CN104478878B - A kind of method that separation from green peel of walnut prepares alkaloid - Google Patents
A kind of method that separation from green peel of walnut prepares alkaloid Download PDFInfo
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- CN104478878B CN104478878B CN201410650315.0A CN201410650315A CN104478878B CN 104478878 B CN104478878 B CN 104478878B CN 201410650315 A CN201410650315 A CN 201410650315A CN 104478878 B CN104478878 B CN 104478878B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
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- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/22—Bridged ring systems
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- C07—ORGANIC CHEMISTRY
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
- C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
- C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
- C07D451/06—Oxygen atoms
- C07D451/10—Oxygen atoms acylated by aliphatic or araliphatic carboxylic acids, e.g. atropine, scopolamine
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- 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
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Abstract
The present invention relates to a kind of method that separation from green peel of walnut prepares alkaloid, with green peel of walnut as raw material, through ultrasonic microwave abstraction instrument aid in treatment after, alkaloid is separated and prepared using multitple extraction and HSCCC, wherein, use methyl alcohol：Water ratio makees extractant for 4: 1 mixed liquor, and liquid ratio is 10: 1, through ultrasonic microwave abstraction instrument aid in treatment 15min after, using obtaining chloroform methanol water alkaloids extract after multitple extraction；Use chloroform methanol.HSCCC methods do not need solid phase carrier, can eliminate the loss that the Irreversible Adsorption due to sample on solid phase carrier is caused with degraded, high with the rate of recovery；Can multiple sample introduction, its separation process all keeps very stable, favorable reproducibility；Gradient elution and anti-phase wash-out are capable of achieving, can also carry out repeating sample introduction, separative efficiency is high, and fractional dose is larger.
Description
Technical field
Alkaloid is prepared the present invention relates to Alkaloid separation preparation field, more particularly to a kind of separation from green peel of walnut
Method.
Background technology
Walnut (Juglans regia L) rascal, also known as pericarpium juglandis, is the walnut outside immature green of one thick layer
Pericarp.Wherein contain alkaloid, naphthoquinones class, flavonoids, diaryl heptane class, polysaccharide, small molecule terpene etc..Alkaloid is one
The important nitrogenous organic matter from living nature (based on plant) of class, is primarily present in each organ and group in plant
In knitting.Equal congener may contain the alkaloid of identical structure type, may be higher in certain organ content of plant, phase
To other tissues of walnut.In walnut is respectively organized, alkaloid is organized apparently higher than other in fresh rascal.
Alkaloid is little in plant, but huge number, and structure and nature difference are very big.What alkaloid was usually taken
Extraction separation method is usually to be determined according to its solubility.Except quaternary ammonium base and number molecular weight is relatively low or polar functionalities compared with
Outside many alkaloids, alkaloid is generally insoluble or is insoluble in water, but can be dissolved in the organic solvents such as chloroform, methyl alcohol, ethanol greatly.
Based on this characteristic, generally extracted using non-polar solven, polar solvent and mixed solvent.The extraction of common alkaloid
Method has cold soaking, diacolation, ultrasonic wave, surname extraction, circumfluence distillation etc., but has that recovery rate is low, separates not exclusively and purity
Relatively low problem.
In view of drawbacks described above, creator of the present invention passes through research and practice obtains this creation finally.
The content of the invention
It is an object of the invention to provide a kind of method that separation from green peel of walnut prepares alkaloid, it is used to overcome above-mentioned
Technological deficiency.
To achieve the above object, the present invention provides a kind of method that separation from green peel of walnut prepares alkaloid, with walnut
Rascal is raw material, through ultrasonic microwave abstraction instrument aid in treatment after, alkaloid separate using multitple extraction and HSCCC and
Prepare, wherein, with methyl alcohol: water ratio makees extractant for 4: 1 mixed liquor, and liquid ratio is 10: 1, through ultrasonic microwave abstraction instrument
After aid in treatment 15min, using acquisition chloroform methanol water alkaloids extract after multitple extraction；Use chloroform methanol.
