CN104327066B - Method for rapidly and efficiently extracting carboline alkaloids - Google Patents
Method for rapidly and efficiently extracting carboline alkaloids Download PDFInfo
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- CN104327066B CN104327066B CN201410521507.1A CN201410521507A CN104327066B CN 104327066 B CN104327066 B CN 104327066B CN 201410521507 A CN201410521507 A CN 201410521507A CN 104327066 B CN104327066 B CN 104327066B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/10—Spiro-condensed systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
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Abstract
The invention relates to a method for extracting carboline alkaloids from plant herbs. The method comprises: (1) soaking the selected herbs with ethanol, carrying out percolate extraction and pressure reducing concentration to obtain an ethanol extract, and sequentially adopting petroleum ether, ethyl acetate and n-butanol to carry out extraction treatments on the obtained ethanol extract; (2) carrying out crude extraction on the ethyl acetate extraction layer and/or the n-butanol extraction layer through silica gel column chromatography, and adopting a TLC thin layer chromatography method to track, wherein the eluent is selected from a chloroform-methanol system, a petroleum ether-acetone system and a petroleum ether-ethyl acetate system; (3) carrying out chromatography separation on the crude extract through an MCI gel CHP-20P column, and adopting a TLC thin layer chromatography method to track; (4) carrying out chromatography separation on the extract obtained in the step (3) through an Sephadex LH-20 dextran gel column, wherein a methanol-chloroform solution is adopted as an eluent; and (5) purifying the extract obtained in the step (4) through HPLC to obtain the carboline alkaloid product, wherein the chromatography column is the reversed-phase column, and the mobile phase is the methanol-water solution. With the method of the present invention, the alkaloid with the high purity (more than 99%) can be obtained through purification, and the extraction yield of the method of the present invention is much higher than the extraction yield of the conventional method.
Description
Technical field
The present invention relates to a kind of method that can be used for card Berlin Alkaloid in quick, high efficiency extraction plant.
Background technology
Chinese medicine be the mankind's thousands of years since during being struggled with disease and the pain of injury, by continuous cognitive, continuous reality
Trample, the treasure that accumulation is obtained, is to condense mankind's more than one thousand years crystallization of wisdom.Chinese medicine is general essentially from plant, animal
Deng species is more.Great physician's Li Shizhen (1518-1593 A.D.) of the Ming Dynasty exists《Classified Materia Meidca》On the basis of revised, weaved into and met the epoch
The monumental work of development need-《Compendium of Materia Medica》, this book includes 1892 kinds of Chinese herbal medicine, subsidiary formula more than 11000,《A Supplement to the Compendium of Materia Medica》
1021 kinds of Chinese herbal medicines are supplemented again.According to correlation study data display, in more than 20000 kinds of higher plant in the world, once carried out
Cross activity research or screening active ingredients less than 15%.Therefore, the potentiality of Natural products research work are huge.
In ancient times, due to being restricted by scientific and technological level at that time, the drug effect and its mechanism of action of many Chinese herbal medicines are failed to understand
Really.And in recent years, as people progressively deepen to the attention degree of own health, the cry more and more higher of back to nature, and
Scientific and technical develops rapidly, and the drug efficacy study of the natural products such as Chinese herbal medicine and Chinese patent drug is obtained for the weight of height in the whole world
Depending on.The New Drug Research work of various countries is also carried out without any confusion tightened around natural products.
The active component of natural products is found, and it is that natural product chemistry grinds further to disclose its drug effect and its mechanism of action
Study carefully the key problem in field, and the key point of this problem is to seek advanced to efficiently separate purification analysis method.In other words, day
Material in right product efficiently separate and Structural Identification be natural products activity research basis, be natural product chemistry research
Intermediate portions in field.
