CN105198751A - Method for preparing diterpene ester compound euphorbia factor L2 - Google Patents
Method for preparing diterpene ester compound euphorbia factor L2 Download PDFInfo
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- CN105198751A CN105198751A CN201510594625.XA CN201510594625A CN105198751A CN 105198751 A CN105198751 A CN 105198751A CN 201510594625 A CN201510594625 A CN 201510594625A CN 105198751 A CN105198751 A CN 105198751A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/80—Acids; Esters in position 3
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/803—Processes of preparation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
- C07C2602/14—All rings being cycloaliphatic
- C07C2602/32—All rings being cycloaliphatic the ring system containing at least eleven carbon atoms
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a method for preparing a diterpene ester compound euphorbia factor L2. The two-dimensional liquid-phase chromatographic technology is adopted, so that the euphorbia factor L2 is extracted from moleplant seed; through reasonable selection of a chromatographic column, a dissolved solution and flow equality, optimization of the flow speed, the elution mode, a collection zone and other process conditions and orthogonality of a first dimension and a second dimension, complementary separation is achieved, so that the whole separation and purification method is feasible and high in operability, and the purity of the obtained euphorbia factor L2 is as high as over 98%.
Description
Technical field
The present invention relates to production of chemicals field, the preparation method of especially a kind of diterpene alkoxide compound root of Beijing euphorbia factor L2.
Background technology
The dry mature seed of a thousand pieces of gold subsystem euphorbia plant Euphorbia lathyris Euphorbialathyris is one of China's traditional Chinese medicine material.Semen Euphorbiae is warm in nature, and taste is pungent, slightly poisonous, and primary efficacy is detumescence of relieving oedema or abdominal distension through diuresis or purgation, Po Xue Xiao Disorder, being used for the treatment of oedema, phlegm and retained fluid, stagnant turgor, constipation and anuresis, blood stasis through closing, controlling stubborn dermatitis, wart outward.EuphorbiaFactorL2 (root of Beijing euphorbia factor L2) is diterpene alkoxide compound, and molecular formula is C
38h
42o
9, molecular weight is 642.283, and its structure is as follows:
At present for the refining main method adopting conventional column chromatography and silica gel column chromatography and crystallization phases to combine of EuphorbiaFactorL2, its production cycle is long, product yield is low, and utilizes preparative chromatography separation and purification to obtain EuphorbiaFactorL2 monomer not yet to have pertinent literature to report.Increasing research shows, many rings diterpene is extensively exist in euphorbia.This platymiscium can yet be regarded as one potential, contain the secondary metabolite storehouse of enriching diterpene compound.No matter in the active lead compound of discovery, or inquiring into plant source of students synthesis mechanism further, illustrate in euphorbiaceous chemical classification etc., the research of Euphorbia many rings diterpene all will embody important meaning.
