CN108084131A - It is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying - Google Patents
It is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying Download PDFInfo
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- CN108084131A CN108084131A CN201711387948.7A CN201711387948A CN108084131A CN 108084131 A CN108084131 A CN 108084131A CN 201711387948 A CN201711387948 A CN 201711387948A CN 108084131 A CN108084131 A CN 108084131A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
- C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D309/10—Oxygen atoms
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
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Abstract
The present invention relates to the enrichment and purification methods of the hydroxyl radical carthamin yellow carthamus A contained in the technical field of Chinese medical extract more particularly to a kind of Chinese medicine.The performance purified using polysaccharide-based medium for the efficient separation and concentration of active ingredient in Chinese medicine, the present invention provides it is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying, effectively such medicinal active ingredient can be refined, and effective enrichment can be achieved at the same time.To providing high-quality, high-purity, high extraction, standard is unified, the hydroxyl radical carthamin yellow carthamus A stablized has important application value and economic value, while standardization, standardization to promoting Chinese Medicine Industry, there is an extremely important effect.
Description
Technical field
The present invention relates to Chinese medical extract technical field more particularly to it is a kind of to hydroxyl radical carthamin yellow carthamus A be enriched with it is pure
The lower polysaccharide-based medium of the method for change, predominantly solvent regulation and control is for the adsorption and de-adsorption of specific molecular structure alkaloids molecule
Process, and it is related to design and application for novel polysaccharide base dielectric structure.
Background technology
Hydroxyl radical carthamin yellow carthamus A (hydroxysafflor yellow A, HSYA) is a kind of chalcone glycosides compound,
The main constituents of water-soluble red colouring matter in safflower.Safflower is feverfew safflower Carthamus tinctorius L.
Dry flower, the Eastern Han Dynasty before nearly 2000, Zhang Zhongjing exist《Synopsis Golden Chamber》In just describe the medical value of safflower.According to
2010 editions《Pharmacopoeia of People's Republic of China》It records, with invigorate blood circulation, the effect of removing blood stasis and analgesics.And research table in recent years
Bright, the hydroxyl radical carthamin yellow carthamus A in safflower is a kind of important pharmacological component in safflower, and it is small to be found to have anti-blood
Plate aggregation, inhibition thrombosis mitigate the effects that cardiac-cerebral ischemia damage, antioxidation activity, antitumor activity, and in molecular layer
Face, cell level give more detailed explanation.Therefore it is effective as a kind of compound with good pharmacological activity
Separation and Extraction and enriching and purifying process just seem particularly significant.
Generally, the separation and Extraction of hydroxyl radical carthamin yellow carthamus A and enriching and purifying process, have more at present in process aspect
In detail, systematic research.In extraction process, often using the mixture of water or water/alcohol as Extraction medium.Late enrichment purifies
Process generally use macroreticular resin as chromatography media carry out Image processing, further purification work can have some apply Portugal
Example of the glycan medium as chromatography media.But macroreticular resin has as a kind of polymer matrix medium for the extract of safflower
There is stronger non-specific adsorption, hydroxyl radical carthamin yellow carthamus A can also have stronger absorption on medium, and what is directly affected is final
The rate of recovery.And the chromatography media of glucan is using most commercialization medium Sephadex LH-20, this kind of media applications scope
Greatly, but a kind of glucan base, and the chromatography media Jing Guo chemical modification are used as, on the basis of polysaccharide-based, further repaiied
Decorations are so that the hydrophobicity of medium enhances, since hydroxyl radical carthamin yellow carthamus A has very strong hydrophily, in this kind of hydrophobicity
Polysaccharide medium in effective adsorption capacity be decreased obviously, it is red in hydroxyl also just to greatly limit this kind of polysaccharide medium
Application in anthoxanthin A concentration and separations.
