CN104610214B - Method for rapidly preparing six compounds in Stellera chamaejasme L. - Google Patents
Method for rapidly preparing six compounds in Stellera chamaejasme L. Download PDFInfo
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- CN104610214B CN104610214B CN201310539312.5A CN201310539312A CN104610214B CN 104610214 B CN104610214 B CN 104610214B CN 201310539312 A CN201310539312 A CN 201310539312A CN 104610214 B CN104610214 B CN 104610214B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- 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
- C07D311/42—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms in positions 2 and 4
- C07D311/44—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms in positions 2 and 4 with one hydrogen atom in position 3
- C07D311/54—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms in positions 2 and 4 with one hydrogen atom in position 3 substituted in the carbocyclic ring
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- 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
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- 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
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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Abstract
The invention relates to a new method for rapidly preparing six highly pure compounds (comprising isodaphnoretin, sikokianin B, sikokianin C, (+)-chameajasmenin B, isochameajasmenin B and isochameajasmenin A) in Stellera chamaejasme L.. The method adopts a 95% ethanol extract product of a Stellera chamaejasme L. medicinal material as a raw material, adopts normal phase silica gel column chromatography and reverse phase ODS column chromatography to carry out preliminary separation and enrichment, and adopts preparative high performance liquid chromatography as a final separation means, and the compounds with the purity of 98% or more can be obtained through one-step high performance liquid phase preparation, corresponding fraction collection and direct reduced pressure concentration. The steps of the method are simple, and the above products have high purity and good color, so the method is very suitable for large scale production.
Description
Technical field
The present invention relates to a kind of new technology quickly preparing 6 kinds of compounds in Stellera chamaejasme L..Mainly include positive and negative phase post layer
Analysis separation and concentration and quick high performance liquid chromatography remove impurity preparation.Compound 1（Isodaphnoretin）For dicoumarol, compound
2（Sikokianin B）、3（Sikokianin C）、4（The peaceful B of (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)）、5（The peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)）、6（The peaceful A of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)）For double flavanones.Its
In, compound 1 is to prepare first in Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) platymiscium, and compound 2,6 is the Novel isomeric of such pair of flavanone.This 6 kinds changes
The chemical constitution of compound is as follows respectively：
Background technology
Stellera chamaejasme L.（Stellerachamaejasme L.）It is thymelaeceae Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) platymiscium, popular name Gelsemium elegans Benth., it is distributed in
In, Russia, illiteracy, towards etc. state, mainly originate from northern each provinces and regions and southwest in China, be born in 2600～4200 meters of dryings of height above sea level and
The careless slope of high mountain on the sunny side, lawn or river shoal tableland.Its bitter is pungent, mild-natured, very toxic, enter lung spleen Liver Channel, root is used as medicine, and relieves oedema or abdominal distension through diuresis or purgation and dispels
The effect of expectorant, removing mass parasite killing.Modern pharmacology research shows that Stellera chamaejasme L. has good anti-tumor activity, and current research is also sent out
Existing its has the effect such as antiviral, antibacterial, convulsion and immunomodulating.At present, the chemistry that isolation identification goes out from Stellera chamaejasme L.
Composition mainly has Diterpeneses, sesquiterpenoidss, flavonoid, Coumarinses, lignanoid and Phenylpropanoid Glycosides glycoside etc., and wherein, flavonoid is many
For double flavanones, it is the index chemical composition of this medical material, is also one of its main active.Sikokianin C（Compound 3）
Plasmodium falciparum chloroquine drug resistance strain is had significantly kills except effect, its IC50Value is up to 0.56 μ g/ml（Nunome S,et
al.PLANTA MED,2004,70(1)）；（+）The peaceful B of-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)（Compound 4）The increasing of kinds of tumor cells can be suppressed in vitro
Grow, the IC to human A549 cell lines50Value is up to 1.08 μm of ol/L（Zhang C, et al.ACTA PHARMACOL SIN,
2013,34（2））；The peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)（Compound 5）Show stronger antifungal and resisting mitosis activity, the MIC to Pyricularia oryzae
It is worth for 6.25 μ g/mL（Yang G, et al.CHEM PHARM BULL, 2005,53（7））；Separation preparation to compound 2,6
Have not been reported with pharmacological research.In addition, as quality control index composition it is necessary to there be available chemical reference substance supply in a large number, but
Rarely has offer to the coumarin including this 6 kinds of compounds and double flavanone reference substance in the market.Therefore, to auspicious
In fragrant Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material this 6 kinds of compounds simultaneously, the research of high efficiency preparation method significant.