Further, the detailed process is：
Step a, with english walnut exocarp as raw material, adds 10 times of methyl alcohol of volume: water is 4: 1 mixed liquor, micro- in ultrasonic wave
Ripple auxiliary extraction instrument is processed in three stages, and ultrasonic frequency is 50KHz, pattern 15: 10, motor speed 900rmin-1；
Centrifuging and taking supernatant, rotary evaporator is evaporated to the 1/10 of original volume, uses 2molL-1H2SO4It is 2 to be acidified to PH
~3, chloroform is extracted 3 times, and it is 10 or so that upper strata aqueous acid layer ammoniacal liquor is alkalized to PH, with chloroform: methanol ratio is 3: 1
Mixed liquor is extracted 2 times, then is extracted 1 time with chloroform, takes chloroform: the extract solution of methyl alcohol；
Rotary evaporation, leaves and takes and is evaporated with crucible a little, is dissolved with methyl alcohol after calculating recovery rate；
Step b, thin-layer chromatography isolating alkaloids；Weighing sodium carboxymethylcellulose adds appropriate pure water to dissolve, plus thin layer
Analysis silica gel, adds microcrystalline cellulose again after mixing, ultrasonic wave cleaning bubble removing, chart board film-making is standby；
With chloroform: methanol ratio is that 16: 1 mixed liquor makees solvent；
Step c, HSCCC separation prepares alkaloid, is compared with methyl alcohol, to green peel of walnut alkaloid sample and 6 kinds of standards
Alkaloid；Detected with UV, visible light luminosity meter at 190-400nm, observed absorption peak, and derive crest data；
Petroleum ether: chloroform: methyl alcohol: 2molL-1H2SO4It 2: 4: 2: 2V/V is dicyandiamide solution that ratio is, in proportion by 4 kinds
Solvent is added in separatory funnel, and concussion is sufficiently mixed solution, stands overnight at room temperature, using upper and lower phase is preceding got, is surpassed
Sound deaerates 30min with standby；
It is lower with 20mLmin after by ultrasound degassing-1Speed pump into HSCCC separating pipes, treat the lower phase full of whole
Individual pipeline, setting separating pipe turns clockwise, slow regulation main frame revolution to 800rmin-1, while with 2mLmin-1Flow velocity
Phase in injection, two-phase enters sample solution up to after balancing by sampling valve, while gathering corresponding data, is detected by ultraviolet detection
Effluent, corresponding composition is collected according to chromatographic peak.
Compared with prior art the beneficial effects of the present invention are：HSCCC technologies employ the molten of different physicochemical characteristics
Agent system and multifarious operating condition, can be to be extracted not from complicated natural products semifinished product with stronger adaptability
The active ingredient of same characteristic (such as opposed polarity) provides advantage.
HSCCC methods do not need solid phase carrier, can eliminate because Irreversible Adsorption of the sample on solid phase carrier and degraded are made
Into loss, it is high with the rate of recovery；Can multiple sample introduction, its separation process all keeps very stable, favorable reproducibility；Achievable gradient is washed
De- and anti-phase wash-out, can also carry out repeating sample introduction, and separative efficiency is high, and fractional dose is larger.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of separation of the invention and preparation method；
Fig. 2 a are matrine uv scan figure of the invention；
Fig. 2 b are fourth bromic acid east Liang alkali uv scan figures of the invention；
Fig. 2 c are aconitine uv scan figure of the invention；
Fig. 2 d are jamaicin uv scan figure of the invention；
Fig. 2 e are catechin uv scan figure of the invention；
Fig. 2 f are gallic acid uv scan figure of the invention；
Fig. 3 is alkaloids extract uv scan figure of the invention；
Fig. 4 is the separating resulting figure of alkaloid sample of the invention.
Specific embodiment
Below in conjunction with accompanying drawing, the technical characteristic above-mentioned and other to the present invention and advantage are described in more detail.
