CN106832039B - Utilize the method for adverse current chromatogram quick separating polysaccharide from dragon fruit - Google Patents
Utilize the method for adverse current chromatogram quick separating polysaccharide from dragon fruit Download PDFInfo
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- CN106832039B CN106832039B CN201710215336.3A CN201710215336A CN106832039B CN 106832039 B CN106832039 B CN 106832039B CN 201710215336 A CN201710215336 A CN 201710215336A CN 106832039 B CN106832039 B CN 106832039B
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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 method of the invention discloses a kind of using adverse current chromatogram from dragon fruit quick separating polysaccharide, comprising the following steps: boiling water extracts after dragon fruit dry plate crushes；Leaching liquor filters after being cooled to room temperature, and filtrate is successively concentrated, depigmentation, takes off albumen, and is concentrated again, obtains concentrate；It is added to absolute ethanol and is precipitated in concentrate, resulting sediment is freeze-dried, and dragon fruit Thick many candies are obtained；It takes 0.25~0.35g dragon fruit Thick many candies to be dissolved in the lower phase of the aqueous two-phase solvent system of 25 ± 5ml, and injects adverse current chromatogram；The adjusting of pH is carried out to the upper phase of aqueous two-phase solvent system, to obtain mobile phase I, mobile phase II, mobile phase III respectively；It takes mobile phase I, mobile phase II, mobile phase III successively to be eluted respectively, collects the eluent of identical Rf value under every kind of mobile phase, concentrated, dialysis, freeze-drying by the 3 class eluents difference of collection, to obtain 3 kinds of dragon fruit polysaccharide respectively.
Description
Technical field
The invention belongs to food processing utilization and functional food manufacturing fields, mainly apply chromatographic process and chemical method
Extracted from biomaterial with utility value or containing bioactive ingredients isolating biologically active substance or it is functional at
Point, activity mechanism or exploitation new product etc. to study this ingredient provide technical support.
Background technique
Dragon fruit (Hylocereus undulates Britt) is known as " prickly-pear cactus ", " lucky fruit ", is a kind of nutrition
Abundant, suitable for people of all ages fruit, contains the multiple nutritional components such as polysaccharide, organic acid, dietary fiber and potassium, calcium, magnesium, phosphorus.According to report
Road, dragon fruit polysaccharide promote intestines peristalsis, in terms of have good function.
The extraction separation of functional polysaccharide is the key that development functionality food.The current existing extraction system about polysaccharide
Preparation Method mainly uses the methods of gel column chromatography, UF membrane, ion-exchange chromatography, and limitation is used material
(filler or film) price, at high cost, speed is slow, and the amount separated every time is limited.Therefore, existing separation of polysaccharides technology is difficult
To meet the isolated needs of dragon fruit polysaccharide.
Has the report that laminarin, lentinan, spirulina polysaccharide are separated using adverse current chromatogram at present, but according to report
Adverse current chromatogram separation condition and method be not suitable for separate dragon fruit polysaccharide.
Summary of the invention
The technical problem to be solved in the present invention is to provide it is a kind of it is pollution-free, rapidly and efficiently, it is lower-cost from dragon fruit
The method that extraction prepares polysaccharide.
Adverse current chromatogram quick separating polysaccharide from dragon fruit is utilized in order to solve the above technical problem, the present invention provides a kind of
Method, comprising the following steps:
(1) it is sliced after removing the peel dragon fruit, is freeze-dried the dragon fruit dry plate that water content≤0.05% is made, by dragon fruit
(100 DEG C) 2~3h of extraction of boiling water after dry plate crushes；
(2) leaching liquor (10~30 DEG C) being cooled to room temperature to filter afterwards, filtrate is successively concentrated, depigmentation, takes off albumen,
And be concentrated again, obtain concentrate；
(3) it is added to absolute ethanol and is precipitated in concentrate, resulting sediment is freeze-dried, and flue is obtained
Fruit Thick many candies；
(4) it takes 0.25~0.35g dragon fruit Thick many candies to be dissolved in the lower phase of the aqueous two-phase solvent system of 25 ± 5ml, and infuses
Enter and (be slowly injected into) adverse current chromatogram (such as selecting HSCCC-1000 type counter-current chromatograph)；
The aqueous two-phase solvent system is by following mass content at being grouped as: 4~6% Macrogol 6000s
(PEG6000), 16~20% polyvinylpyrrolidone (PVP), 7~9% potassium dihydrogen phosphate (KH2PO4), 3~5% sulphur
Sour potassium (K2SO4), and as the water of surplus；
Aqueous two-phase solvent system natural layering, to form the upper phase of aqueous two-phase solvent system, aqueous two-phase solvent system
Lower phase.
