CN108014517A - A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying - Google Patents
A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying Download PDFInfo
- Publication number
- CN108014517A CN108014517A CN201711387963.1A CN201711387963A CN108014517A CN 108014517 A CN108014517 A CN 108014517A CN 201711387963 A CN201711387963 A CN 201711387963A CN 108014517 A CN108014517 A CN 108014517A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- based medium
- enrichment
- purifying
- enriching
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to the enrichment and purification method of the Protoberberine Alkoloids 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 a kind of method using polysaccharide-based medium to Protoberberine Alkoloids 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 Protoberberine Alkoloids stablized has important application value and economic value, while to promoting standardization, the standardization of Chinese Medicine Industry, there is an extremely important effect.
Description
Technical field
The present invention relates to the technical field of Chinese medical extract, more particularly to it is a kind of Protoberberine Alkoloids is enriched with it is pure
Adsorption and de-adsorption of the lower polysaccharide-based medium of the method for change, predominantly solvent regulation and control for specific molecular structure alkaloids molecule
Process, and it is related to design and application for novel polysaccharide base dielectric structure.
Background technology
Protoberberine Alkoloids (proberberine) is a kind of important quaternary ammonium type biology base molecule, this Alkaloid
Molecule is extensive in distributed in nature, and huge number.Meanwhile the pharmacological action scope of this Alkaloid molecule is wide, in analgesia, resist
The field such as bacterium, antimalarial, hypoglycemic, antitumor, suffers from excellent pharmacological activity.Meanwhile in chemical constitution, this Alkaloid
Parent nucleus can be summarized as four six-membered ring structures that two isoquinolin rings are formed, and chemical property is relatively stable, it is easily prepared and
The synthesis of derivant structure.The presence of isoquinolin causes molecule to have certain hydrophobic property in molecule, while connection two is different
The N atoms of quinoline structure, exist so that this Alkaloid molecule has certain parent in this quasi-molecule in the form of quaternary ammonium salt
It is water-based.Feature in these structures and chemical property, result in this Alkaloid molecule in separation, the method for being enriched with, purifying
There is very outstanding feature.
Divided from structure, the Protoberberine Alkoloids contained in Chinese medicine or natural plants mainly includes jamaicin, original
Jamaicin, berberrubine, Palmatine, jateorrhizine, epiberberine, jateorrhizine etc., among these with the pharmacological activity of jamaicin
That studies is the most thorough.Research shows, it is with preferable antibacterial effect, and has a broad antifungal spectrum, in vitro to a variety of gram positive bacterias
Has bacteriostasis, infected by influenza, Amoeba, Leptospira, some dermatophytes also have certain inhibitory action.Together
When jamaicin be primarily present in Berberis and coptis plant, in the coptis, radix scutellariae, Cortex Phellodendri, radix berberidis etc. in medicinal plant
There is higher content distribution.
Generally, in the separation and concentration purge process of alkaloids molecule, chromatography media is mostly silica gel or macropore
Resin is adsorbed, but both suffer from stronger non-specific adsorption for alkaloids molecule, cause the target in subtractive process
Loss of the biological base molecule in subtractive process is very big, often causes the reduction of yield and low during component analysis contains
Measure the missing of component.These are all alkaloids molecules, and institute is not particularly in Protoberberine Alkoloids quasi-molecule subtractive process
Obtain the problem of not facing.Meanwhile the presence of substantial amounts of non-specific adsorption, it can also cause the chromatography efficiency of chromatography media to reduce.
On the other hand, although ion-exchange type or the mixed type polysaccharide-based medium containing ion exchange mode are widely developed,
And application and chromatographic purifying field, but due to the presence of quaternary ammonium structure in Protoberberine Alkoloids, if directly utilization from
Sub- switch mode adsorbs this quasi-molecule, and rear elution process will inevitably use the elution bar of high ionic strength
Part, the processing procedure for waste liquid in production technology will be very unfavorable, equally draw in the selection of chromatography media structure
Play special attention.
Polysaccharide-based medium is typically used among gel chromatography technique, it, 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, are prepared, and as media applications among technique is chromatographed.With fine jade
Exemplified by lipolysaccharide, it can form relatively stable polysaccharide-based skeleton 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, it contains substantial amounts of protein, polysaccharide and the very big chemical combination of some other nature difference
Thing molecule, the dielectric material with effectively enrichment, purification capacity is generally required in purge 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, it is former for effective enriching and purifying using design and application with certain hydrophobic polysaccharide-based medium
Berberine-type alkaloid is this kind of to have fine water miscible 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, it is former small to stablize
Bark of a cork tree alkaline alkaloid has important application value and economic value, promotes standardization, the standardization of Chinese Medicine Industry, has extremely
Important effect.