Polysaccharide-based medium is typically used among gel chromatography technique, can be wider with relatively stable structure
PH in the range of used, while there is more excellent mechanical performance, be applicable to be used for multiple times process without changing it
The chromatographic properties of itself.Nineteen fifty-nine, the first polysaccharide-based medium, cross-link dextran have been produced out, ever after, agar
A variety of polysaccharide matrixes such as sugar, cellulose are gradually synthesized, prepared, and as media applications among technique is chromatographed.With fine jade
Exemplified by lipolysaccharide, relatively stable polysaccharide-based skeleton can be formed under gel state, between polysaccharide molecule chain structure, through further
Chemical crosslinking after can form relatively stable apertures dielectric material.The hydrophily of this kind of structural framework and for biological big point
The good compatibility of son so that this kind of medium has protein and other good purification effect.Meanwhile in
The extract of medicine or other natural plants contains substantial amounts of protein, polysaccharide and the very big chemical combination of some other nature difference
Object molecule generally requires the dielectric material with effectively enrichment, purification capacity in purification process.So in Chinese medicine or other days
The extract separation and concentration purification art of right plant, the application of polysaccharide-based medium have good application value and real situation.
Therefore, using the design and application of the polysaccharide-based medium with compared with strongly hydrophilic, for effective enriching and purifying hydroxyl
Base carthamus tinctorius yellow colour A is this kind of to have fine water-soluble compound molecule, will be likely to effectively to improve this kind of molecule of pharmaceutical and exists
Total extraction efficiency during enriching and purifying.To provide high-quality, high-purity, high extraction, standard is unified, the hydroxyl stablized
Carthamus tinctorius yellow colour A has important application value and economic value, promotes standardization, the standardization of Chinese Medicine Industry, has extremely
Important role.
The content of the invention
The present invention is directed to foregoing problems, it is proposed that the method for effective enriching and purifying hydroxyl radical carthamin yellow carthamus A, and by molten
Lower control of the polysaccharide-based medium for the adsorption and de-adsorption process of specific molecular structure alkaloids molecule of agent regulation and control, utilizes spy
Determine the polysaccharide-based media implementation of structure this enriching and purifying process.
The present invention propose it is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying, utilize polysaccharide
Base medium is effectively adsorbed and eluted for hydroxyl radical carthamin yellow carthamus A scattered in a liquid state.
In the enrichment and purification method proposed by the present invention, hydroxyl radical carthamin yellow carthamus A is enriched in a manner of non-column chromatography pure
Change, including：
Polysaccharide-based medium is sufficiently mixed with being dispersed with the liquid of hydroxyl radical carthamin yellow carthamus A；
It is static after polysaccharide-based medium precipitation after, remove supernatant liquor；
The polysaccharide-based medium is cleaned using cleaning solution, cleaning dosage and the polysaccharide-based medium volume ratio of cleaning solution are 0.1:
1~100:1；
By the polysaccharide-based medium after cleaning in eluent soaking flushing, collect eluent.
In the enrichment and purification method proposed by the present invention, to hydroxyl radical carthamin yellow carthamus A enriching and purifying in a manner of column chromatography,
Including：
By polysaccharide-based Filled Dielectrics in the column tube of a diameter of 0.5mm to 500mm, it is compacted through filling；
Added in into column tube be dispersed with hydroxyl radical carthamin yellow carthamus A treat enriching and purifying solution；
Mobile phase is added in, flow rate of mobile phase is 0.1mL/min to 100.0mL/min, and UV detector Detection wavelength is
350nm to 450nm collects elution solution.
In the enrichment and purification method proposed by the present invention, cleaning solution, eluent or mobile phase are water, methanol, ethyl alcohol, just
Propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, ether, acetone, methyl ethyl ketone, acetonitrile, N, N- bis-
One or both of methylformamide, dimethyl sulfoxide (DMSO) any of the above combines.
In the enrichment and purification method proposed by the present invention, ion object is further added in during soaking flushing or in mobile phase
Matter, ionic strength are 0.1mmol/L between 1mol/L.
