CN104610214A - Method for rapidly preparing six compounds in Stellera chamaejasme L. - Google Patents

Method for rapidly preparing six compounds in Stellera chamaejasme L. Download PDF

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CN104610214A
CN104610214A CN201310539312.5A CN201310539312A CN104610214A CN 104610214 A CN104610214 A CN 104610214A CN 201310539312 A CN201310539312 A CN 201310539312A CN 104610214 A CN104610214 A CN 104610214A
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methanol
column chromatography
langdu
peaceful
chloroform
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CN104610214B (en
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肖红斌
王志鑫
高明哲
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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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
    • 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
    • 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

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

A kind of method preparing 6 kinds of compounds in Stellera chamaejasme L. fast
Technical field
The present invention relates to a kind of novel process preparing 6 kinds of compounds in Stellera chamaejasme L. fast.Mainly comprise the enrichment of positive and negative phase column chromatography for separation and the preparation of high performance liquid chromatography removal of impurities fast.Compound 1(Isodaphnoretin) be temparin, compound 2(wild goose skin element B), 3(wild goose skin element C), 4((+) the peaceful B of-root of langdu), the peaceful B of the different root of langdu of 5(), the peaceful A of the different root of langdu of 6() be pair flavanones.Wherein, compound 1 is prepared first in stellera plant, and compound 2,6 is the Novel isomeric of such pair of flavanone.The chemical structure of these 6 kinds of compounds is as follows respectively:
Background technology
Stellera chamaejasme L. (Stellerachamaejasme L.) is thymelaeceae stellera plant, popular name Graceful Jessamine Herb, in being distributed in, Russia, illiteracy, towards etc. state, mainly originate from northern each provinces and regions and southwest in China, be born in height above sea level 2600 ~ 4200 meters of dryings and face south high mountain grass slope, lawn or tableland, river shoal.Its bitter is pungent, and property is put down, and very toxic, enter lung spleen Liver Channel, root is used as medicine, and having relieves oedema or abdominal distension through diuresis or purgation eliminates the phlegm, effect of broken long-pending desinsection.Modern pharmacology research shows that Stellera chamaejasme L. has good anti-tumor activity, and current research also finds that it has the effects such as antiviral, antibacterial, anticonvulsion and immunomodulatory.At present, the chemical composition that isolation identification goes out from Stellera chamaejasme L. mainly contains diterpenes, sesquiterpenoids, flavonoid, coumarins, lignanoid and Phenylpropanoid Glycosides glycoside etc., and wherein, flavonoid mostly is two flavanone, being the index chemical composition of this medicinal material, is also one of its main active ingredient.Wild goose skin element C(compound 3) to have the strain of plasmodium falciparum chloroquine resistance and significantly kill except effect, its IC 50value can reach 0.56 μ g/ml(Nunome S, et al.PLANTA MED, 2004,70 (1)); The peaceful B(compound 4 of (+)-root of langdu) propagation of kinds of tumor cells can be suppressed in vitro, to the IC of human A549 cell lines 50value can reach 1.08 μm of ol/L(Zhang C, et al.ACTA PHARMACOL SIN, 2013,34(2)); The different root of langdu peaceful B(compound 5) show stronger antimycotic and antimitotic activity, be 6.25 μ g/mL(Yang G, et al.CHEM PHARM BULL, 2005,53(7 to the MIC value of Pyricularia oryzae)); The separation preparation of compound 2,6 and pharmacological research be have not been reported.In addition, as quality control index composition, chemical reference substance supply available in a large number must be had, but rarely have the tonka bean camphor and two flavanone reference substance that comprise these 6 kinds of compounds in the market and provide.Therefore, to these 6 kinds of compounds in Stellera chamaejasme L. medicinal material simultaneously, the research of high efficiency preparation method is significant.