The content of the invention
The present invention be directed to foregoing problems, it is proposed that the effectively method of enriching and purifying Protoberberine Alkoloids, and by
Lower control of the polysaccharide-based medium for the adsorption and de-adsorption process of specific molecular structure alkaloids molecule of solvent regulation and control, utilizes
This enriching and purifying process of the polysaccharide-based media implementation of specific structure.
The present invention proposes a kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying, utilizes polysaccharide
Base medium, is effectively adsorbed and is eluted for Protoberberine Alkoloids scattered in a liquid state.
In the enrichment and purification method proposed by the present invention, Protoberberine Alkoloids is enriched with a manner of non-column chromatography pure
Change, including：
Polysaccharide-based medium is sufficiently mixed with being dispersed with the liquid of Protoberberine Alkoloids；
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, Protoberberine Alkoloids is enriched with a manner of column chromatography pure
Change, including：
By polysaccharide-based Filled Dielectrics in the column tube of a diameter of 0.5mm to 500mm, it is compacted through filling；
Added into column tube be dispersed with Protoberberine Alkoloids treat enriching and purifying solution；
Mobile phase is added, 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, ethanol, 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 thing is further added during soaking flushing or in mobile phase
Matter, its 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, bar-shaped,
One kind in line style, netted, irregular shape, 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 any of the above combination of one or both of hydroxyl, amino, methyl, and Cn is
Link the carbon structure between X and R, n is the number of the carbon atom in carbon structure, and n is 0 to 20, and carbon atom is bonded in carbon structure
Mode 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 original stablized
Berberine-type alkaloid has important application value and economic value, promotes standardization, the standardization of Chinese Medicine Industry, there is pole
Its important effect.
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
Deng macromolecular polysaccharide molecule, this quasi-molecule is free of electric charge in itself, and derives from a wealth of sources, and 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 necessarily in medium
Other composition components, if the mixing of two or more polysaccharide is (by taking agarose/glucan as an example), or add 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, processing and the control to pattern are more easy
In realization.Spherical, bar-shaped, 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, the feature of enrichment of the medium to Protoberberine Alkoloids and elution process to 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.Hydroxyl, amino and other groups are waited while medium is modified, some hydrophobising groups can be entered to modification knot
Among structure, be conducive to the effective adsorption and enrichment of Protoberberine Alkoloids in water or in water/alcohol mixture, therefore to polysaccharide
The structure of base medium proposes following require：
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 one kind in oxygen atom, nitrogen-atoms, one end of X atoms with it is more
Sugared structure is connected, and R is the one or more in hydroxyl, amino, methyl, and Cn is the carbon structure linked between X and R, and n is carbon structure
In carbon atom number, n is 0 to 20, and the bonded mode of carbon atom is straight line or non-directional branched structure in carbon structure.
Polysaccharide-based medium should pass through the abundant of solution and soak before the use, activated media, therefore when in use, medium
Weigh using volume as Dose standard, unit is mL or L.
Protoberberine Alkoloids described in the present invention, is related to what is extracted from Chinese medicine or other natural plants
Product, such as jamaicin, protoberberine, berberrubine, Palmatine, jateorrhizine, epiberberine, jateorrhizine, and it is different with two
Quinoline quaternary ammonium salt structure is parent nucleus, the other derivatives obtained by derivatization or chemical modification, but preferably jamaicin, former barberry
The natural origin such as alkali, berberrubine, Palmatine, jateorrhizine, epiberberine, jateorrhizine compound carries out enriching and purifying.
Protoberberine Alkoloids should carry out further enriching and purifying under liquid-phase condition, consider protoberberine type biology
Dissolubility of the alkali in pure water solution is poor, therefore when Protoberberine Alkoloids disperses, the selection of liquid phase can be pure water
Or the mixed liquor of water and other polar solvents.Other polar solvents can be methanol, ethanol, 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-
The mixed solution that one or more in sulfone, preferably water, ethanol or both are mixed with arbitrary proportion.
Under the process conditions of non-column chromatography, after Protoberberine Alkoloids in the liquid phase scattered, can with solution or
The form of suspension 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).
