CN102432419B - Method for extracting and separating beta-elemene from Eupatorium adenophorum - Google Patents
Method for extracting and separating beta-elemene from Eupatorium adenophorum Download PDFInfo
- Publication number
- CN102432419B CN102432419B CN201110457523.5A CN201110457523A CN102432419B CN 102432419 B CN102432419 B CN 102432419B CN 201110457523 A CN201110457523 A CN 201110457523A CN 102432419 B CN102432419 B CN 102432419B
- Authority
- CN
- China
- Prior art keywords
- elemene
- beta
- volatile oil
- raw material
- solvent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Plant Substances (AREA)
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 extract the method for separating beta-elemene from malignant weed Weed Eupatorium adenophorum.
Background technology
Hemp Eupatorium (Eupatorium adenophorum Spreng), another name destroys grass, liberation grass, Herba Eupatorii Odorati etc., is composite family feverwort.This plant is widely distributed on Yunnan Province of China, Guangxi, Sichuan and other places, and resource is very abundant, belongs to the torrid zone, subtropics invasive weed plant, produces to local husbandry and causes very big harm, and local ecological safety is caused to great threat.
Beta-elemene, Latin is called β-Elemenum, English β-elemene by name, chemistry (1S, 2S, 4R) Elemene by name.Structural formula is as follows:
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 off tumor cells and suppresses tumor growth effect; can also make body obtain obvious immunoprotective effec by activating immune system; have anticancer spectrum wide, efficient, without hepatorenal damage, without the feature such as bone marrow depression, few side effects; be the mankind from now on one period internal therapy tumour choice drug, domestic and international market demand is large.At present, beta-elemene mainly depends on from Traditional Chinese medicine Rhizoma curcumae, myrrh, lemongrass, Herba Solidaginis and extracts, and not yet finds that there is to extract from Hemp Eupatorium to separate the report that obtains beta-elemene.Because Traditional Chinese medicine Rhizoma curcumae, myrrh, lemongrass, Herba Solidaginis are Chinese medicine, cost is relatively high, 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.
The present inventor finds in Hemp Eupatorium that beta-elemene content is far above Traditional Chinese medicine Rhizoma curcumae, myrrh, lemongrass, Herba Solidaginis, and Hemp Eupatorium is invasive weed plant, aboundresources, and its raw materials cost is also low more than these raw materials.Therefore the present invention wants to provide a kind of method of effectively extracting beta-elemene from Hemp Eupatorium, for significantly reducing the production cost of beta-elemene and developing, utilize, administer this worldwide malignant weed plant of Hemp Eupatorium and opened up new approach.
Summary of the invention
Technical problem solved by the invention is to provide a kind of method of extracting separating beta-elemene from Hemp Eupatorium.
Detect discovery through contriver: the beta-elemene in Hemp Eupatorium is mainly distributed in its leaf and stem and (accounts for the more than 90% of herb total amount).Wherein, with content in leaf the highest (being about 0.4-0.6%), and content is extremely low or substantially do not have in other organs such as root, flower.Comparative analysis result shows, in Hemp Eupatorium leaf, beta-elemene content is far above the content of Traditional Chinese medicine Rhizoma curcumae, myrrh, lemongrass, Herba Solidaginis.
The raw material using in extracting method of the present invention is mainly at least one or its mixing in stem, the leaf of Hemp Eupatorium, can not affect extraction separating beta-elemene if be mixed with other organs such as root, flower, fruit in raw material yet, but can affect the yield of extracting method, therefore be preferably mixed into raw material with at least one or its in stem, the leaf of Hemp Eupatorium.
Extracting method of the present invention is as follows:
A, raw material prepare: be mainly mixed into raw material, fresh goods making beating or drying and crushing with at least one or its in stem, the leaf of Hemp Eupatorium;
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, collect elutriant, removes the solvent in elutriant, obtains beta-elemene crude product.
Further, the beta-elemene crude product that the volatile oil that step B can also be obtained or step C obtain is further refined, and obtains the beta-elemene highly finished product that purity is higher.
Be that purification step D is: the beta-elemene crude product that the volatile oil that step B is obtained or step C obtain, adopts molecular distillation or preparative high performance liquid chromatography system to refine and obtain highly purified beta-elemene.
Adopt stem, the leaf step B extraction of aforesaid method processing Hemp Eupatorium conventionally can obtain the volatile oil that beta-elemene content is greater than 20%; Adopt the method such as column chromatography to process conventionally can to obtain beta-elemene content to be greater than 40%, even up to 60% beta-elemene crude product; Finally, further refined volatile oil or beta-elemene crude product, collects the purifying composition containing beta-elemene according to " target content ", conventionally can obtain beta-elemene content and be greater than 90%, even up to 98% beta-elemene highly finished product.
