CN102432419A - Method for extracting and separating beta-elemene from Eupatorium adenophorum - Google Patents
Method for extracting and separating beta-elemene from Eupatorium adenophorum Download PDFInfo
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Abstract
The invention belongs to the field of plant extraction, and aims to provide a method for extracting and separating beta-elemene from a noxious weed plant Eupatorium adenophorum. The method provided by the invention comprises the following steps: A. raw material preparation: carrying out pulping, or drying and pulverizing on fresh stems and leaves of Eupatorium adenophorum as a raw material; B. volatile oil extraction: acquiring volatile oil from the raw material; C. column chromatography: carrying out column chromatography on the volatile oil acquired in the step B, collecting an eluate, and removing solvent in the eluate to acquire a crude beta-elemene product; and D. refinement: refining the volatile oil acquired in the step B or the crude product acquired in the step C by a molecular distillation or preparation-type high efficiency liquid chromatography system to acquire the high-purity beta-elemene. The invention has the advantages of high product purity, high yield and low cost, and provides a brand-new, cheap and effective way for the development and treatment of Eupatorium adenophorum and the acquisition of beta-elemene.
Description
Technical field
The invention belongs to field of plant extraction, be specifically related to the method for extraction separation beta-elemene from malignant weed plant Hemp Eupatorium.
Background technology
Hemp Eupatorium (Eupatorium adenophorum Spreng), another name are destroyed grass, liberation grass, Herba Eupatorii Odorati etc., are the composite family feverwort.This plant distributes extensively on Yunnan Province of China, Guangxi, Sichuan and other places, and resource is very abundant, belongs to the torrid zone, subtropics invasive weed plant, causes very big harm for local husbandry production, and local ecological security is caused very big threat.
Beta-elemene, Latin is called β-Elemenum, English β-elemene by name, chemistry (1S, 2S, 4R) 1-methyl isophthalic acid-vinyl-2,4-diisopropenyl hexanaphthene by name.Structural formula is following:
Beta-elemene is that China passes through a series of State Commission for Restructuring the Economic Systems projects such as " the Seventh Five-Year Plan ", " eight or five ", " 95 "; It is the anti-cancer agent that independent research goes out from traditional Chinese medicine; Be applied to clinically, and be put into national essential drugs and national medical insurance medication catalogue.Clinical application shows; Beta-elemene not only has very strong killing tumour cell and suppress the tumor growth effect; Can also make body obtain tangible immunoprotection effect through activating immune system; Have characteristics such as anticancer spectrum is wide, efficient, no hepatorenal damage, no bone marrow depression, few side effects, be the mankind from now on one period the internal therapy tumour choice drug, the domestic and international market demand is big.At present, beta-elemene mainly depends on from Chinese medicine curcuma zedoary, myrrh, lemongrass, Herba Solidaginis and extracts, and finds to have from Hemp Eupatorium extraction separation to obtain the report of beta-elemene as yet.Because Chinese medicine curcuma zedoary, myrrh, lemongrass, Herba Solidaginis are Chinese medicine, cost is higher relatively, and wherein the content of beta-elemene is not high, and the cost that extracts purifying is higher, causes the price of beta-elemene very expensive, has seriously hindered applying of this medicine.
Contriver of the present invention finds in the Hemp Eupatorium beta-elemene content far above Chinese medicine curcuma zedoary, myrrh, lemongrass, Herba Solidaginis, and Hemp Eupatorium is the invasive weed plant, and aboundresources, its raw materials cost are also low more than these raw materials.So the present invention desires to provide a kind of method of from Hemp Eupatorium, effectively extracting beta-elemene, for the production cost and the exploitation that reduce beta-elemene significantly, utilize, administer this worldwide malignant weed plant of Hemp Eupatorium and opened up new approach.
Summary of the invention
The technical problem that the present invention solved provide a kind of from Hemp Eupatorium the method for extraction separation beta-elemene.
Detect discovery through the contriver: the beta-elemene in the Hemp Eupatorium mainly is distributed in its leaf and the stem and (accounts for more than 90% of herb total amount).Wherein, with content in the leaf the highest (being about 0.4-0.6%), and content is extremely low or do not have basically in other organs such as root, flower.Comparative analysis is the result show, beta-elemene content is far above the content of Chinese medicine curcuma zedoary, myrrh, lemongrass, Herba Solidaginis in the Hemp Eupatorium leaf.