Summary of the invention
The present invention, on the basis that early-stage Study Chemical Components in Stellera Chamaejasme L systematic position is analyzed, provides the novel process that one prepares 6 kinds of compounds (Isodaphnoretin, wild goose skin element B, wild goose skin element C, the peaceful B of (+)-root of langdu, the peaceful B of the different root of langdu and the peaceful A of the different root of langdu) in Stellera chamaejasme L. medicinal material fast.With Stellera chamaejasme L. medicinal material for raw material, through purification on normal-phase silica gel column chromatography, anti-phase ODS column chromatography initial gross separation enrichment, be final separation means with preparative high performance liquid chromatography, by a step high performance liquid phase preparation, the stream part of collecting containing each compound is directly evaporated to the dry compound that can obtain purity and be greater than 98%.Concrete steps are as follows:
(1) purification on normal-phase silica gel column chromatography:
Get Stellera chamaejasme L. medicinal material 95% extraction using alcohol medicinal extract, dissolve with the mixed solution of one or both in chloroform, methyl alcohol, under constantly stirring, add 200 ~ 300 order column chromatography silica gels with the quality such as medicinal extract, mix sample, fully dry under normal temperature; Dry method loading afterwards, normal pressure descended 200 ~ 300 order silicagel columns, successively with chloroform, chloroform-methanol 100:1, chloroform-methanol 50:1, chloroform-methanol 20:1 wash-out, collected chloroform-methanol 20:1 and flowed part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography:
By above-mentioned one-level rough segmentation thing with after dissolve with methanol, adding with the particle diameter of the quality such as one-level rough segmentation thing is that sample mixed by 40 ~ 50 μm of ODS column chromatography fillers, fully dries and be processed into powdery under normal temperature; Dry method loading afterwards, cross 40 ~ 50 μm of ODS chromatography column (column length 40cm, internal diameter 8cm), medium lift pump pressurizes, and pressure is 5 ~ 10MPa, and flow control is at 50 ~ 100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water wash-out, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
(3) high performance liquid chromatography preparation:
Above-mentioned secondary rough segmentation thing is dissolved with DMSO, is mixed with the need testing solution that concentration is 50 ~ 100mg/ml, through 0.45 μm of filtering with microporous membrane; Filtrate adopts column length 25-50cm, the high performance liquid preparative chromatography post of diameter 2-10cm is separated, each sampling volume is 0.5 ~ 2.5ml, with volumetric concentration 70% ~ 80% methanol-water or 45% ~ 55% acetonitrile-aqueous solution for eluent system, flow rate control is at 10 ~ 100ml/min, online ultraviolet detection, determined wavelength is set as 295nm, collect the stream part containing compound 1,2,3,4,5,6 respectively, negative pressure drying obtains 6 kinds of powder, is compound 1,2,3,4,5,6 sterling that purity is greater than 98%.
In aforesaid method, the filler of high performance liquid phase preparative column is C 18bonded Phase, its particle diameter is 5 ~ 10 μm, the preferred 295nm of ultraviolet detection wavelength, concrete determined wavelength per sample in target compound signal overload condition and determine.
The present invention prepares 6 kinds of compounds (a kind of temparin, 5 kinds of two flavanones) from Stellera chamaejasme L. material simultaneously, and tool has the following advantages and improves:
1. the present invention adopts three-step approach, first carries out preliminary concentration by positive and negative phase two step column chromatography to target compound, and then adopt a step high performance liquid phase preparation method carry out removal of impurities respectively and refine, technique is simpler.
2. the present invention is last separation means with preparative high performance liquid phase, and separation efficiency is high, can ensure the purity of reference substance.
3. the high performance liquid phase preparation method of the present invention's development is fast preparation method, and single needle disengaging time, between 40-60 minute, is applicable to the preparation of chemical reference substance in enormous quantities very much; Add the recycling preparing solvent simultaneously, the cost of batch preparation can be reduced.
4. the present invention adopts UV-detector on-line checkingi, can directly observe the separation case of each reference substance, can direct selective collection high purity sample, thus realize purity ensure under high-recovery.
In a word, present invention process step is simple, and product purity is high, color and luster good, is very applicable to scale operation.
Accompanying drawing explanation
Fig. 1 is the HPLC analysis of spectra (295nm) of Stellera chamaejasme L. medicinal material secondary rough segmentation thing and 6 kinds of compounds (Isodaphnoretin, wild goose skin element B, wild goose skin element C, the peaceful B of (+)-root of langdu, the peaceful B of the different root of langdu, the peaceful A of the different root of langdu),
Fig. 1 a: be secondary rough segmentation thing figure;
Fig. 1 b: be Isodaphnoretin figure;
Fig. 1 c: be wild goose skin element B figure;
Fig. 1 d: be wild goose skin element C figure;
Fig. 1 e: be the peaceful B figure of (+)-root of langdu;
Fig. 1 f: be the peaceful B figure of the different root of langdu;
Fig. 1 g: be the peaceful A figure of the different root of langdu.