Alkaloid be present in nature (predominantly plant, but have exist in animal) in the nitrogenous alkalescence of a class
Organic compound, there is the property like alkali, so being also called counterfeit alkali in the past.Most of cyclic structures for having a complexity, include nitrogen more
In ring, there is significant bioactivity, be one of important active ingredient in Chinese herbal medicine.Alkaloid has cyclic structure, is insoluble in
Water, can have certain optical activity and absorption spectrum with acid with forming salt, there is bitter taste mostly.In colorless crystals, minority is liquid
Body.Alkaloid has thousands of kinds, is synthesized by different amino acid or its direct derivative, is one of secondary metabolites, opposite
The toxic or strong physiological action of thing body.
There is traditional separation method in natural product chemistry：Solvent extraction method, extraction, precipitation, crystallization and filtering.With
Natural product chemistry and isolation technics are continued to develop, efficient novel isolation technics natural product chemistry research at home and abroad
In be widely used, including：Chromatographic separation technology, supercritical liquid extraction technique, membrane separation technique, macropore tree
Fat adsorption separation technology, molecular distillation technique etc..
The architectural feature of card Berlin Alkaloid is, its precursor structure is the basic framework of C6-N1-C5, and its side chain can be with
The polarity for having various changes, whole compound changes as side chain connects the polarity of group.
Traditional card Berlin extracting method, the method for depending on silica gel column chromatography or recrystallization.But this method mainly for
In the compound that polarity is medium or polarity is less than normal, if the polarity of compound is larger, this separation and Extraction side of silica gel column chromatography
The effect and efficiency of method be not then good；And if select recrystallization method, how to choose suitable solvent is more scabrous asking
Topic.Additionally, the card Berlin alkaloid obtained with silica gel column chromatography separation and Extraction, its purity are nor very well.
The content of the invention
During the invention reside in overcoming the shortcomings of that existing isolation technics is separated for card Berlin series alkaloid, in tradition
On the basis of isolation technics, a kind of new separation and Extraction purification process of feature extraction according to card Berlin series alkaloid.
The present invention provides a kind of method that card Berlin Alkaloid is extracted from vegetable drug, including step：(1) choose
Medicinal material is soaked with ethanol, seepage pressure effects, and ethanol extract is obtained after being concentrated under reduced pressure, and resulting ethanol extract uses petroleum ether, second successively
Acetoacetic ester and n-butanol carry out extraction processing；(2) by the ethyl acetate extract layer in (1) and/or extracting n-butyl alcohol layer silica gel
Column chromatography carries out coarse extraction, and eluant, eluent is selected from chloroform-methanol system, petroleum ether-acetone system and petroleum ether-ethyl acetate body
System, is tracked with TLC thin-layered chromatography；(3) by the CE in (2) with MCI gel CHP-20P column chromatography for separation, with
Methanol-water solution is eluant, eluent, is tracked with TLC thin-layered chromatography；(4) extract that will be obtained in (3) is with Sephadex
LH-20 sephadex column chromatography for separation, with methanol-chloroform solution as eluant, eluent, is tracked with TLC thin-layered chromatography；And
(5) extract obtained in (4) is obtained into card Berlin Alkaloid product with the purifying of HPLC efficient liquid phases, wherein chromatographic column is anti-
Xiang Zhu, mobile phase is methanol-water solution.
According to the method for the specific embodiment of the invention, wherein the medicinal material is white bur, in vain thorn stalk, Vietnam's Common Leafflower Herb, tiger
Skin nanmu or plain rice tree.
According to the method for the specific embodiment of the invention, purified through the step (5), corresponded in the white bur, in vain
Thorn stalk, Vietnam's Common Leafflower Herb, leaf or seed of Common Daphniphyllum and plain rice tree, respectively obtain card Berlin Alkaloid of following structural formula：
According to the method for the specific embodiment of the invention, the wherein eluant, eluent in step (2) is selected from 100:1~3:1 chloroform-
Methanol system, 5:1~1:1 petroleum ether-acetone system and 4:1~1:1 petroleum ether-ethyl acetate system wherein step (3)
In with 3:1~1:3 methanol-water solution is eluant, eluent；With 1 in step (4):1~1:3 methanol-chloroform solution is eluant, eluent；
Mobile phase in step (5) is 1:4~4:1 methanol-water solution system, the reversed-phase column is C18-ODS-A posts.