Employed raw material is mainly the stem of Hemp Eupatorium, at least a or its mixing in the leaf in the process for extracting of the present invention; Can not influence the extraction separation beta-elemene if be mixed with other organs such as root, flower, fruit in the raw material yet; But can influence the yield of process for extracting, so preferably be mixed into raw material with at least a in the stem of Hemp Eupatorium, the leaf or its.
Process for extracting of the present invention is following:
A, raw material prepare: mainly be mixed into raw material with at least a in the stem of Hemp Eupatorium, the leaf or its, bright article are pulled an oar or drying and crushing;
B, volatile oil extract: from steps A gained raw material, extract and obtain volatile oil;
C, column chromatography: the volatile oil that step B is obtained carries out column chromatography, collects elutriant, removes the solvent in the elutriant, obtains the beta-elemene bullion.
Further, the beta-elemene bullion that volatile oil that can also step B be obtained or step C obtain carries out further refining, obtains the higher beta-elemene highly finished product of purity.
Be that purification step D is: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain, adopt short-path distillation or preparative high performance liquid chromatography system make with extra care the highly purified beta-elemene of acquisition.
Adopting aforesaid method to handle the stem of Hemp Eupatorium, leaf step B extracts and can obtain beta-elemene content usually greater than 20% volatile oil; Adopt method such as column chromatography to handle and can obtain beta-elemene content usually greater than 40%, even up to 60% beta-elemene bullion; Finally, further refined volatile oil or beta-elemene bullion are collected the purifying composition that contains beta-elemene according to " target content ", can obtain beta-elemene content usually greater than 90%, even up to 98% beta-elemene highly finished product.
Can adopt one of following five kinds of methods to extract volatile oil when wherein, extracting volatile oil:
Method one: steam distillation:
(1) raw material through processing of step A carries out air distillation, obtains distillate;
(2) in distillate, add boiling point less than 60 ℃ non-polar organic solvent, upper organic phase is collected in extraction, removes the organic solvent in the upper organic phase, promptly gets volatile oil.Wherein, preferably primary solvent during distillation with water, add and improve the extraction system boiling point and prevent the blistered material of saponification.As, adding terepthaloyl moietie is the temperature that improves distillation azeotropic liquid, adding ethanol is to eliminate the saponification foaming phenomenon that produces in the distillation, adds zeolite and be bumping when preventing to distill.So existing, known, conventional, foreseeable, material replacement terepthaloyl moietie with above-mentioned function capable of using, ethanol, zeolite are to reach identical purpose.
Method two: pickling process:
(1) raw material through processing of step A adds at least a or its arbitrary proportion blended organic solvent lixiviate, filtration in acetone, sherwood oil or the methyl alcohol, gets vat liquor;
(2) vat liquor removes the ethanol that the back of desolvating adds concentration 20-80%v/v, and the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
Method three: Continuous Countercurrent Extraction method:
(1) raw material through processing of step A adds at least a or its arbitrary proportion blended organic solvent Continuous Countercurrent Extraction in acetone, sherwood oil or the methyl alcohol, gets extracting solution;
(2) ethanol of adding concentration 20-80%v/v behind the solvent in the removal extracting solution, the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
Method four: microwave extraction method:
(1) raw material through processing of step A adds at least a or its arbitrary proportion blended organic solvent in acetone, sherwood oil or the methyl alcohol, in microwave extracting apparatus, adopts power 300-800W extraction 300-800s, gets extracting solution;
(2) ethanol of adding concentration 20-80%v/v behind the extracting solution removal solvent, the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
Method five: supercritical extraction:
(1) through the raw material of processing of step A, uses supercritical CO
2The fluid extraction device extracts, extracting pressure 20-30MPa, and extraction temperature 30-50 ℃, extraction time 1.5-3h gets extraction product;
(2) ethanol of adding concentration 20-80%v/v in the extraction product, the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
Adopting boiling point during above-mentioned five kinds of volatile oil extraction methods extraction is because if boiling point is higher than 60 ℃ less than 60 ℃ non-polar organic solvent; Remove and can beta-elemene be removed in the lump when desolvating, so the selection boiling point is less than 60 ℃ non-polar organic solvent extracting and separating beta-elemene.