Wherein, while extracting volatile oil, can adopt one of following Lung biopsy to extract volatile oil:
Method one: steam distillation:
(1) carry out air distillation through the raw material of processing of step A, obtain distillate;
(2) non-polar organic solvent that adds boiling point to be less than 60 ℃ in distillate, extraction, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil.Wherein, when distillation, preferably take water as primary solvent, add and improve extraction system boiling point and prevent the material that saponification is bubbled.As, adding ethylene glycol is the temperature that improves distillation azeotropic liquid, and adding ethanol is to eliminate the saponification foaming phenomenon producing in distillation, and adding zeolite is bumping while preventing from distilling.Replace ethylene glycol, ethanol, zeolite to reach identical object therefore can utilize existing, known, conventional, foreseeable, to there is above-mentioned function material.
Method two: pickling process:
(1) the organic solvent lixiviate, the filtration that add at least one or its arbitrary proportion in acetone, sherwood oil or methyl alcohol to mix through the raw material of processing of step A, obtain vat liquor;
(2) vat liquor is except adding the ethanol of concentration 20-80%v/v after desolventizing, and the non-polar organic solvent extraction that adds boiling point to be less than 60 ℃, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil.
Method three: Continuous Countercurrent Extraction method:
(1) the organic solvent Continuous Countercurrent Extraction that adds at least one or its arbitrary proportion in acetone, sherwood oil or methyl alcohol to mix through the raw material of processing of step A, obtains extracting solution;
(2) remove the ethanol that adds concentration 20-80%v/v after the solvent in extracting solution, the non-polar organic solvent extraction that adds boiling point to be less than 60 ℃, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil.
Method four: microwave extraction method:
(1) organic solvent that adds at least one or its arbitrary proportion in acetone, sherwood oil or methyl alcohol to mix through the raw material of processing of step A, in microwave extracting apparatus, adopts power 300-800W extraction 300-800s, obtains extracting solution;
(2) extracting solution is removed the ethanol that adds concentration 20-80%v/v after solvent, and the non-polar organic solvent extraction that adds boiling point to be less than 60 ℃, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil.
Method five: supercritical extraction:
(1) through the raw material of processing of step A, use supercritical CO
2fluid extraction device extracts, extracting pressure 20-30MPa, and extraction temperature 30-50 ℃, extraction time 1.5-3h, obtains extraction product;
(2) in extraction product, add the ethanol of concentration 20-80%v/v, the non-polar organic solvent extraction that adds boiling point to be less than 60 ℃, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil.
If the non-polar organic solvent that adopts boiling point to be less than 60 ℃ when above-mentioned five kinds of volatile oil extraction methods extraction is higher than 60 ℃ because of boiling point, during except desolventizing, beta-elemene can be removed in the lump, therefore the non-polar organic solvent extracting and separating beta-elemene of selecting boiling point to be less than 60 ℃.
Wherein, the adoptable method of step C column chromatography is as follows:
Method 1: macroporous resin chromatography column on the volatile oil that step B is obtained, the solvent elution that at least one in water, methyl alcohol, ethanol, acetone or its arbitrary proportion mix, flow velocity is 1-4% column volume/min, collect the elutriant containing beta-elemene, remove solvent, add after the ethanol of concentration 20-80%v/v, the non-polar organic solvent extraction that is less than 60 ℃ with boiling point, collect upper organic phase, remove the organic solvent in upper organic phase, obtain beta-elemene crude product.
Or employing method 2: silica gel column chromatography or silver ions silica gel column chromatography on the volatile oil that step B is obtained, the non-polar organic solvent wash-out that is less than 60 ℃ with boiling point, flow velocity is 1-4% column volume/min, collects the elutriant containing beta-elemene, except desolventizing, obtain beta-elemene crude product.
Wherein, while obtaining beta-elemene highly finished product, the crude product that the volatile oil that step B can be obtained or step D obtain adopts one of following method to refine, and obtains highly purified beta-elemene.When refining, normally collect containing the purifying composition of beta-elemene according to " target content ", conventionally can obtain beta-elemene content and be greater than 90%, even up to 98% beta-elemene highly finished product.
Process for purification 1: when molecular distillation method: the beta-elemene crude product that the volatile oil that step B is obtained or step C obtain carries out molecular distillation, collection beta-elemene reaches the distillate of target content, obtains beta-elemene highly finished product.
Process for purification 2: highly effective liquid phase chromatographic system preparation method: the beta-elemene crude product that the volatile oil that step B is obtained or step C obtain separates by preparative high performance liquid chromatography system, collect the effluent liquid that beta-elemene reaches target content, except desolventizing obtains beta-elemene highly finished product.