, should be with settled solution after Protoberberine Alkoloids in the liquid phase scattered under the process conditions of column chromatography
Form 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 Protoberberine Alkoloids according to aforementioned ratio, it is sufficiently mixed in certain proportion.Mixing
Protoberberine Alkoloids 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 silicic acid anhydride, therefore Protoberberine Alkoloids will be to during redistributing
Polysaccharide-based media fraction is enriched with.The liquid phase on upper strata is removed at this time, adds a small amount of cleaning solution and polysaccharide-based medium is cleaned.Cleaning
Liquid can be water, methanol, ethanol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, ether, third
One or more in ketone, methyl ethyl ketone, acetonitrile, n,N-Dimethylformamide, dimethyl sulfoxide (DMSO), but preferred water.Cleaned
Journey can remove the non-Protoberberine Alkoloids material that polysaccharide-based media fraction is dispersed in non-adsorbed effect.Afterwards, adopt
The Protoberberine Alkoloids in polysaccharide-based medium is eluted with certain hydrophobic fat-soluble solvent.Eluent can be with
For water, methanol, ethanol, normal propyl alcohol, isopropanol, n-butanol, isobutanol, the tert-butyl alcohol, ethylene glycol, glycerine, ether, acetone, methyl
One or more in ethyl ketone, acetonitrile, n,N-Dimethylformamide, dimethyl sulfoxide (DMSO), preferably methanol, ethanol, acetonitrile and with
The mixture of upper three kinds of solvents.Due to containing quaternary ammonium cations structure in Protoberberine Alkoloids, in elution process
The material of certain ionic strength can be added, ion exchange is offset and Protoberberine Alkoloids is adsorbed in polysaccharide-based medium
The influence of effect, improves elution efficiency.A certain amount of ion, ionic strength for 0.1mmol/L between 1mol/L, there is ion
The material of intensity is formic acid, acetic acid, trifluoroacetic acid, boric acid, hydrochloric acid, sulfuric acid, phosphoric acid, sodium chloride, potassium chloride, ammonium chloride, sulfuric acid
Sodium, 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, sodium hydroxide, potassium hydroxide, triethylamine, diisopropyl ethyl amine, trishydroxymethylaminomethane
In one or more, preferably sodium dihydrogen phosphate, potassium dihydrogen phosphate, ionic strength are preferably 0.01mol/L to 0.1mol/L.
The concentration of Protoberberine Alkoloids in eluent is analyzed with purity using HPLC, and analysis method is use
Reverse-phase chromatography is analyzed, chromatographic column filler C18, mobile phase：Acetonitrile, acetic acid/triethylamine aqueous solution that pH is 6.4, elution
Gradient：20% acetonitriles of 0-15min, 16-25min 21%-29% acetonitriles, 26-35min 30%-55% acetonitriles, 36-45min
56%-70% acetonitriles, 46-70min 71%-80% acetonitriles, Detection wavelength 282nm, sample size：10 microlitres, flow velocity：
1.0mL/min, 30 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.Original will be contained by the way of the automatic loading of manual or chromatograph
The settled solution of Berberine-type alkaloid is added in chromatographic column.The selection of eluent and non-column chromatography phase described before
Together.
The concentration of Protoberberine Alkoloids in eluent is analyzed with purity using HPLC, analysis method with before
Described process is identical.
Contrast through the sample before and after polysaccharide-based medium enriching and purifying, concentration contrasted 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 Protoberberine Alkoloids requirement obtained through enrichment and purification method described in the present invention is original
2.0 times to 20.0 times of concentration, purity are 25.00% to 99.99%.The source of Protoberberine Alkoloids passes through for natural plants
Extracting solution after extraction process or the product being chemically synthesized, are jamaicin, protoberberine, berberrubine, Palmatine, medicine root
Alkali, epiberberine, jateorrhizine and other satisfactions：
Wherein R2, R3, R9, R10 are respectively point of one or more of structures in hydrogen atom, hydroxyl, methoxyl group, ethyoxyl
One or more in son.