Wherein, the adoptable method of step C column chromatography is following:
Method 1: macroporous resin chromatography column on the volatile oil that step B is obtained, at least a or its arbitrary proportion blended solvent elution in water, methyl alcohol, ethanol, the acetone, flow velocity is 1-4% column volume/min; Collection contains the elutriant of beta-elemene, removes solvent, behind the ethanol of adding concentration 20-80%v/v; With the non-polar organic solvent extraction of boiling point less than 60 ℃; Collect upper organic phase, remove the organic solvent in the upper organic phase, promptly get the beta-elemene bullion.
Or employing method 2: silica gel column chromatography or silver ions silica gel column chromatography on the volatile oil that step B is obtained; With the non-polar organic solvent wash-out of boiling point less than 60 ℃, flow velocity is 1-4% column volume/min, collects the elutriant that contains beta-elemene; Remove and desolvate, obtain the beta-elemene bullion.
Wherein, when obtaining the beta-elemene highly finished product, the bullion that volatile oil that can step B be obtained or step D obtain adopts one of following method to make with extra care, and obtains highly purified beta-elemene.Normally collect the purifying composition contain beta-elemene when refining, can obtain beta-elemene content usually greater than 90% according to " target content ", even up to 98% beta-elemene highly finished product.
Process for purification 1: during molecular distillation method: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain carries out short-path distillation, collects the distillate that beta-elemene reaches target content, promptly gets the beta-elemene highly finished product.
Process for purification 2: highly effective liquid phase chromatographic system preparation method: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain separates through the preparative high performance liquid chromatography system; Collect the effluent that beta-elemene reaches target content, removing desolvates promptly gets the beta-elemene highly finished product.
Process for extracting gained beta-elemene elaboration purity of the present invention is at least greater than 90%; Even can be up to 98%; Productive rate can be up to 70%; For effectively utilizing malignant weed plant Hemp Eupatorium that a kind of new approach is provided, more obtain beta-elemene a kind of brand-new, cheap, effective means is provided.
Embodiment
The contriver adopts the performance liquid chromatography micrometric measurement to find: the beta-elemene in the Hemp Eupatorium mainly is distributed in its leaf and the stem and (accounts for more than 90% of herb total amount).Wherein, with content in the leaf the highest (being about 0.4-0.6%), and content is extremely low or do not have basically in other organs such as root, flower.And comparative analysis is the result show, beta-elemene content is far above the content of Chinese medicine curcuma zedoary, myrrh, lemongrass, Herba Solidaginis in the Hemp Eupatorium leaf.
Process for extracting of the present invention is following:
A, raw material prepare: with stem, the leaf of Hemp Eupatorium at least a or its be mixed into raw material, bright article are pulled an oar or drying and crushing;
B, extraction volatile oil: from steps A gained raw material, obtain volatile oil;
C, column chromatography: the volatile oil that step B is obtained carries out column chromatography, collects elutriant, removes solvent in the elutriant, obtains the beta-elemene bullion.
Purification step D is: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain, adopt short-path distillation or preparative high performance liquid chromatography system make with extra care the highly purified beta-elemene of acquisition.
Can adopt one of following five kinds of methods to extract volatile oil when one, extracting volatile oil:
Method one, steam distillation: adding entry through the raw material of processing of step A is that main solvent carries out air distillation, obtains containing the distillate of beta-elemene; In the gained distillate, add boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether less than 60 ℃ non-polar organic solvent, extract at least 2 times, collect upper organic phase, remove the organic solvent in the upper organic phase, promptly get volatile oil.Wherein, preferably primary solvent during distillation with water, add terepthaloyl moietie, ethanol.Because of regional reason boiling point is low, add the temperature that terepthaloyl moietie can improve distillation azeotropic liquid; Adding ethanol is the saponification foaming phenomenon that produces in the distillation in order to eliminate; Also can add zeolite and be bumping when preventing to distill.So existing, known, conventional, foreseeable, material replacement terepthaloyl moietie with above-mentioned function capable of using, ethanol, zeolite are to reach identical purpose.