Summary of the invention
Technical problem to be solved by this invention is the preparation method providing a kind of diterpene alkoxide compound root of Beijing euphorbia factor L2.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2, concrete steps are as follows:
(1) getting Semen Euphorbiae (commercial) is raw material, carries out pulverizing and obtains Semen Euphorbiae powder;
(2) get Semen Euphorbiae powder and carry out organic reagent extraction, organic reagent is ethyl acetate, collects the supernatant after extracting, and filters, concentrated, dry;
(3) precipitation step (2) obtained repeats 1 ~ 5 time and extracts, and collects the supernatant after extracting, filters, concentrated, dry, obtains QJZ primary extract;
(4) get step (3) to obtain QJZ primary extract moving phase and all dissolve, described moving phase is mobile phase A or Mobile phase B or both combinations, be separated through preparation liquid phase DAC forward chromatographic column, binary-mobile phase system is adopted to carry out wash-out separation, described mobile phase A and Mobile phase B adopt the combination of one or more in normal hexane, ethanol, type of elution gradient is washed, determined wavelength is 210nm, collect object peak, component composition QJZ-5 is obtained after drying, 60 DEG C of air dry ovens dry up, and carry out next step separation and purification;
(5) get step (4) to obtain QJZ-5 component moving phase and all dissolve, described moving phase is mobile phase A or Mobile phase B or both combinations, be separated through preparative liquid chromatography post, binary-mobile phase system is adopted to carry out wash-out separation, described mobile phase A and Mobile phase B adopt normal hexane, ethyl acetate, ethanol, methylene dichloride, the combination of one or more in methyl alcohol, type of elution isocratic elution, determined wavelength is 210nm, collect object peak, monomeric compound QJZ-5-1 is obtained after drying, 60 DEG C of air dry ovens dry up, QJZ-5-1 is final Active Target monomer diterpene alkoxide compound.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, in described step (2), the temperature of organic solvent extraction is 40 ~ 60 DEG C, and extraction time is 4 ~ 6h, leaves standstill a night.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, the weightmeasurement ratio of Semen Euphorbiae powder described in described step (3) or precipitation and organic reagent counts 1:(3 ~ 8 by Kg:L), Semen Euphorbiae powder or precipitation are carried out mixing lixiviate with organic reagent.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, the chromatographic column type that in the sepn process of described step (4), one dimension liquid chromatography adopts is purification on normal-phase silica gel axial pressure chromatographic column (InnovalSilica150 × 250mm, 10 μm, 100
), flow velocity: 600ml/min, determined wavelength: 210nm, during use, column temperature is room temperature or 25-40 DEG C.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, getting QJZ primary extract 60-80% normal hexane-dissolve with ethanol in the sepn process of described step (4) is 1ml/ml to concentration, after mixing, be separated through preparation liquid phase DAC forward chromatographic column with after 0.45 μm of organic membrane suction filtration.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, in the sepn process of described step (4), type of elution is specially 100% normal hexane isocratic elution 5min, 100% normal hexane-70% ethanol gradient elution 35-50min, 100% ethanol 10min.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, the chromatographic column type that in the sepn process of described step (5), two-dimensional liquid chromatography adopts is ACCOHXAmide chromatographic column (10 × 250mm, 10 μm, 100
), flow velocity: 2-4ml/min, determined wavelength: 210nm, during use, column temperature is room temperature or 25-40 DEG C.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, getting QJZ-5 component 50% normal hexane-ethanol 40 DEG C in the sepn process of described step (5) ultrasonic is 50-75mg/ml to being just dissolved to concentration, with 0.45 μm of organic membrane filter.
Preferably, the preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, in the sepn process of described step (5), type of elution is specially 100% normal hexane-90% ethanol gradient elution 30-40min, 100% ethanol 10min.
The invention has the beneficial effects as follows:
The preparation method of above-mentioned diterpene alkoxide compound root of Beijing euphorbia factor L2, separation efficiency is high, with short production cycle, process stabilizing, easy and simple to handle, with low cost, the high purity separation preparation of a large amount of EuphorbiaFactorL2 (root of Beijing euphorbia factor L2) monomer can be realized, specifically, two-dimensional liquid chromatography technology is adopted to carry out extracting EuphorbiaFactorL2 from Semen Euphorbiae, pass through chromatographic column, lysate, flow equal choose reasonable, flow velocity, type of elution, collect the optimization of the processing condition such as section, and first orthogonality tieed up of the peacekeeping second and complementation that reaches is separated, make whole separation purification method practical, operability is high, the EuphorbiaFactorL2 purity obtained is the highest more than 98%.
