 3- dextran chain
Cigarette potato polysaccharide causes the apoptotic protective effect of hepg2 to tbc.
Liver function biochemistry detection result shows, application the present invention α -1,3- dextran chain cigarette potato polysaccharide treatment three-(2,
3- dibromopropyl) isocyanates cause hepatic injury after, glutamate pyruvate transaminase, glutamic oxaloacetic transaminase, GOT=total serum protein, serum albumin, urine
Plain nitrogen, creatinine, cholesterol and triglyceride etc. are compared tbc contamination group and are all greatly improved.
Liver function biochemistry detection result shows, application the present invention α -1,3- dextran chain cigarette potato polysaccharide treatment three-(2,
3- dibromopropyl) isocyanates cause hepatic injury after, alkali phosphatase (alp), alanine aminotransferase (alt), aspartic acid turns ammonia
Enzyme (ast), total protein (tp), albumin (alb), total bilirubin (tbil), TOTAL BILE ACID TBA (tba), T-CHOL (tcho), three
Acyl glycerol (tg), glucose (glu) is compared tbc contamination group and is all greatly improved.
Cigarette potato polysaccharide treatment three-(2,3- dibromopropyl) isocyanates of the α -1,3- dextran chain of the application present invention cause liver
After damage, liver/body ratio is compared tbc contamination group and is improved significantly.
Histopathologic examination's result shows, cavity and cell infiltration in liver, it could be assumed that the application present invention
α -1,3- dextran chain cigarette potato polysaccharide treatment three-(2,3- dibromopropyl) isocyanates cause hepatic injury after, liver is had and changes
It is apt to and protective effect.
Show to by tbc induction hepg2 apoptotic apoptosis rate testing result, α -1 of the application present invention, 3- glucosan
After cigarette potato polysaccharide treatment three-(2, the 3- dibromopropyl) isocyanates of chain cause hepatic injury, apoptosis rate has obvious reduction.
Brief description
Fig. 1 is the light that cigarette potato crude polysaccharides prepared by test one step one carry out UV scanning in wavelength 200-400nm interval
Spectrogram；
Fig. 2 is test one Plays sephadex chromatography standard curve；
Fig. 3 is the infrared scan collection of illustrative plates of the cigarette potato crude polysaccharides of test one step one preparation；
Fig. 4 is the methylated sugar residue gas chromatogram of the cigarette potato polysaccharide with α -1,3- dextran chain of test one preparation
Figure, peak 1 correspond to methylated sugar residue 1, and peak 2 correspond to methylated sugar residue 2, and peak 3 correspond to methylated sugar residue 3；
Fig. 5 is the mass spectrum of methylated sugar residue 1；
Fig. 6 is the mass spectrum of methylated sugar residue 2；
Fig. 7 is the mass spectrum of methylated sugar residue 3；
Fig. 8 is liver histopathology section (x400) photo of matched group in test five；
Fig. 9 is the liver histopathology section of 100mg/kg bw dosage Sweet Potato Polysaccharide intervention group of the present invention in test five
(x400) photo；
Figure 10 is the liver histopathology section of 50mg/kg bw dosage Sweet Potato Polysaccharide intervention group of the present invention in test five
(x400) photo；
Figure 11 is liver histopathology section (x400) photo of tbc contamination group in test five.
Specific embodiment
Specific embodiment one: present embodiment has α -1, the cigarette potato polysaccharide of 3- dextran chain, its structural formula for one kind
For:
Wherein n is
340～345, relative molecular mass is 2.40 × 105Da～2.46 × 105Da, contains 1 → 3 and 1 → 6 liang in constitutional repeating unit
Plant glycosidic bond, main chain is 1 → 3 glycosidic bond, side chain is made up of 1 → 6 glycosidic bond, saccharide residue is α configuration, degree of branching is 0.33.
Specific embodiment two: present embodiment is many for the cigarette potato in specific embodiments one with α -1,3- dextran chain
The preparation method of sugar, particularly as follows:
First, cigarette potato crude polysaccharides are prepared: cigarette potato 25 is cleaned up, peeling, stripping and slicing, naturally dry, pulverize, cross 50 mesh sieves,
Obtain cigarette potato starch end, immersion 24h～48h in the ethanol that mass fraction is 95% is put at cigarette potato starch end and carries out defat, after taking-up certainly
So dry, then carry out hot water extraction, sucking filtration obtains extracting solution, and extracting solution is carried out concentrating under reduced pressure, obtains concentrated solution i, will concentrate
Liquid i puts into immersion 12h～24h in the ethanol solution that mass fraction is 95%, and sucking filtration must precipitate i, obtains after precipitating i vacuum drying
To precipitation powder, precipitation powder is put in distilled water, obtain the crude polysaccharides solutions i that concentration is 2mg/ml, then use sevag method
Deproteinization, takes supernatant, and supernatant is carried out concentrating under reduced pressure, obtains concentrated solution ii, concentrated solution ii is put into mass fraction and is
Soak 12h～24h in 95% ethanol solution, sucking filtration must precipitate ii, then precipitation ii is vacuum dried, obtains cigarette potato thick
Polysaccharide；The volume ratio of the ethanol that the quality at described cigarette potato starch end is 95% with mass fraction is 1g:(5ml～10ml)；Described
Concentrated solution i is put in the ethanol solution that mass fraction is 95% soak 12h～24h in concentrated solution i with mass fraction be