Method two, pickling process: the raw material through processing of step A adds organic solvent lixiviates 2-4 time such as acetone, sherwood oil or methyl alcohol, filters; United extraction liquid is also removed solvent; The ethanolic soln that adds 20-80% with organic solvent extractions such as sherwood oil at least 2 times, is collected upper organic phase; Remove the organic solvent in the upper organic phase, promptly get volatile oil.
Method three, Continuous Countercurrent Extraction method: the raw material through processing of step A adds organic solvent Continuous Countercurrent Extraction such as acetone, sherwood oil or methyl alcohol 3-4 time, obtains extracting solution.Remove the solvent in the extracting solution; The ethanolic soln that adds 20-80% extracts 2 times with the non-polar organic solvent of boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether less than 60 ℃ at least, collects upper organic phase; Remove the organic solvent in the upper organic phase, promptly get volatile oil.
Method four, microwave extraction method: the raw material through processing of step A adds organic solvents such as acetone, sherwood oil or methyl alcohol, and in microwave extracting apparatus, the power of 300-800W is extraction 300-800s down, extracts 2-4 time, filters united extraction liquid.Remove the solvent in the extracting solution; The ethanolic soln that adds 20-80% extracts 2 times with the non-polar organic solvent of boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether less than 60 ℃ at least, collects upper organic phase; Remove the organic solvent in the upper organic phase, promptly get volatile oil.
Method five, supercritical extraction:, use supercritical CO through the raw material of processing of step A
2The fluid extraction device extracts, extracting pressure 20-30MPa, extraction temperature 30-50 ℃, extraction time 1.5-3h.In extraction product, add the ethanolic soln of 20-80%, with the non-polar organic solvent of boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether less than 60 ℃; At least extract 2 times; Collect upper organic phase, remove the organic solvent in the upper organic phase, promptly get volatile oil.
Through comparing, above-mentioned five kinds of methods all can obtain volatile oil (beta-elemene content＞20%) from raw material.But best process for extracting is Continuous Countercurrent Extraction method and steam distillation, wherein: and advantages such as the Continuous Countercurrent Extraction method has the extraction yield height, and solvent load is less, but beta-elemene content is lower in the extraction gained volatile oil; That steam distillation has is easy and simple to handle, extract beta-elemene content advantage of higher in the gained volatile oil, and volatile oil purity is generally 30%, even can reach 40%, but extraction yield is on the low side.
Wherein, the Continuous Countercurrent Extraction condition is: the acetone that adds material quantity (g) 3-7 times of volume (mL) is made solvent, soaks 1h at least under the room temperature, and constant temperature (20-30 ℃) gas bath vibration (100-140r/min) Continuous Countercurrent Extraction at least 1 time is no less than 1.5h at every turn.Parameters optimization is: the acetone that adds material quantity (g) 5 times of volumes (mL) is made solvent, soaks 4h under the room temperature, constant temperature (25 ℃) gas bath vibration (120r/min) Continuous Countercurrent Extraction 3 times, the 1st extraction 2.5h, the 2nd extraction 3.5h, the 3rd extraction 3h.
The steam distillation extraction conditions is: get the raw material that steps A obtains, put into matrass, add terepthaloyl moietie, small amount of ethanol, zeolite that water, 1-3 that material quantity (g) 3-7 doubly measures (mL) doubly measure (mL), 80-150 ℃ distillate is collected in the air distillation of heating.Parameters optimization is: get the raw material that steps A obtains, put into matrass, add the water of material quantity (g) 5 times of amounts (mL), terepthaloyl moietie, small amount of ethanol, the zeolite of 2 times of amounts (mL), 80-150 ℃ distillate is collected in the air distillation of heating.