Extracting method gained beta-elemene fine work 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 Weed Eupatorium adenophorum that a kind of new approach is provided, more obtain beta-elemene a kind of completely newly, cheap, effective means is provided.
Embodiment
Contriver adopts high performance liquid chromatography micrometric measurement to find: the beta-elemene in Hemp Eupatorium is mainly distributed in its leaf and stem and (accounts for the more than 90% of herb total amount).Wherein, with content in leaf the highest (being about 0.4-0.6%), and content is extremely low or substantially do not have in other organs such as root, flower.And comparative analysis result shows, in Hemp Eupatorium leaf, beta-elemene content is far above the content of Traditional Chinese medicine Rhizoma curcumae, myrrh, lemongrass, Herba Solidaginis.
Extracting method of the present invention is as follows:
A, raw material prepare: be mixed into raw material, fresh goods making beating or drying and crushing with the stem of Hemp Eupatorium, leaf at least one or its;
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, collect elutriant, removes solvent in elutriant, obtains beta-elemene crude product.
Purification step D is: the beta-elemene crude product that the volatile oil that step B is obtained or step C obtain, adopts molecular distillation or preparative high performance liquid chromatography system to refine and obtain highly purified beta-elemene.
One, while extracting volatile oil, can adopt one of following Lung biopsy to extract volatile oil:
Method one, steam distillation: adding water through the raw material of processing of step A is that main solvent carries out air distillation, obtains the distillate containing beta-elemene; The non-polar organic solvent that adds the boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether to be less than 60 ℃ in gained distillate, extracts at least 2 times, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil.Wherein, when distillation, preferably take water as primary solvent, add ethylene glycol, ethanol.Because regional reason boiling point is low, add ethylene glycol can improve the temperature of distillation azeotropic liquid; Adding ethanol is the saponification foaming phenomenon producing in distillation in order to eliminate; Also can add zeolite is bumping while preventing from distilling.Replace ethylene glycol, ethanol, zeolite to reach identical object therefore can utilize existing, known, conventional, foreseeable, to there is above-mentioned function material.
Method two, pickling process: add organic solvent lixiviates 2-4 time such as acetone, sherwood oil or methyl alcohol through the raw material of processing of step A, filter, united extraction liquid is also removed solvent, add the ethanolic soln of 20-80%, with organic solvent extractions such as sherwood oil at least 2 times, collect upper organic phase, remove the organic solvent in upper organic phase, obtain volatile oil.
Method three, Continuous Countercurrent Extraction method: add the organic solvent Continuous Countercurrent Extraction such as acetone, sherwood oil or methyl alcohol through the raw material of processing of step A 3-4 time, obtain extracting solution.Remove the solvent in extracting solution, add the ethanolic soln of 20-80%, the non-polar organic solvent that is less than 60 ℃ with boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether extracts at least 2 times, collects upper organic phase, remove the organic solvent in upper organic phase, obtain volatile oil.
Method four, microwave extraction method: add the organic solvents such as acetone, sherwood oil or methyl alcohol through the raw material of processing of step A, in microwave extracting apparatus, under the power of 300-800W, extract 300-800s, extract 2-4 time, filter united extraction liquid.Remove the solvent in extracting solution, add the ethanolic soln of 20-80%, the non-polar organic solvent that is less than 60 ℃ with boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether extracts at least 2 times, collects upper organic phase, remove the organic solvent in upper organic phase, obtain volatile oil.
Method five, supercritical extraction: through the raw material of processing of step A, use supercritical CO
2fluid 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%, the non-polar organic solvent that is less than 60 ℃ with boiling points such as sherwood oil, hexanaphthene, normal hexane, benzene, ether, at least extract 2 times, collect upper organic phase, remove the organic solvent in upper organic phase, obtain volatile oil.
Through comparing, above-mentioned Lung biopsy all can obtain volatile oil (beta-elemene content > 20%) from raw material.But best extracting method is Continuous Countercurrent Extraction method and steam distillation, wherein: the advantages such as it is high that Continuous Countercurrent Extraction method has extraction yield, and solvent load is less, but in extraction gained volatile oil, beta-elemene content is lower; That steam distillation has advantages of is easy and simple to handle, it is higher to extract in gained volatile oil beta-elemene content, and volatile oil purity is generally 30%, even can reach 40%, but extraction yield is on the low side.
Wherein, Continuous Countercurrent Extraction condition is: add the acetone of 3-7 times of volume of material quantity (g) (mL) to make solvent, under room temperature, soak at least 1h, 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: add the acetone of 5 times of volumes of material quantity (g) (mL) to make solvent, under room temperature, soak 4h, constant temperature (25 ℃) gas bath vibration (120r/min) Continuous Countercurrent Extraction 3 times, extract 2.5h the 1st time, extract 3.5h the 2nd time, extract 3h the 3rd time.