Content of the invention
The present invention is on the basis of early-stage Study Chemical Components in Stellera Chamaejasme L system separates and analyzes, there is provided a kind of quick system
6 kinds of compounds in standby Stellera chamaejasme L. medical material（Isodaphnoretin, sikokianin B, sikokianin C, (+) the peaceful B of-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) and
The peaceful A of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)）New technology.With Stellera chamaejasme L. medical material as raw material, tentatively divide through normal-phase silica gel column chromatography, anti-phase ODS column chromatography
From enrichment, with preparative high performance liquid chromatography for being finally recovered means, prepared by a step efficient liquid phase, collect and contain each compound
Stream part be directly evaporated to and dry can obtain the compound that purity is more than 98%.Comprise the following steps that：
（1）Normal-phase silica gel column chromatography：
Take Stellera chamaejasme L. medical material 95% ethanol extraction extractum, with the mixed liquor dissolving of one of chloroform, methanol or two kinds,
Under being stirred continuously, add 200～300 mesh column chromatography silica gels with quality such as extractum, mix sample, fully dry under room temperature；Afterwards
Dry method loading, normal pressure descended 200～300 mesh silicagel columns, successively with chloroform, chloroform-methanol 100：1st, chloroform-methanol 50：1st, chlorine
Imitation-carbinol 20：1 eluting, collects chloroform-methanol 20：1 stream part, after negative pressure concentration and recovery solvent, obtains one-level rough segmentation thing.
（2）Anti-phase ODS column chromatography：
After above-mentioned one-level rough segmentation thing is dissolved with methanol, the particle diameter with quality such as one-level rough segmentation things is added to be 40～50 μm
Sample mixed by ODS column chromatography filler, fully dries and process powdering under room temperature；Dry method loading afterwards, crosses 40～50 μm of ODS chromatographies
Post（Column length 40cm, internal diameter 8cm）, medium lift pump pressurizes, and pressure is 5～10MPa, flow-control in 50～100ml/min, successively with
15%th, 30%, 45%, 60%, 70% methanol-water eluting, collects 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtains two grades slightly
Divide thing.
（3）Prepared by high performance liquid chromatography：
Above-mentioned two grades of rough segmentation things are dissolved with DMSO, is configured to the need testing solution that concentration is 50～100mg/ml, warp
0.45 μm of filtering with microporous membrane；Filtrate is carried out point using the high performance liquid preparative chromatography post of column length 25-50cm, diameter 2-10cm
From each sampling volume is 0.5～2.5ml, with volumetric concentration 70%～80% methanol-water or 45%～55% acetonitrile-aqueous solution is
Eluent system, flow speed control in 10～100ml/min, online ultraviolet detection, Detection wavelength is set as 295nm, respectively collect contain
Have the stream part of compound 1,2,3,4,5,6, negative pressure drying obtains 6 kinds of powder, as purity be more than 98% compound 1,2,3,4,5,
6 sterlings.
In said method, the filler that efficient liquid phase prepares post is C18Bonded Phase, its particle diameter is 5～10 μm, ultraviolet detection ripple
Long preferred 295nm, depending on concrete Detection wavelength is according to the overload condition of target compound signal in sample.
The present invention prepares 6 kinds of compounds from Stellera chamaejasme L. material simultaneously（1 kind of dicoumarol, 5 kinds of double flavanones）, have
Following advantage and progress：
1. the present invention adopts three-step approach, first passes through positive and negative phase two step column chromatography and carries out preliminary concentration to target compound, so
Afterwards remove impurity is carried out respectively using a step efficient liquid phase preparation method and refine, technique is simpler.