The present invention with green peel of walnut as raw material, through ultrasonic microwave abstraction instrument aid in treatment after, using multitple extraction and
HSCCC is separated and prepared to alkaloid.Result shows：Make extractant with methanol-water (4: 1), liquid ratio is 10: 1, through super
After sound wave Microwave Extraction Apparatus aid in treatment 15min, using acquisition chloroform methanol water alkaloids extract after multitple extraction；Use chloroform
Methyl alcohol (16: 1) makees solvent, and more clearly multiple monomer images are obtained through TLC；HSCCC (dicyandiamide solutions：N-hexane, chlorine
Imitative, methyl alcohol, 2molL-1H2SO4, volume ratio is 2: 4: 2: each monomer is collected at 313nm 2).Conclusion：With reference to
Ultraviolet spectrum characteristic, each monomer that will be collected carries out TLC images and compares with various alkaloid standard items, preliminary to confirm wherein 4
Individual monomer is respectively gallic acid, jamaicin, matrine and catechin.
Refer to shown in Fig. 1, the process of the method that the separation from green peel of walnut of the invention prepares alkaloid is：
Step a, with english walnut exocarp as raw material, adds 10 times of methyl alcohol of volume: water is 4: 1 mixed liquor, micro- in ultrasonic wave
Ripple auxiliary extraction instrument is processed in three stages, and ultrasonic frequency is 50KHz, pattern 15: 10, motor speed 900rmin-1。
Centrifuging and taking supernatant, rotary evaporator is evaporated to the 1/10 of original volume, uses 2molL-1H2SO4It is 2 to be acidified to PH
~3, chloroform is extracted 3 times, and it is 10 or so that upper strata aqueous acid layer ammoniacal liquor is alkalized to PH, with chloroform: methanol ratio is 3: 1
Mixed liquor is extracted 2 times, then is extracted 1 time with chloroform, takes chloroform: the extract solution of methyl alcohol.
Rotary evaporation, leaves and takes and is evaporated with crucible a little, is dissolved with methyl alcohol after calculating recovery rate.
Step b, thin-layer chromatography isolating alkaloids；Weighing sodium carboxymethylcellulose adds appropriate pure water to dissolve, plus thin layer
Analysis silica gel, adds microcrystalline cellulose again after mixing, ultrasonic wave cleaning bubble removing, chart board film-making is standby.
With chloroform: methanol ratio is that 16: 1 mixed liquor makees solvent.
Step c, HSCCC separation prepares alkaloid, is compared with methyl alcohol, to green peel of walnut alkaloid sample and 6 kinds of standards
Alkaloid (matrine, fourth bromic acid east Liang alkali, aconitine, jamaicin, catechin, gallic acid) is at 190-400nm with ultraviolet
It can be seen that photometer is detected, absorption peak is observed, and derive crest data.
Petroleum ether: chloroform: methyl alcohol: 2molL-1H2SO4(2: 4: 2: 2V/V) it is dicyandiamide solution, in proportion by 4 kinds of solvents
Add in separatory funnel, concussion is sufficiently mixed solution, stands overnight at room temperature, using preceding getting upper phase (fixing phase) and lower phase
(mobile phase), 30min is with standby for ultrasound degassing.
By the lower phase (mobile phase) after ultrasound degassing with 20mLmin-1Speed pump into HSCCC separating pipes, treat down
Whole pipeline is mutually full of, setting separating pipe turns clockwise, slow regulation main frame revolution to 800rmin-1, while with 2mL
min-1The upper phase of flow velocity injection, two-phase enters sample solution (20mL being less than, without solid particle) up to after balancing by sampling valve, while
Corresponding data are gathered, effluent is detected by ultraviolet detection (254nm), corresponding composition is collected according to chromatographic peak.
Illustrated below by embodiment.
Step a, coarse extraction and the identification of alkaloid；
3 parts of 20g Cortex walnuts powder is weighed, 10 times of methyl alcohol of volume: water mixed liquid (4: 1) are added, in ultrasonic microwave auxiliary
Abstraction instrument is processed in three stages, per stage 5min, common 15min.Under conditions of ultrasonic wave is constant, it is 45 to set temperature
DEG C, 50 DEG C, 55 DEG C, ultrasonic power is 800,850,900W, microwave power is 150,200,250W, ultrasonic frequency is
50KHz, pattern 15: 10, motor speed 900rmin-1.Centrifuging and taking supernatant, rotary evaporator is evaporated to the 1/10 of original volume.