Extraction by steam distillation condition is: get the raw material that steps A obtains, put into matrass, water, the 1-3 that adds material quantity (g) 3-7 doubly to measure (mL) doubly measures the ethylene glycol of (mL), a small amount of ethanol, zeolite, the air distillation of heating, collects the distillate of 80-150 ℃.Parameters optimization is: get the raw material that steps A obtains, put into matrass, add the water of 5 times of amounts of material quantity (g) (mL), the ethylene glycol of 2 times of amounts (mL), a small amount of ethanol, zeolite, the air distillation of heating, collects the distillate of 80-150 ℃.
Two, column chromatography method is as follows:
When column chromatography, 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 beta-elemene crude product, and beta-elemene purity conventionally can > 40%.Adoptable method is as follows:
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, collect the elutriant containing beta-elemene, remove solvent, add 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 upper organic phase, obtain beta-elemene crude product.
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 sherwood oil wash-out, flow velocity is 1-4% column volume/min, collects the elutriant containing beta-elemene, except desolventizing, obtains beta-elemene crude product.
Concrete, the chromatography condition of optimization is: take HP20 as chromatography media, successively carry out wash-out, flow velocity 2% column volume/min with distilled water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the dehydrated alcohol of 2 times of column volumes respectively.Carry out column chromatography by this condition, the purity > 60% of beta-elemene crude product, rate of recovery > 85%.
Three, process for purification is as follows:
When refining, contriver finds to take molecular distillation method, the highly effective liquid phase chromatographic system preparation method all can be from volatile oil or crude product, separates and obtains highly purified beta-elemene.
The preferred method of molecular distillation is: the crude product that the volatile oil that step B is obtained or step C obtain carries out vacuum fractionation, collect the cut of 76-78 ℃/40Pa, be sterling (purity > 90%, productive rate > 70%).
Highly effective liquid phase chromatographic system is prepared one of preferred method: the crude product that the volatile oil that step B is obtained or step C obtain separates with following highly effective liquid phase chromatographic system: Shimadzu high performance liquid chromatograph LC-10A, C18 preparative column (10mm × 150mm), eluent is ethanol/water/acetonitrile=70/20/10 (V/V), flow velocity is 2mL/min, and detection wavelength is 210nm; Sampling volume 1mL.Collect beta-elemene effluent liquid, add sherwood oil in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, add Na
2sO
4dry 10-24h, 60 ℃ of vacuum rotatings are drained solvent, obtain sterling (purity > 98%, productive rate > 75%).
Prepare example by the present invention below beneficial effect of the present invention is described.
Embodiment 1 distillation method is extracted separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect Hemp Eupatorium stem and leaf, under the sun, naturally dry, 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 distilled water 500mL, ethylene glycol 200mL, ethanol 50mL, zeolite 3-5 grain, the air distillation of heating, collects the distillate of 80-150 ℃.In the distillate obtaining, add sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain volatile oil (purity > 40%).
C, high performance liquid phase preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Shimadzu high performance liquid chromatograph LC-10A, C18 preparative column (10mm × 150mm), eluent is ethanol/water/acetonitrile=70/20/10 (V/V), flow velocity is 2mL/min, and detection wavelength is 210nm; Sampling volume 1mL.Collect beta-elemene effluent liquid, add sherwood oil in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, add Na
2sO
4dry 10-24h, 60 ℃ of vacuum rotatings are drained solvent, obtain sterling (purity > 98%, productive rate > 75%).
Embodiment 2 distillation method are extracted separating beta-elemene from Hemp Eupatorium
A, raw material prepare: collect Hemp Eupatorium stem and leaf, break into pulpous state with pulverizer.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or distilled water 500mL, ethylene glycol 200mL, ethanol 50mL, zeolite 3-5 grain, the air distillation of heating, collects the distillate of 80-150 ℃.In the distillate obtaining, add sherwood oil (boiling range 30-60 ℃) in 1: 1.5 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain volatile oil (purity > 35%).
C, high performance liquid phase preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Shimadzu high performance liquid chromatograph LC-10A, C18 preparative column (10mm × 150mm), eluent is ethanol/water/acetonitrile=70/20/10 (V/V), flow velocity is 2mL/min, and detection wavelength is 210nm; Sampling volume 1mL.Collect beta-elemene effluent liquid, add sherwood oil in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, add Na
2sO
4dry 10-24h, 60 ℃ of vacuum rotatings are drained solvent, obtain sterling (purity > 97%, productive rate > 73%).