(5), 5.0mL 0.05M HCl solution, 5.0mL are separately added into the upper phase of the aqueous two-phase solvent system of every 500mL
0.15M HCl solution, 2.78mL 0.01M NaOH solution, thus respectively as mobile phase I, mobile phase II, mobile phase III；
The mobile phase I, mobile phase II, mobile phase III of 300 ± 30mL is taken successively to be eluted respectively, mobile phase I, mobile phase
II, the flow velocity of mobile phase III is 3.0 ± 0.2mL/min；
The eluent for collecting identical Rf value under every kind of mobile phase, by 3 class eluents of collection difference concentrated, dialysis,
Freeze-drying, to obtain 3 kinds of dragon fruit polysaccharide respectively.
Remarks explanation:
The lower identical Rf value collected of mobile phase I is 0.28；That is, collecting 23~45；
The lower identical Rf value collected of mobile phase II is 0.57；That is, collecting 53~77；
The lower identical Rf value collected of mobile phase III is 0.85.That is, collecting 111~138 pipes.
Freeze-drying is that 120~240min is freeze-dried under the conditions of 50~100Pa in -60~-80 DEG C.
As it is of the invention using adverse current chromatogram from dragon fruit the method for quick separating polysaccharide improvement:
In the step (1): be crushed to 200~300 meshes, liquid material mass ratio when extraction be 35~50L/1kg (compared with
Good is 38~45L/1kg)；
Resulting residue repeats above-mentioned boiling water extraction 2~3 times after extraction, merges all leaching liquors.
As it is of the invention using adverse current chromatogram from dragon fruit quick separating polysaccharide further improvements in methods:
The step (2) are as follows: filtrate is first concentrated and (is concentrated with Rotary Evaporators, vacuum degree 0.05Mpa, temperature 32~35
DEG C) to 1/10~1/5 (preferably 1/6~1/8) of original volume, the work for accounting for 0.002~0.003 times of weight of concentrate is then added
Property carbon uniformly mix, stand 3~5h adsorpting pigment, filtering is added in resulting filtrate II and accounts for the three of II 0.01 times of volumes of filtrate
Monoxone is mixed, is shaken 5~10 minutes (purpose is to make albuminous degeneration that deproteination be precipitated), centrifugation (3000~3500 ×
G), the 1/5~1/3 of resulting supernatant concentration (being concentrated with Rotary Evaporators) most original volume；Obtain concentrate.
As it is of the invention using adverse current chromatogram from dragon fruit quick separating polysaccharide further improvements in methods:
The step (3) are as follows: the dehydrated alcohol concussion 3 of 2~5 volumes (preferably 3~5 volumes times) is added in concentrate
~5 minutes, 24 ± 2h (dragon fruit polysaccharide is precipitated sufficiently) then is analysed in 4 ± 1 DEG C of alcohol, centrifugation (2000~3000 × g centrifugation 3
~5 minutes), resulting sediment is freeze-dried (- 50~-80 DEG C of dry 2~8h), obtains dragon fruit Thick many candies.
As it is of the invention using adverse current chromatogram from dragon fruit quick separating polysaccharide further improvements in methods:
The aqueous two-phase solvent system is by following mass content at being grouped as: 5% Macrogol 6000 (PEG6000),
18% polyvinylpyrrolidone (PVP), 8% potassium dihydrogen phosphate (KH2PO4), 4% potassium sulfate (K2SO4), 65% water.
That is, PEG6000:PVP:KH2PO4:K2SO4:H2O=0.5:1.8:0.8:0.4:6.5.