2. the present invention is with preparative efficient liquid phase for the means that are finally separating, separation efficiency high it is ensured that reference substance pure
Degree.
3. the efficient liquid phase preparation method of present invention development is fast preparation method, and single needle disengaging time is in 40-60 minute
Between, it is especially suitable for the preparation of high-volume chemical reference substance；Add the recycling preparing solvent, it is possible to decrease batch is made simultaneously
Standby cost.
4. the present invention adopts UV-detector on-line checking, and the separation situation of each reference substance can directly be observed, can be straight
Connect selective collection high-purity sample, thus realizing the high-recovery under purity ensures.
In a word, present invention process step is simple, and product purity is high, color and luster is good, is especially suitable for large-scale production.
Brief description
Fig. 1 is two grades of rough segmentation things of Stellera chamaejasme L. medical material and 6 kinds of compounds（Isodaphnoretin, sikokianin B, sikokianin C,
The peaceful B of (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the peaceful A of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae)）HPLC analysis of spectra（295nm）,
Fig. 1 a：For two grades of rough segmentation thing figures；
Fig. 1 b：For Isodaphnoretin figure；
Fig. 1 c：For sikokianin B figure；
Fig. 1 d：For sikokianin C figure；
Fig. 1 e：For (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) peaceful B figure；
Fig. 1 f：For the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) peaceful B figure；
Fig. 1 g：For the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) peaceful A figure.
Its integration report is as follows respectively：
| Name | Retention Time | Area | %Area | |
| 1 | - | 4.39 | 36.5 | 1.77 |
| 2 | Isodaphnoretin | 6.21 | 2030.1 | 98.23 |
| Name | Retention Time | Area | %Area | |
| 1 | - | 4.37 | 63 | 0.66 |
| 2 | Sikokianin B | 14.64 | 9523.4 | 99.34 |
| Name | Retention Time | Area | %Area | |
| 1 | Sikokianin C | 16.21 | 6040.6 | 99.25 |
| 2 | - | 18.34 | 45.4 | 0.75 |
| Name | Retention Time | Area | %Area | |
| 1 | - | 4.37 | 124.2 | 0.60 |
| 2 | The peaceful B of (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) | 28.50 | 20530.6 | 99.40 |
| Name | Retention Time | Area | %Area | |
| 1 | - | 4.37 | 185.6 | 1.01 |
| 2 | The peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) | 36.65 | 18280.2 | 98.99 |
| Name | Retention Time | Area | %Area | |
| 1 | - | 4.37 | 254.6 | 1.28 |
| 2 | The peaceful A of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) | 45.11 | 19579.1 | 98.72 |
（HPLC condition：Chromatographic column is river Shen Chromatorex C18（4.6 × 250mm, 10 μm）, 30 DEG C of column temperature, flow velocity
1ml/min, sampling volume 5 μ l, mobile phase is：0～60min, 50% acetonitrile-water）.
Specific embodiment
In conjunction with embodiment and accompanying drawing, the present invention is described in further details, embodiment is only limitted to the present invention is described, and
Non- limitation of the invention.
Embodiment 1
In the present embodiment, with Stellera chamaejasme L. medical material 95%（Volumetric concentration, similarly hereinafter）Ethanol extraction is raw material, and the first step is just
Phase column chromatography adopts 200～300 mesh silica gel, and second step reversed phase column chromatography adopts 40～50 μm of ODS, the 3rd step high-efficient liquid phase color
Compose for using 5 μm of C18Bonded Phase prepares post, and the methanol-water solution using volumetric concentration 75% is eluent system, divides through three steps
From obtaining 6 kinds of pure compounds.Concrete technology step is as follows：
（1）Normal-phase silica gel column chromatography：Take Stellera chamaejasme L. medical material 95% ethanol extraction extractum, with one of chloroform, methanol or
Two kinds of mixed liquor dissolving, under being stirred continuously, adds 200～300 mesh column chromatography silica gels with quality such as extractum, mixes sample, often
Fully dry under temperature；Dry method loading afterwards, normal pressure descended 200～300 mesh silicagel columns, successively with chloroform, chloroform-methanol 100：1
（Volume ratio, similarly hereinafter）, chloroform-methanol 50：1st, chloroform-methanol 20：1 eluting, collects chloroform-methanol 20：1 stream part, negative pressure concentrates
After recycling design, obtain one-level rough segmentation thing.