Accompanying drawing explanation
Shown in Fig. 1 is the one dimension high performance preparative liquid chromatography figure that example 3 of the present invention extracts QJZ-5 from Semen Euphorbiae;
Shown in Fig. 2 is the two-dimensional highly effective preparative liquid chromatography figure that example 3 of the present invention extracts No. 1, the separating obtained cut of one dimension high performance preparative liquid chromatography of Semen Euphorbiae QJZ-5 from Semen Euphorbiae;
Shown in Fig. 3 is the analysis mode high-efficient liquid phase chromatogram of the example 3 object monomer that the present invention obtains;
Shown in Fig. 4 is the nuclear magnetic resonance spectroscopy figure of QJZ-5-1 of the present invention, wherein (a) is 1D1HNMR spectrum (CDCl3, data collection of illustrative plates 400MHz), b () is 1D13CNMR spectrum (CDCl3, data collection of illustrative plates 100MHz), d () is HSQC data collection of illustrative plates, (c) is DEPT135 data collection of illustrative plates, and (e) is COSY data collection of illustrative plates.
Embodiment
In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with the drawings and the specific embodiments, technical scheme of the present invention is described in further detail.Except as otherwise noted, all Science and Technology terms that the present invention uses have the identical meanings usually understood with the technical field of the invention personnel.
Embodiment 1
One, the acquisition of QJZ primary extract
1, pulverize:
Getting Semen Euphorbiae is raw material, puts into pulverizer and pulverizes.
2, organic reagent extracts:
Carry out organic reagent to Semen Euphorbiae powder to extract, extract continuously for three times, each extraction time is 6h (after being wherein heated to 60 degree, reheating 6h).Concrete:
First time organic reagent extraction: 2.5kg Semen Euphorbiae powder adds 20L organic reagent, organic reagent is ethyl acetate, is stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extract 6h, extract after terminating, collect supernatant liquor, with filter paper filtering, be transferred in 60 DEG C of constant temperature blast drying ovens dry after supernatant is concentrated in Rotary Evaporators, obtain QJZ primary extract, precipitate stand-by;
Second time organic reagent extracts: first time extracts precipitation and adds 15L organic reagent, organic reagent is ethyl acetate, be stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extracts 6h, after extraction terminates, the method extracted by first time processes, and obtains QJZ primary extract, precipitates stand-by;
Third time organic reagent extraction: second time is extracted precipitation and added, organic reagent, organic reagent is ethyl acetate, is stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extract 6h, extract after terminating, the method extracted by first time processes, obtain QJZ8 primary extract, precipitation is dried;
Two, the acquisition of QJZ-5
QJZ primary extract component moving phase is dissolved completely, is mixed with the solution of 1mL/mL, through preparation liquid phase DAC150 forward chromatographic column (250mm × 150mm, 10 μm, 100
) be separated, determined wavelength 210nm, A, B binary-mobile phase system is adopted to carry out wash-out separation, mobile phase A and Mobile phase B adopt the combination of normal hexane and ethanol, type of elution is 100% normal hexane 5min, 100% normal hexane-70% ethanol gradient elution 40min, 100% ethanol 10min, flow velocity is 600mL/min, sample size 170mL.Collect 18-22min cut, obtain component QJZ-5 after drying.
Three, the acquisition of QJZ-5-1
By QJZ-5 component with 50% ethanol-hexane solution dissolve completely, be mixed with the solution of 60mg/mL, through preparative liquid chromatography post (ACCOHDiol, 250mm × 150mm, 10 μm, 100
) be separated, determined wavelength 210nm, adopt A, B binary-mobile phase system to carry out wash-out separation, mobile phase A and Mobile phase B adopt the combination of normal hexane and ethanol, and type of elution is 100%-85% normal hexane-ethanol isocratic elution 21min, 100% ethanol 9min, flow velocity is 3mL/min, sample size 80 μ L, collects 19-24min cut, through liquid-phase chromatographic analysis, purity is 92%.In two dimension preparation EuphorbiaFactorL2 crude product, EuphorbiaFactorL2 content is 38%.
Embodiment 2
One, the acquisition of QJZ primary extract
1, pulverize:
Getting Semen Euphorbiae is raw material, puts into pulverizer and pulverizes.