According to the inventive method, the alkaloid for obtaining purity (more than 99%) very high can be purified, and extract yield is remote
Higher than existing method.
Brief description of the drawings
Fig. 1 is the nmr spectrum that embodiment 1 obtains card Berlin Alkaloid；And
Fig. 2 is the LC-ESI-MS spectrograms that embodiment 1 obtains card Berlin Alkaloid.
Specific embodiment
The present invention is discussed further below in conjunction with example.Implementation below is preferably in implementation method
One kind, not limitation of the present invention.It is other it is any without departing from the change made under Spirit Essence of the invention and principle,
Modify, substitute, combine, simplify, should be equivalent substitute mode, be included within protection scope of the present invention.Spy is not made
The experiment reagent and method of different explanation, refer both to conventional reagent and method.
In particular by embodiment 1-5 and comparative example 1-2, the characteristics of exemplary illustration the inventive method and advantage.
Embodiment 1：
The extraction and purification of β-card Berlin -4- methoxyl group -2- hexenoic acid methyl esters：
(1), initial processing steps：4.1 kilograms of white burs (picking up from Chaidamu Basin, Qinghai Province).
Soaked three days with the ethanol of 25L 95%, room temperature seepage pressure effects are repeated two more times, extract solution merges, being concentrated under reduced pressure will
Ethanol all evaporates, and obtains ethanol extract 230g.By medicinal extract with deionized water dissolving (about 3L) after, successively with petroleum ether, acetic acid second
Ester and n-butanol (each 3L × 3) are extracted.Then, ethyl acetate and n-butanol extracting liquid are concentrated under reduced pressure respectively, respectively obtain second
Acetoacetic ester medicinal extract 68.3g, n-butanol medicinal extract 153.2g.
The medicinal extract obtained after ethyl acetate extraction part concentration, is dissolved with 150mL chloroforms, admixes 100~200 mesh of 120g
Thick silica gel, naturally volatilization removes solvent, is made the medicinal extract silica white of yellow.Weigh the thick silica gel of 2Kg100~200 mesh, wet method dress
Post, loading uses petroleum ether-ethyl acetate solvent system.Gradient elution, eluant strength scope is：Pure petroleum ether, petroleum ether：
Ethyl acetate=3:1, relevant fractions, using TLC thin-layer chromatography tracking and monitorings, ten are merged into according to monitoring result by pure acetone
Group section.
Each group section reuses silica gel column chromatography.Eluent system is petroleum ether-acetone, chloroform-methanol, petroleum ether-acetic acid
Ethyl ester, chloroform-acetone, chlorofonn-ethylacetate.
(2), MCI gel CHP-20P column chromatographies：The result followed the trail of according to TLC thin-layer chromatographys after early stage treatment, by card
Component screening where the Alkaloid of Berlin goes out.Then MCI gel CHP-20P column chromatography for separation is used, it is water-soluble with 50% methyl alcohol
Liquid is eluant, eluent, removes the material of big polarity, using TLC thin-layer chromatography tracking and monitorings, relevant fractions is closed according to monitoring result
And.
(3), Sephadex LH-20 sephadexes column chromatography：It will be purified by MCI gel CHP-20P column chromatographies
Component afterwards, uses chloroform：Methyl alcohol=1:1 solution dissolving, and with chloroform：Methyl alcohol=1:1 solution is eluant, eluent, is used
Sephadex LH-20 sephadex column chromatographies are separated.Sephadex LH-20 sephadex column column volumes are
100ml, then elution volume be 400ml.
Removed using Sephadex LH-20 sephadex columns and larger impurity is differed with alkaloid character, use TLC
Thin-layer chromatography tracking and monitoring, merges relevant fractions according to monitoring result, and is concentrated under reduced pressure, and boils off solvent.
(4), HPLC efficient liquid phases purifying：The sample 500mg that will be processed more than, is dissolved with methyl alcohol.