Embodiment 3 distillation method are extracted separating beta-elemene from Hemp Eupatorium
A, raw material prepare: collect Hemp Eupatorium stem and leaf, break into pulpous state with pulverizer.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or distilled water 500mL, ethylene glycol 200mL, ethanol 50mL, zeolite 3-5 grain, the air distillation of heating, collects the distillate of 80-150 ℃.In the distillate obtaining, add hexanaphthene in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain volatile oil (purity > 37%).
C, column chromatography: the volatile oil that step B is obtained, upper 300mL HP20 macroporous resin chromatography column, successively carries out wash-out, flow velocity 6mL/min with distilled water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the dehydrated alcohol of 600mL respectively.The elutriant of collecting beta-elemene content > 60%, adds sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, repeatedly extracts three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain crude product (purity > 60%).
D, molecular distillation: the crude product that step C is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 73%).
Embodiment 4 distillation method are extracted separating beta-elemene from Hemp Eupatorium
A, raw material prepare: collect Hemp Eupatorium stem and leaf, break into pulpous state with pulverizer.
The extraction of B, volatile oil: get the raw material 100g that steps A obtains, put into matrass, add deionized water or distilled water 500mL, ethylene glycol 200mL, ethanol 50mL, zeolite 3-5 grain, the air distillation of heating, collects the distillate of 80-150 ℃.In the distillate obtaining, add sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain volatile oil (purity > 35%).
C, molecular distillation: the volatile oil that step B is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 70%).
Embodiment 5 Continuous Countercurrent Extraction methods are extracted separating beta-elemene from Hemp Eupatorium
A, raw material prepare: collect fresh Hemp Eupatorium stem and leaf, break into pulpous state with pulverizer.
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, 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure, except desolventizing, adds 50% ethanol 100mL, then uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, column chromatography: the volatile oil that step B is obtained, upper 300mL HP20 macroporous resin chromatography column, successively carries out wash-out, flow velocity 6mL/min with distilled water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the dehydrated alcohol of 600mL respectively.The elutriant of collecting beta-elemene content > 60%, adds sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, repeatedly extracts three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain crude product (purity > 60%).
D, molecular distillation: the crude product that step C is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 70%).
Embodiment 6 Continuous Countercurrent Extraction methods are extracted separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, under the sun, naturally dry, 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, 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure, except desolventizing, adds 50% ethanol 100mL, then uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, column chromatography: the volatile oil that step B is obtained, upper 300mL HP20 macroporous resin chromatography column, successively carries out wash-out, flow velocity 6mL/min with distilled water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the dehydrated alcohol of 600mL respectively.The elutriant of collecting beta-elemene content > 60%, adds sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, repeatedly extracts three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain crude product (purity > 60%).
D, molecular distillation: the crude product that step C is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 70%).
Embodiment 7 Continuous Countercurrent Extraction methods are extracted separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, under the sun, naturally dry, 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, 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure, except desolventizing, adds 50% ethanol 100mL, then uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, column chromatography: the volatile oil that step B is obtained, upper 300mL HP20 macroporous resin chromatography column, successively carries out wash-out, flow velocity 6mL/min with distilled water, 80% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, the dehydrated alcohol of 600mL respectively.The elutriant of collecting beta-elemene content > 60%, adds sherwood oil (boiling range 30-60 ℃) in 1: 1 ratio, repeatedly extracts three times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotatings are drained solvent, obtain crude product (purity > 60%).
D, molecular distillation: the crude product that step C is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 70%).
Embodiment 8 Continuous Countercurrent Extraction methods are extracted separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, under the sun, naturally dry, 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, 4-8h, temperature 20-25 ℃ are extracted in concussion (rotating speed 60-100r/min).Filter united extraction liquid, concentrating under reduced pressure, except desolventizing, adds 50% ethanol 100mL, then uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 3 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, high performance liquid phase preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Shimadzu high performance liquid chromatograph LC-10A, C18 preparative column (10mm × 150mm), eluent is ethanol/water/acetonitrile=70/20/10 (V/V), flow velocity is 2mL/min, and detection wavelength is 210nm; Sampling volume 1mL.Collect beta-elemene effluent liquid, add sherwood oil in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, add Na
2sO
4dry 10-24h, 60 ℃ of vacuum rotatings are drained solvent, obtain sterling (purity > 98%, productive rate > 75%).
Embodiment 9 pickling processes are extracted separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, under the sun, naturally dry, pulverize, cross 40 mesh sieves.