The volume ratio of 95% ethanol solution is 1:(3～4)；Described puts into the ethanol solution that mass fraction is 95% by concentrated solution ii
Concentrated solution ii in middle immersion 12h～24h and mass fraction are the volume ratio of 95% ethanol solution is 1:(3～4)；
2nd, purification: the cigarette potato crude polysaccharides that step one is obtained are put in distilled water, with 0.45 μm of filtering with microporous membrane,
Obtain crude polysaccharides solutions i i that concentration is 2mg/ml, carry out initial gross separation with deae-52 cellulose chromatographic column, after being separated
Eluent i, the eluent i after separation is carried out flowing water successively and dialyses 1 day, distilled water is dialysed 2 days, then carries out concentrating under reduced pressure and true
Vacuum freecing-dry, obtains cigarette potato polysaccharide component, and cigarette potato polysaccharide component is put in distilled water, with 0.45 μm of microporous filter membrane mistake
Filter, obtains the cigarette potato polysaccharide component solution that concentration is 2mg/ml, then uses sephadexg-200 chromatographic column to cigarette potato polysaccharide component
Solution is isolated and purified, the eluent ii after being separated, and the eluent ii after separation is carried out concentrating under reduced pressure and vacuum is cold
Lyophilizing is dry, obtains with α -1, the cigarette potato polysaccharide of 3- dextran chain；
What the flowing water described in step 2 was dialysed concretely comprises the following steps: it is 8000 dongle that eluent i is loaded molecular cut off
Pause in the bag filter of～11000 dalton, bag filter is placed in the beaker of 1000ml, room temperature down-flow water persistently soaks dialysis 1
My god；
What the distilled water described in step 2 was dialysed concretely comprises the following steps: the bag filter after flowing water dialysis treatment is placed in and fills
In the beaker of 1000ml distilled water, distilled water immersion is dialysed 2 days, and wherein every 6h～12h changes No. 1 distilled water；
The condition that described deae-52 cellulose chromatographic column carries out initial gross separation is: carry out sodium chloride solution gradient elution,
Described concentration of sodium chloride solution is followed successively by 0.5mol/l and 1.0mol/l, and the volume of sodium chloride solution used is deae- every time
52 celluloses chromatograph 1.5 times～2 times of column volume, and it is 1ml/min that constant flow pump controls elution flow rate, the loading of crude polysaccharides solutions i i
Measure as 2ml, collect 1 pipe with the every 3ml of automatic fraction collector, the eluent collecting the 20th pipe to the 60th pipe is eluting after separating
Liquid i；Described deae-52 cellulose chromatography column internal diameter is 2.6cm, and length is 55cm；
The condition that described sephadexg-200 chromatographic column is isolated and purified to cigarette potato crude polysaccharides component solution is: washes
De- liquid is distilled water, and the volume of distilled water used chromatographs 1.5 times～2 times of column volume for sephadexg-200, and constant flow pump controls
The flow velocity of eluting is 0.5ml/min, and the applied sample amount of cigarette potato crude polysaccharides component solution is 1ml, is received with the every 3ml of automatic fraction collector
Collection 1 pipe, the eluent of collection the 25th pipe to the 50th pipe is the eluent ii after separating；Described sephadexg-200 chromatographic column
Internal diameter is 1.6cm, and length is 80cm.
Specific embodiment three: present embodiment from unlike specific embodiment two: the hot water described in step one
The condition of extraction is: Extracting temperature is 65 DEG C～85 DEG C, extraction time 1.5h～3h, described naturally dry after cigarette potato 25 with
The mass ratio of water is 1:(10～20), extraction time is 2 times～4 times.Other identical with specific embodiment two.
Specific embodiment four: unlike one of present embodiment and specific embodiment two and three: described in step one
The process of concentrating under reduced pressure be: extracting solution is put in rotary evaporation bottle, is 0.05mpa～0.08mpa and water-bath temperature in pressure
Spend for extracting solution being compressed under conditions of 55 DEG C～65 DEG C the 15%～30% of original volume.Other with specific embodiment two and
One of three is identical.
Specific embodiment five: unlike one of present embodiment and specific embodiment two to four: described in step one
The Deproteinated process of sevag method be: the crude polysaccharides solutions i for 2mg/ml for the concentration is mixed with sevag liquid equal-volume, concussion is filled
Point mix, be then 3000r min in rotating speed-1Centrifugation 10min, takes supernatant；Supernatant is repeated sevag method Deproteinated
Process 5 times～10 times.Other identical one of with specific embodiment two to four.
Specific embodiment six: unlike one of present embodiment and specific embodiment two to five: institute in step one
The vacuum drying process of i that will precipitate stated is: will precipitate i and load in mortar, and mortar will be placed in vacuum drying oven, baking temperature
For 50 DEG C～70 DEG C, pressure is 0.02mpa～0.05mpa, and drying time is 12h～24h；
The vacuum drying process of ii that will precipitate described in step one is: will precipitate ii and load in mortar, mortar is placed in
In vacuum drying oven, baking temperature is 50 DEG C～70 DEG C, and pressure is 0.02mpa～0.05mpa, and drying time is 12h～24h.
Other identical one of with specific embodiment two to five.