Purified using HPLC efficient liquid phases, its specific purification condition is：Mobile phase selection is 45% methanol aqueous solution,
Chromatographic column is C18-ODS-A (50 μm) reversed-phase column, and room temperature condition is 25 DEG C, and the compound at retention time 16min is to be wanted
Target compound, finally give target compound 350mg, recovery rate is 0.5%, and purity is 99.9%.
Traditional extracting method, it is simple using this method of silica gel column chromatography, recovery rate be about 0.07% (details referring to
The Tetrahedron Letters.1999,40 such as Duan J A (13):2593-2596).The extraction of the remote ultra-traditional method of this method
Rate.
It is below the collection of illustrative plates parameter of product.
1H NMR(400MHz,DMSO-d6)δ(ppm):12.19(1H,s,H/D exchangeable),8.71(1H,d,J
=16.0Hz), 8.61 (1H, d, J=4.9Hz), 8.55 (1H, d, J=4.9Hz), 8.35 (1H, d, J=8.0Hz), 7.83
(1H, d, J=8.3Hz), 7.63 (1H, ddd, J1=8.2Hz, J2=7.1Hz, J3=1.1Hz), 7.35 (1H, ddd, J1=
8.0Hz, J2=7.2Hz, J3=1.2Hz), 6.95 (1H, d, J=16.0Hz), 3.82 (3H, s)；LC-ESI-MS:M/z=
281.1[M+H]+(positive).
Embodiment 2：
The extraction and purification of (3S, 4R) -1- β-card Berlin -3,4,5- trihydroxy ketone
(1), initial processing steps：3.6 kilograms of white thorns stalk (picking up from Chaidamu Basin, Qinghai Province) are soaked with the ethanol of 25L 95%
Three days, room temperature seepage pressure effects were repeated two more times, and extract solution merges, and ethanol extract is obtained after being concentrated under reduced pressure.By medicinal extract deionized water
After dissolving (about 3L), extracted with petroleum ether, ethyl acetate and n-butanol (each 3L × 3) successively.Then, ethyl acetate and positive fourth
Alcohol extract is concentrated under reduced pressure respectively, respectively obtains ethyl acetate extract 43.8g, n-butanol medicinal extract 103.2g.N-butanol leaching afterwards
The process step of cream is identical with step (1) in embodiment 1.
(2), MCI gel CHP-20P column chromatographies：The result followed the trail of according to TLC thin-layer chromatographys afterwards, by the life of card Berlin class
Component screening where alkaloids goes out.Then MCI gel CHP-20P column chromatography for separation is used, is wash-out with 45% methanol aqueous solution
Agent, removes the material of big polarity, using TLC thin-layer chromatography tracking and monitorings, relevant fractions is merged according to monitoring result.
(3), Sephadex LH-20 sephadexes column chromatography：It will be purified by MCI gel CHP-20P column chromatographies
Component afterwards, uses chloroform：Methyl alcohol=1:1 solution dissolving, and with chloroform：Methyl alcohol=1:1 solution is eluant, eluent, is used
Sephadex LH-20 sephadex column chromatographies are separated, and the amount according to component selects suitable column volume, and elutes body
Long-pending selection is identical with the method in embodiment 1.In this step, Sephadex LH-20 sephadex column column volumes are
120ml, then elution volume be 480ml.Removed using Sephadex LH-20 sephadex columns and differed with alkaloid character
Larger impurity, using TLC thin-layer chromatography tracking and monitorings, method for subsequent processing is in the same manner as in Example 1.
(4), HPLC efficient liquid phases purifying：The sample 600mg that will be processed more than, is dissolved with methyl alcohol, high using HPLC
Effect liquid phase purifying, is purified using HPLC efficient liquid phases, and its specific purification condition is：Mobile phase selection is water-soluble for 43% methyl alcohol
Liquid, chromatographic column is C18-ODS-A (50 μm) reversed-phase column, and room temperature condition is 25 DEG C, and the compound at retention time 14.8min is
Desired target compound, finally gives target compound 380mg, and recovery rate is 0.37%, and purity is 99.9%.Traditional carries
Method is taken, typically just simple using this method of silica gel column chromatography, recovery rate is about 0.09%, and this method exceeds conventional method
Recovery rate.