B, pickling process: the raw material 100g of processing of step A,, add 500ml acetone extraction 3 times, each 2 hours, filter, united extraction liquid is also removed acetone, adds 50% ethanol 100mL, then uses 100mL sherwood oil (boiling range 30-60 ℃) extraction 2 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, high performance liquid phase preparation: the volatile oil that step B is obtained separates with following highly effective liquid phase chromatographic system: Shimadzu high performance liquid chromatograph LC-10A, C18 preparative column (10mm × 150mm), eluent is ethanol/water/acetonitrile=70/20/10 (V/V), flow velocity is 2mL/min, and detection wavelength is 210nm; Sampling volume 1mL.Collect beta-elemene effluent liquid, add sherwood oil in 1: 1 ratio, repeatedly extract three times.Separate and merge petroleum ether layer, add Na
2sO
4dry 10-24h, 60 ℃ of vacuum rotatings are drained solvent, obtain sterling (purity > 98%, productive rate > 75%).
Wherein, when step B adopts sherwood oil or methyl alcohol lixiviate, sterling purity and productive rate are suitable with employing acetone extraction.
Embodiment 10 microwave extraction methods extract separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, under the sun, naturally dry, 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 extracting solution, add 50% ethanol 100mL, then use 100mL sherwood oil (boiling range 30-60 ℃) extraction 2 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, molecular distillation: the volatile oil that step B is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 70%).
Wherein, when step B adopts sherwood oil or methyl alcohol lixiviate, sterling purity and productive rate are suitable with employing acetone extraction.
Embodiment 11 supercritical extractions extract separating beta-elemene from Hemp Eupatorium
A, raw material are prepared: collect fresh Hemp Eupatorium stem and leaf, under the sun, naturally dry, pulverize, cross 40 mesh sieves.
B, supercritical extraction: the raw material 100g of processing of step A,, use supercritical CO
2fluid extraction device extracts, and controls extracting pressure 20-30MPa, extraction temperature 30-50 ℃, extraction time 2h.In extraction product, add 50% ethanol 100mL, then use 100mL sherwood oil (boiling range 30-60 ℃) extraction 2 times.Separate and merge petroleum ether layer, 60 ℃ of vacuum rotating solvent evaporated, obtain volatile oil (purity > 20%).
C, molecular distillation: the volatile oil that step B is obtained carries out vacuum fractionation, the cut of 76-78 ℃/40Pa of collection, is sterling (purity > 90%, productive rate > 70%).
Claims (4)
1. the method for extracting separating beta-elemene from Hemp Eupatorium, is characterized in that: step is as follows:
A, raw material prepare: be mainly mixed into raw material, fresh goods making beating or drying and crushing with at least one or its in stem, the leaf of Hemp Eupatorium;
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, collect elutriant, removes the solvent in elutriant, obtains beta-elemene crude product;
Wherein, when step B adopts wet distillation to extract volatile oil, method is as follows:
(1) carry out air distillation through the raw material of processing of step A, obtain distillate;
(2) non-polar organic solvent that adds boiling point to be less than 60 ℃ in distillate, extraction, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil;
In step C, column chromatography adopts following method separation and purification:
Method 1: macroporous resin chromatography column on the volatile oil that step B is obtained, the solvent elution that at least one in water, methyl alcohol, ethanol, acetone or its arbitrary proportion mix, flow velocity is 1-4% column volume/min, collect the elutriant containing beta-elemene, remove solvent, add after the ethanol of concentration 20-80%v/v, the non-polar organic solvent extraction that is less than 60 ℃ with boiling point, collect upper organic phase, remove the organic solvent in upper organic phase, obtain beta-elemene crude product;
Or method 2: silica gel column chromatography or silver ions silica gel column chromatography on the volatile oil that step B is obtained, the non-polar organic solvent wash-out that is less than 60 ℃ with boiling point, flow velocity is 1-4% column volume/min, collects the elutriant containing beta-elemene, except desolventizing, obtain beta-elemene crude product;
When distillation, take water as primary solvent, in solvent systems, add and improve solvent systems boiling point and prevent that saponification from bubbling, preventing the material of bumping; Wherein, the material of described raising solvent systems boiling point is ethylene glycol; The described material that prevents that saponification from bubbling is ethanol; The described material that prevents bumping is zeolite;
Extraction by steam distillation condition is: get the raw material that steps A obtains, put into matrass, prepare in following ratio: every 1g raw material adds 3-7mL water, 1-3mL ethylene glycol, a small amount of ethanol, zeolite, and the air distillation of heating, collects the distillate of 80-150 ℃.
2. the method for extracting separating beta-elemene from Hemp Eupatorium according to claim 1, it is characterized in that: extraction by steam distillation condition is: get the raw material that steps A obtains, put into matrass, prepare in following ratio: every 1g raw material adds 5mL water, 2mL ethylene glycol, a small amount of ethanol, zeolite, the air distillation of heating, collects the distillate of 80-150 ℃.