Embodiment 1：
Polysaccharide-based medium selects the agarose microbeads of hydroxypropyl modification.The agarose in 20% ethanol water will be stored in
Microballoon is dispersed in water again, takes 1mL agarose microbeads.Aqueous solution of the configuration containing jamaicin sample, concentration 0.1mg/mL,
Take 5mL to be used as and treat enriching and purifying solution.It will treat that enriching and purifying solvent is mixed with agarose microbeads, and by concussion, ultrasound, make both
It is sufficiently mixed, it is rear to stand.After agarose microbeads completely precipitation, supernatant liquor is removed.5mL is added into agarose microbeads
Water, stands after concussion cleaning, removes the water of cleaning.Agarose microbeads are transferred in chromatographic column, stand compacting.Add 2mL
Ethanol is eluted as eluent.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 aqueous solution containing jamaicin sample is configured, concentration 0.1mg/mL, takes 5mL to be used as and wait to be enriched with
Purification solution.It will treat that enriching and purifying solvent is mixed with dextran microspheres, and by concussion, ultrasound, be sufficiently mixed both, it is rear quiet
Put.After dextran microspheres completely precipitation, supernatant liquor is removed.5mL water is added into dextran microspheres, it is quiet after concussion cleaning
Put, remove the water of cleaning.Dextran microspheres are transferred in chromatographic column, stand compacting.2mL ethanol is added as eluent,
Eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 3：
Polysaccharide-based medium selects the agarose microbeads of hydroxyisobutyl modification.The fine jade in 20% ethanol water will be stored in
Lipolysaccharide microballoon is dispersed in water again, takes 1mL agarose microbeads.The aqueous solution containing protoberberine sample is configured, concentration is
0.1mg/mL, takes 5mL 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, surpassing
Sound, is sufficiently mixed both, rear to stand.After agarose microbeads completely precipitation, supernatant liquor is removed.Into agarose microbeads
5mL water is added, is stood after concussion cleaning, removes the water of cleaning.Agarose microbeads are transferred in chromatographic column, stand compacting.
2mL ethanol is added as eluent, is eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 4：
Polysaccharide-based medium selects the agarose microbeads of hydroxyisobutyl modification.The fine jade in 20% ethanol water will be stored in
Lipolysaccharide microballoon is dispersed in water again, takes 1mL agarose microbeads.The sample for treating enriching and purifying is, using jateorrhizine as raw material, passes through
The new derivatives of the proto-berberine parent nucleus reacted into ether.0.1mg/mL will be configured to containing the sample for needing enriching and purifying
Aqueous solution, take 5mL as treating enriching and purifying solution.It will treat that enriching and purifying solvent is mixed with agarose microbeads, by shaking, surpassing
Sound, is sufficiently mixed both, rear to stand.After agarose microbeads completely precipitation, supernatant liquor is removed.Into agarose microbeads
5mL water is added, is stood after concussion cleaning, removes the water of cleaning.Agarose microbeads are transferred in chromatographic column, stand compacting.
2mL ethanol is added as eluent, is eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 5：
Polysaccharide-based medium selects the agarose microbeads of hydroxypropyl 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 installed on AKTA purifier protein purification systems after filling.Configuration is containing small
The aqueous solution of bark of a cork tree alkali sample, concentration 0.1mg/mL, takes 50mL to be used as and treats enriching and purifying solution.Using AKTA purifier certainly
Dynamic loading system carries out loading.It will treat that enriching and purifying solution is added in chromatographic column with the loading speed of 2mL/min.End of the sample
Afterwards, with the flow velocity of 2mL/min, 20mL aqueous solutions is added and are eluted.After elution, with the flow velocity of 2mL/min, 10mL is added
Ethanol, is eluted.Eluent carries out the analysis of content and purity using HPLC.
Embodiment 6：The analysis method of hydroxyl radical carthamin yellow carthamus A in eluent.Analyzed using reverse-phase chromatography, chromatograph
For Agilent 1260, chromatographic column is Agilent HC-C18, mobile phase：Acetonitrile, acetic acid/triethylamine aqueous solution that pH is 6.4,
Gradient：20% acetonitriles of 0-15min, 16-25min 21%-29% acetonitriles, 26-35min 30%-55% acetonitriles, 36-
45min 56%-70% acetonitriles, 46-70min 71%-80% acetonitriles, Detection wavelength 282nm, sample size：10 microlitres, stream
Speed：1.0mL/min, 30 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 variation such as any type of equivalent substitution or modification, belong to the content without departing from technical scheme, still
Belong within protection scope of the present invention.
Claims (8)
- A kind of 1. method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying, it is characterised in that：Utilize polysaccharide Base medium, is effectively adsorbed and is eluted for Protoberberine Alkoloids scattered in a liquid state.