The report of its integration 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 Wild goose skin element B 14.64 9523.4 99.34
Name Retention Time Area %Area
1 Wild goose skin element 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 (+)-root of langdu 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 root of langdu 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 root of langdu 45.11 19579.1 98.72
(HPLC condition: chromatographic column is Shen, river Chromatorex C 18(4.6 × 250mm, 10 μm), column temperature 30 DEG C, flow velocity 1ml/min, sampling volume 5 μ l, moving phase is: 0 ~ 60min, 50% acetonitrile-water).
Embodiment
Now be described in further details the present invention with accompanying drawing, embodiment is only limitted to the present invention is described in conjunction with the embodiments, but not limitation of the invention.
Embodiment 1
In the present embodiment, with Stellera chamaejasme L. medicinal material 95%(volumetric concentration, lower with) ethanol extraction is raw material, the first step normal phase column chromatography adopts 200 ~ 300 order silica gel, second step reversed phase column chromatography adopts 40 ~ 50 μm of ODS, and employing 5 μm of C prepared by the 3rd step high performance liquid chromatography 18bonded Phase preparative column, the methanol-water solution adopting volumetric concentration 75% is eluent system, is separated namely obtains 6 kinds of pure compounds through three steps.Concrete technology step is as follows:
(1) purification on normal-phase silica gel column chromatography: get Stellera chamaejasme L. medicinal material 95% extraction using alcohol medicinal extract, dissolve with the mixed solution of one or both in chloroform, methyl alcohol, under constantly stirring, add 200 ~ 300 order column chromatography silica gels with the quality such as medicinal extract, mix sample, fully dry under normal temperature; Dry method loading afterwards, normal pressure descended 200 ~ 300 order silicagel columns, successively with chloroform, chloroform-methanol 100:1(volume ratio, down together), chloroform-methanol 50:1, chloroform-methanol 20:1 wash-out, collect chloroform-methanol 20:1 and flow part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography: by above-mentioned one-level rough segmentation thing with after dissolve with methanol, adding with the particle diameter of the quality such as one-level rough segmentation thing is that sample mixed by 40 ~ 50 μm of ODS column chromatography fillers, fully dries and be processed into powdery under normal temperature; Dry method loading afterwards, cross 40 ~ 50 μm of ODS chromatography column (column length 40cm, internal diameter 8cm), medium lift pump pressurizes, and pressure is 5 ~ 10MPa, flow control is at 50 ~ 100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water (volumetric concentration, lower same) wash-out, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
(3) high performance liquid chromatography preparation: above-mentioned secondary rough segmentation thing is dissolved with DMSO, is mixed with the need testing solution that concentration is 50mg/ml, through 0.45 μm of filtering with microporous membrane; Chromatographic column is Chromatorex C 18preparative column, particle diameter 5 μm, size 250 × 20mm; Sampling volume is 0.5ml, is eluent system with the methanol-water solution (volumetric concentration, lower same) that volumetric concentration is 75%, flow rate control at 20ml/min, online ultraviolet detection, determined wavelength is set as 295nm, collect the stream part containing compound 1,2,3,4,5,6 respectively, negative pressure drying obtains 6 kinds of light brown powders, is compound 1,2,3,4,5,6 sterling (quality about 1.5mg, the 3.6mg successively that purity is greater than 98%, 1.5mg, 6.0mg, 5.2mg, 6.6mg).
