CN104710502B - A kind of method utilizing ionic liquid extract triptolide and B prime - Google Patents

A kind of method utilizing ionic liquid extract triptolide and B prime Download PDF

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CN104710502B
CN104710502B CN201510117363.8A CN201510117363A CN104710502B CN 104710502 B CN104710502 B CN 104710502B CN 201510117363 A CN201510117363 A CN 201510117363A CN 104710502 B CN104710502 B CN 104710502B
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extraction
triptolide
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liquid
hsccc
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CN104710502A (en
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蔡美强
苏洁
金米聪
宋志军
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Zhejiang Gongshang University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

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Abstract

The present invention relates to a kind of utilize ionic liquid high efficiency extraction triptolide and the method for NSC-163063, the method comprises the following steps: 1) ultrasound-enhanced extraction: extracting solution be volume ratio be the ethanol of 1:0.1～2.0:0.5～2.0, water and glyoxaline ion liquid I mixed liquor, sonification power density is 10～200W/cm2, frequency is 10～100kHz；2) organic solvent extraction: obtain triptolide and NSC-163063 extraction phase with chloroform back extraction after using petroleum ether degreasing；3) HSCCC is isolated and purified: select HSCCC dicyandiamide solution, using ionic liquid II as Mobile Phase Additives, through HSCCC isolated and purified triptolide and NSC-163063 solution；4) vacuum lyophilization.The method significantly reduce the use of organic solvent, significantly reduce the pollution during extraction and harm, improving extraction effect and extraction efficiency, and ionic liquid physical ability is greatly shortened the disengaging time of HSCCC, the triptolide obtained and the purity of NSC-163063 are all higher than 95%.