（2）Anti-phase ODS column chromatography：After above-mentioned one-level rough segmentation thing is dissolved with methanol, add and the quality such as one-level rough segmentation thing
Particle diameter mix sample for 40～50 μm of ODS column chromatography fillers, fully dry under room temperature and process powdering；Dry method loading afterwards, mistake
40～50 μm of ODS chromatographic columns（Column length 40cm, internal diameter 8cm）, medium lift pump pressurizes, and pressure is 5～10MPa, flow-control 50～
100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water（Volumetric concentration, similarly hereinafter）Eluting, collects 70% methanol-water
Stream part, after negative pressure concentration and recovery solvent, obtains two grades of rough segmentation things.
（3）Prepared by high performance liquid chromatography：Above-mentioned two grades of rough segmentation things are dissolved with DMSO, being configured to concentration is 50mg/ml's
Need testing solution, through 0.45 μm of filtering with microporous membrane；Chromatographic column is Chromatorex C18Prepare post, 5 μm of particle diameter, size 250
×20mm；Sampling volume is 0.5ml, the methanol-water solution being 75% with volumetric concentration（Volumetric concentration, similarly hereinafter）For eluent system,
Flow speed control in 20ml/min, online ultraviolet detection, Detection wavelength is set as 295nm, collect respectively containing compound 1,2,3,
4th, 5,6 stream part, negative pressure drying obtains 6 kinds of light brown powders, compound 1,2,3,4,5,6 sterling that as purity is more than 98%（Matter
Amount about 1.5mg successively, 3.6mg, 1.5mg, 6.0mg, 5.2mg, 6.6mg）.
Embodiment 2
In the present embodiment, with Stellera chamaejasme L. medical material 95% ethanol extraction as raw material, first step normal phase column chromatography adopts 200
～300 mesh silica gel, second step reversed phase column chromatography adopts 40～50 μm of ODS, and the 3rd step high performance liquid chromatography preparation adopts 10 μm
C18Bonded Phase prepares post, and the acetonitrile-aqueous solution using 50% is eluent system, separates through three steps and obtains 6 kinds of pure compounds.
Concrete technology step is as follows：
（1）Normal-phase silica gel column chromatography：Take Stellera chamaejasme L. medical material 95% ethanol extraction extractum, with one of chloroform, methanol or
Two kinds of mixed liquor dissolving, under being stirred continuously, adds 200～300 mesh column chromatography silica gels with quality such as extractum, mixes sample, often
Fully dry under temperature；Dry method loading afterwards, normal pressure descended 200～300 mesh silicagel columns, successively with chloroform, chloroform-methanol 100：1、
Chloroform-methanol 50：1st, chloroform-methanol 20：1 eluting, collects chloroform-methanol 20：1 stream part, after negative pressure concentration and recovery solvent, obtains one
Level rough segmentation thing.
（2）Anti-phase ODS column chromatography：After above-mentioned one-level rough segmentation thing is dissolved with methanol, add and the quality such as one-level rough segmentation thing
Particle diameter mix sample for 40～50 μm of ODS column chromatography fillers, fully dry under room temperature and process powdering；Dry method loading afterwards, mistake
40～50 μm of ODS chromatographic columns（Column length 40cm, internal diameter 8cm）, medium lift pump pressurizes, and pressure is 5～10MPa, flow-control 50～
100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water eluting, collects 70% methanol-water stream part, negative pressure concentrates back
After receiving solvent, obtain two grades of rough segmentation things.