- 2. enrichment and purification method according to claim 1, it is characterised in that given birth in a manner of non-column chromatography to protoberberine type Alkaloids enriching and purifying, including：Polysaccharide-based medium is sufficiently mixed with being dispersed with the liquid of Protoberberine Alkoloids；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, it is characterised in that to protoberberine type biology in a manner of column chromatography Alkali enriching and purifying, including：By polysaccharide-based Filled Dielectrics in the column tube of a diameter of 0.5mm to 500mm, it is compacted through filling；Added into column tube be dispersed with Protoberberine Alkoloids treat enriching and purifying solution；Add mobile phase, flow rate of mobile phase be 0.1mL/min to 100.0mL/min, UV detector Detection wavelength be 350nm extremely 450nm, collects elution solution.
- 4. the enrichment and purification method according to Claims 2 or 3, it is characterised in that cleaning solution, eluent or mobile phase are Water, methanol, ethanol, 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, it is characterised in that into one during soaking flushing or in mobile phase Step adds ionic species, its ionic strength is 0.1mmol/L between 1mol/L.
- 6. enrichment and purification method according to claim 5, it is characterised 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, it is characterised 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 One kind in spherical, bar-shaped, line style, netted, irregular shape, the characteristic size of medium is 20 nanometers to 500 microns.
- 8. enrichment and purification method according to claim 1, it is characterised 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, and R is one or both of hydroxyl, amino, methyl any of the above Combination, Cn are the carbon structure linked between X and R, and n is the number of the carbon atom in carbon structure, and n is 0 to 20, and carbon is former in carbon structure The bonded mode of son is straight line or non-directional branched structure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711387963.1A CN108014517A (en) | 2017-12-20 | 2017-12-20 | A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711387963.1A CN108014517A (en) | 2017-12-20 | 2017-12-20 | A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108014517A true CN108014517A (en) | 2018-05-11 |
Family
ID=62074372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711387963.1A Pending CN108014517A (en) | 2017-12-20 | 2017-12-20 | A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108014517A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004337A (en) * | 2018-12-14 | 2020-04-14 | 泰州医药城国科化物生物医药科技有限公司 | Preparation method and application of fast carboxymethylated high-hydrophilicity agarose derivative |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651432A (en) * | 2004-02-02 | 2005-08-10 | 郭曙平 | Method of refining plamatine from herba fibranreae recisae total ulkuloid and its application |
| CN101129364A (en) * | 2007-08-29 | 2008-02-27 | 成都军区昆明总医院 | Use of protoberberine alkaloid in preparing against bacterial resistance medicament |
| CN101491596A (en) * | 2007-04-18 | 2009-07-29 | 北京和润创新医药科技发展有限公司 | Method for separating berberine total alkaloid from extract liquid of traditional Chinese medicine |
| CN102050843A (en) * | 2010-11-22 | 2011-05-11 | 四川中测科技投资有限公司 | Method for extracting alkaloid and catechin from tea |
| CN102600247A (en) * | 2012-04-11 | 2012-07-25 | 宁波德沃生物科技有限公司 | Corydalis saxicola bunting alkaloid extract and preparation method thereof, as well as extraction method of dehydrocavidine |
| CN104327066A (en) * | 2014-09-30 | 2015-02-04 | 华东理工大学 | Method for rapidly and efficiently extracting carboline alkaloids |
| CN105541856A (en) * | 2015-12-24 | 2016-05-04 | 利穗科技(苏州)有限公司 | Method for extracting high-purity sanguinarine and chelerythrine by using chromatographic technique |
| CN105541831A (en) * | 2016-01-15 | 2016-05-04 | 辽宁中医药大学 | Medicine root berberrubine derived from amur cork-tree processed product and separation preparation method and application thereof |
-
2017
- 2017-12-20 CN CN201711387963.1A patent/CN108014517A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651432A (en) * | 2004-02-02 | 2005-08-10 | 郭曙平 | Method of refining plamatine from herba fibranreae recisae total ulkuloid and its application |
| CN101491596A (en) * | 2007-04-18 | 2009-07-29 | 北京和润创新医药科技发展有限公司 | Method for separating berberine total alkaloid from extract liquid of traditional Chinese medicine |