Use 2molL-1H2SO4It is 2~3 to be acidified to PH, and chloroform is extracted 3 times, and it is 10 that upper strata aqueous acid layer ammoniacal liquor is alkalized to PH
Left and right, with chloroform: methyl alcohol (3: 1) is extracted 2 times, then is extracted 1 time with chloroform, takes chloroform: the extract solution of methyl alcohol.Rotary evaporation, stays
Take and be evaporated with crucible a little, dissolved with methyl alcohol after calculating recovery rate.
Step b, thin-layer chromatography isolating alkaloids；
Weighing sodium carboxymethylcellulose 60g adds appropriate pure water to dissolve, plus tlc silica gel 20g, again plus micro- after mixing
Crystalline cellulose 2.5g, ultrasonic wave cleaning bubble removing, chart board film-making is standby.
The different solvent of different proportion, chloroform: methyl alcohol (15: 1 are prepared respectively；15.5∶1；16∶1∶；16.5∶1)；Chlorine
It is imitative: ethanol (15: 1；15.5∶1；16∶1∶；16.5∶1)；Hexamethylene: ethyl acetate: normal propyl alcohol: dimethyl amine (30: 25: 0.9:
0.1), launch through thin-layer chromatography, ultra-violet analysis, it is determined that optimal solvent.And will self-control silica gel plate and standard silica gel plate and difference
Solvent acquired results are contrasted, and determine used silica gel plate.
Photographic analysis is carried out to lamellae under wavelength 254nm, optimal expansion lamellae is preserved after contrast.
Step c, HSCCC separation prepares alkaloid；
Compared with methyl alcohol, to green peel of walnut alkaloid sample and 6 kinds of standard biological alkali (matrine, fourth bromic acid east Liang alkali,
Aconitine, jamaicin, catechin, gallic acid) detected with UV, visible light luminosity meter at 190-400nm, observation absorbs
Peak value, and derive crest data.
Select petroleum ether: chloroform: methyl alcohol: 2molL-1H2SO4(2: 4: 2: 2V/V) it is dicyandiamide solution, it is molten by 4 kinds in proportion
Agent add separatory funnel in, concussion be sufficiently mixed solution, stand overnight at room temperature, using preceding get upper phase (fixing phase) and under
Phase (mobile phase), 30min is with standby for ultrasound degassing.By the lower phase (mobile phase) after ultrasound degassing with 20mLmin-1Speed
In pumping into HSCCC separating pipes, treat lower phase full of whole pipeline, setting separating pipe turns clockwise, slow regulation main frame revolution to
800r·min-1, while with 2mLmin-1The upper phase of flow velocity injection, two-phase (is less than by sampling valve up to after balancing into sample solution
20mL, without solid particle), while gathering corresponding data, effluent is detected by ultraviolet detection (254nm), according to chromatogram
Collect corresponding composition in peak.
Interpretation of result：
1st, the identification of middle Johnson ＆ Johnson's alkaloids crystal；The precipitation reaction of alkaloid is in acid condition using most of alkaloids
Under, with some precipitating reagents reaction the generation insoluble double salt of weak acid or complex compound sediment.Alkaloid precipitation reaction is general in diluted acid water
Carried out in solution.This is that alkaloid precipitation reagent is also soluble in water because alkaloid is soluble in water into salt with acid, and in sour water
Relatively stablize, and product is insoluble in water, thus be conducive to the observation for carrying out result of reaction.
Commonly used in prerun, extracting and developing and Structural Identification in alkaloid be alkaloid precipitation reaction and colour developing it is anti-
Should.
Take the thick product that get sample containing middle Johnson ＆ Johnson's alkaloids a little, Dragendorff's reagent observation is added after acidification, there is brown color to sink
Form sediment and occur, be shown to be alkaloid.
2nd, the thin-layer chromatography of alkaloid；
The uv scan result of alkaloid standard items, refers to shown in Fig. 2 a-2f, and it is six kinds of alkaloid standard items
Uv scan figure, table 1 is the absorption crests of six kinds of alkaloids.