Embodiment 2
In the present embodiment, with Stellera chamaejasme L. medicinal material 95% ethanol extraction for raw material, the first step normal phase column chromatography adopts 200 ~ 300 order silica gel, and second step reversed phase column chromatography adopts 40 ~ 50 μm of ODS, and employing 10 μm of C prepared by the 3rd step high performance liquid chromatography 18bonded Phase preparative column, the acetonitrile-aqueous solution adopting 50% is eluent system, is separated namely obtains 6 kinds of pure compounds through three steps.Concrete technology step is as follows:
(1) purification on normal-phase silica gel column chromatography: get Stellera chamaejasme L. medicinal material 95% extraction using alcohol medicinal extract, dissolve with the mixed solution of one or both in chloroform, methyl alcohol, under constantly stirring, add 200 ~ 300 order column chromatography silica gels with the quality such as medicinal extract, mix sample, fully dry under normal temperature; Dry method loading afterwards, normal pressure descended 200 ~ 300 order silicagel columns, successively with chloroform, chloroform-methanol 100:1, chloroform-methanol 50:1, chloroform-methanol 20:1 wash-out, collected chloroform-methanol 20:1 and flowed part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography: by above-mentioned one-level rough segmentation thing with after dissolve with methanol, adding with the particle diameter of the quality such as one-level rough segmentation thing is that sample mixed by 40 ~ 50 μm of ODS column chromatography fillers, fully dries and be processed into powdery under normal temperature; Dry method loading afterwards, cross 40 ~ 50 μm of ODS chromatography column (column length 40cm, internal diameter 8cm), medium lift pump pressurizes, and pressure is 5 ~ 10MPa, and flow control is at 50 ~ 100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water wash-out, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
(3) high performance liquid chromatography preparation: above-mentioned secondary rough segmentation thing is dissolved with DMSO, is mixed with the need testing solution that concentration is 60mg/ml, through 0.45 μm of filtering with microporous membrane; Chromatographic column is Chromatorex C 18preparative column, particle diameter 10 μm, size 300 × 50mm; Sampling volume is 1ml, take volumetric concentration as the acetonitrile-aqueous solution of 50% is eluent system, and flow rate control is at 40ml/min, online ultraviolet detection, determined wavelength is set as 295nm, and collect the stream part containing compound 1,2,3,4,5,6 respectively, negative pressure drying obtains 6 kinds of light brown powders, be purity be greater than 98% compound 1,2,3,4,5,6 sterling (quality is about 3.6mg successively, 8.6mg, 3.6mg, 14.4mg, 12.4mg, 15.5mg).
Embodiment 3
In the present embodiment, with Stellera chamaejasme L. medicinal material 95% ethanol extraction for raw material, the first step normal phase column chromatography adopts 200 ~ 300 order silica gel, and second step reversed phase column chromatography adopts 40 ~ 50 μm of ODS, and employing 5 μm of C prepared by the 3rd step high performance liquid chromatography 18bonded Phase preparative column, the methanol-water solution adopting 70% is eluent system, is separated namely obtains 6 kinds of pure compounds through three steps.Concrete technology step is as follows:
(1) purification on normal-phase silica gel column chromatography: get Stellera chamaejasme L. medicinal material 95% extraction using alcohol medicinal extract, dissolve with the mixed solution of one or both in chloroform, methyl alcohol, under constantly stirring, add 200 ~ 300 order column chromatography silica gels with the quality such as medicinal extract, mix sample, fully dry under normal temperature; Dry method loading afterwards, normal pressure descended 200 ~ 300 order silicagel columns, successively with chloroform, chloroform-methanol 100:1, chloroform-methanol 50:1, chloroform-methanol 20:1 wash-out, collected chloroform-methanol 20:1 and flowed part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography: by above-mentioned one-level rough segmentation thing with after dissolve with methanol, adding with the particle diameter of the quality such as one-level rough segmentation thing is that sample mixed by 40 ~ 50 μm of ODS column chromatography fillers, fully dries and be processed into powdery under normal temperature; Dry method loading afterwards, cross 40 ~ 50 μm of ODS chromatography column (column length 40cm, internal diameter 8cm), medium lift pump pressurizes, and pressure is 5 ~ 10MPa, and flow control is at 50 ~ 100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water wash-out, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
(3) high performance liquid chromatography preparation: above-mentioned secondary rough segmentation thing is dissolved with DMSO, is mixed with the need testing solution that concentration is 70mg/ml, through 0.45 μm of filtering with microporous membrane; Chromatographic column is Chromatorex C 18preparative column, particle diameter 5 μm, size 500 × 60mm; Sampling volume is 1.4ml, with volumetric concentration be 70% methanol-water solution for eluent system, flow rate control is at 60ml/min, online ultraviolet detection, determined wavelength is set as 295nm, and collect the stream part containing compound 1,2,3,4,5,6 respectively, negative pressure drying obtains 6 kinds of light brown powders, be purity be greater than 98% compound 1,2,3,4,5,6 sterling (quality is about 5.8mg successively, 14.1mg, 5.8mg, 23.5mg, 20.4mg, 25.8mg).