Two, column chromatography method is following:
During column chromatography, the inventor takes silica gel, silver ions silica gel, D101 macroporous resin, HP20 macroporous resin to do chromatography media all can from the volatile oil of Hemp Eupatorium, to separate and obtain the beta-elemene bullion, beta-elemene purity usually can＞40%.Adoptable method is following:
Method one, Amberlyst process: macroporous resin chromatography column on the volatile oil that step B is obtained, water, methyl alcohol, ethanol, acetone equal solvent or its mixing solutions wash-out, flow velocity is 1-4% column volume/min; Collection contains the elutriant of beta-elemene, removes solvent, adds the ethanolic soln of 20-80%; With organic solvent extractions such as sherwood oil at least 2 times; Separate upper organic phase, remove the organic solvent in the upper organic phase, promptly get the beta-elemene bullion.
Method two, silica gel method: silica gel column chromatography or silver ions silica gel column chromatography on the volatile oil that step B is obtained, use the sherwood oil wash-out, flow velocity is 1-4% column volume/min, collects the elutriant that contains beta-elemene, removes and desolvates, and obtains the beta-elemene bullion.
Concrete, the chromatography condition of optimization is: be chromatography media with HP20, successively carry out wash-out, flow velocity 2% column volume/min with zero(ppm) water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the absolute ethyl alcohol of 2 times of column volumes respectively.Carry out column chromatography by this condition, the purity of beta-elemene bullion＞60%, the recovery＞85%.
Three, process for purification is following:
When refining, the contriver finds to take molecular distillation method, highly effective liquid phase chromatographic system preparation method all can from volatile oil or bullion, separate obtaining highly purified beta-elemene.
The short-path distillation preferable methods is: the bullion that volatile oil that step B is obtained or step C obtain carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Highly effective liquid phase chromatographic system prepares one of preferable methods: the bullion that volatile oil that step B is obtained or step C obtain separates with following highly effective liquid phase chromatographic system: Tianjin, island high performance liquid chromatograph LC-10A; The C18 preparative column (10mm * 150mm); Eluent is ethanol/water/acetonitrile=70/20/10 (V/V); Flow velocity is 2mL/min, and the detection wavelength is 210nm; Sampling volume 1mL.Collect the beta-elemene effluent, add sherwood oil, extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, add Na
2SO
4Dry 10-24h, solvent is drained in 60 ℃ of vacuum rotations, obtains pure article (purity＞98%, productive rate＞75%).
Below prepare instance through the present invention beneficial effect of the present invention be described.
Embodiment 1 distillation method extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or zero(ppm) water 500mL, terepthaloyl moietie 200mL, ethanol 50mL, zeolite 3-5 grain, 80-150 ℃ distillate is collected in the air distillation of heating.In the distillate that obtains, add sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, extract repeatedly three times.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains volatile oil (purity＞40%).
C, performance liquid preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Tianjin, island high performance liquid chromatograph LC-10A; The C18 preparative column (10mm * 150mm); Eluent is ethanol/water/acetonitrile=70/20/10 (V/V); Flow velocity is 2mL/min, and the detection wavelength is 210nm; Sampling volume 1mL.Collect the beta-elemene effluent, add sherwood oil, extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, add Na
2SO
4Dry 10-24h, solvent is drained in 60 ℃ of vacuum rotations, obtains pure article (purity＞98%, productive rate＞75%).
Embodiment 2 distillation method extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect Hemp Eupatorium stem and leaf, break into pulpous state with kibbler.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or zero(ppm) water 500mL, terepthaloyl moietie 200mL, ethanol 50mL, zeolite 3-5 grain, 80-150 ℃ distillate is collected in the air distillation of heating.In the distillate that obtains, add sherwood oil (boiling range 30-60 ℃) in 1: 1.5 ratio, extract repeatedly three times.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains volatile oil (purity＞35%).
C, performance liquid preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Tianjin, island high performance liquid chromatograph LC-10A; The C18 preparative column (10mm * 150mm); Eluent is ethanol/water/acetonitrile=70/20/10 (V/V); Flow velocity is 2mL/min, and the detection wavelength is 210nm; Sampling volume 1mL.Collect the beta-elemene effluent, add sherwood oil, extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, add Na
2SO
4Dry 10-24h, solvent is drained in 60 ℃ of vacuum rotations, obtains pure article (purity＞97%, productive rate＞73%).