Specific embodiment seven: unlike one of present embodiment and specific embodiment two to six: institute in step 2
The concentrating under reduced pressure step carrying out the eluent i after separation successively in concentrating under reduced pressure and vacuum lyophilization stated is: will separate
Eluent i afterwards puts in rotary evaporation bottle, and in pressure be 0.05mpa～0.08mpa and bath temperature is 55 DEG C～65 DEG C
Under the conditions of eluent i is compressed to the 15%～30% of original volume, obtain concentrated solution；
The process of vacuum lyophilization is: concentrated solution is loaded in the ampoule bottle of 2ml, ampoule bottle is placed in vacuum freezing and does
In dry machine, set freeze-dried product temperature as -25 DEG C～-40 DEG C, cooling time is 12h～24h, pressure 5pa～40pa, solution
Eutectoid temperature is 20 DEG C～40 DEG C, and the parsing time is 8h～18h.Other identical one of with specific embodiment two to six.
Specific embodiment eight: unlike one of present embodiment and specific embodiment two to seven: institute in step 2
The concentrating under reduced pressure step carrying out the eluent ii after separation successively in concentrating under reduced pressure and vacuum lyophilization stated is: will separate
Eluent ii afterwards puts in rotary evaporation bottle, and in pressure be 0.05mpa～0.08mpa and bath temperature is 55 DEG C～65 DEG C
Under the conditions of eluent ii is compressed to the 15%～30% of original volume, obtain concentrated solution；
The process of vacuum lyophilization is: concentrated solution is loaded in the ampoule bottle of 2ml, ampoule bottle is placed in vacuum freezing and does
In dry machine, set freeze-dried product temperature as -25 DEG C～-40 DEG C, cooling time is 12h～24h, pressure 5pa～40pa, solution
Eutectoid temperature is 20 DEG C～40 DEG C, and the parsing time is 8h～18h.Other identical one of with specific embodiment two to seven.
By tests below checking effect of the present invention:
Test one: this test the cigarette potato polysaccharide for having α -1,3- dextran chain preparation method particularly as follows:
First, cigarette potato crude polysaccharides are prepared: cigarette potato 25 is cleaned up, peeling, stripping and slicing, naturally dry, pulverize, cross 50 mesh sieves,
Obtain cigarette potato starch end, immersion 48h in the ethanol that mass fraction is 95% is put at cigarette potato starch end and carries out defat, naturally dry in the air after taking-up
Dry, then carry out hot water extraction, sucking filtration obtains extracting solution, and extracting solution is carried out concentrating under reduced pressure, obtains concentrated solution i, and concentrated solution i is put
Enter immersion 24h in the ethanol solution that mass fraction is 95%, sucking filtration must precipitate i, be precipitated powder by after precipitation i vacuum drying
End, precipitation powder is put in distilled water, obtains the crude polysaccharides solutions i that concentration is 2mg/ml, then uses sevag method deproteinization,
Take supernatant, supernatant is carried out concentrating under reduced pressure, obtain concentrated solution ii, concentrated solution ii is put into the ethanol that mass fraction is 95%
Soak 24h in solution, sucking filtration must precipitate ii, then precipitation ii is vacuum dried, obtains cigarette potato crude polysaccharides；Described cigarette potato
The quality of powder and mass fraction are the volume ratio of 95% ethanol is 1g:5ml；Described puts into mass fraction by concentrated solution i
Volume ratio for soaking the ethanol solution that the concentrated solution i in 24h is 95% with mass fraction in 95% ethanol solution is 1:3；
The described concentrated solution ii soaking in 24h in the ethanol solution that mass fraction is 95% that puts into concentrated solution ii with mass fraction is
The volume ratio of 95% ethanol solution is 1:3；
2nd, purification: the cigarette potato crude polysaccharides that step one is obtained are put in distilled water, with 0.45 μm of filtering with microporous membrane,
Obtain crude polysaccharides solutions i i that concentration is 2mg/ml, carry out initial gross separation with deae-52 cellulose chromatographic column, after being separated
Eluent i, the eluent i after separation is carried out flowing water successively and dialyses 1 day, distilled water is dialysed 2 days, then carries out concentrating under reduced pressure and true
Vacuum freecing-dry, obtains cigarette potato polysaccharide component, and cigarette potato polysaccharide component is put in distilled water, with 0.45 μm of microporous filter membrane mistake
Filter, obtains the cigarette potato polysaccharide component solution that concentration is 2mg/ml, then uses sephadexg-200 chromatographic column to cigarette potato polysaccharide component
Solution is isolated and purified, the eluent ii after being separated, and the eluent ii after separation is carried out concentrating under reduced pressure and vacuum is cold
Lyophilizing is dry, obtains with α -1, the cigarette potato polysaccharide of 3- dextran chain；
What the flowing water described in step 2 was dialysed concretely comprises the following steps: it is 8000 dongle that eluent i is loaded molecular cut off
Pause in the bag filter of～11000 dalton, bag filter is placed in the beaker of 1000ml, room temperature down-flow water persistently soaks dialysis 1
My god；
What the distilled water described in step 2 was dialysed concretely comprises the following steps: the bag filter after flowing water dialysis treatment is placed in and fills
In the beaker of 1000ml distilled water, distilled water immersion is dialysed 2 days, and wherein every 12h changes No. 1 distilled water；
The condition that described deae-52 cellulose chromatographic column carries out initial gross separation is: carry out sodium chloride solution gradient elution,
Described concentration of sodium chloride solution is followed successively by 0.5mol/l and 1.0mol/l, and the volume of sodium chloride solution used is deae- every time
52 celluloses chromatograph 1.5 times～2 times of column volumes, and it is 1ml/min that constant flow pump controls elution flow rate, crude polysaccharides solutions i i upper
Sample amount is 2ml, collects 1 pipe with the every 3ml of automatic fraction collector, and the eluent collecting the 20th pipe to the 60th pipe is washing after separating
De- liquid i；Described deae-52 cellulose chromatography column internal diameter is 2.6cm, and length is 55cm；
The condition that described sephadexg-200 chromatographic column is isolated and purified to cigarette potato crude polysaccharides component solution is: washes
De- liquid is distilled water, and the volume of distilled water used chromatographs 1.5 times～2 times of column volume for sephadexg-200, and constant flow pump controls
The flow velocity of eluting is 0.5ml/min, and the applied sample amount of cigarette potato crude polysaccharides component solution is 1ml, is received with the every 3ml of automatic fraction collector
Collection 1 pipe, the eluent of collection the 25th pipe to the 50th pipe is the eluent ii after separating；Described sephadexg-200 chromatographic column
Internal diameter is 1.6cm, and length is 80cm.