（3）Prepared by high performance liquid chromatography：Above-mentioned two grades of rough segmentation things are dissolved with DMSO, being configured to concentration is 60mg/ml's
Need testing solution, through 0.45 μm of filtering with microporous membrane；Chromatographic column is Chromatorex C18Prepare post, 10 μm of particle diameter, size 300
×50mm；Sampling volume be 1ml, with volumetric concentration be 50% acetonitrile-aqueous solution as eluent system, flow speed control is in 40ml/
Min, online ultraviolet detection, Detection wavelength is set as 295nm, collects the stream part containing compound 1,2,3,4,5,6, negative pressure respectively
Compound 1,2,3,4,5,6 sterling that dry 6 kinds of light brown powders, as purity are more than 98%（Quality about 3.6mg successively,
8.6mg, 3.6mg, 14.4mg, 12.4mg, 15.5mg）.
Embodiment 3
In the present embodiment, with Stellera chamaejasme L. medical material 95% ethanol extraction as raw material, first step normal phase column chromatography adopts 200
～300 mesh silica gel, second step reversed phase column chromatography adopts 40～50 μm of ODS, and the 3rd step high performance liquid chromatography preparation adopts 5 μm of C18
Bonded Phase prepares post, and the methanol-water solution using 70% is eluent system, separates through three steps and obtains 6 kinds of pure compounds.Tool
Body technology step is as follows：
（1）Normal-phase silica gel column chromatography：Take Stellera chamaejasme L. medical material 95% ethanol extraction extractum, with one of chloroform, methanol or
Two kinds of mixed liquor dissolving, under being stirred continuously, adds 200～300 mesh column chromatography silica gels with quality such as extractum, mixes sample, often
Fully dry under temperature；Dry method loading afterwards, normal pressure descended 200～300 mesh silicagel columns, successively with chloroform, chloroform-methanol 100：1、
Chloroform-methanol 50：1st, chloroform-methanol 20：1 eluting, collects chloroform-methanol 20：1 stream part, after negative pressure concentration and recovery solvent, obtains one
Level rough segmentation thing.
（2）Anti-phase ODS column chromatography：After above-mentioned one-level rough segmentation thing is dissolved with methanol, add and the quality such as one-level rough segmentation thing
Particle diameter mix sample for 40～50 μm of ODS column chromatography fillers, fully dry under room temperature and process powdering；Dry method loading afterwards, mistake
40～50 μm of ODS chromatographic columns（Column length 40cm, internal diameter 8cm）, medium lift pump pressurizes, and pressure is 5～10MPa, flow-control 50～
100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water eluting, collects 70% methanol-water stream part, negative pressure concentrates back
After receiving solvent, obtain two grades of rough segmentation things.
（3）Prepared by high performance liquid chromatography：Above-mentioned two grades of rough segmentation things are dissolved with DMSO, being configured to concentration is 70mg/ml's
Need testing solution, through 0.45 μm of filtering with microporous membrane；Chromatographic column is Chromatorex C18Prepare post, 5 μm of particle diameter, size 500
×60mm；Sampling volume is 1.4ml, and with volumetric concentration for 70% methanol-water solution as eluent system, flow speed control is in 60ml/
Min, online ultraviolet detection, Detection wavelength is set as 295nm, collects the stream part containing compound 1,2,3,4,5,6, negative pressure respectively
Compound 1,2,3,4,5,6 sterling that dry 6 kinds of light brown powders, as purity are more than 98%（Quality about 5.8mg successively,
14.1mg, 5.8mg, 23.5mg, 20.4mg, 25.8mg）.