Product spectrum data is as follows
1H NMR(400MHz,DMSO-d6)δ(ppm):8.50 (1H, d, J=4.9Hz), 8.43 (1H, d, J=4.9Hz),
8.30 (1H, d, J=7.8Hz), 7.81 (1H, d, J=8.2Hz), 7.59 (1H, t, J=4.3Hz), 7.30 (1H, t, J=
4.9Hz), 4.72 (1H, m), 4.46 (1H, s), 4.14 (1H, m), 3.56 (2H, dd, J1=15.6Hz, J2=9.0Hz), 3.27
(3H, dd, J1=15.5Hz, J2=3.4Hz), 3.16 (1H, d, J=4.8Hz), 1.23 (1H, s)；LC-ESI-MS:M/z=
301.1[M+H]+(positive).
Embodiment 3：
The extraction and purification of O- acetyl group nitrains：
(1), initial processing steps：7.6 kilograms of Vietnam's Common Leafflower Herbs (picking up from Nanning) soak three with the ethanol of 25L 95%
My god, room temperature seepage pressure effects are repeated two more times, and extract solution merges, and ethanol extract is obtained after being concentrated under reduced pressure.Medicinal extract is molten with deionized water
After solution (about 3L), extracted with petroleum ether, ethyl acetate and n-butanol (each 3L × 3) successively.Then, ethyl acetate and n-butanol
Extract solution is concentrated under reduced pressure respectively, respectively obtains ethyl acetate extract 83.8g, n-butanol medicinal extract 183.7g.Ethyl acetate leaching afterwards
The process step of cream is identical with step (1) in embodiment 1.
(2), MCI gel CHP-20P column chromatographies：The result followed the trail of according to TLC thin-layer chromatographys afterwards, by the life of card Berlin class
Component screening where alkaloids goes out.Then MCI gel CHP-20P column chromatography for separation is used, is wash-out with 50% methanol aqueous solution
Agent, removes the material of big polarity, using TLC thin-layer chromatography tracking and monitorings, relevant fractions is merged according to monitoring result.
(3), Sephadex LH-20 sephadexes column chromatography：It will be purified by MCI gel CHP-20P column chromatographies
Component afterwards, is dissolved, and with pure methyl alcohol as eluant, eluent, entered using Sephadex LH-20 sephadex column chromatographies with methyl alcohol
Row is separated, and the amount according to component selects suitable column volume, and the selection of elution volume is identical with the method in embodiment 1.This
In step, Sephadex LH-20 sephadex columns column volume is 150ml, then elution volume is 600ml or 750ml.Make
With Sephadex LH-20 sephadex columns remove larger impurity is differed with alkaloid character, using TLC thin-layer chromatographies with
Track is monitored, and method for subsequent processing is in the same manner as in Example 1.
(4), HPLC efficient liquid phases purifying：The sample 750mg that will be processed more than, is dissolved with methyl alcohol, high using HPLC
Effect liquid phase is purified, and its specific purification condition is：Mobile phase selection is 41% methanol aqueous solution, and chromatographic column is C18-ODS-A
(50 μm) reversed-phase column, room temperature condition is 25 DEG C, and compound is desired target compound at retention time 15.4min, finally
Target compound 580mg is obtained, recovery rate is 0.69%, and purity is 99.9%.
Traditional extracting method, simply simple using this method of silica gel column chromatography, recovery rate is about 0.36%, details ginseng
See the Tetrahedron such as T.S.Tulyaganov Letters.1999,40 (13):2593-2596Chemistry of
Natural Compounds.2005,41(5):Recovery rate of the 578-579. this method beyond conventional method.
It is below product spectrum data.