In the enrichment and purification method proposed by the present invention, the ionic species for formic acid, acetic acid, trifluoroacetic acid, boric acid,
Hydrochloric acid, sulfuric acid, phosphoric acid, sodium chloride, potassium chloride, ammonium chloride, sodium sulphate, potassium sulfate, ammonium sulfate, niter cake, potassium acid sulfate, sulphur
Sour hydrogen ammonium, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, lithium hydroxide, sodium hydroxide, hydroxide
One or both of potassium, triethylamine, diisopropyl ethyl amine, trishydroxymethylaminomethane any of the above combines.
In the enrichment and purification method proposed by the present invention, the main component of polysaccharide-based medium is cellulose, glucan, fine jade
One or both of lipolysaccharide, starch any of the above combines, and proportion of the main component in total media dry powder is 20%-
100%；When main component is glucan, not including hydroxypropyl glucan medium；The pattern of polysaccharide-based medium for it is spherical, rodlike,
Line style, netted, in irregular shape one kind, the characteristic size of medium is 20 nanometers to 500 microns.
In the enrichment and purification method proposed by the present invention, the polysaccharide of the polysaccharide-based medium is to be modified by derivatization,
The molecular structure of polyose modification part by derivatization modification is-X-Cn-R types, and wherein X is one in oxygen atom, nitrogen-atoms
Kind, one end of X atoms is connected with polysaccharide structures, and R is more than one or both of hydroxyl, amino, carboxyl, sulfonic group, methyl
Any combination, Cn are the carbon structure between connection X and R, and n is the number of the carbon atom in carbon structure, and n is 0 to 20, in carbon structure
The bonded mode of carbon atom is straight line or non-directional branched structure.
Compared with prior art, beneficial effects of the present invention：The present invention will be likely to effectively improve this kind of molecule of pharmaceutical
Total extraction efficiency during enriching and purifying.To provide high-quality, high-purity, high extraction, standard is unified, the hydroxyl stablized
Base carthamus tinctorius yellow colour A has important application value and economic value, promotes standardization, the standardization of Chinese Medicine Industry, there is pole
Its important role.
Specific embodiment
Enrichment and purification method proposed by the present invention will be described in more detail below, which show the excellent of the present invention
Select embodiment, it should be appreciated that those skilled in the art can change invention described herein, and still realize having for the present invention
Sharp effect.Therefore, description below is appreciated that for the widely known of those skilled in the art, and is not intended as to this hair
Bright limitation.
Involved in invention to the derived polysaccharide raw material for preparing of polysaccharide-based medium be cellulose, glucan, agarose, starch
Macromolecular polysaccharides molecule is waited, for this quasi-molecule in itself without charge, and derive from a wealth of sources, preparation process is more mature.Polysaccharide molecule exists
Preferable mechanical performance can be obtained after certain chemical crosslinking, can there is stable pattern and certain anti-pressure ability, profit
Application in terms of standardization preparation and column chromatography.
In order to further improve the mechanical performance of polysaccharide-based medium, in addition to crosslinking agent, can also contain centainly in medium
Other composition components, as two or more polysaccharide mixes (by taking agarose/glucan as an example) or add in other composite materials
(such as polyamide, polyvinyl alcohol, polyacrylic acid), but generally polysaccharide component ratio shared in total media is in 20%-
Between 100%, preferably between 60%-100%.
The pattern of polysaccharide-based medium is influenced be subject to preparation method, and can further influence the operation mould of enriching and purifying process
Formula, since polysaccharide molecule often possesses preferable dissolubility in aqueous solution, processing and be more easily to the control of pattern
In realization.Spherical, rodlike, line style, netted and irregular shape can be realized in preparation process, consider gelation and micella
The method that the processes such as change are more often related in being prepared for polysaccharide-based medium, and chondritic is relatively stable in these methods, here
With it is spherical be preferred.
Under conditions of non-column chromatography, medium is to the enrichment of hydroxyl radical carthamin yellow carthamus A and elution process to the feature of medium
Size considers the preparation present situation of polysaccharide-based medium without particular/special requirement, the characteristic size of medium 20 nanometers to 500 microns it
Between.