The condition of the hot water extraction described in step one is: Extracting temperature is 80 DEG C, extraction time 2h, and described dries in the air naturally
Cigarette potato 25 after dry is 1:10 with the mass ratio of water, and extraction time is 3 times.
The process of the concentrating under reduced pressure described in step one is: extracting solution is put in rotary evaporation bottle, is 0.07mpa in pressure
Under conditions of being 60 DEG C with bath temperature, extracting solution is compressed to the 20% of original volume.
The Deproteinated process of sevag method described in step one is: by the crude polysaccharides solutions i for 2mg/ml for the concentration and sevag
Liquid equal-volume mixes, and concussion fully mixes, and is then 3000r min in rotating speed-1Centrifugation 10min, takes supernatant；By supernatant
Repeat the Deproteinated process of sevag method 8 times.
The vacuum drying process of i that will precipitate described in step one is: will precipitate i and load in mortar, mortar is placed in very
In empty drying baker, baking temperature is 60 DEG C, and pressure is 0.03mpa, and drying time is 12h；
The vacuum drying process of ii that will precipitate described in step one is: will precipitate ii and load in mortar, mortar is placed in
In vacuum drying oven, baking temperature is 60 DEG C, and pressure is 0.03mpa, and drying time is 12h.
The decompression eluent i after separation being carried out successively in concentrating under reduced pressure and vacuum lyophilization described in step 2
Concentration step is: the eluent i after separation is put in rotary evaporation bottle, in pressure be 0.07mpa and bath temperature is 60 DEG C
Under conditions of eluent i is compressed to the 20% of original volume, obtain concentrated solution；
The process of vacuum lyophilization is: concentrated solution is loaded in the ampoule bottle of 2ml, ampoule bottle is placed in vacuum freezing and does
In dry machine, set freeze-dried product temperature as -40 DEG C, cooling time is 24h, pressure 20pa, and resolution temperature is 35 DEG C, parsing
Time is 12h.
Described in step 2, the eluent ii after separation is carried out subtracting in concentrating under reduced pressure and vacuum lyophilization successively
Pressure concentration step is: the eluent ii after separation is put in rotary evaporation bottle, in pressure be 0.07mpa and bath temperature is 60
Under conditions of DEG C, eluent ii is compressed to the 20% of original volume, obtains concentrated solution；
The process of vacuum lyophilization is: concentrated solution is loaded in the ampoule bottle of 2ml, ampoule bottle is placed in vacuum freezing and does
In dry machine, set freeze-dried product temperature as -40 DEG C, cooling time is 24h, pressure 20pa, and resolution temperature is 35 DEG C, parsing
Time is 12h.
Characterize and detect:
First, cigarette potato crude polysaccharides uv scan:
In wavelength 200-400nm interval, UV scanning, scanning figure are carried out to the cigarette potato crude polysaccharides testing step one preparation
Spectrum is as shown in figure 1, by spectrogram as can be seen that not having obvious characteristic peak between 260-280nm, illustrate to test a step one
Do not contain nucleic acid and protein component in the cigarette potato crude polysaccharides of preparation, deproteinization completely, to testing a step one after preliminary purification
It is 70.73% that the cigarette potato crude polysaccharides of preparation are calculated polyoses content using Phenol sulfuric acid procedure detection.
2nd, the mensure of the cigarette potato polysaccharide relative molecular weight with α -1,3- dextran chain of test one preparation:
1st, polysaccharide relative molecular mass standard curve making
Precision weighs each 50mg of blue dextran t20, t70, t100, t2000, and constant volume is in 100ml volumetric flask, dense respectively
Degree is 0.5mg/ml, takes 1ml respectively, is splined on sephadexg-200 chromatographic column, pumps into single steaming water elution with constant flow pump, uses
Part automatic collector every 2ml/ pipe is collected, phenolsulfuric acid tracing detection, volume v when collecting and combining standard sugar liquid complete eluting.
It is designated as v respectively1、v2、v3And v0.With v/v0For abscissa, relative molecular mass logarithm lgm is vertical coordinate drafting polydextran gel color
Spectrum standard curve, calculates regression equation.
Collect complete eluting known molecular amount blue dextran t20, t70, t100 and t2000 be respectively 25ml, 16ml,
15ml and 8ml.Vertical coordinate is respectively 3.30103,4.845098,5,6.30103 by the blue dextran lgm of known molecular amount.
Abscissa is by v/v0It is respectively 3.125,2,1.25,1.Calculating equation of linear regression is y=-1.4082x+7.6781, phase relation
Number r2=0.9993.