3. the method for extracting separating beta-elemene from Hemp Eupatorium, is characterized in that: step is as follows:
A, raw material prepare: be mainly mixed into raw material, fresh goods making beating or drying and crushing with at least one or its in stem, the leaf of Hemp Eupatorium;
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, collect elutriant, removes the solvent in elutriant, obtains beta-elemene crude product;
D, refining: the beta-elemene crude product that the volatile oil that step B is obtained or step C obtain, adopts preparative high performance liquid chromatography system to refine and obtain beta-elemene highly finished product;
Wherein, when step B adopts wet distillation to extract volatile oil, method is as follows:
(1) carry out air distillation through the raw material of processing of step A, obtain distillate;
(2) non-polar organic solvent that adds boiling point to be less than 60 ℃ in distillate, extraction, collects upper organic phase, removes the organic solvent in upper organic phase, obtains volatile oil;
In step C, column chromatography adopts following method separation and purification:
Method 1: macroporous resin chromatography column on the volatile oil that step B is obtained, the solvent elution that at least one in water, methyl alcohol, ethanol, acetone or its arbitrary proportion mix, flow velocity is 1-4% column volume/min, collect the elutriant containing beta-elemene, remove solvent, add after the ethanol of concentration 20-80%v/v, the non-polar organic solvent extraction that is less than 60 ℃ with boiling point, collect upper organic phase, remove the organic solvent in upper organic phase, obtain beta-elemene crude product;
Or method 2: silica gel column chromatography or silver ions silica gel column chromatography on the volatile oil that step B is obtained, the non-polar organic solvent wash-out that is less than 60 ℃ with boiling point, flow velocity is 1-4% column volume/min, collects the elutriant containing beta-elemene, except desolventizing, obtain beta-elemene crude product;
Step D adopts highly effective liquid phase chromatographic system preparation method method as follows: the beta-elemene crude product that the volatile oil that step B is obtained or step C obtain separates by preparative high performance liquid chromatography system, collect the effluent liquid that beta-elemene reaches target content, the solvent of removing in effluent liquid obtains beta-elemene highly finished product, and condition is as follows:
Shimadzu high performance liquid chromatograph LC-10A; C18 preparative column: 10mm × 150mm, eluent is ethanol/water/acetonitrile, its volume ratio is 70/20/10; Flow velocity is 2mL/min; Detection wavelength is 210nm; Sampling volume 1mL; Collect beta-elemene effluent liquid, add sherwood oil in 1:1 ratio, repeatedly extract three times; Separate and merge petroleum ether layer, add Na
2sO
4dry 10-24h, 60 ℃ of vacuum rotatings are drained solvent, obtain sterling;
When distillation, take water as primary solvent, in solvent systems, add and improve solvent systems boiling point and prevent that saponification from bubbling, preventing the material of bumping; Wherein, the material of described raising solvent systems boiling point is ethylene glycol; The described material that prevents that saponification from bubbling is ethanol; The described material that prevents bumping is zeolite;
Extraction by steam distillation condition is: get the raw material that steps A obtains, put into matrass, prepare in following ratio: every 1g raw material adds 3-7mL water, 1-3mL ethylene glycol, a small amount of ethanol, zeolite, and the air distillation of heating, collects the distillate of 80-150 ℃.