As it is of the invention using adverse current chromatogram from dragon fruit quick separating polysaccharide further improvements in methods, it is described
In step (5):
It is detected with thin layer chromatography, collects the eluent of identical Rf value under every kind of mobile phase；
It is concentrated into the 1/4~1/6 of original volume；
The dialysis is through the dialysis of 8000~14000Da dialysis membrane.
As it is of the invention using adverse current chromatogram from dragon fruit quick separating polysaccharide further improvements in methods, step
(1) slice are as follows: by sarcocarp section to 3~5mm of thickness, freeze-drying are as follows: in -50~-55 DEG C of temperature, 50~100Pa
Vacuum degree is dried to moisture content≤0.05% (drying time is about for 24 hours).
The present invention obtains polysaccharide extraction liquid and albumen using the polysaccharide components of alcohol precipitation in (100 DEG C) of hot water extraction plants
Mixed liquor；Albumen, pigment are taken off, freeze-drying obtains polysaccharide crude；It is separated again using adverse current chromatogram method；It is molten with aqueous two-phase
The lower phase of agent system is as stationary phase；Using the upper phase of aqueous two-phase separation system as mobile phase.
In the present invention, obtained dragon fruit polysaccharide is tested and analyzed with high performance liquid chromatography, chromatographic condition is
ELSD 2000 evaporates photodetector, TSK-GEL3000PWXLLiquid-phase chromatographic column uses H2O is mobile phase, flow velocity 0.65mL/min.
Analysis detection, content (purity) >=97% are carried out to collected sample.
The present invention has following technical advantage: the present invention and traditional UF membrane, gel post separation, ionic energy transfer side
Method is compared, and has the characteristics that separating rate is fast, at low cost, sample volume is big, the present invention establishes the adverse current of dragon fruit separation of polysaccharides
Chromatography aqueous two-phase system and pH gradient separation method, make adverse current chromatogram can apply to the separation of dragon fruit polysaccharide, expand adverse current
The application range of chromatographic isolation polysaccharide, the resulting dragon fruit polysaccharide of the present invention have significant functions of loosening bowel relieving constipation.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is process flow chart of the invention.
Fig. 2 is the HSCCC separating spectrum of embodiment 1.
Fig. 3 is the separating obtained dragon fruit polysaccharide HPLC map of embodiment 1.
Fig. 4 is the HSCCC separating spectrum of embodiment 2.
Fig. 5 is the HSCCC separating spectrum of embodiment 3.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1, a kind of method for preparing polysaccharide from separation in dragon fruit (are quickly divided from dragon fruit using adverse current chromatogram
Method from polysaccharide), successively follow the steps below:
(1) dragon fruit peeling is taken into pulp, by sarcocarp section to 3~5mm of thickness, in -50~-55 DEG C of temperature, 50~
It is freeze-dried under the vacuum degree of 100Pa, until the dragon fruit dry plate of water content≤0.05% is made, (drying time is about
24h)。
200g dragon fruit dry plate is put into pulper and was crushed to 200~300 meshes, obtains dragon fruit dry powder, is added
It to the large beaker for filling 8.0L distilled water, is heated to boiling, is kept for 100 DEG C, boiling extracts 3h；
Above-mentioned boiling water is repeated with residue substitution dragon fruit dry plate resulting after extraction to extract 2 times, merges all leaching liquors.
(2) leaching liquor after merging filters after being cooled to room temperature, and filtrate is successively concentrated, depigmentation, takes off albumen, and again
It is secondary to be concentrated, obtain concentrate；
It is specific as follows:
By filtrate concentration (being concentrated with Rotary Evaporators, vacuum degree 0.05Mpa, 32~35 DEG C of temperature) to the 1/6 of original volume,
The activated carbon mixing 5min for accounting for 0.002 times of weight of concentrate is added, stands 3h adsorpting pigment, filtering, resulting filtrate II
The trichloroacetic acid that middle addition accounts for II 0.01 times of volumes of filtrate is mixed, shaking 5~10 minutes, (purpose is that albuminous degeneration is precipitated
Deproteination), it is centrifuged (3000~3500 × g), resulting supernatant concentration (is concentrated, 42 DEG C of temperature, vacuum with Rotary Evaporators
Spend 100Pa) most original volume 1/3；Obtain concentrate.