2, organic reagent extracts:
Carry out organic reagent to Semen Euphorbiae powder to extract, extract continuously for three times, each extraction time is 6h (after being wherein heated to 60 degree, reheating 6h).Concrete:
First time organic reagent extraction: 2.5kg Semen Euphorbiae powder adds 20L organic reagent, organic reagent is ethyl acetate, is stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extract 6h, extract after terminating, collect supernatant liquor, with filter paper filtering, be transferred in 60 DEG C of constant temperature blast drying ovens dry after supernatant is concentrated in Rotary Evaporators, obtain QJZ primary extract, precipitate stand-by;
Second time organic reagent extracts: first time extracts precipitation and adds 15L organic reagent, organic reagent is ethyl acetate, be stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extracts 6h, after extraction terminates, the method extracted by first time processes, and obtains QJZ primary extract, precipitates stand-by;
Third time organic reagent extraction: second time is extracted precipitation and added, organic reagent, and organic reagent is ethyl acetate, is stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extracts 6h, extracts after terminating, the method extracted by first time processes, and obtain QJZ primary extract, precipitation is dried;
Two, the acquisition of QJZ-5
By QJZ primary extract component with 30% ethanol-hexane solution dissolve completely, be mixed with the solution of 1mL/mL, through preparation liquid phase DAC150 forward chromatographic column (250mm × 150mm, 10 μm, 100
) be separated, determined wavelength 210nm, A, B binary-mobile phase system is adopted to carry out wash-out separation, mobile phase A and Mobile phase B adopt the combination of normal hexane and ethanol, type of elution is 100% normal hexane 5min, 100% normal hexane-87% ethanol gradient elution 40min, 100% ethanol 10min, flow velocity is 600mL/min, sample size 150mL.Collect 19-23min cut, obtain component QJZ-5 after drying.
Three, the acquisition of QJZ-5-1
By QJZ-5 component with 50% ethanol-hexane solution dissolve completely, be mixed with the solution of 60mg/mL, through preparative liquid chromatography post (ACCOHDiol, 250mm × 150mm, 10 μm, 100
) be separated, determined wavelength 210nm, adopts A, B binary-mobile phase system to carry out wash-out separation, and mobile phase A and Mobile phase B adopt the combination of normal hexane and ethanol, type of elution is 100%-90% normal hexane-ethanol gradient elution 30min, 100% ethanol 9min, flow velocity is 3mL/min, sample size 80 μ L, collect, 21-25min cut, through liquid-phase chromatographic analysis, purity is 94%.In two dimension preparation EuphorbiaFactorL2 crude product, EuphorbiaFactorL2 content is 41%.
Embodiment 3
One, the acquisition of QJZ primary extract
1, pulverize:
Getting Semen Euphorbiae is raw material, puts into pulverizer and pulverizes.
2, organic reagent extracts:
Carry out organic reagent to Semen Euphorbiae powder to extract, extract continuously for three times, each extraction time is 6h (after being wherein heated to 60 degree, reheating 6h).Concrete:
First time organic reagent extraction: 2.5kg Semen Euphorbiae powder adds 20L organic reagent, organic reagent is ethyl acetate, is stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extract 6h, extract after terminating, collect supernatant liquor, with filter paper filtering, be transferred in 60 DEG C of constant temperature blast drying ovens dry after supernatant is concentrated in Rotary Evaporators, obtain QJZ primary extract, precipitate stand-by;
Second time organic reagent extracts: first time extracts precipitation and adds 15L organic reagent, organic reagent is ethyl acetate, be stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extracts 6h, after extraction terminates, the method extracted by first time processes, and obtains QJZ primary extract, precipitates stand-by;
Third time organic reagent extraction: second time is extracted precipitation and added, organic reagent, and organic reagent is ethyl acetate, is stirred well to feed liquid homogeneous, without obvious bulk thing, open water bath, extracts 6h, extracts after terminating, the method extracted by first time processes, and obtain QJZ primary extract, precipitation is dried;
Two, the acquisition of QJZ-5
By QJZ primary extract component with 30% ethanol-hexane solution dissolve completely, be mixed with the solution of 1mL/mL, through preparation liquid phase DAC150 forward chromatographic column (250mm × 150mm, 10 μm, 100
) be separated, determined wavelength 210nm, A, B binary-mobile phase system is adopted to carry out wash-out separation, mobile phase A and Mobile phase B adopt the combination of normal hexane and ethanol, type of elution is 100% normal hexane 5min, 100% normal hexane-87% ethanol gradient elution 40min, 100% ethanol 10min, flow velocity is 600mL/min, sample size 150mL.As shown in Figure 1, collect 19-23min cut, obtain component QJZ-5 after drying.