4. the method for extracting separating beta-elemene from Hemp Eupatorium according to claim 3, it is characterized in that: extraction by steam distillation condition is: get the raw material that steps A obtains, put into matrass, prepare in following ratio: every 1g raw material adds 5mL water, 2mL ethylene glycol, a small amount of ethanol, zeolite, the air distillation of heating, collects the distillate of 80-150 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110457523.5A CN102432419B (en) | 2011-12-31 | 2011-12-31 | Method for extracting and separating beta-elemene from Eupatorium adenophorum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110457523.5A CN102432419B (en) | 2011-12-31 | 2011-12-31 | Method for extracting and separating beta-elemene from Eupatorium adenophorum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102432419A CN102432419A (en) | 2012-05-02 |
| CN102432419B true CN102432419B (en) | 2014-05-14 |
Family
ID=45980815
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110457523.5A Active CN102432419B (en) | 2011-12-31 | 2011-12-31 | Method for extracting and separating beta-elemene from Eupatorium adenophorum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102432419B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106480004B (en) * | 2016-10-27 | 2019-05-24 | 杭州师范大学 | A kind of sesquiterpene synthase, gene, carrier, engineering bacteria and its application in Eupatorium adenophorum source |
| CN112535180A (en) * | 2020-11-30 | 2021-03-23 | 湖南人文科技学院 | Eupatorium adenophorum spreng extract and application thereof in preventing and controlling paddy field weeds |
| CN114668072A (en) * | 2022-03-10 | 2022-06-28 | 昆明悦馨生物科技有限公司 | Comprehensive utilization method of Eupatorium adenophorum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6464839B1 (en) * | 1997-04-14 | 2002-10-15 | Yuan Da International Group Limited | Beta-elemene, method to prepare the same and uses thereof |
| CN101402544A (en) * | 2008-11-14 | 2009-04-08 | 沈阳万爱普利德医药科技有限公司 | Industrial production method of high-purity beta-elemi alkene bulk medicament |
| CN101402543A (en) * | 2008-11-14 | 2009-04-08 | 沈阳万爱普利德医药科技有限公司 | Beta-elemi alkene bulk medicament and method of preparing its preparations |
-
2011
- 2011-12-31 CN CN201110457523.5A patent/CN102432419B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6464839B1 (en) * | 1997-04-14 | 2002-10-15 | Yuan Da International Group Limited | Beta-elemene, method to prepare the same and uses thereof |
| CN101402544A (en) * | 2008-11-14 | 2009-04-08 | 沈阳万爱普利德医药科技有限公司 | Industrial production method of high-purity beta-elemi alkene bulk medicament |
| CN101402543A (en) * | 2008-11-14 | 2009-04-08 | 沈阳万爱普利德医药科技有限公司 | Beta-elemi alkene bulk medicament and method of preparing its preparations |
Non-Patent Citations (2)
| Title |
|---|
| "紫茎泽兰不同生长部位中β-榄香烯的含量测定";韦会平等;《安徽农业科学》;20111110;第39卷(第32期);第19768-19769,19870页 * |
| 韦会平等."紫茎泽兰不同生长部位中β-榄香烯的含量测定".《安徽农业科学》.2011,第39卷(第32期),第19768-19769,19870页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102432419A (en) | 2012-05-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110304994B (en) | Method for extracting high-purity cannabidiol from industrial cannabis sativa | |
| CN102976909B (en) | Method for extracting and purifying 6-gingerol from ginger | |
| CN103789094A (en) | Method for extracting agilawood essential oil from agilawood chips | |
| CN102351819B (en) | Extraction, purification and preparation method of high-purity salvianolic acid B | |
| CN102432582A (en) | Preparation method of proanthocyanidin | |
| CN102219814A (en) | Method for extracting aucubin from eucommia ulmoides oliver seed draff | |
| CN102731592B (en) | A kind of method extracting oleuropein and Tridemethylsciadopitysin from olive leaf | |
| CN102432419B (en) | Method for extracting and separating beta-elemene from Eupatorium adenophorum | |
| CN101328201A (en) | Method for extracting betulin from birch bark | |
| CN102432420B (en) | Method for extracting and separating beta-elemene from Lantana camara | |
| CN101967119A (en) | Method for purifying arecoline | |
| CN111848358A (en) | Method for preparing high-purity cannabidiol by combining macroporous resin enrichment with dynamic axial compression column system | |
| CN104211690B (en) | Method for separating and purifying mangiferin from aquilaria sinensis leaves | |
| CN103467262B (en) | Method for preparing 9-oxonerolidol from camphor tree plants | |
| CN100564364C (en) | The method of Ligustrazine, forulic acid and volatile oil in a kind of comprehensively extracting and purifying Ligusticum wallichii | |
| CN106699819B (en) | The preparation method of Penta-O-galloyl-D-glucopyranose chemical reference substance | |
| CN101880301B (en) | Novel method for preparing paeoniflorin extract | |
| CN103524578B (en) | A kind of method of extraction and isolation paeoniflorin compound from tree peony stamen | |
| CN102964462B (en) | Wedelia prostrate polysaccharide as well as preparation method and application of wedelia prostrate polysaccharide | |
| CN102293804B (en) | Optimized scheme for comprehensively utilizing silybum mariamum | |
| CN105646424A (en) | A method of extracting luteolin | |
| CN103012510A (en) | Preparation method of 1,2,3,4,6-pentagalloylglucose reference substances | |
| CN101974008A (en) | Process for extracting and purifying podophyllotoxin from Dysosma difformis | |
| CN103254164A (en) | Preparation method of atractylenolide I | |
| CN102091127B (en) | New technology for extracting Chinese magnoliavine fruit lignanoid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201119 Address after: 214500, 6 Industrial Park, Xinqiao Town, Taizhou, Jiangsu, Jingjiang Patentee after: Jingjiang Huatai Machinery Manufacturing Co., Ltd Address before: 617000 Airport Road, Panzhihua, Sichuan, No. 10 Patentee before: PANZHIHUA University |
|
| TR01 | Transfer of patent right |