Under conditions of column chromatography, the characteristic size of operating pressure and medium has very close relationship, due to polysaccharide
Base medium for high pressure operating condition and do not apply to, consider normally in middle pressure or normal pressure to the scope in the eyes of the work between middle pressure
Lower work, therefore the characteristic size of medium is between 20 nanometers to 500 microns, preferably 1 micron to 100 microns of scope.
Polysaccharide-based medium has great amount of hydroxy group in itself, can carry out further chemical modification, realizes various features chemistry base
The modification of group.Carboxyl, the polysaccharide-based medium of amido modification can further increase the hydrophily of medium, be conducive in water or water/
By the effective adsorption and enrichment of hydroxyl radical carthamin yellow carthamus A in alcohol mixture, while appropriate connection group is needed to link carboxyl, amido
With polysaccharide in itself, therefore to the structure of polysaccharide-based medium following requirement is proposed：
Polysaccharide is by derivatization modification or unmodified, by the more of derivatization modification in polysaccharide-based medium main component
The molecular structure of sugar-modified part is-X-Cn-R types, and wherein X is oxygen atom, one kind in nitrogen-atoms, one end of X atoms with it is more
Sugared structure is connected, and R is one or more of hydroxyl, amino, carboxyl, sulfonic group, methyl, and Cn is the carbon knot between connection X and R
Structure, n are the number of the carbon atom in carbon structure, and n is 0 to 20, and the bonded mode of carbon atom is straight line or non-rectilinear in carbon structure
Branched structure.
Polysaccharide-based medium should pass through the abundant immersion of solution before the use, activated media, therefore when in use, medium
It weighs using volume as Dose standard, unit is mL or L.
Hydroxyl radical carthamin yellow carthamus A described in the present invention, for the product extracted from Chinese medicine safflower.
Hydroxyl radical carthamin yellow carthamus A should carry out further enriching and purifying under liquid-phase condition, consider hydroxyl radical carthamin yellow carthamus A
Have preferable dissolubility in pure water solution, therefore when hydroxyl radical carthamin yellow carthamus A disperses, the selection of liquid phase can be pure water or
The mixed liquor of water and other highly polar solution.Other polar solvents can be methanol, ethyl alcohol, normal propyl alcohol, isopropanol, n-butanol,
Isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, ether, acetone, methyl ethyl ketone, acetonitrile, N,N-dimethylformamide, dimethyl are sub-
One or more of sulfone, preferably water.
It, can be with solution or outstanding after hydroxyl radical carthamin yellow carthamus A in the liquid phase scattered under the process conditions of non-column chromatography
The form of turbid exists, and scattered concentration is 0.01mg/mL to 50mg/mL, preferably 0.01mg/mL to 20mg/mL.
Under the process conditions of non-column chromatography, the dosage of polysaccharide-based medium is 0.01mL/ (mL liquid volumes) to 10mL/
(mL liquid volumes), preferable amount are 0.1mL/ (mL liquid volumes) to 2mL/ (mL liquid volumes).
It, should be with the shape of settled solution after hydroxyl radical carthamin yellow carthamus A in the liquid phase scattered under the process conditions of column chromatography
Formula exists, and scattered concentration is 0.01mg/mL to 50mg/mL, preferably 0.01mg/mL to 5mg/mL.
Under the process conditions of column chromatography, the dosage of polysaccharide-based medium is 0.01mL/ (mL liquid volumes) to 10mL/ (mL
Liquid volume), preferable amount is 0.5mL/ (mL liquid volumes) to 2mL/ (mL liquid volumes).
For the method for non-column chromatography, the present invention is to mix the method for immersion as main description main body.I.e. in liquid phase state
Under, by polysaccharide-based medium and hydroxyl radical carthamin yellow carthamus A according to aforementioned ratio, it is sufficiently mixed in certain proportion.Mixing
Hydroxyl radical carthamin yellow carthamus A will be redistributed between liquid phase and polysaccharide-based medium afterwards.Due to described in the present invention
Polysaccharide-based medium have passed through certain hydrophilicity-imparting treatment, therefore hydroxyl radical carthamin yellow carthamus A will be to during redistributing
Polysaccharide-based media fraction is enriched with.The liquid phase on upper strata is removed at this time, adds in a small amount of cleaning solution and polysaccharide-based medium is cleaned.Cleaning
Liquid can be water, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, ether, third
One or more of ketone, methyl ethyl ketone, acetonitrile, n,N-Dimethylformamide, dimethyl sulfoxide (DMSO), but preferably acetone, acetonitrile.