The absorption crest of 1 six kinds of alkaloids of table
| Alkaloid | Matrine | Fourth bromic acid east Liang alkali | Aconitine | Jamaicin | Catechin | Gallic acid |
| Absorbing wavelength (nm) | 214 | 208 | 230 | 349 | 280 | 316 |
Alkaloids extract uv scan result, Fig. 3 is alkaloids extract uv scan figure, biological alkali carries
Liquid is taken compared with alkaloid standard items uv scan result to rear discovery, may be contained in green peel of walnut alkaloids extract
Aconitine, gallic acid, catechin and matrine.
Compared through TLC methods, it was demonstrated that contain aconitine, gallic acid, catechin and hardship in green peel of walnut alkaloids extract
Ginseng alkali, its spot colors, Rf values are also similar.
3rd, HSCCC is separated and prepared
The selection of HSCCC dicyandiamide solutions
With n-hexane, ethyl acetate, chloroform, methyl alcohol, 2molL-1H2SO4(volume ratio is 2: 5: 2: is 3: 4) solvent body
System, upper phase is mobile phase, and lower phase is fixing phase, just reversed turn, must descend phase retention rate less than 40% under 280nm, also proves the body
System is not suitable for isolating alkaloids sample.
With n-hexane, chloroform, methyl alcohol, 2mo1L-1H2SO4(volume ratio is 2: 4: 2: is 2) dicyandiamide solution, upper phase is stream
Dynamic phase, lower phase is fixing phase, just reversed turn, phase retention rate must be descended to be more than 40% under 313nm, it was demonstrated that the system is adapted to alkaloid sample
The separation of product.
With n-hexane: chloroform: methyl alcohol: 2molL-1H2SO4 constitutes dicyandiamide solution, mixes according to various ratios, and volume is
10mL, after being fully layered after dissolving in separatory funnel, take up and down mutually respectively point plate (silica gel plate, solvent is：Chloroform: methyl alcohol
=16: 1), spot size is approximately equivalent in two-phase is defined, select n-hexane: chloroform: methyl alcohol: 2molL-1H2SO4=2: 4:
Be optimum solvent system at 2: 2.
The determination of HSCCC dicyandiamide solutions phase up and down
Take n-hexane: chloroform: methyl alcohol: 2molL-1H2SO4, dicyandiamide solution is constituted according to 2: 4: 2: 2 mixing, cumulative volume is
1000mL.It is layered after fully being dissolved in separatory funnel, upper phase is methyl alcohol, aqueous acid, for dissolving and extracting alkaloid；Under
It is mutually chloroform, hexane solution, for isolating alkaloids.Upper phase is mainly fixing phase, and lower phase is mobile phase.
HSCCC method separation prepares alkaloid
Under 280nm and 313nm wavelength, green peel of walnut alkaloids extract is gathered respectively with HSCCC.
And carried out uv scan and TLC methods to collection liquid and compare to show to contain in green peel of walnut alkaloids extract
Aconitine, gallic acid, catechin and matrine.
Conclusion：
With green peel of walnut as raw material, through ultrasonic microwave abstraction instrument aid in treatment, Johnson ＆ Johnson in being obtained using multitple extraction method
Alkaloids CE.With chloroform: methyl alcohol (16: 1) is optimal solvent, detected by TLC, obtain more clear alkaloid figure
Image patch point.
Alkaloid monomer that TLC methods are detected and gallic acid, aconitine, fourth bromic acid east Liang alkali, jamaicin, kuh-seng
The alkaloid such as alkali, catechin standard items carry out uv scan respectively.Compare and find：The rhizome of Chinese monkshood may be contained in green peel of walnut
4 kinds of alkaloids such as alkali, gallic acid, catechin and matrine, its spot colors, Rf values are also similar..
With n-hexane, chloroform, methyl alcohol, 2molL-1H2SO4(volume ratio is 2: 4: 2: is 2) dicyandiamide solution, upper phase is solid
Determine phase, lower phase is mobile phase, and sample liquid is have collected respectively under 280nm and 313nm wavelength with HSCCC.And purple has been carried out to sample liquid
External spectrum is scanned and the detection of TLC methods, is shown in green peel of walnut alkaloids extract containing aconitine, gallic acid, catechin and
Matrine.