Description

A kind of method utilizing ionic liquid extract triptolide and B prime
Technical field
The present invention relates to technology of pharmaceutical engineering field, particularly relate to a kind of utilize ionic liquid high efficiency extraction triptolide and the method for NSC-163063.
Background technology
Radix Tripterygii Wilfordii (TripterygiumwilfordiiHook.f.) cry again Caulis Fibraureae, for Celastraceae tripterygium plant, has the effects such as expelling wind and removing dampness, removing obstruction in the collateral to relieve pain, reducing swelling and alleviating pain, removing toxic substances parasite killing.It is mainly used in damp and hot tuberosity, the long-pending poison of carcinoma, treats lepra reaction, rheumatoid arthritis etc. with it clinically, have antitumor, antiinflammatory etc. to act on.nullTriptolide is (also known as Triptolide or Radix Tripterygii Wilfordii lactone alcohol，Triptolide，And NSC-163063 is (also known as Tripdiolide or NSC-163063 CASNo.38748-32-2)，Tripdiolide，CASNo.38647-10-8) it is two kinds produced in the Radix Tripterygii Wilfordii plant native chemical things with higher physiologically active，Because it has special chemical constitution and mechanism of action，The most clinically to autoimmune disease (such as lupus erythematosus、Rheumatoid arthritis、Ankylosing spondylitis and anaphylactoid purpura etc.) there is unique curative effect，It is widely used in the autoimmune diseasees such as treatment rheumatoid arthritis at present，Its instant effect、Determined curative effect，Almost without the similar Chinese medicine that can substitute completely，But in use，The toxic reaction event that Radix Tripterygii Wilfordii causes frequently is reported，One of Chinese herbal medicine that since becoming nearly half a century, generation Times of toxicosis is most.Radix Tripterygii Wilfordii contains multiple effective active composition, there is other unrivaled clinical effects of a lot of medicines, triptolide and NSC-163063 are as one of the main active of Radix Tripterygii Wilfordii medicament, therefore, the preparation method research carrying out triptolide and NSC-163063 has broad application prospects.The structural formula of triptolide and NSC-163063 is as follows:
The method extracting triptolide and NSC-163063 at present from Radix et Rhizoma Tripterygii, stem, leaf mainly uses volatile organic solvent to extract, and uses liquid/liquid extraction binding silica gel column chromatography or liquid/liquid extraction to combine high-speed countercurrent chromatography (HSCCC) and carry out isolated and purified.The method that preparative chromatography separates preparation high-purity triptolide with recrystallization method is combined as the Chinese invention patent " a kind of method of quickly and massively separating high purity triptolide from Radix Tripterygii Wilfordii " of Patent No. ZL200810046163.8 (Authorization Notice No. is CN101367862) provides a kind of column chromatography chromatogram.This patent, with Radix Tripterygii Wilfordii stem and leaf as raw material, uses ethanol or methanol warm macerating, organic solvent back extraction, uses efficiently preparation liquid phase separation and two-phase solvent purified by crystallisation, obtain the triptolide of purity more than 99.5% after column chromatography for separation.The Chinese invention patent " a kind of method of separating triptolide from tripterygium leaf " of Patent No. ZL200810164190.5 (Authorization Notice No. is CN101445545) provides a kind of silica gel column chromatography and combines the method that high speed adverse current chromatogram (HSCCC) method refines Radix Tripterygii Wilfordii lactone alcohol.This patent is with tripterygium leaf as raw material, and by ethanol extraction, ethyl acetate back extraction, silica gel column chromatography uses HSCCC to be refining to obtain purity Radix Tripterygii Wilfordii lactone alcohol more than 95% after separating.Number of patent application is that the Chinese invention patent " preparation method of a kind of prepared from active ingredients of tripterygium wilfordii " of 201210230388.5 (Publication No. 103524591A) provides the method that triptolide is prepared in the separation from Radix et Rhizoma Tripterygii of a kind of silica gel column chromatography.This patent is with Radix et Rhizoma Tripterygii as raw material, and the double-aqueous phase system using polymerinorganic saline solution and ethanol to be formed extracts, and then uses water-insoluble organic solvents back extraction, is then passed through silica gel column chromatography and separates, obtains triptolide after recrystallization.Number of patent application is that the Chinese invention patent " a kind of method separating NSC-163063 from tripterygium leaf " of 201310237971.3 (Publication No. 104231032A) provides a kind of silica gel column chromatography and combines the method that HSCCC method refines NSC-163063.This patent is with tripterygium leaf as raw material, and by ethanol extraction, normal hexane defat, silica gel column chromatography uses HSCCC to be refining to obtain purity NSC-163063 more than 95% after separating.These inventions have employed substantial amounts of organic solvent and carry out extracting separation preparation, although document before is compared in these inventions all preferable novelty, some shortens extraction time, and some decreases solvent consumption, some improves extraction ratio, but all there is safety and the problem of environmental pollution of organic solvent.