Embodiment 4
In the present embodiment, with Stellera chamaejasme L. medical material 95% ethanol extraction as raw material, first step normal phase column chromatography adopts 200
～300 mesh silica gel, second step reversed phase column chromatography adopts 40～50 μm of ODS, and the 3rd step high performance liquid chromatography preparation adopts 10 μm
C18Bonded Phase prepares post, is eluent system using 45% acetonitrile-aqueous solution, separates through three steps and obtains 6 kinds of pure compounds.Tool
Body technology step is as follows：
（1）Normal-phase silica gel column chromatography：Take Stellera chamaejasme L. medical material 95% ethanol extraction extractum, with one of chloroform, methanol or
Two kinds of mixed liquor dissolving, under being stirred continuously, adds 200～300 mesh column chromatography silica gels with quality such as extractum, mixes sample, often
Fully dry under temperature；Dry method loading afterwards, normal pressure descended 200～300 mesh silicagel columns, successively with chloroform, chloroform-methanol 100：1、
Chloroform-methanol 50：1st, chloroform-methanol 20：1 eluting, collects chloroform-methanol 20：1 stream part, after negative pressure concentration and recovery solvent, obtains one
Level rough segmentation thing.
（2）Anti-phase ODS column chromatography：After above-mentioned one-level rough segmentation thing is dissolved with methanol, add and the quality such as one-level rough segmentation thing
Particle diameter mix sample for 40～50 μm of ODS column chromatography fillers, fully dry under room temperature and process powdering；Dry method loading afterwards, mistake
40～50 μm of ODS chromatographic columns（Column length 40cm, internal diameter 8cm）, medium lift pump pressurizes, and pressure is 5～10MPa, flow-control 50～
100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water eluting, collects 70% methanol-water stream part, negative pressure concentrates back
After receiving solvent, obtain two grades of rough segmentation things.
（3）Prepared by high performance liquid chromatography：Above-mentioned two grades of rough segmentation things are dissolved with DMSO, being configured to concentration is 80mg/ml's
Need testing solution, through 0.45 μm of filtering with microporous membrane；Chromatographic column prepares post, 10 μm of particle diameter, size for Chromatorex C18
600×80mm；Sampling volume is 1.8ml, and with volumetric concentration for 45% acetonitrile-aqueous solution as eluent system, flow speed control exists
90ml/min, online ultraviolet detection, Detection wavelength is set as 295nm, collects the stream containing compound 1,2,3,4,5,6 respectively
Part, negative pressure drying obtains 6 kinds of light brown powders, compound 1,2,3,4,5,6 sterling that as purity is more than 98%（Quality is successively about
8.6mg, 20.7mg, 8.6mg, 34.5mg, 29.9mg, 38.0mg）.
The physical parameter of above-mentioned 6 kinds of compounds and Structural Identification result are as follows：
Compound 1, white powder.UV（MeOH,）λmaxnm：205,350.MS（+ESI）m/z：353.068（[M+H]+）.
Molecular weight 352, molecular formula is C19H12O7.This compound identification is Isodaphnoretin.
Compound 2, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）Non- appearance.
MS（+ESI）m/z：557.148（[M+H]+）.Molecular weight 556, molecular formula is C31H24O10.This compound is Novel isomeric, name
For sikokianin B.
Compound 3, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）Non- appearance.
MS（+ESI）m/z：557.148（[M+H]+）.Molecular weight 556, molecular formula is C31H24O10.This compound identification is sikokianin C.
Compound 4, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）-4.2
（346）, 0（325）,+13.8（302）,+1.0（274）,+3.2（253）, 0（240）, -2.7（233）, -0.3（225）, -7.3
（217）.MS（+ESI）m/z：571.165（[M+H]+）.Molecular weight 570, point
Minor is C32H26O10.This compound identification is（+）The peaceful B of-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
Compound 5, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.2mg/ml, MeOH）Non- appearance.
MS（+ESI）m/z：571.165（[M+H]+）.Molecular weight 570, molecular formula is C32H26O10.This compound identification is that the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) is peaceful
B.
Compound 6, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）-13.0
(309), 0 (299) ,+19.4 (286), 0 (260), -1.1 (254), 0 (247) ,+1.6 (241), 0 (234), -14.1 (217).