| CN101129364A (en) * | 2007-08-29 | 2008-02-27 | 成都军区昆明总医院 | Use of protoberberine alkaloid in preparing against bacterial resistance medicament |
| CN102050843A (en) * | 2010-11-22 | 2011-05-11 | 四川中测科技投资有限公司 | Method for extracting alkaloid and catechin from tea |
| CN102600247A (en) * | 2012-04-11 | 2012-07-25 | 宁波德沃生物科技有限公司 | Corydalis saxicola bunting alkaloid extract and preparation method thereof, as well as extraction method of dehydrocavidine |
| CN104327066A (en) * | 2014-09-30 | 2015-02-04 | 华东理工大学 | Method for rapidly and efficiently extracting carboline alkaloids |
| CN105541856A (en) * | 2015-12-24 | 2016-05-04 | 利穗科技(苏州)有限公司 | Method for extracting high-purity sanguinarine and chelerythrine by using chromatographic technique |
| CN105541831A (en) * | 2016-01-15 | 2016-05-04 | 辽宁中医药大学 | Medicine root berberrubine derived from amur cork-tree processed product and separation preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 《化工百科全书》编辑委员会: "《化工百科全书 第14卷》", 31 July 1997, 化学工业出版社 * |
| 刘军红等: "生物碱提取、分离和纯化的研究进展", 《时珍国医国药》 * |
| 徐东翔: "《植物资源化学》", 30 September 2004, 湖南科学技术出版社 * |
| 蒋伟哲等: "制备色谱系统从岩黄连中分离岩黄连碱", 《中草药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004337A (en) * | 2018-12-14 | 2020-04-14 | 泰州医药城国科化物生物医药科技有限公司 | Preparation method and application of fast carboxymethylated high-hydrophilicity agarose derivative |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Simultaneous separation and purification of flavonoids and oleuropein from Olea europaea L.(olive) leaves using macroporous resin | |
| Zhang et al. | Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography | |
| Wang et al. | Zirconium metal-organic framework assisted miniaturized solid phase extraction of phenylurea herbicides in natural products by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry | |
| CN105175240B (en) | Novel tobacco sesquiterpene H with antiviral activity is prepared with supercritical fluid chromatography | |
| Bai et al. | Highly selective separation and purification of chicoric acid from Echinacea purpurea by quality control methods in macroporous adsorption resin column chromatography | |
| CN103058871B (en) | Separation and purification method of tobacco chlorogenic acid | |
| CN108014517A (en) | A kind of method using polysaccharide-based medium to Protoberberine Alkoloids enriching and purifying | |
| CN104974122B (en) | Coumarin compound originated from tobacco, and preparation method and application thereof | |
| CN101016357A (en) | Preparation of screening type adsorption resin and application thereof in separating panaxoside monomer Rb1 | |
| CN103613684A (en) | Separation and purification method for N-acetylated chitohexaoses with different acetylation degrees | |
| CN106040202B (en) | A kind of boronate benzoyl-β-cyclodextrin bonded silica gel and application thereof | |
| Li et al. | Separation of flavonoid and alkaloid using collagen fiber adsorbent | |
| Qiao et al. | Rigid crosslink improves the surface area and porosity of β-cyclodextrin beads for enhanced adsorption of flavonoids | |
| CN102329355B (en) | The preparation method of a kind of Myricitroside and pharmaceutical composition thereof | |
| CN103937013A (en) | Agar gel microspheres and preparation method thereof | |
| CN108084131A (en) | It is a kind of using polysaccharide-based medium to the method for hydroxyl radical carthamin yellow carthamus A enriching and purifying | |
| CN104497632B (en) | A kind of method utilizing the red wood carthamin that continues of macroporous resin-active carbon compound adsorbent purification | |
| CN107573362A (en) | A kind of method of the separating-purifying sirolimus from zymotic fluid | |
| CN106860631B (en) | A kind of method for efficiently separating purifying jujuboside | |
| CN109939131B (en) | Preparation method and application of high-purity ginkgo total flavonol glycosides | |
| CN101575277B (en) | Method for preparing high purity irisquinone, pallason A | |
| CN105601793B (en) | The preparation method and application of bluish dogbane flavones Purification Resin | |
| CN110938053B (en) | Extraction method of ginkgo element | |
| CN110066306A (en) | A kind of Isorhamnetin-3-O-neohespeidoside preparative liquid chromatography separation method | |
| CN102863485B (en) | Process for purifying arctiin crude product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180511 |
|
| RJ01 | Rejection of invention patent application after publication |