Embodiment 3 distillation method extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect Hemp Eupatorium stem and leaf, break into pulpous state with kibbler.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or zero(ppm) water 500mL, terepthaloyl moietie 200mL, ethanol 50mL, zeolite 3-5 grain, 80-150 ℃ distillate is collected in the air distillation of heating.In the distillate that obtains, add hexanaphthene in 1: 1 ratio, extract repeatedly three times.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains volatile oil (purity＞37%).
C, column chromatography: with the volatile oil that step B obtains, last 300mL HP20 macroporous resin chromatography column successively carries out wash-out, flow velocity 6mL/min with zero(ppm) water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the absolute ethyl alcohol of 600mL respectively.Collect the elutriant of beta-elemene content＞60%, add sherwood oil (boiling range 30-60 ℃), extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains bullion (purity＞60%).
D, short-path distillation: the bullion that step C is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞73%).
Embodiment 4 distillation method extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect Hemp Eupatorium stem and leaf, break into pulpous state with kibbler.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or zero(ppm) water 500mL, terepthaloyl moietie 200mL, ethanol 50mL, zeolite 3-5 grain, 80-150 ℃ distillate is collected in the air distillation of heating.In the distillate that obtains, add sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, extract repeatedly three times.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains volatile oil (purity＞35%).
C, short-path distillation: the volatile oil that step B is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Embodiment 5 Continuous Countercurrent Extraction methods extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, break into pulpous state with kibbler.
B, Continuous Countercurrent Extraction: get steps A raw material 100g, put into triangular flask, Continuous Countercurrent Extraction three times adds acetone 500mL at every turn, and 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure removes and desolvates, and adds 50% ethanol 100mL, uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, column chromatography: with the volatile oil that step B obtains, last 300mL HP20 macroporous resin chromatography column successively carries out wash-out, flow velocity 6mL/min with zero(ppm) water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the absolute ethyl alcohol of 600mL respectively.Collect the elutriant of beta-elemene content＞60%, add sherwood oil (boiling range 30-60 ℃), extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains bullion (purity＞60%).
D, short-path distillation: the bullion that step C is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Embodiment 6 Continuous Countercurrent Extraction methods extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
B, Continuous Countercurrent Extraction: get steps A raw material 100g, put into triangular flask, Continuous Countercurrent Extraction three times adds sherwood oil 500mL at every turn, and 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure removes and desolvates, and adds 50% ethanol 100mL, uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, column chromatography: with the volatile oil that step B obtains, last 300mL HP20 macroporous resin chromatography column successively carries out wash-out, flow velocity 6mL/min with zero(ppm) water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the absolute ethyl alcohol of 600mL respectively.Collect the elutriant of beta-elemene content＞60%, add sherwood oil (boiling range 30-60 ℃), extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains bullion (purity＞60%).
D, short-path distillation: the bullion that step C is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Embodiment 7 Continuous Countercurrent Extraction methods extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
B, Continuous Countercurrent Extraction: get steps A raw material 100g, put into triangular flask, Continuous Countercurrent Extraction three times adds methyl alcohol 500mL at every turn, and 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure removes and desolvates, and adds 50% ethanol 100mL, uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, column chromatography: with the volatile oil that step B obtains, last 300mL HP20 macroporous resin chromatography column successively carries out wash-out, flow velocity 6mL/min with zero(ppm) water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the absolute ethyl alcohol of 600mL respectively.Collect the elutriant of beta-elemene content＞60%, add sherwood oil (boiling range 30-60 ℃), extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, solvent is drained in 60 ℃ of vacuum rotations, obtains bullion (purity＞60%).
D, short-path distillation: the bullion that step C is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Embodiment 8 Continuous Countercurrent Extraction methods extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
B, Continuous Countercurrent Extraction: get steps A raw material 100g, put into triangular flask, Continuous Countercurrent Extraction three times adds methyl alcohol 500mL at every turn, and 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure removes and desolvates, and adds 50% ethanol 100mL, uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, performance liquid preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Tianjin, island high performance liquid chromatograph LC-10A; The C18 preparative column (10mm * 150mm); Eluent is ethanol/water/acetonitrile=70/20/10 (V/V); Flow velocity is 2mL/min, and the detection wavelength is 210nm; Sampling volume 1mL.Collect the beta-elemene effluent, add sherwood oil, extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, add Na
2SO
4Dry 10-24h, solvent is drained in 60 ℃ of vacuum rotations, obtains pure article (purity＞98%, productive rate＞75%).