MS（+ESI）m/z：571.165（[M+H]+）.Molecular weight 570, molecular formula is C32H26O10.This compound is Novel isomeric, name
For the peaceful A of the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
The NMR hydrogen spectrum of each compound, carbon modal data see table：
Note：1. solvent is DMSO-d6.2.na represents that signal does not occur or inconspicuous.
Claims (4)
1. a kind of method quickly preparing 6 kinds of compounds in Stellera chamaejasme L. it is characterised in that：With Stellera chamaejasme L. medical material volumetric concentration
It is raw material for 95% ethanol extraction, be enriched with through normal-phase silica gel column chromatography, anti-phase ODS column chromatography initial gross separation successively, finally lead to
Cross a step high performance liquid chromatography preparation, collect the stream part containing each compound, be directly evaporated to respectively and dry can obtain purity
6 kinds of compounds more than 98%；They be respectively Isodaphnoretin, sikokianin B, sikokianin C, (+) the peaceful B of-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) be peaceful
The B and peaceful A of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), wherein structural formula is
(1) normal-phase silica gel column chromatography：
Stellera chamaejasme L. medical material volumetric concentration is taken to be 95% ethanol extraction extractum, with the mixing of one of chloroform, methanol or two kinds
Liquid dissolves, and under being stirred continuously, adds 200～300 mesh column chromatography silica gels with quality such as extractum, mixes sample, fully dry in the air under room temperature
Dry；Dry method loading afterwards, normal pressure descended 200～300 mesh silicagel columns, successively with chloroform, chloroform-methanol volumetric concentration than for 100：
1st, chloroform-methanol 50：1st, chloroform-methanol 20：1 eluting, collects chloroform-methanol 20：1 stream part, after negative pressure concentration and recovery solvent, obtains
One-level rough segmentation thing；
(2) anti-phase ODS column chromatography：
After above-mentioned one-level rough segmentation thing is dissolved with methanol, the particle diameter with quality such as one-level rough segmentation things is added to be 40～50 μm of ODS posts
Chromatographic stuffing mixes sample, fully dries and process powdering under room temperature；Dry method loading afterwards, crosses 40～50 μm of ODS chromatographic columns, its
Middle column length 40cm, internal diameter 8cm, medium lift pump pressurize, pressure be 5～10MPa, flow-control in 50～100ml/min, successively with body
Long-pending concentration is 15%, 30%, 45%, 60%, 70% methanol-water eluting, collects 70% methanol-water stream part, negative pressure concentration and recovery
After solvent, obtain two grades of rough segmentation things；
(3) high performance liquid chromatography preparation：
Above-mentioned two grades of rough segmentation things are dissolved with DMSO, is configured to the need testing solution that concentration is 50～100mg/ml, through 0.45 μm
Filtering with microporous membrane；Filtrate carries out separating using the high performance liquid preparative chromatography post of column length 25-50cm, diameter 2-10cm, every time
Sampling volume is 0.5～2.5ml, with volumetric concentration 70%～80% methanol-water or 45%～55% acetonitrile-aqueous solution as eluting
System, flow speed control in 10～100ml/min, online ultraviolet detection, Detection wavelength is set as 295nm, respectively collect containing change
Compound Isodaphnoretin, sikokianin B, sikokianin C, (+) stream part of the peaceful B of-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) and the peaceful A of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), negative pressure does
Dry 6 kinds of powder, that is, respectively purity be more than 98% compound Isodaphnoretin, sikokianin B, sikokianin C, (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) is peaceful
B, the peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) and the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) peaceful A sterling.
2. method according to claim 1 it is characterised in that：The filler that first step positive post separation is adopted be 200～
300 mesh column chromatography silica gels；The filler that the anti-phase post separation of second step is adopted is 40～50 μm of column chromatography ODS.
3. method according to claim 1 it is characterised in that：The post of preparing that 3rd step efficient liquid phase preparation process adopts is filled out
Expect for C18Bonded phase packings, particle diameter is 5～10 μm.
4. method according to claim 1 it is characterised in that：Efficient liquid phase preparation process is using using the inspection of online ultraviolet
Survey, its Detection wavelength 295nm.
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