Table 1 dextran standards gel chromatography data determination
Fig. 2 is dextran standards gel chromatography standard curve.
2nd, sample Measuring Molecule Weight
Precision weigh test one preparation there is α -1, the cigarette potato polysaccharide 50mg of 3- dextran chain, constant volume is in 100ml capacity
In bottle, concentration is 0.5mg/ml, and according to the method loading of step 1, eluting, when calculating complete eluting, volume v utilizes standard curve
Calculate corresponding relative molecular mass respectively, obtain elution volume and be respectively 13ml, test the tool of a preparation through regression equation calculation
The relative molecular mass having the cigarette potato polysaccharide of α -1,3- dextran chain is 2.45 × 105da.
3rd, the cigarette potato polysaccharide IR spectrum scanning with α -1,3- dextran chain of test one preparation
Take prepared by the test one of 1mg to have α -1, the cigarette potato polysaccharide dried powder of 3- dextran chain, with 200mgkbr powder
End mixes, and grinds, be carefully placed on filter paper, be pressed into thin slice with tablet machine, then go up fourier infrared in agate mortar
Spectrophotometer, in 4000-400cm-1Interval carries out infrared scan, scanning spectra such as Fig. 3.
From the figure 3, it may be seen that in 3200cm-1-2800cm-1And 1400cm-1-1200cm-1, prepared by test one has α -1,3-
The cigarette potato polysaccharide of dextran chain has characteristic absorption peak, and what test one preparation was described has α -1, the cigarette potato polysaccharide tool of 3- dextran chain
There is saccharide feature, in 960cm-1-730cm-1For polysaccharide characteristic absorption peak, 3604cm-1There is the strong absworption peak of o-h stretching vibration,
1773cm-1It is the stretching vibration peak of-cho or c=o, 2860cm-1There is c-h stretching vibration peak, have characteristic absorption in 1033cm-1
Prepared by peak explanation test one has α -1, and the cigarette potato polysaccharide of 3- dextran chain is pyranoid form sugar ring, in 833cm-1There is absworption peak,
Prove that the cigarette potato polysaccharide glycosidic bond configuration with α -1,3- dextran chain of test one preparation contains α-glycosidic bond.
4th, the cigarette potato polysaccharide methylation analysis with α -1,3- dextran chain of test one preparation
Take prepared by dry test one has the cigarette potato polysaccharide 0.1mg of α -1,3- dextran chain in reaction tube → be dissolved in
The anhydrous dmso of 0.3ml → be filled with nitrogen, ultrasonic water bath 30min → addition 10mg sodium hydroxide powder → be filled with nitrogen, ultrasonic water
Bath 20min → ice bath solidifies → is filled with nitrogen to sample solution, plus 0.1ml iodomethane → seal reaction 1h → be filled with nitrogen → plus
Single steam water 0.2ml → plus chloroform extraction 2 times, each 1ml → combined chloroform liquid, add anhydrous sodium sulfate, overnight → be filtered to remove
Anhydrous sodium sulfate, evaporated under reduced pressure chloroform → obtain methylate → addition 2mol/l trifluoroacetic acid 0.5ml, 100 DEG C of reaction 2h
→ evaporated under reduced pressure, plus methanol 0.1ml, are filled with nitrogen → addition 5mg sodium borohydride, and 2ml is mono- to steam water, reacts 2h → Deca 1mol/l
Acetic acid neutralizes → adds 0.1ml methanol, is evaporated → adds anhydrous pyridine and each 0.5ml of Glacial acetic acid acid anhydride, and reaction 2h → decompression is steamed
Do → add 0.5ml chloroform and dissolve → carry out gc-ms analysis.
Gc-ms analysis condition: chromatographic column is ec-1column, packed column 0.25mm × 25m, temperature programming 100～250
DEG C, carrier gas is nitrogen, ei source 70ev, sample size 1 μ l.
Fig. 4 is the methylated sugar residue gas chromatogram of the cigarette potato polysaccharide with α -1,3- dextran chain of test one preparation
It can be seen that there being three peaks, correspond to three methylated sugar residues 1,2 and 3, Fig. 5,6 and 7 respectively is that three methylated sugars are residual to figure
The mass spectrum of base it can be seen that three saccharide residue fragments are methylated sugar residue 1:2 respectively, 3,4,6-me4-glc, methylated sugar
Residue 2:2,4-me2-glc and methylated sugar residue 3:2,4,6-me3-glc thus it is speculated that go out test one preparation there is α -1,3- Portugal
The connected mode of the cigarette potato polysaccharide of polysaccharide chains is 1-glc, 1,3,6-glc and 1,3-glc.
As shown in Figure 4, the mol ratio of methylated sugar residue 2 and 3 can be inferred that as testing a system for 23.84 and 15.06
Standby have α -1, the cigarette potato polysaccharide of 3- dextran chain main chain, and mol ratio 4.58 methylated sugar residue 1 be worth little for side chain
Composition.
The structure of the cigarette potato polysaccharide with α -1,3- dextran chain of test one preparation: main chain is by 1 → 3 and 1 → 3 → 6 liang
Kind of alpha-glucanses replace and constitute, and have side chain, be made up of 1-glc on backbone portion 6.
So, prepared by test one has α -1, the structural formula of the cigarette potato polysaccharide of 3- dextran chain:
N is 340, weight
1 → 3 and 1 → 6 two kinds of glycosidic bonds are contained, main chain is 1 → 3 glycosidic bond, side chain is made up of 1 → 6 glycosidic bond, sugar in complex structure unit
Residue is α configuration, and degree of branching is 0.33.