Embodiment 4
In the present embodiment, with Stellera chamaejasme L. medicinal material 95% ethanol extraction for raw material, the first step normal phase column chromatography adopts 200 ~ 300 order silica gel, and second step reversed phase column chromatography adopts 40 ~ 50 μm of ODS, and employing 10 μm of C prepared by the 3rd step high performance liquid chromatography 18bonded Phase preparative column, adopts 45% acetonitrile-aqueous solution to be eluent system, is separated namely obtains 6 kinds of pure compounds through three steps.Concrete technology step is as follows:
(1) purification on normal-phase silica gel column chromatography: get Stellera chamaejasme L. medicinal material 95% extraction using alcohol medicinal extract, dissolve with the mixed solution of one or both in chloroform, methyl alcohol, under constantly stirring, add 200 ~ 300 order column chromatography silica gels with the quality such as medicinal extract, mix sample, fully dry under normal temperature; Dry method loading afterwards, normal pressure descended 200 ~ 300 order silicagel columns, successively with chloroform, chloroform-methanol 100:1, chloroform-methanol 50:1, chloroform-methanol 20:1 wash-out, collected chloroform-methanol 20:1 and flowed part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography: by above-mentioned one-level rough segmentation thing with after dissolve with methanol, adding with the particle diameter of the quality such as one-level rough segmentation thing is that sample mixed by 40 ~ 50 μm of ODS column chromatography fillers, fully dries and be processed into powdery under normal temperature; Dry method loading afterwards, cross 40 ~ 50 μm of ODS chromatography column (column length 40cm, internal diameter 8cm), medium lift pump pressurizes, and pressure is 5 ~ 10MPa, and flow control is at 50 ~ 100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water wash-out, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
(3) high performance liquid chromatography preparation: above-mentioned secondary rough segmentation thing is dissolved with DMSO, is mixed with the need testing solution that concentration is 80mg/ml, through 0.45 μm of filtering with microporous membrane; Chromatographic column is Chromatorex C18 preparative column, particle diameter 10 μm, size 600 × 80mm; Sampling volume is 1.8ml, with volumetric concentration be 45% acetonitrile-aqueous solution for eluent system, flow rate control is at 90ml/min, online ultraviolet detection, determined wavelength is set as 295nm, and collect the stream part containing compound 1,2,3,4,5,6 respectively, negative pressure drying obtains 6 kinds of light brown powders, be purity be greater than 98% compound 1,2,3,4,5,6 sterling (quality is about 8.6mg successively, 20.7mg, 8.6mg, 34.5mg, 29.9mg, 38.0mg).
Physical parameter and the Structural Identification result of above-mentioned 6 kinds of compounds are as follows:
Compound 1, white powder.UV（MeOH，）λ maxnm：205，350。MS（+ESI）m/z：353.068（[M+H] +）。Molecular weight 352, molecular formula is C 19h 12o 7.This compound identification is Isodaphnoretin.
Compound 2, light brown powder.UV（MeOH，）λ maxnm：215，295。CD(c=0.1mg/ml, MeOH) do not go out peak.MS（+ESI）m/z：557.148（[M+H] +）。Molecular weight 556, molecular formula is C 31h 24o 10.This compound is Novel isomeric, called after wild goose skin element B.
Compound 3, light brown powder.UV（MeOH，）λ maxnm：215，295。CD(c=0.1mg/ml, MeOH) do not go out peak.MS（+ESI）m/z：557.148（[M+H] +）。Molecular weight 556, molecular formula is C 31h 24o 10.This compound identification is wild goose skin element C.
Compound 4, light brown powder.UV（MeOH，）λ maxnm：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, molecular formula is C 32h 26o 10.This compound identification is the peaceful B of (+)-root of langdu.
Compound 5, light brown powder.UV（MeOH，）λ maxnm：215，295。CD(c=0.2mg/ml, MeOH) do not go out peak.MS（+ESI）m/z：571.165（[M+H] +）。Molecular weight 570, molecular formula is C 32h 26o 10.This compound identification is the peaceful B of the different root of langdu.
Compound 6, light brown powder.UV（MeOH，）λ maxnm：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 C 32h 26o 10.This compound is Novel isomeric, the peaceful A of the called after root of langdu.