Three, the acquisition of QJZ-5-1
By QJZ-5 component with 50% ethanol-hexane solution dissolve completely, be mixed with the solution of 60mg/mL, through preparative liquid chromatography post (ACCOHDiol, 250mm × 150mm, 10 μm, 100
) be separated, determined wavelength 210nm, adopts A, B binary-mobile phase system to carry out wash-out separation, and mobile phase A and Mobile phase B adopt the combination of normal hexane and ethanol, type of elution is 99%-97% normal hexane-ethanol isocratic elution 21min, 100% ethanol 9min, flow velocity is 3mL/min, sample size 100 μ L, collect 22-26min cut, through liquid-phase chromatographic analysis, as shown in Figure 2, purity is 98.5%.In two dimension preparation EuphorbiaFactorL2 crude product, EuphorbiaFactorL2 content is 45%.
Embodiment 4
The confirmation of QJZ-5-1 structure
1, HPLC carries out purity check:
By QJZ-5-1 component with 50% normal hexane-ethanolic soln all dissolve, be configured to the solution of 10mg/mL, carry out purity check through HPLC.YMC forward chromatographic column (250mm × 4.6mm, 5 μm, 100
), determined wavelength 210nm, testing conditions is: moving phase is normal hexane and ethanol, 100%-90% normal hexane-ethanol gradient degree wash-out 30min, flow velocity is 0.6mL/min, sample size: 5 μ L, temperature 30 DEG C, detect through high performance liquid phase, as shown in Figure 3, purity reaches 98.5%.
2, nuclear magnetic resonance spectroscopy:
Carry out nuclear magnetic resonance spectroscopy respectively to QJZ-5-1, BrukerA.GAVIII400PLUS, biotechnology (Tianjin) company limited of vast alliance, nuclear magnetic spectrogram as shown in Figure 4.
The material that the embodiment of the present invention relates to, reagent and experimental installation, if no special instructions, be the common commercially available prod meeting field of medicine preparation.
The present invention adopts two-dimensional liquid chromatography technology separation to obtain EuphorbiaFactorL2 (root of Beijing euphorbia factor L2) as can be seen from the above-described embodiment, with normal hexane-ethanol for moving phase, with positive InnovalSilica for one dimension preparative chromatography post, component cutting is carried out to Extract of Euphorbiae, collecting target components is EuphorbiaFactorL2 crude product, again with hydrophilic ACCOHXamide for two-dimentional preparative chromatography post, component cutting is carried out to EuphorbiaFactorL2 crude product, collect, rotary evaporation concentrates, obtain highly purified EuphorbiaFactorL2, by optimum technique and Parameter Conditions, making that EuphorbiaFactorL2 purity in product is the highest can up to 98.5%, whole process is stablized, easy and simple to handle, Semen Euphorbiae aboundresources simultaneously, easy acquisition, be applicable to the requirement of scale operation.