Table 2 methylation analysis results
Effect detection to the cigarette potato polysaccharide intervention tbc cause hepatic injury with α -1,3- dextran chain of test one preparation:
The cigarette potato polysaccharide of α -1,3- dextran chain intervenes the external nursing wistar rat test process of tbc cause hepatic injury such as
Under:
The wistar rat for 60g～70g for the body weight is randomly divided into four groups, every group 20, male and female half and half, remember respectively for four groups
For negative control group, positive controls (tbc contamination group), the cigarette potato polysaccharide low dosage intervention group of α -1,3- dextran chain and α -1,
The cigarette potato polysaccharide high dose intervention group of 3- dextran chain, wherein four groups processed as follows:
Negative control group: feed normal diet daily, freely ingest；
Positive controls (tbc contamination group): contaminated with tbc gavage daily, the dosage of tbc is calculated as with rat body weight
0.625g/kg, feeds normal diet, freely ingests；
α -1, the cigarette potato polysaccharide low dosage intervention group of 3- dextran chain: first contaminated with tbc gavage daily, the agent of tbc
Amount is calculated as 0.625g/kg with rat body weight, α -1 after contamination, the normal diet of the cigarette potato polysaccharide of 3- dextran chain, α -1, and 3- Portugal gathers
The dosage of the cigarette potato polysaccharide of sugar chain is calculated as 50mg/kg with wistar rat body weight daily, freely ingests；
α -1, the cigarette potato polysaccharide high dose intervention group of 3- dextran chain: first contaminated with tbc gavage daily, the agent of tbc
Amount is calculated as 0.625g/kg with rat body weight, α -1 after contamination, the normal diet of the cigarette potato polysaccharide of 3- dextran chain, α -1, and 3- Portugal gathers
The dosage of the cigarette potato polysaccharide of sugar chain is calculated as 100mg/kg with wistar rat body weight daily, freely ingests；Adopt in process of the test
Consistent with protein content in casein adjustment each group animal feed, test continuous 30 days.
Test two: the monitoring to growing state and result:
During the nursing of 30 days, measure a body weight weekly, calculate a weight gain.
In terms of diet regimen amount, weigh and spill appetite and surpluses, calculate a food ration.
Obtain feeding trial result as shown in table 3, as can be seen from Table 3 tbc contamination group, test two: low dosage is done
Pre- group of (50mg/kg bw；Body weight, bw), α -1, the cigarette potato polysaccharide high dose intervention group (1000mg/ of 3- dextran chain
kg·bw；Body weight, bw) and the body weight increase of negative control group present obvious gradient relation, male group and female
Two dosage intervention group of group all have obvious difference with tbc contamination group, and there is significant difference.
3 three ten days feeding trial results of table
Note:?Compare with matched group, p < 0.05.
Test three: liver function biochemistry detection and result:
After the nursing of 30 days terminates, abdominal aortic blood separation serum under the conditions of Animal Anesthesia, is divided automatically using biochemical
Analyzer measures liver function biochemical indicator, obtains the blood parameters testing result as shown in table 4 and table 5.
1. table 4 gives glutamate pyruvate transaminase, glutamic oxaloacetic transaminase, GOT (inclusion middle dose group), total serum protein, cholesterol and sweet
The testing result of oily three esters, from table 4, it can be seen that α -1 of 100mg/kg bw, the cigarette potato polysaccharide intervention group of 3- dextran chain
Male rat glutamate pyruvate transaminase, glutamic oxaloacetic transaminase, GOT, total serum protein, cholesterol and triglyceride all have pole relative to tbc contamination group
Big improvement, and there is statistical significance.The intervention group female rats of cigarette two dosage of potato polysaccharide of α -1,3- dextran chain
The total serum protein of cigarette potato polysaccharide intervention group of the α -1,3- dextran chain of glutamic oxaloacetic transaminase, GOT and 100mg/kg bw, the white egg of serum
In vain, blood urea nitrogen, creatinine, cholesterol and triglyceride all have great improvement relative to tbc contamination group.
Table 4 blood parameters testing result a
Note:?Compare with matched group, p < 0.05；??Compare with matched group, p < 0.01.
2. table 5 gives alkali phosphatase (alp), alanine aminotransferase (alt), aspartate transaminase (ast), total egg
In vain (tp), albumin (alb), total bilirubin (tbil), TOTAL BILE ACID TBA (tba), T-CHOL (tcho), triacylglycerol (tg),
The testing result of glucose (glu), as can be seen from Table 5
Table 5 blood parameters testing result b
Note:?Compare with matched group, p < 0.05.
After the test feeding trial of four: three ten days terminates, each internal organs of animal are weighed, and calculate dirty body ratio, the results are shown in Table 6,
As can be seen from Table 6, α -1 of the 50mg/kg bw and 100mg/kg bw of male rat and female rats contamination group, 3- Portugal gathers
The cigarette potato polysaccharide intervention group of sugar chain is improved significantly compared with tbc contamination group with the liver/body odds ratio of matched group, there are statistics poor
Different, and have dose-effect relationship.
After table 6 contamination surrounding, liver/body compares result
Note:?Compare with matched group, p < 0.05.