1H NMR(400MHz,DMSO-d6)δ(ppm):8.65 (1H, d, J=4.9Hz), 8.53 (1H, d, J=4.9Hz),
8.40 (1H, d, J=7.8Hz), 7.87 (1H, d, J=8.2Hz), 7.69 (1H, t, J=4.3Hz), 7.40 (1H, t, J=
4.9Hz), 4.92 (1H, m), 4.64 (1H, s), 4.34 (1H, m), 3.76 (2H, dd, J1=15.6Hz, J2=9.0Hz), 3.67
(3H, dd, J1=15.5Hz, J2=3.4Hz), 3.46 (1H, d, J=4.8Hz), 1.53 (1H, s)；LC-ESI-MS:M/z=
309.2[M+H]+(positive).
Embodiment 4：
The extraction and purification of acetyl group Ke Maka Berlin：
(1), initial processing steps：9.3 kilograms of leaf or seed of Common Daphniphyllum (picking up from Fujian) are soaked three days with the ethanol of 25L 95%, room temperature
Seepage pressure effects, are repeated two more times, and extract solution merges, and ethanol extract is obtained after being concentrated under reduced pressure.By medicinal extract with deionized water dissolving (about
After 3L), extracted with petroleum ether, ethyl acetate and n-butanol (each 3L × 3) successively.Then, ethyl acetate and n-butanol are extracted
Liquid is concentrated under reduced pressure respectively, respectively obtains ethyl acetate extract 106.3g, n-butanol medicinal extract 197.1g.Ethyl acetate extract afterwards
Process step is identical with step (1) in embodiment 1.
(2), MCI gel CHP-20P column chromatographies：The result followed the trail of according to TLC thin-layer chromatographys afterwards, by the life of card Berlin class
Component screening where alkaloids goes out.Then MCI gel CHP-20P column chromatography for separation is used, is wash-out with 40% methanol aqueous solution
Agent, removes the material of big polarity, using TLC thin-layer chromatography tracking and monitorings, relevant fractions is merged according to monitoring result.
(3), Sephadex LH-20 sephadexes column chromatography：It will be purified by MCI gel CHP-20P column chromatographies
Component afterwards, is dissolved, and with methyl alcohol as eluant, eluent, carried out using Sephadex LH-20 sephadexes column chromatographies with methyl alcohol
Separate, the amount according to component selects suitable column volume, and the selection of elution volume is identical with the method in embodiment 1.This step
In rapid, Sephadex LH-20 sephadex columns column volume is 180ml, then elution volume is 720ml or 900ml.Use
Sephadex LH-20 sephadex columns remove and larger impurity are differed with alkaloid character, are tracked using TLC thin-layer chromatographies
Monitoring, merges relevant fractions according to monitoring result, and method for subsequent processing is in the same manner as in Example 1.
(4), HPLC efficient liquid phases purifying：The sample 750mg that will be processed more than, is dissolved with methyl alcohol, high using HPLC
Effect liquid phase is purified, and its specific purification condition is：Mobile phase selection is 40% methanol aqueous solution, and chromatographic column is C18-ODS-A
(50 μm) reversed-phase column, room temperature condition is 25 DEG C, and the compound at retention time 16.3min is desired target compound, most
Target compound 530mg is obtained eventually, and recovery rate is 0.49%, and purity is 99.9%.Traditional extracting method, recovery rate is
0.39%, recovery rate of this method beyond conventional method.
It is below product spectrum data.