Cleaning process can remove the non-hydroxyl carthamus tinctorius yellow colour A substance that polysaccharide-based media fraction is dispersed in non-adsorbed effect.It
Afterwards, the hydroxyl radical carthamin yellow carthamus A in polysaccharide-based medium is washed using the mixed solvent of water or water and other highly polar solvents
It is de-.Eluent can be water, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, second
One or more of ether, acetone, methyl ethyl ketone, acetonitrile, n,N-Dimethylformamide, dimethyl sulfoxide (DMSO), preferably water or water
With the mixture of ethyl alcohol.For the enrichment elution process of hydroxyl radical carthamin yellow carthamus A, it is strong that certain ion can be added in elution process
The substance of degree, offsets influence of the ion exchange to hydroxyl radical carthamin yellow carthamus A suction-operated in polysaccharide-based medium, and raising is washed
De- efficiency.A certain amount of ion, ionic strength are 0.1mmol/L between 1mol/L, are formic acid, acetic acid, trifluoroacetic acid, boron
Acid, hydrochloric acid, sulfuric acid, phosphoric acid, sodium chloride, potassium chloride, ammonium chloride, sodium sulphate, potassium sulfate, ammonium sulfate, niter cake, hydrogen sulfate
Potassium, ammonium hydrogen sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, lithium hydroxide, sodium hydroxide, hydrogen
One or more of potassium oxide, triethylamine, diisopropyl ethyl amine, trishydroxymethylaminomethane, preferably sodium dihydrogen phosphate, phosphorus
Acid dihydride potassium, ionic strength are preferably 0.01mol/L to 0.1mol/L.
The concentration of hydroxyl radical carthamin yellow carthamus A in eluent is analyzed with purity using HPLC, and analysis method is use
Reverse-phase chromatography is analyzed, chromatographic column filler C18, mobile phase：Methanol, 0.18% acetic acid aqueous solution, using isocratic elution
Mode, 20% methanol and 80%0.18% acetic acid aqueous solution, Detection wavelength 403nm, sample size：10 microlitres, flow velocity：1.0mL/
Min, 25 degrees Celsius of column temperature.
For column chromatography method, method described in the invention concentrates on, and polysaccharide-based medium is filled to column chromatography column tube
In, column tube a diameter of 0.5mm to 500mm, preferably 10mm to 50mm.Medium is after filling is compacted, in order to ensure effectively to be enriched with
Purification effect, medium filling height should ensure that in more than 30mm.It will contain hydroxyl by the way of the automatic loading of manual or chromatograph
The settled solution of base carthamus tinctorius yellow colour A is added in chromatographic column.The selection of eluent and non-column chromatography phase described before
Together.
The concentration of hydroxyl radical carthamin yellow carthamus A in eluent is analyzed with purity using HPLC, analysis method with before
Described process is identical.
It compares through the sample before and after polysaccharide-based medium enriching and purifying, concentration is compared using the HPLC results analyzed,
Enrichment ratio is obtained, and the purity after enriching and purifying is calculated using the HPLC spectrograms after enriching and purifying.