Above-mentioned detailed description of carrying out with reference to the preparation method of embodiment to this kind of diterpene alkoxide compound root of Beijing euphorbia factor L2; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (9)
1. a preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2, is characterized in that: concrete steps are as follows:
(1) getting Semen Euphorbiae is raw material, carries out pulverizing and obtains Semen Euphorbiae powder;
(2) get Semen Euphorbiae powder and carry out organic reagent extraction, organic reagent is ethyl acetate, collects the supernatant after extracting, and filters, concentrated, dry;
(3) precipitation step (2) obtained repeats 1 ~ 5 time and extracts, and collects the supernatant after extracting, filters, concentrated, dry, obtains QJZ primary extract;
(4) get step (3) to obtain QJZ primary extract moving phase and all dissolve, described moving phase is mobile phase A or Mobile phase B or both combinations, be separated through preparation liquid phase DAC forward chromatographic column, binary-mobile phase system is adopted to carry out wash-out separation, described mobile phase A and Mobile phase B adopt the combination of one or more in normal hexane, ethanol, type of elution gradient is washed, determined wavelength is 210nm, collect object peak, component composition QJZ-5 is obtained after drying, 60 DEG C of air dry ovens dry up, and carry out next step separation and purification;
(5) get step (4) to obtain QJZ-5 component moving phase and all dissolve, described moving phase is mobile phase A or Mobile phase B or both combinations, be separated through preparative liquid chromatography post, binary-mobile phase system is adopted to carry out wash-out separation, described mobile phase A and Mobile phase B adopt normal hexane, ethyl acetate, ethanol, methylene dichloride, the combination of one or more in methyl alcohol, type of elution isocratic elution, determined wavelength is 210nm, collect object peak, monomeric compound QJZ-5-1 is obtained after drying, 60 DEG C of air dry ovens dry up, QJZ-5-1 is final Active Target monomer diterpene alkoxide compound.
2. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, is characterized in that: in described step (2), the temperature of organic solvent extraction is 40 ~ 60 DEG C, and extraction time is 4 ~ 6h, leaves standstill a night.
3. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: the weightmeasurement ratio of Semen Euphorbiae powder described in described step (3) or precipitation and organic reagent counts 1:(3 ~ 8 by Kg:L), Semen Euphorbiae powder or precipitation are carried out mixing lixiviate with organic reagent.
4. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: the chromatographic column type that in the sepn process of described step (4), one dimension liquid chromatography adopts is purification on normal-phase silica gel axial pressure chromatographic column, flow velocity: 600ml/min, determined wavelength: 210nm, during use, column temperature is room temperature or 25-40 DEG C.
5. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: getting QJZ primary extract 60-80% normal hexane-dissolve with ethanol in the sepn process of described step (4) is 1ml/ml to concentration, after mixing, be separated through preparation liquid phase DAC forward chromatographic column with after 0.45 μm of organic membrane suction filtration.
6. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: in the sepn process of described step (4), type of elution is specially 100% normal hexane isocratic elution 5min, 100% normal hexane-70% ethanol gradient elution 35-50min, 100% ethanol 10min.
7. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: the chromatographic column type that in the sepn process of described step (5), two-dimensional liquid chromatography adopts is ACCOHXAmide chromatographic column, flow velocity: 2-4ml/min, determined wavelength: 210nm, during use, column temperature is room temperature or 25-40 DEG C.
8. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: getting QJZ-5 component 50% normal hexane-ethanol 40 DEG C in the sepn process of described step (5) ultrasonic is 50-75mg/ml to being just dissolved to concentration, with 0.45 μm of organic membrane filter.
9. the preparation method of diterpene alkoxide compound root of Beijing euphorbia factor L2 according to claim 1, it is characterized in that: in the sepn process of described step (5), type of elution is specially 100% normal hexane-90% ethanol gradient elution 30-40min, 100% ethanol 10min.