After the test feeding trial of five: three ten days terminates, take the standby histopathology of doing of partial liver to detect, obtain as Fig. 8
Liver histopathology section (x400) photo shown in～11, from Fig. 8～11 as can be seen that it is found that contamination group and low dosage
The cigarette potato polysaccharide intervention group of α -1,3- dextran chain is relative to the cigarette potato polysaccharide intervention group of high dose α -1,3- dextran chain and comparison
Group finds that in histopathologic examination cavity and cell infiltration in liver, shows α -1, the cigarette potato polysaccharide of 3- dextran chain
Intervention group has improvement and protective effect to liver.
After the test feeding trial of six: three ten days terminates, prepare liver homogenate detection superoxide dismutase under cryogenic
Enzyme (sod), catalase (cat), glutathion peroxidase (gshpx), the index such as malonaldehyde (mda), the results are shown in Table
7, as can be seen from Table 7, relatively above-mentioned two groups of α -1 of indices, the cigarette potato polysaccharide intervention group of 3- dextran chain compares contamination group
All it is greatly improved.
Table 7 Antioxidant Indexes testing result
Note:?Compare with matched group, p < 0.05；??Compare with matched group, p < 0.01.
Sweet Potato Polysaccharide is intervened tbc and causes the internal hepg2 apoptosis inhibition test process of hepatic injury as follows:
With hepg2 cell as target cell, with tbc induction the apoptotic dosage of hepg2 process cell after, then with difference agent
Amount and different disposal time observe α -1, and the cigarette potato polysaccharide of 3- dextran chain causes the apoptotic protective effect of hepg2 to tbc,
Illustrate it and there is dosage effect and Time-effect relationship.Obtain the cigarette potato polysaccharide intervention of α -1,3- dextran chain as shown in table 8
Apoptosis rate result to hepg-2 cell, as can be seen from Table 8, α -1, the apoptosis rate of the cigarette potato polysaccharide intervention group of 3- dextran chain
Relatively tbc contamination group, its apoptosis rate has obvious reduction.
The cigarette potato polysaccharide of table 8 flow cytomery α -1,3- dextran chain intervenes the apoptosis rate to hepg-2 cell
Note:?Compare with matched group, p < 0.05,??With matched group ratio.
Claims (8)
1. one kind has α -1, the cigarette potato polysaccharide of 3- dextran chain it is characterised in that having α -1, the cigarette potato polysaccharide of 3- dextran chain
Constitutional repeating unit in contain 1 → 3 and 1 → 6 two kinds of glycosidic bonds, main chain be 1 → 3 glycosidic bond, side chain is by 1 → 6 glycosidic bond group
Become, saccharide residue is α configuration, degree of branching is 0.33, and structural formula is:
Wherein n be 340～
345, relative molecular mass is 2.40 × 105Da～2.46 × 105da.
2. one kind as claimed in claim 1 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain it is characterised in that
The preparation method with the cigarette potato polysaccharide of α -1,3- dextran chain is:
First, cigarette potato crude polysaccharides are prepared: cigarette potato 25 is cleaned up, peeling, stripping and slicing, naturally dry, pulverize, cross 50 mesh sieves, obtain cigarette
Potato starch end, immersion 24h～48h in the ethanol that mass fraction is 95% is put at cigarette potato starch end and carries out defat, naturally dry in the air after taking-up
Dry, then carry out hot water extraction, sucking filtration obtains extracting solution, and extracting solution is carried out concentrating under reduced pressure, obtains concentrated solution i, and concentrated solution i is put
Enter immersion 12h～24h in the ethanol solution that mass fraction is 95%, sucking filtration must precipitate i, sunk after precipitation i vacuum drying
Starch flour, precipitation powder is put in distilled water, obtains the crude polysaccharides solutions i that concentration is 2mg/ml, then uses sevag method to take off egg
In vain, take supernatant, supernatant is carried out concentrating under reduced pressure, obtain concentrated solution ii, it is 95% that concentrated solution ii is put into mass fraction
Soak 12h～24h in ethanol solution, sucking filtration must precipitate ii, then precipitation ii is vacuum dried, obtains cigarette potato crude polysaccharides；
The volume ratio of the ethanol that the quality at described cigarette potato starch end is 95% with mass fraction is 1g:(5ml～10ml)；Described will be dense
It is 95% with mass fraction that contracting liquid i puts into the concentrated solution i soaking in 12h～24h in the ethanol solution that mass fraction is 95%
The volume ratio of ethanol solution is 1:(3～4)；Described putting into concentrated solution ii in the ethanol solution that mass fraction is 95% is soaked
Concentrated solution ii in 12h～24h and mass fraction are the volume ratio of 95% ethanol solution is 1:(3～4)；
2nd, purification: the cigarette potato crude polysaccharides that step one is obtained are put in distilled water, with 0.45 μm of filtering with microporous membrane, obtain
Concentration is crude polysaccharides solutions i i of 2mg/ml, carries out initial gross separation with deae-52 cellulose chromatographic column, the eluting after being separated
Liquid i, the eluent i after separation is carried out flowing water successively and dialyses 1 day, distilled water is dialysed 2 days, then carries out concentrating under reduced pressure and vacuum is cold
Lyophilizing is dry, obtains cigarette potato polysaccharide component, cigarette potato polysaccharide component is put in distilled water, with 0.45 μm of filtering with microporous membrane, obtains
To the cigarette potato polysaccharide component solution for 2mg/ml for the concentration, then use sephadexg-200 chromatographic column to cigarette potato polysaccharide component solution
Isolated and purified, the eluent ii after being separated, the eluent ii after separation is carried out concentrating under reduced pressure and vacuum freezing is done