Embodiment 9 pickling processes extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
B, pickling process: the raw material 100g of processing of step A,, add the 500ml acetone extraction 3 times, each 2 hours, filter, united extraction liquid is also removed acetone, adds 50% ethanol 100mL, uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 2 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, performance liquid preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Tianjin, island high performance liquid chromatograph LC-10A; The C18 preparative column (10mm * 150mm); Eluent is ethanol/water/acetonitrile=70/20/10 (V/V); Flow velocity is 2mL/min, and the detection wavelength is 210nm; Sampling volume 1mL.Collect the beta-elemene effluent, add sherwood oil, extract repeatedly three times in 1: 1 ratio.Separate and merge petroleum ether layer, add Na
2SO
4Dry 10-24h, solvent is drained in 60 ℃ of vacuum rotations, obtains pure article (purity＞98%, productive rate＞75%).
Wherein, when step B adopted sherwood oil or methyl alcohol lixiviate, pure article purity and productive rate were suitable with the employing acetone extraction.
Embodiment 10 microwave extraction methods extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
B, microwave extracting: the raw material 100g of processing of step A,, in microwave extracting apparatus, power 800W extracts 400s, adds acetone extract 3 times, filters united extraction liquid.Remove the solvent in the extracting solution, add 50% ethanol 100mL, use 100mL sherwood oil (boiling range 30-60 ℃) extraction 2 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, short-path distillation: the volatile oil that step B is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Wherein, when step B adopted sherwood oil or methyl alcohol lixiviate, pure article purity and productive rate were suitable with the employing acetone extraction.
Embodiment 11 supercritical extractions extraction separation beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, dry naturally under the sun, pulverize, cross 40 mesh sieves.
B, supercritical extraction: the raw material 100g of processing of step A,, use supercritical CO
2The fluid extraction device extracts, control extracting pressure 20-30MPa, extraction temperature 30-50 ℃, extraction time 2h.In extraction product, add 50% ethanol 100mL, use 100mL sherwood oil (boiling range 30-60 ℃) extraction 2 times again.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotation solvent evaporated obtain volatile oil (purity＞20%).
C, short-path distillation: the volatile oil that step B is obtained carries out vacuum fractionation, collects the cut of 76-78 ℃/40Pa, is pure article (purity＞90%, productive rate＞70%).
Claims (10)
1. the method for extraction separation beta-elemene from Hemp Eupatorium, it is characterized in that: step is following:
A, raw material prepare: mainly be mixed into raw material with at least a in the stem of Hemp Eupatorium, the leaf or its, bright article are pulled an oar or drying and crushing;
B, volatile oil extract: from steps A gained raw material, extract and obtain volatile oil;
C, column chromatography: the volatile oil that step B is obtained carries out column chromatography, collects elutriant, removes the solvent in the elutriant, obtains the beta-elemene bullion.
2. the method for extraction separation beta-elemene from Hemp Eupatorium, it is characterized in that: step is following:
A, raw material prepare: mainly be mixed into raw material with at least a in the stem of Hemp Eupatorium, the leaf or its, bright article are pulled an oar or drying and crushing;
B, volatile oil extract: from steps A gained raw material, extract and obtain volatile oil;
C, column chromatography: the volatile oil that step B is obtained carries out column chromatography, collects elutriant, removes the solvent in the elutriant, obtains the beta-elemene bullion;
D, refining: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain, adopt short-path distillation or preparative high performance liquid chromatography system make with extra care acquisition beta-elemene highly finished product.