(3) dehydrated alcohol that 3 volumes times are added in concentrate shakes 3~5 minutes, then (makes for 24 hours in 4 ± 1 DEG C of alcohol analysis
Dragon fruit polysaccharide is sufficiently precipitated), 2000~3000 × g is centrifuged 3~5 minutes, and resulting sediment is under the conditions of -80 DEG C, 100Pa
It is freeze-dried 120min, obtains dragon fruit Thick many candies 4.51g.
(4) by PEG6000-PVP-KH2PO4–K2SO4–H2O is mixed according to 0.5:1.8:0.8:0.4:6.5 (w/w), as
Aqueous two-phase solvent system.
Aqueous two-phase solvent system natural layering, to form the upper phase of aqueous two-phase solvent system, aqueous two-phase solvent system
Lower phase.
It takes 0.35g dragon fruit Thick many candies to be dissolved in the lower phase of 25ml aqueous two-phase solvent system, can be used auxiliary in ultrasonoscope
Hydrotropy solution 10min.Start counter-current chromatograph (HSCCC-1000 type counter-current chromatograph), revolving speed 800rpm, with compacted after velocity-stabilization
Dynamic pump, which is slowly injected into counter-current chromatograph, to be separated.
(5), 5.0mL 0.05M HCl solution, 5.0mL are separately added into the upper phase of the aqueous two-phase solvent system of every 500mL
0.15M HCl solution, 2.78mL 0.01M NaOH solution, thus respectively as mobile phase I (pH value 6.25), mobile phase II
(pH value 4.70), mobile phase III (pH value 7.90)；
Take the mobile phase I, mobile phase II, mobile phase III of 300mL successively to be eluted respectively, mobile phase I, mobile phase II,
The flow velocity of mobile phase III is 3.0mL/min, and every 2min collects 1 pipe, collects 150 pipes altogether.
Efflux is detected with thin layer chromatography, collects the eluent of identical Rf value under every kind of mobile phase, that is, is collected
24th~45 pipe (Rf value 0.28) collects that 58~77 pipes (Rf value 0.57), collecting 119~138 pipes, (Rf value is
0.85)；
Remarks: every 2min collects a pipe；
3 class eluents of collection are concentrated into the 1/5 of original volume respectively, 8000~14000Da dialysis membrane is dialysed 12h, cold
Dry (- 80 DEG C, 100Pa under the conditions of be freeze-dried 120min) are lyophilized, respectively correspond to obtain 3 kinds of dragon fruit homogeneous polysaccharides ----
0.1033g polysaccharides-I, 0.0847g polysaccharide II, 0.0763g polysaccharide III, i.e. yield are respectively 29.51%, 24.20%, 21.80%.
Yield is detected according to phend-sulphuric acid, and content can reach 97% or more.
Embodiment 2, a kind of method that the separation from dragon fruit prepares polysaccharide, successively follow the steps below:
(1), the preparation method is the same as that of Example 1 for dragon fruit dry plate.
180g dragon fruit dry plate is put into pulper and was crushed to 200~300 meshes, obtains dragon fruit dry powder, is added
It to the large beaker for filling 7.0L distilled water, is heated to boiling, is kept for 100 DEG C, boiling extracts 2.5h；
Above-mentioned boiling water is repeated with residue substitution dragon fruit dry plate resulting after extraction to extract 2 times, merges all leaching liquors.
(2) leaching liquor after merging filters after being cooled to room temperature, and filtrate is successively concentrated, depigmentation, takes off albumen, and again
It is secondary to be concentrated, obtain concentrate；
It is specific as follows:
By filtrate concentration (being concentrated with Rotary Evaporators, vacuum degree 0.05Mpa, 32~35 DEG C of temperature) to the 1/7 of original volume,
The activated carbon mixing 5min for accounting for 0.002 times of weight of concentrate is added, stands 3h adsorpting pigment, filtering, resulting filtrate II
The trichloroacetic acid that middle addition accounts for II 0.01 times of volumes of filtrate is mixed, shaking 5~10 minutes, (purpose is that albuminous degeneration is precipitated
Deproteination), it is centrifuged (3000~3500 × g), resulting supernatant concentration (is concentrated, 42 DEG C of temperature, vacuum with Rotary Evaporators
Spend 100Pa) most original volume 1/3；Obtain concentrate.