1H NMR(400MHz,DMSO-d6)δ(ppm):1.30-1.80 (8H, m), 2.21 (3H, s), 2.77 (2H, t, J=
6.0Hz), 2.85-3.12 (2H, m), 3.72 (2H, t, J=6.0Hz), 7.08 (2H, m), 7.23 (1H, m), 7.42 (1H, m),
8.26(1H,brs)；LC-ESI-MS:M/z=283.3 [M+H]+(positive)
Embodiment 5：
The extraction and purification of tetrahydrochysene amine nitrain：
(1), initial processing steps：7.3 kilograms of plain rice trees (picking up from Nanning) are soaked three days with the ethanol of 25L 95%,
Room temperature seepage pressure effects, are repeated two more times, and extract solution merges, and ethanol extract is obtained after being concentrated under reduced pressure.By medicinal extract deionized water dissolving
After (about 3L), extracted with petroleum ether, ethyl acetate and n-butanol (each 3L × 3) successively.Then, ethyl acetate and n-butanol are carried
Take liquid to be concentrated under reduced pressure respectively, respectively obtain ethyl acetate extract 86.3g, n-butanol medicinal extract 127.1g.Ethyl acetate extract afterwards
Process step it is identical with step (1) in embodiment 1.
(2), MCI gel CHP-20P column chromatographies：The result followed the trail of according to TLC thin-layer chromatographys afterwards, by the life of card Berlin class
Component screening where alkaloids goes out.Then MCI gel CHP-20P column chromatography for separation is used, is wash-out with 50% methanol aqueous solution
Agent, removes the material of big polarity, using TLC thin-layer chromatography tracking and monitorings, relevant fractions is merged according to monitoring result.
(3), Sephadex LH-20 sephadexes column chromatography：It will be purified by MCI gel CHP-20P column chromatographies
Component afterwards, is dissolved, and with methyl alcohol as eluant, eluent, carried out using Sephadex LH-20 sephadexes column chromatographies with methyl alcohol
Separate, the amount according to component selects suitable column volume, and the selection of elution volume is identical with the method in embodiment 1.This step
In rapid, Sephadex LH-20 sephadex columns column volume is 200ml, then elution volume is 800ml or 1000ml.Use
Sephadex LH-20 sephadex columns remove and larger impurity are differed with alkaloid character, are tracked using TLC thin-layer chromatographies
Monitoring, merges relevant fractions according to monitoring result.
(4), HPLC efficient liquid phases purifying：The sample 700mg that will be processed more than, is dissolved with methyl alcohol, high using HPLC
Effect liquid phase is purified, and its specific purification condition is：Mobile phase selection is 35% methanol aqueous solution, and chromatographic column is C18-ODS-A
(50 μm) reversed-phase column, room temperature condition is 25 DEG C, and the compound at retention time 15.1min is desired target compound, most
Target compound 550mg is obtained eventually, and recovery rate is 0.64%, and purity is 99.9%.Traditional extracting method, recovery rate is
0.27%, this method exceeds the recovery rate of conventional method beyond the recovery rate of conventional method.
It is below product profiling results.
1H NMR(400MHz,DMSO-d6)δ(ppm):7.30(2H,m),7.48(3H,m),7.52(1H,m),7.62
(1H, m), 7.94 (1H, dd, J1=8Hz, J2=2Hz), 8.10 (1H, d, J=6Hz), 8.19 (1H, dd, J1=9Hz, J2=
2Hz), 8.51 (1H, d, J=7Hz), 8.67 (1H, d, J=5Hz) .LC-ESI-MS:M/z=300.3 [M+H]+
(positive).
Comparative example 1
Raw material：It is identical with raw material in embodiment 1, it is the white bur for picking up from the Caidamu Basin, weight is 4.5 kilograms.
Experimentation：Initial processing steps are in the same manner as in Example 1, obtain ethyl acetate extract 70g.Then with tradition
Extracting method, i.e., be eluant, eluent with chloroform-methanol, eluant strength ratio is chloroform：Methyl alcohol=10:1, simply use
The method of silica gel column chromatography, separates repeatedly, and period is not high using the MCI gel CHP-20P column chromatographies and HPLC in the present invention
Effect liquid phase.
Extract result：Target compound 54.6mg is obtained, recovery rate is 0.078%.
Comparative example 2
Raw material：It is identical with raw material in embodiment 2, it is the white bur for picking up from the Caidamu Basin, weight is 4 kilograms.