3. according to claim 1 and 2 from Hemp Eupatorium the method for extraction separation beta-elemene, it is characterized in that: when step B adopted wet distillation to extract volatile oil, method was following:
(1) raw material through processing of step A carries out air distillation, obtains distillate;
(2) in distillate, add boiling point less than 60 ℃ non-polar organic solvent, upper organic phase is collected in extraction, removes the organic solvent in the upper organic phase, promptly gets volatile oil;
Be primary solvent with water preferably during distillation, add in the solvent systems and improve the solvent systems boiling point and prevent saponification foaming, the material of bumping.
4. according to claim 1 and 2 from Hemp Eupatorium the method for extraction separation beta-elemene, it is characterized in that: when step B adopted pickling process to extract volatile oil, method was following:
(1) raw material through processing of step A adds at least a or its arbitrary proportion blended organic solvent lixiviate, filtration in acetone, sherwood oil or the methyl alcohol, gets vat liquor;
(2) vat liquor removes the ethanol that the back of desolvating adds concentration 20-80%v/v, and the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
5. according to claim 1 and 2 from Hemp Eupatorium the method for extraction separation beta-elemene, it is characterized in that: when step B adopted the Continuous Countercurrent Extraction method to extract volatile oil, method was following:
(1) raw material through processing of step A adds at least a or its arbitrary proportion blended organic solvent Continuous Countercurrent Extraction in acetone, sherwood oil or the methyl alcohol, gets extracting solution;
(2) ethanol of adding concentration 20-80%v/v behind the solvent in the removal extracting solution, the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
6. according to claim 1 and 2 from Hemp Eupatorium the method for extraction separation beta-elemene, it is characterized in that: when step B adopted microwave extraction method to extract volatile oil, method was following:
(1) raw material through processing of step A adds at least a or its arbitrary proportion blended organic solvent in acetone, sherwood oil or the methyl alcohol, in microwave extracting apparatus, adopts power 300-800W extraction 300-800s, gets extracting solution;
(2) ethanol of adding concentration 20-80%v/v behind the extracting solution removal solvent, the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
7. according to claim 1 and 2 from Hemp Eupatorium the method for extraction separation beta-elemene, it is characterized in that: when step B adopted supercritical fluid extraction volatile oil, method was following:
(1) through the raw material of processing of step A, extract with the supercritical carbon dioxide extraction device, extracting pressure 20-30MPa, extraction temperature 30-50 ℃, extraction time 1.5-3h gets extraction product;
(2) ethanol of adding concentration 20-80%v/v in the extraction product, the adding boiling point extracts less than 60 ℃ non-polar organic solvent, collects upper organic phase, removes the organic solvent in the upper organic phase, promptly gets volatile oil.
8. according to claim 1 and 2 from Hemp Eupatorium the method for extraction separation beta-elemene, it is characterized in that: column chromatography adopts following method separation and purification among the step C:
Method 1: macroporous resin chromatography column on the volatile oil that step B is obtained, at least a or its arbitrary proportion blended solvent elution in water, methyl alcohol, ethanol, the acetone, flow velocity is 1-4% column volume/min; Collection contains the elutriant of beta-elemene, removes solvent, behind the ethanol of adding concentration 20-80%v/v; With the non-polar organic solvent extraction of boiling point less than 60 ℃; Collect upper organic phase, remove the organic solvent in the upper organic phase, promptly get the beta-elemene bullion;
Or method 2: silica gel column chromatography or silver ions silica gel column chromatography on the volatile oil that step B is obtained; With the non-polar organic solvent wash-out of boiling point less than 60 ℃, flow velocity is 1-4% column volume/min, collects the elutriant that contains beta-elemene; Remove and desolvate, obtain the beta-elemene bullion.
9. according to claim 2 from Hemp Eupatorium the method for extraction separation beta-elemene; It is characterized in that: when step D adopts molecular distillation method; Method is following: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain carries out short-path distillation; Collect the distillate that beta-elemene reaches target content, promptly get the beta-elemene highly finished product.
10. beta-elemene process for purification according to claim 2; It is characterized in that: when step D adopts the highly effective liquid phase chromatographic system preparation method; Method is following: the beta-elemene bullion that volatile oil that step B is obtained or step C obtain separates through the preparative high performance liquid chromatography system; Collect the effluent that beta-elemene reaches target content, the solvent of removing in the effluent promptly gets the beta-elemene highly finished product.
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