(3) dehydrated alcohol that 3 volumes times are added in concentrate shakes 3~5 minutes, then (makes for 24 hours in 4 ± 1 DEG C of alcohol analysis
Dragon fruit polysaccharide is sufficiently precipitated), 2000~3000 × g is centrifuged 3~5 minutes, and resulting sediment is under the conditions of -80 DEG C, 100Pa
It is freeze-dried 120min, obtains dragon fruit Thick many candies 3.97g.
(4)~(5),
0.25g dragon fruit Thick many candies are taken to be dissolved in the lower phase of 30ml aqueous two-phase solvent system, efflux thin layer chromatography
It is detected, collects the 23rd~41,53~72,111~131 pipes of identical Rf value, the step of remaining is equal to embodiment 1
(4)~(5)；
Obtain 3 kinds of dragon fruit homogeneous polysaccharides ----polysaccharides-I, polysaccharide II, polysaccharide III, respectively 0.0743g, 0.0607g,
0.0550g (i.e. yield is respectively 29.72%, 24.28%, 22.00%), content reaches 97% or more.
Embodiment 3, a kind of method that the separation from dragon fruit prepares polysaccharide, successively follow the steps below:
(1), the preparation method is the same as that of Example 1 for dragon fruit dry plate.
180g dragon fruit dry plate is put into pulper and was crushed to 200~300 meshes, obtains dragon fruit dry powder, is added
It to the large beaker for filling 8.0L distilled water, is heated to boiling, is kept for 100 DEG C, boiling extracts 3h；
Above-mentioned boiling water is repeated with residue substitution dragon fruit dry plate resulting after extraction to extract 2 times, merges all leaching liquors.
(2) leaching liquor after merging filters after being cooled to room temperature, and filtrate is successively concentrated, depigmentation, takes off albumen, and again
It is secondary to be concentrated, obtain concentrate；
It is specific as follows:
By filtrate concentration (being concentrated with Rotary Evaporators, vacuum degree 0.05Mpa, 32~35 DEG C of temperature) to the 1/8 of original volume,
The activated carbon mixing 5min for accounting for 0.002 times of weight of concentrate is added, stands 3h adsorpting pigment, filtering, resulting filtrate II
The trichloroacetic acid that middle addition accounts for II 0.01 times of volumes of filtrate is mixed, shaking 5~10 minutes, (purpose is that albuminous degeneration is precipitated
Deproteination), it is centrifuged (3000~3500 × g), resulting supernatant concentration (is concentrated, 42 DEG C of temperature, vacuum with Rotary Evaporators
Spend 50Pa) most original volume 1/5.
(3) dehydrated alcohol that 5 volumes times are added in concentrate shakes 3~5 minutes, then (makes for 24 hours in 4 ± 1 DEG C of alcohol analysis
Dragon fruit polysaccharide is sufficiently precipitated), 2000~3000 × g is centrifuged 3~5 minutes, and resulting sediment is under the conditions of -80 DEG C, 100Pa
It is freeze-dried 120min, obtains dragon fruit Thick many candies 4.09g.
(4)~(5),
0.30g dragon fruit Thick many candies are taken to be dissolved in the lower phase of 25mL aqueous two-phase solvent system, by efflux thin-layer chromatography
The step of method is detected, and the 23rd~44,57~75,116~137 pipes of identical Rf value are collected, remaining is equal to embodiment 1
(4)~(5)；
Obtain 3 kinds of dragon fruit homogeneous polysaccharides ----polysaccharides-I, polysaccharide II, polysaccharide III, respectively 0.0886g, 0.0730g,
0.0647g (that is, yield is respectively 29.53%, 24.33%, 21.57%), content reaches 98% or more.