Its concentration of hydroxyl radical carthamin yellow carthamus A requirement obtained through enrichment and purification method described in the present invention is original dense
2.0 times to 20.0 times of degree, purity are 25.00% to 99.99%.The source of hydroxyl radical carthamin yellow carthamus A is natural plants through carrying
The extracting solution that takes that treated or the product being chemically synthesized, molecular structural formula are：
Embodiment 1
Polysaccharide-based medium selects the agarose microbeads of sulfonic group modification.The agarose in 20% ethanol water will be stored in
Microballoon is dispersed in water again, takes 1mL agarose microbeads.Configure the ethanol solution containing hydroxyl radical carthamin yellow carthamus A sample, concentration
For 0.1mg/mL, 5mL is taken to be used as and treats enriching and purifying solution.It will treat that enriching and purifying solvent is mixed with agarose microbeads, by shaking,
Ultrasound is sufficiently mixed the two, rear to stand.After agarose microbeads completely precipitation, supernatant liquor is removed.To agarose microbeads
Middle addition 5mL acetonitriles, stand after concussion cleaning, remove the acetonitrile of cleaning.Agarose microbeads are transferred in chromatographic column, are stood
Compacting.2mL water is added in as eluent, is eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 2
Polysaccharide-based medium selects the dextran microspheres without chemical modification.Dextran microspheres powder is fully molten in water
It is swollen, take 1mL dextran microspheres.The ethanol solution containing hydroxyl radical carthamin yellow carthamus A sample is configured, concentration 0.1mg/mL takes 5mL
As treating enriching and purifying solution.It will treat that enriching and purifying solvent is mixed with dextran microspheres, and by concussion, ultrasound, make the two fully
Mixing, it is rear to stand.After dextran microspheres completely precipitation, supernatant liquor is removed.5mL acetonitriles are added in into dextran microspheres,
It is stood after concussion cleaning, removes the acetonitrile of cleaning.Dextran microspheres are transferred in chromatographic column, stand compacting.Add in 2mL water
As eluent, eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 3
Polysaccharide-based medium selects the agarose microbeads of sulfonic group modification.The agarose in 20% ethanol water will be stored in
Microballoon is dispersed in water again, takes 20mL agarose microbeads.Agarose microbeads are seated in AxiChrom 16* using column filler
In 100mm chromatographic columns, chromatographic column is mounted on AKTA purifier protein purification systems after filling.Configuration contains hydroxyl
The ethanol solution of base carthamus tinctorius yellow colour A sample, concentration 0.1mg/mL take 50mL to be used as and treat enriching and purifying solution.Utilize AKTA
The automatic loading systems of purifier carry out loading.It will treat that enriching and purifying solution is added to chromatographic column with the loading speed of 2mL/min
In.After end of the sample, with the flow velocity of 2mL/min, add in 30% acetonitrile solutions of 20mL and eluted.After elution, with
The flow velocity of 2mL/min adds in 10mL water, is eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 4
The analysis method of hydroxyl radical carthamin yellow carthamus A in eluent.It is analyzed using reverse-phase chromatography, chromatograph is
Agilent 1260, chromatographic column be Agilent HC-C18, mobile phase：Methanol, 0.18% acetic acid aqueous solution, using isocratic elution
Mode, 20% methanol and 80%0.18% acetic acid aqueous solution, Detection wavelength 403nm, sample size：10 microlitres, flow velocity：
1.0mL/min, 25 degrees Celsius of column temperature.
The preferred embodiment of the present invention is above are only, does not play the role of any restrictions to the present invention.Belonging to any
Those skilled in the art, in the range of technical scheme is not departed from, to the invention discloses technical solution and
Technology contents make the variations such as any type of equivalent substitution or modification, belong to the content without departing from technical scheme, still
Within belonging to the scope of protection of the present invention.
Claims (8)
1. it is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying, it is characterised in that：Utilize polysaccharide-based
Medium is effectively adsorbed and eluted for hydroxyl radical carthamin yellow carthamus A scattered in a liquid state.
2. enrichment and purification method according to claim 1, which is characterized in that hydroxyl safflower yellow in a manner of non-column chromatography
Plain A enriching and purifyings, including：
Polysaccharide-based medium is sufficiently mixed with being dispersed with the liquid of hydroxyl radical carthamin yellow carthamus A；
It is static after polysaccharide-based medium precipitation after, remove supernatant liquor；
The polysaccharide-based medium is cleaned using cleaning solution, cleaning dosage and the polysaccharide-based medium volume ratio of cleaning solution are 0.1:1~
100:1；
By the polysaccharide-based medium after cleaning in eluent soaking flushing, collect eluent.