Experimentation：Initial processing steps are in the same manner as in Example 1, obtain n-butanol medicinal extract 105.3g.Then with biography
The extracting method of system, i.e., be eluant, eluent with chloroform-methanol, and eluant strength ratio is chloroform：Methyl alcohol=8:1, only make
With the method for silica gel column chromatography, separate repeatedly, period does not use the MCI gel CHP-20P column chromatographies and HPLC in the present invention
Efficient liquid phase.
Extract result：Target compound 4.6mg is obtained, recovery rate is 0.0044%.
Conclusion：It is single that embodiment and the contrast of comparative example can be seen that traditional extraction process means, substantially by silica gel
Column chromatography a kind of this method is extracted, and once runs into the larger compound of polarity, then recovery rate can be greatly lowered (such as right
Ratio 2).And the method for the present invention, with reference to various separating and extracting process, especially MCI gel CHP-20P column chromatographies and HPLC
The use of efficient liquid phase so that the compound in face of general polarity is quickly and efficiently to separate；And it is bigger than normal to face polarity
During compound, MCI gel CHP-20P column chromatographies and HPLC efficient liquid phases can be separated pointedly, so that still can be preferable
Ground reaches separating effect.Additionally, the ultraviolet detection in HPLC efficient liquid phases, the compound that isolates and purifies can be intuitive to see
How is purity.Method provided by the present invention can be widely used in the mask work of card Berlin Alkaloid.
Above-described embodiment is not limited for the present invention preferably implementation method, but embodiments of the present invention by above-described embodiment
System.The deformation made under other any spirit and principle for not departing from the present invention, is considered as protection scope of the present invention.
Claims (5)
1. a kind of method that card Berlin Alkaloid is extracted from vegetable drug, it is characterised in that methods described includes following step
Suddenly：
(1) medicinal material chosen is soaked with ethanol, seepage pressure effects, then it is concentrated under reduced pressure after ethanol extract, gained ethanol extract is successively
Extraction processing is carried out with petroleum ether, ethyl acetate and n-butanol；
(2) the ethyl acetate extract layer in step (1) and/or extracting n-butyl alcohol layer are carried out into coarse extraction with silica gel column chromatography, is washed
De- agent is selected from chloroform-methanol system, petroleum ether-acetone system and petroleum ether-ethyl acetate system, is entered with TLC thin-layered chromatography
Row is followed the trail of；
(3) it is wash-out with methanol-water solution by the CE in step (2) with MCI gel CHP-20P column chromatography for separation
Agent, is tracked with TLC thin-layered chromatography；
(4) extract that will be obtained in step (3) is with Sephadex LH-20 sephadex column chromatography for separation, with methyl alcohol-chlorine
Imitative solution is eluant, eluent, is tracked with TLC thin-layered chromatography；And
(5) extract that will be obtained in step (4) is purified with HPLC efficient liquid phases, obtains card Berlin Alkaloid product, wherein
Chromatographic column is reversed-phase column, and mobile phase is methanol-water solution；
Wherein, the medicinal material is white bur, in vain thorn stalk, Vietnam's Common Leafflower Herb, leaf or seed of Common Daphniphyllum or plain rice tree；
Correspond in the white bur, pierce stalk, Vietnam's Common Leafflower Herb, leaf or seed of Common Daphniphyllum or plain rice tree, card Berlin described in step (5) in vain
Alkaloid product is followed successively by following compounds：
2. method according to claim 1, the wherein eluant, eluent in step (2) is selected from 100: 1~3: 1 chloroform-methanol body
System, 5: 1~1: 1 petroleum ether-acetone system and 4: 1~1: 1 petroleum ether-ethyl acetate system.
3. method according to claim 1, with 3: 1~1: 3 methanol-water solution is eluant, eluent wherein in step (3).
4. method according to claim 1, with 1: 1~1: 3 methanol-chloroform solution is eluant, eluent wherein in step (4).
5. method according to claim 1, the wherein mobile phase in step (5) for 1: 4~4: 1 methanol-water solution body
System, the reversed-phase column is C18-ODS-A posts.
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