Performance test:
In order to examine the defecating feces excretion of dragon fruit polysaccharide, 3 kinds of dragon fruit polysaccharide (polysaccharides-I, polysaccharide II, polysaccharide III) is devised
Respectively to the experiment of Constipation Model mouse defecating feces excretion.Male mouse of kunming is randomly divided into blank group, positive controls and 3
A dragon fruit polysaccharide administration group, Blackfungus polyhexose administration group, every group 12.Administration group orally gives 0.50g/kg bw's respectively
Dragon fruit polysaccharide I, II, III, Blackfungus polyhexose；Blank group and model group give the distilled water of 20mL/kg bw；Positive controls
Give the Maren wan of 0.50g/kg bw.After continuous gavage 8d, melena time of row for the first time of each group mouse is observed, defecation grain in 5h
Number and measurement excrement water content.Acquired results are specific as follows:
The influence of 3 kinds of dragon fruit Polysaccharides on Mice defecation indexs
The melena time is arranged for the first time, increases defecation grain number in 5h and improves excrement the result shows that dragon fruit polysaccharide can significantly shorten
Water content, 3 kinds of dragon fruit polysaccharide have significant functions of loosening bowel relieving constipation, and polysaccharide II effect is best.
Comparative example 1-1, the aqueous two-phase solvent system in embodiment 1 is changed to PEG6000-KH2PO4–K2SO4–H2O(2.3:
0.8:0.4:6.5, w/w)；Remaining is equal to embodiment 1, then matched solvent cannot function as inverse due to that cannot form two-phase up and down
The solvent system of flow chromatography, so not being suitable for separating dragon fruit polysaccharide.
Comparative example 1-2, the aqueous two-phase solvent system in embodiment 1 is changed to PEG6000-PVP-KH2PO4–K2SO4–H2O
(0.8:1.5:0.8:0.4:6.5, w/w)；Remaining is equal to embodiment 1, the solubility of the mobile phase of generation to dragon fruit polysaccharide
Not enough, dragon fruit polysaccharide cannot be efficiently separated.
Comparative example 1-3, the aqueous two-phase solvent system in embodiment 1 is changed to PEG6000-PVP-KH2PO4–K2SO4–H2O
(0.2:2.1:0.8:0.4:6.5, w/w), remaining is equal to embodiment 1, then matched solvent is due to that cannot form two-phase up and down, no
Solvent system of the energy as adverse current chromatogram, so not being suitable for separating dragon fruit polysaccharide.
Comparative example 2-1, embodiment 1 is changed as follows:
2.5mL 0.05M HCl solution, 2.5mL 0.15M HCl solution, 1.39mL are separately added on every 500mL in phase
0.01M NaOH solution, thus respectively as mobile phase I (pH value 6.55), mobile phase II (pH value 5.70), mobile phase III
(pH value 7.30)；Remaining is equal to embodiment 1.Acquired results show: it is more cannot to efficiently separate 3 kinds of dragon fruits for mobile phase at this time
Sugar, the 3 kinds of polysaccharide collected are still mixture.
Comparative example 2-2, embodiment 1 is changed as follows:
10mL 0.05M HCl solution, 10mL 0.15M HCl solution, 5.56mL are separately added on every 500mL in phase
0.01M NaOH solution, thus respectively as mobile phase I (pH value 5.25), mobile phase II (pH value 3.60), mobile phase III
(pH value 9.15)；Remaining is equal to embodiment 1.
Acquired results show that only polysaccharide I can be separated a little, yield 3.61%, remaining 2 kinds of polysaccharide cannot achieve separation.
Comparative example 3-1, the PEG6000- (NH that the aqueous two-phase solvent system in embodiment 1 has been reported instead4)2SO4-
H2O (2.0:0.8:7.2, w/w), remaining is equal to embodiment 1, the results showed that this aqueous two-phase system cannot make dragon fruit polysaccharide real
Now separate.