3. enrichment and purification method according to claim 1, which is characterized in that Sydroxy carthamin in a manner of column chromatography
A enriching and purifyings, including：
By polysaccharide-based Filled Dielectrics in the column tube of a diameter of 0.5mm to 500mm, it is compacted through filling；
Added in into column tube be dispersed with hydroxyl radical carthamin yellow carthamus A treat enriching and purifying solution；
Add in mobile phase, flow rate of mobile phase be 0.1mL/min to 100.0mL/min, UV detector Detection wavelength for 350nm extremely
450nm collects elution solution.
4. the enrichment and purification method according to Claims 2 or 3, which is characterized in that cleaning solution, eluent or mobile phase are
Water, methanol, ethyl alcohol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, ether, acetone, methyl second
One or both of base ketone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide (DMSO) any of the above combines.
5. the enrichment and purification method according to Claims 2 or 3, which is characterized in that into one during soaking flushing or in mobile phase
Step adds in ionic species, and ionic strength is 0.1mmol/L between 1mol/L.
6. enrichment and purification method according to claim 5, which is characterized in that the ionic species are formic acid, acetic acid, trifluoro
Acetic acid, boric acid, hydrochloric acid, sulfuric acid, phosphoric acid, sodium chloride, potassium chloride, ammonium chloride, sodium sulphate, potassium sulfate, ammonium sulfate, niter cake,
Potassium acid sulfate, ammonium hydrogen sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, lithium hydroxide, hydroxide
One or both of sodium, potassium hydroxide, triethylamine, diisopropyl ethyl amine, trishydroxymethylaminomethane any of the above combines.
7. enrichment and purification method according to claim 1, which is characterized in that the main component of polysaccharide-based medium is fiber
One or both of element, glucan, agarose, starch any of the above combines, and main component is shared by total media dry powder
Ratio is 20%-100%；When main component is glucan, not including hydroxypropyl glucan medium；The pattern of polysaccharide-based medium is
Spherical, rodlike, line style, netted, in irregular shape one kind, the characteristic size of medium is 20 nanometers to 500 microns.
8. enrichment and purification method according to claim 1, which is characterized in that the polysaccharide of the polysaccharide-based medium is by spreading out
Biochemical modification, the molecular structure of the polyose modification part by derivatization modification is-X-Cn-R types, and wherein X is oxygen atom, nitrogen is former
One kind in son, one end of X atoms are connected with polysaccharide structures, R for hydroxyl, amino, carboxyl, sulfonic group, one kind in methyl or
Two or more any combination, Cn are the carbon structure between connection X and R, and n is the number of the carbon atom in carbon structure, and n is 0 to 20,
The bonded mode of carbon atom is straight line or non-directional branched structure in carbon structure.
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| CN201711387948.7A CN108084131A (en) | 2017-12-20 | 2017-12-20 | It is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying |
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| CN201711387948.7A CN108084131A (en) | 2017-12-20 | 2017-12-20 | It is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying |
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| CN111359262A (en) * | 2018-12-25 | 2020-07-03 | 泰州医药城国科化物生物医药科技有限公司 | Method for adsorbing curcumin by adopting anion ion exchange agarose filler |
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| CN101195647A (en) * | 2006-12-06 | 2008-06-11 | 杨炜 | Hydroxyl carthamus tinctorius yellow colour A, preparation method and application thereof |
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| CN101195647A (en) * | 2006-12-06 | 2008-06-11 | 杨炜 | Hydroxyl carthamus tinctorius yellow colour A, preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111359262A (en) * | 2018-12-25 | 2020-07-03 | 泰州医药城国科化物生物医药科技有限公司 | Method for adsorbing curcumin by adopting anion ion exchange agarose filler |
| CN111359262B (en) * | 2018-12-25 | 2022-04-29 | 泰州医药城国科化物生物医药科技有限公司 | Method for adsorbing curcumin by adopting anion ion exchange agarose filler |
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