Dry, obtain with α -1, the cigarette potato polysaccharide of 3- dextran chain；
Described in step 2 flowing water dialysis concretely comprise the following steps: by eluent i load molecular cut off be 8000 dalton～
In the bag filter of 11000 dalton, bag filter is placed in the beaker of 1000ml, room temperature down-flow water persistently soaks dialysis 1 day；
What the distilled water described in step 2 was dialysed concretely comprises the following steps: the bag filter after flowing water dialysis treatment is placed in and fills 1000ml
In the beaker of distilled water, distilled water immersion is dialysed 2 days, and wherein every 6h～12h changes No. 1 distilled water；
The condition that described deae-52 cellulose chromatographic column carries out initial gross separation is: carry out sodium chloride solution gradient elution, described
Concentration of sodium chloride solution be followed successively by 0.5mol/l and 1.0mol/l, the volume of sodium chloride solution used is that deae-52 is fine every time
1.5 times～2 times of dimension element chromatography column volume, it is 1ml/min that constant flow pump controls elution flow rate, and the applied sample amount of crude polysaccharides solutions i i is
2ml, collects 1 pipe with the every 3ml of automatic fraction collector, and the eluent of collection the 20th pipe to the 60th pipe is the eluent i after separating；
Described deae-52 cellulose chromatography column internal diameter is 2.6cm, and length is 55cm；
The condition that described sephadexg-200 chromatographic column is isolated and purified to cigarette potato crude polysaccharides component solution is: eluent
For distilled water, the volume of distilled water used chromatographs 1.5 times～2 times of column volume for sephadexg-200, and constant flow pump controls eluting
Flow velocity be 0.5ml/min, the applied sample amount of cigarette potato crude polysaccharides component solution is 1ml, collects 1 with the every 3ml of automatic fraction collector
Pipe, the eluent of collection the 25th pipe to the 50th pipe is the eluent ii after separating；Described sephadexg-200 chromatography column internal diameter
For 1.6cm, length is 80cm.
3. one kind according to claim 2 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain, and its feature exists
The condition of the hot water extraction described in step one is: Extracting temperature is 65 DEG C～85 DEG C, and extraction time 1.5h～3h is described
Naturally the cigarette potato 25 after drying is 1:(10～20 with the mass ratio of water), extraction time is 2 times～4 times.
4. one kind according to claim 2 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain, and its feature exists
The process of the concentrating under reduced pressure described in step one is: extracting solution is put in rotary evaporation bottle, pressure for 0.05mpa～
Extracting solution is compressed to the 15%～30% of original volume by 0.08mpa and bath temperature under conditions of being 55 DEG C～65 DEG C.
5. one kind according to claim 2 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain, and its feature exists
The Deproteinated process of sevag method described in step one is: by the crude polysaccharides solutions i for 2mg/ml for the concentration and the bodies such as sevag liquid
Long-pending mixing, concussion fully mixes, and is then 3000r min in rotating speed-1Centrifugation 10min, takes supernatant；Supernatant is repeated
The Deproteinated process of sevag method 5 times～10 times.
6. one kind according to claim 2 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain, and its feature exists
The vacuum drying process of i that will precipitate described in step one is: will precipitate i and load in mortar, mortar is placed in vacuum drying
In case, baking temperature is 50 DEG C～70 DEG C, and pressure is 0.02mpa～0.05mpa, and drying time is 12h～24h；
The vacuum drying process of ii that will precipitate described in step one is: will precipitate ii and load in mortar, mortar is placed in vacuum
In drying baker, baking temperature is 50 DEG C～70 DEG C, and pressure is 0.02mpa～0.05mpa, and drying time is 12h～24h.
7. one kind according to claim 2 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain, and its feature exists
The concentrating under reduced pressure step eluent i after separation being carried out successively in concentrating under reduced pressure and vacuum lyophilization described in step 2
Suddenly it is: the eluent i after separation is put in rotary evaporation bottle, in pressure be 0.05mpa～0.08mpa and bath temperature is 55
DEG C～65 DEG C under conditions of eluent i is compressed to the 15%～30% of original volume, obtain concentrated solution；
The process of vacuum lyophilization is: concentrated solution is loaded in the ampoule bottle of 2ml, ampoule bottle is placed in vacuum freeze drier
In, set freeze-dried product temperature as -25 DEG C～-40 DEG C, cooling time is 12h～24h, pressure 5pa～40pa, parsing temperature
Spend for 20 DEG C～40 DEG C, the parsing time is 8h～18h.
8. one kind according to claim 2 has α -1, the preparation method of the cigarette potato polysaccharide of 3- dextran chain, and its feature exists
The concentrating under reduced pressure eluent ii after separation being carried out successively in concentrating under reduced pressure and vacuum lyophilization described in step 2
Step is: the eluent ii after separation is put in rotary evaporation bottle, is 0.05mpa～0.08mpa and bath temperature in pressure
For eluent ii being compressed under conditions of 55 DEG C～65 DEG C the 15%～30% of original volume, obtain concentrated solution；
The process of vacuum lyophilization is: concentrated solution is loaded in the ampoule bottle of 2ml, ampoule bottle is placed in vacuum freeze drier
In, set freeze-dried product temperature as -25 DEG C～-40 DEG C, cooling time is 12h～24h, pressure 5pa～40pa, parsing temperature
Spend for 20 DEG C～40 DEG C, the parsing time is 8h～18h.
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