The extraction of TA typically uses extract by solvents, but aqueous acid extracts alkaloid and there is extract solution reclaimed water
Solubility impurity is more, the problem for being difficult to remove；There is also extracting liquid volume larger, concentrate relative difficulty.This article employs organic
Solvent multitple extraction method, using the method that will first be alkalized after acidifying in extraction process, can make alkaloid be in free state, then
Alkaline aqueous solution is extracted with organic solvent again, TA can be obtained by reclaiming organic solvent.It separates foundation；Middle strong basicity
Alkaloid is in free in pH10 or so.Furthermore most alkaloid dissolves in big organic molten of methyl alcohol, ethanol, n-butanol isopolarity
Agent, indissoluble or the organic solvent insoluble in the low polarity such as ethyl acetate, ether, chloroform.
In the detection of TLC methods, select preferable solvent particularly important.Most suitable solvent is obtained for chlorine through test of many times
It is imitative: methyl alcohol=16: 1.Also supported for the foundation of HSCCC method solution systems provides science simultaneously.
HSCCC methods do not need solid phase carrier, can eliminate because Irreversible Adsorption of the sample on solid phase carrier and degraded are made
Into loss, it is high with the rate of recovery；Can multiple sample introduction, its separation process all keeps very stable, favorable reproducibility；Achievable gradient is washed
De- and anti-phase wash-out, can also carry out repeating sample introduction, and separative efficiency is high, and fractional dose is larger.
Claims (1)
1. a kind of method that separation from green peel of walnut prepares alkaloid, it is characterised in that with green peel of walnut as raw material, through ultrasound
After ripple Microwave Extraction Apparatus aid in treatment, alkaloid is separated and prepared using multitple extraction and HSCCC；
The detailed process is：
Step a, with english walnut exocarp as raw material, with methyl alcohol: water ratio makees extractant for 4: 1 mixed liquor, 10 times of volumes of addition
Extractant, is processed in three stages in ultrasonic microwave auxiliary extraction instrument, per stage 5min, common 15min, and ultrasonic frequency is
50KHz, pattern 15: 10, motor speed 900rmin-1；
Centrifuging and taking supernatant, rotary evaporator is evaporated to the 1/10 of original volume, uses 2molL-1H2SO4It is 2~3, chlorine to be acidified to pH
Imitative extraction 3 times, it is 10 or so that upper strata aqueous acid layer ammoniacal liquor is alkalized to pH, with chloroform: methanol ratio is 3: 1 mixed liquor
Extraction 2 times, then extracted 1 time with chloroform, take chloroform: the extract solution of methyl alcohol；
Rotary evaporation, leaves and takes and is evaporated with crucible a little, is dissolved with methyl alcohol after calculating recovery rate；
Step b, thin-layer chromatography isolating alkaloids；Weighing sodium carboxymethylcellulose adds appropriate pure water to dissolve, plus thin-layer chromatography silicon
Glue, adds microcrystalline cellulose again after mixing, ultrasonic wave cleaning bubble removing, chart board film-making is standby；
With chloroform: methanol ratio is that 16: 1 mixed liquor makees solvent；
Step c, HSCCC separation prepares alkaloid, is compared with methyl alcohol, to green peel of walnut alkaloid sample and 6 kinds of standard biologicals
Alkali；Detected with UV, visible light luminosity meter at 190-400nm, observed absorption peak, and derive crest data；
Petroleum ether: chloroform: methyl alcohol: 2molL-1H2SO4It 2: 4: 2: 2V/V is dicyandiamide solution that ratio is, in proportion adds 4 kinds of solvents
Enter in separatory funnel, concussion is sufficiently mixed solution, stands overnight at room temperature, using upper and lower phase is preceding got, ultrasound deaerates
30min is with standby；
It is lower with 20mLmin after by ultrasound degassing-1Speed pump into HSCCC separating pipes, treat the lower phase full of whole pipe
Road, setting separating pipe turns clockwise, slow regulation main frame revolution to 800rmin-1, while with 2mLmin-1Flow velocity injects
Upper phase, two-phase enters sample solution up to after balancing by sampling valve, while gathering corresponding data, is detected by ultraviolet detection and flowed out
Thing, corresponding composition is collected according to chromatographic peak.
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