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| CN201510594625.XA CN105198751B (en) | 2014-11-28 | 2015-09-18 | A kind of diterpene alkoxide compound root of Beijing euphorbia factor L2 preparation method |
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| CN201410707604.XA CN104447339A (en) | 2014-11-28 | 2014-11-28 | Preparation method of phorbol ester compound Euphorbia Factor L2 |
| CN201410707604X | 2014-11-28 | ||
| CN201510594625.XA CN105198751B (en) | 2014-11-28 | 2015-09-18 | A kind of diterpene alkoxide compound root of Beijing euphorbia factor L2 preparation method |
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| CN201410707604.XA Pending CN104447339A (en) | 2014-11-28 | 2014-11-28 | Preparation method of phorbol ester compound Euphorbia Factor L2 |
| CN201510593383.2A Pending CN105152932A (en) | 2014-11-28 | 2015-09-18 | Preparation method of Euphorbia factor L3 |
| CN201510594542.0A Pending CN105218438A (en) | 2014-11-28 | 2015-09-18 | The preparation method of a kind of root of Beijing euphorbia factor L9 |
| CN201510594625.XA Active CN105198751B (en) | 2014-11-28 | 2015-09-18 | A kind of diterpene alkoxide compound root of Beijing euphorbia factor L2 preparation method |
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| CN201510593383.2A Pending CN105152932A (en) | 2014-11-28 | 2015-09-18 | Preparation method of Euphorbia factor L3 |
| CN201510594542.0A Pending CN105218438A (en) | 2014-11-28 | 2015-09-18 | The preparation method of a kind of root of Beijing euphorbia factor L9 |
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| CN109419797A (en) * | 2017-08-31 | 2019-03-05 | 四川大学华西医院 | Application of euphorbia factor L2 in preparation of medicine for treating rheumatoid arthritis |
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| CN104447339A (en) * | 2014-11-28 | 2015-03-25 | 天津耀宇生物技术有限公司 | Preparation method of phorbol ester compound Euphorbia Factor L2 |
| CN109419796B (en) * | 2017-08-31 | 2021-01-08 | 四川大学华西医院 | Application of euphorbia factor L2 in preparation of medicine for treating acute lung injury |
| CN113519541B (en) * | 2020-04-16 | 2022-09-13 | 江苏省中国科学院植物研究所 | Application of euphorbia factor L3 in preparation of pesticide preparation for preventing and treating plant oomycetes diseases |
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| CA2848339A1 (en) * | 2011-10-04 | 2013-04-11 | Indena S.P.A. | Method of isolating ingenol |
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| CN103130752B (en) * | 2013-02-25 | 2015-08-19 | 成都大学 | A kind of method producing root of Beijing euphorbia factor L1 from Euphorbia lathyris fruit fast |
| CN104447339A (en) * | 2014-11-28 | 2015-03-25 | 天津耀宇生物技术有限公司 | Preparation method of phorbol ester compound Euphorbia Factor L2 |
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| CA2848339A1 (en) * | 2011-10-04 | 2013-04-11 | Indena S.P.A. | Method of isolating ingenol |
Non-Patent Citations (3)
| Title |
|---|
| GIOVANNI APPENDINO等: "An Expeditious Procedure for the Isolation of Ingenol from the Seeds of Euphorbia lathyris", 《J. NAT. PROD.》 * |
| WEI JIAO等: "Euphorbia Factor L2: an ester of 7-hydroxylathyrol", 《ACTA CRYST.》 * |
| 成英等: "续随子种子中两种大环二萜类物质的提取工艺", 《时珍国医国药》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109419797A (en) * | 2017-08-31 | 2019-03-05 | 四川大学华西医院 | Application of euphorbia factor L2 in preparation of medicine for treating rheumatoid arthritis |
| CN109419797B (en) * | 2017-08-31 | 2021-01-08 | 四川大学华西医院 | Application of euphorbia factor L2 in preparation of medicine for treating rheumatoid arthritis |
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| CN105218438A (en) | 2016-01-06 |
| CN104447339A (en) | 2015-03-25 |
| CN105198751B (en) | 2017-09-22 |
| CN105152932A (en) | 2015-12-16 |
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