The NMR hydrogen spectrum of each compound, carbon modal data see the following form:
Note: 1. solvent is DMSO-d 6.2.na expression signal does not occur or not obvious.

Claims (5)

1. prepare the method for 6 kinds of compounds in Stellera chamaejasme L. fast for one kind, it is characterized in that: with Stellera chamaejasme L. medicinal material 95% ethanol (volumetric concentration, down together) extract is raw material, successively through purification on normal-phase silica gel column chromatography, anti-phase ODS column chromatography initial gross separation enrichment, finally by a step high performance liquid chromatography preparation, collect the stream part containing each compound, be directly evaporated to dry 6 kinds of compounds that can obtain purity and be greater than 98% respectively; They are respectively Isodaphnoretin, wild goose skin element B, wild goose skin element C, the peaceful B of (+)-root of langdu, the peaceful B of the different root of langdu and the peaceful A of the different root of langdu.
2. method according to claim 1, is characterized in that:
(1) purification on normal-phase silica gel column chromatography:
Get Stellera chamaejasme L. medicinal material 95% ethanol (volumetric concentration, lower with) and extract medicinal extract, dissolve with the mixed solution of one or both in chloroform, methyl alcohol, under constantly stirring, add 200 ~ 300 order column chromatography silica gels with the quality such as medicinal extract, mix sample, fully dry under normal temperature; Dry method loading afterwards, normal pressure descended 200 ~ 300 order silicagel columns, successively with chloroform, chloroform-methanol 100:1(volume ratio, down together), chloroform-methanol 50:1, chloroform-methanol 20:1 wash-out, collect chloroform-methanol 20:1 and flow part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing;
(2) anti-phase ODS column chromatography:
By above-mentioned one-level rough segmentation thing with after dissolve with methanol, adding with the particle diameter of the quality such as one-level rough segmentation thing is that sample mixed by 40 ~ 50 μm of ODS column chromatography fillers, fully dries and be processed into powdery under normal temperature; Dry method loading afterwards, cross 40 ~ 50 μm of ODS chromatography column (column length 40cm, internal diameter 8cm), medium lift pump pressurizes, and pressure is 5 ~ 10MPa, flow control is at 50 ~ 100ml/min, successively with 15%(volumetric concentration, lower with), 30%, 45%, 60%, 70% methanol-water wash-out, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing;
(3) high performance liquid chromatography preparation:
Above-mentioned secondary rough segmentation thing is dissolved with DMSO, is mixed with the need testing solution that concentration is 50 ~ 100mg/ml, through 0.45 μm of filtering with microporous membrane, filtrate adopts column length 25-50cm, the high performance liquid preparative chromatography post of diameter 2-10cm is separated, each sampling volume is 0.5 ~ 2.5ml, with volumetric concentration 70% ~ 80% methanol-water or 45% ~ 55% acetonitrile-aqueous solution for eluent system, flow rate control is at 10 ~ 100ml/min, online ultraviolet detection, determined wavelength is set as 295nm, collect respectively containing compound Isodaphnoretin, wild goose skin element B, wild goose skin element C, the peaceful B of (+)-root of langdu, stream part of the peaceful B of the different root of langdu and the peaceful A of the different root of langdu, negative pressure drying obtains 6 kinds of powder, namely the compound Isodaphnoretin that purity is greater than 98% is respectively, wild goose skin element B, wild goose skin element C, the peaceful B of (+)-root of langdu, the peaceful B of the different root of langdu and the peaceful A sterling of the different root of langdu.
3. method according to claim 2, is characterized in that: it is 200 ~ 300 order column chromatography silica gels that the first step normal phase column is separated the filler adopted, and eluent is the chloroform-methanol of volume ratio 100:0 ~ 20:1; It is 40 ~ 50 μm of column chromatography ODS that second step reversed-phase column is separated the filler adopted, and eluent is the methanol-water solution of volumetric concentration 15% ~ 70%.
4. method according to claim 2, is characterized in that: the preparative column filler that the 3rd step high performance liquid phase preparation process adopts is C 18bonded phase packings, particle diameter is 5 ~ 10 μm.
5. method according to claim 2, is characterized in that: high performance liquid phase preparation process adopts online ultraviolet detection, the preferred 295nm of its determined wavelength.
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