Comparative example 3-2, the PEG1000-K that the aqueous two-phase solvent system in embodiment 1 has been reported instead2HPO4–
KH2PO4–H2O (0.5:1.25:1.25:7.0, w/w), remaining is equal to embodiment 1, the results showed that this aqueous two-phase system cannot make
Dragon fruit polysaccharide realizes separation.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (6)
1. using adverse current chromatogram, quick separating has the method for the polysaccharide of functions of loosening bowel relieving constipation from dragon fruit, it is characterized in that including
Following steps:
(1) it is sliced after removing the peel dragon fruit, is freeze-dried the dragon fruit dry plate that water content≤0.05% is made, by dragon fruit dry plate
Boiling water extracts 2~3h after crushing；
(2) it is filtered after being cooled to room temperature leaching liquor, filtrate is successively concentrated, depigmentation, takes off albumen, and is carried out again dense
Contracting, obtains concentrate；
(3) it is added to absolute ethanol and is precipitated in concentrate, resulting sediment is freeze-dried, and it is thick to obtain dragon fruit
Polysaccharide；
(4) it takes 0.25~0.35g dragon fruit Thick many candies to be dissolved in the lower phase of the aqueous two-phase solvent system of 25 ± 5ml, and injects inverse
Flow chromatography；
The aqueous two-phase solvent system is by following mass content at being grouped as: 5% Macrogol 6000,18% polyethylene pyrrole
Pyrrolidone, 8% potassium dihydrogen phosphate, 4% potassium sulfate, 65% water；
(5), 5.0mL 0.05M HCl solution, 5.0mL are separately added into the upper phase of the aqueous two-phase solvent system of every 500mL
0.15M HCl solution, 2.78mL 0.01M NaOH solution, thus respectively as mobile phase I, mobile phase II, mobile phase III；
Take the mobile phase I, mobile phase II, mobile phase III of 300 ± 30mL successively to be eluted respectively, mobile phase I, mobile phase II,
The flow velocity of mobile phase III is 3.0 ± 0.2mL/min；
The eluent for collecting identical Rf value under every kind of mobile phase, concentrated, dialysis, freezing by the 3 class eluents difference of collection
It is dry, to obtain 3 kinds of dragon fruit polysaccharide respectively.
2. according to claim 1, using adverse current chromatogram, quick separating has the polysaccharide of functions of loosening bowel relieving constipation from dragon fruit
Method, it is characterized in that:
In the step (1): being crushed to 200~300 meshes, liquid material mass ratio when extraction is 35~50L/1kg；
Resulting residue repeats above-mentioned boiling water extraction 2~3 times after extraction, merges all leaching liquors.
3. according to claim 1 or 2, using adverse current chromatogram, quick separating has functions of loosening bowel relieving constipation from dragon fruit
The method of polysaccharide, it is characterized in that the step (2) are as follows: filtrate is first concentrated into the 1/10~1/5 of original volume, then addition accounts for dense
The activated carbon of 0.002~0.003 times of weight of contracting liquid uniformly mixes, and stands 3~5h adsorpting pigment, filters, in resulting filtrate II
The trichloroacetic acid that addition accounts for II 0.01 times of volumes of filtrate is mixed, is shaken 5~10 minutes, is centrifuged, resulting supernatant concentration
Most the 1/5~1/3 of original volume；Obtain concentrate.
4. according to claim 3, using adverse current chromatogram, quick separating has the polysaccharide of functions of loosening bowel relieving constipation from dragon fruit
Method, it is characterized in that the step (3) are as follows: the dehydrated alcohol that 2~5 volumes times are added in concentrate shakes 3~5 minutes,
Then 24 ± 2h, centrifugation are analysed in 4 ± 1 DEG C of alcohol, resulting sediment freeze-drying obtains dragon fruit Thick many candies.
5. according to claim 3, using adverse current chromatogram, quick separating has the polysaccharide of functions of loosening bowel relieving constipation from dragon fruit
Method, it is characterized in that in the step (5):
It is detected with thin layer chromatography, collects the eluent of identical Rf value under every kind of mobile phase；
It is concentrated into the 1/4~1/6 of original volume；
The dialysis is through the dialysis of 8000~14000Da dialysis membrane.
6. according to claim 3, using adverse current chromatogram, quick separating has the polysaccharide of functions of loosening bowel relieving constipation from dragon fruit
Method, it is characterized in that the slice of step (1) are as follows: by sarcocarp section to 3~5mm of thickness, freeze-drying are as follows: in -50~-55 DEG C
Temperature, the vacuum degree of 50~100Pa is dried to moisture content≤0.05%.
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