Due to the special performance of ionic liquid, the most obtain paying close attention to widely in technology of pharmaceutical engineering field, and application of having succeeded in the extraction separation of natural product.As the Chinese invention patent " new method of a kind of efficiently extracting and producing artemisinin by ionic liquid " of Patent No. ZL200810114416.0 (Authorization Notice No. is CN101597296B) utilizes the arteannuin in the glyoxaline ion liquid halide salts mixture ultrasound-enhanced extraction Herba Artemisiae Annuae raw material with water.Have not been reported about the method utilizing ionic liquid to extract triptolide and NSC-163063 from Radix et Rhizoma Tripterygii, stem, leaf now, therefore, application ionic liquid extract triptolide and the research of NSC-163063, separate a large amount of potential safety hazards using organic reagents and cause in preparation process to solving current triptolide and NSC-163063 and be of great practical significance extracting.
Summary of the invention
The technical problem to be solved is to provide a kind of method safely, effectively utilizing ionic liquid extract triptolide and NSC-163063 for prior art.
The present invention solves the technical scheme that above-mentioned technical problem used: a kind of utilize ionic liquid high efficiency extraction triptolide and the method for NSC-163063, it is characterised in that comprise the following steps:
1) ultrasound-enhanced extraction: cross 40～80 mesh sieves after the drying and crushing such as Radix et Rhizoma Tripterygii, stem, leaf, adding volume ratio by solid-to-liquid ratio 1:5～100 is the ethanol of 1:0.1～2.0:0.5～2.0, water and glyoxaline ion liquid I mixed liquor (innovative point 1), in ultrasonic power density 10～200W/cm2And under supersonic frequency 10～100kHz, supersound extraction 5～90min under the conditions of 20～95 DEG C, the extracting solution of triptolide and NSC-163063 must be contained after solid-liquid separation；
2) organic solvent extraction: petroleum ether is joined described step 1) in the extracting solution that obtains, extract 1～5 time by the volume ratio 1～5:1 of extracting solution with petroleum ether, then aqueous phase chloroform aqueous phase by volume: chloroform=1～5:1 extract 1～3 time.Separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 40～60 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.
3) HSCCC is isolated and purified: select HSCCC dicyandiamide solution, by volume the yellow crude of HSCCC dicyandiamide solution and above-mentioned triptolide and NSC-163063 is joined in separatory funnel than 0.2～2:1 by (mL)/weight (mg), concussion, stratification, after balance, by upper and lower phase separately, upper for fixing phase with dicyandiamide solution, the lower phase of dicyandiamide solution is flowing phase, adjust rotating speed, flowing phase is pumped into certain flow rate, spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively；
4) vacuum lyophilization: by above-mentioned each triptolide and NSC-163063 solution, vacuum lyophilization under proper temperature, drying pressure, reclaim organic extractant, respectively obtains the purity triptolide more than 95% and NSC-163063.
As preferably, the step 2 kind in above method, remove the remaining liquid of the extraction after organic facies can be recycled carry out as in step 1 containing triptolide and the extracting solution of NSC-163063, more than this extraction, liquid Main Ingredients and Appearance is above-mentioned glyoxaline ion liquid I.Preferably, the remaining liquid of extraction includes that volume ratio is the ethanol of 1:0.1～2.0:0.5～2.0, water and glyoxaline ion liquid.
As preferably, the imidazole-like ionic in described glyoxaline ion liquid I includes at least one in 1-ethyl-3-methylimidazole villaumite, 1-ethyl-3-methylimidazole bromide, 1-butyl-3-Methylimidazole. bromide, 1-octyl group-3-Methylimidazole. villaumite, 1-pi-allyl-3-Methylimidazole. villaumite.
As preferably, described step 1) in ethanol, the volume ratio of water and glyoxaline ion liquid I mixed liquor be 10:0.5～2:2～10.
As preferably, described HSCCC dicyandiamide solution be volume ratio be 2:3:3:2: 0.05 the normal hexane-ethyl acetate-HSCCC dicyandiamide solution of alcohol-water-ionic liquid II preparation, volume ratio be 2:3:3:4: 0.05 the HSCCC dicyandiamide solution of methyl tertiary butyl ether(MTBE)-n-butyl alcohol-acetonitrile-water-ionic liquid II preparation or HSCCC dicyandiamide solution that volume ratio is 1:2:1: 0.05 chloroform-ethanol-water-ionic liquid II preparation in any one.
As preferably, described glyoxaline ion liquid II is at least one in 1-ethyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methyl imidazolium tetrafluoroborate, 1-pi-allyl-3-Methylimidazole. bromide.
Beneficial effect
Compared with prior art, it is an advantage of the current invention that: the present invention uses the ethanol mixed by a certain percentage, water and glyoxaline ion liquid I mixed liquor are as extracting solution, in conjunction with ultrasound assisted extraction, extract Radix et Rhizoma Tripterygii, stem, triptolide in leaf and NSC-163063, this extracting solution is compared with existing pure organic solvent extraction, it is greatly saved the usage amount of organic solvent, green, environmental protection, significantly reduce pollution and harm that environment and experimenter are brought by traditional volatile organic solvent extracting method, the interpolation of ionic liquid simultaneously, substantially improve extraction effect, improve extraction efficiency.Further, using ionic liquid as HSCCC Mobile Phase Additives in the present invention, triptolide and NSC-163063 can be obtained simultaneously, and the two reaches to be kept completely separate (separating degree is more than 1.5), substantially reduce the disengaging time of HSCCC, the triptolide obtained and the purity of NSC-163063 are all higher than 95%, substantially increase production efficiency.
Accompanying drawing explanation
Fig. 1 is the triptolide and the ultraviolet chromatogram of NSC-163063 that in the embodiment of the present invention 3 prepared by HSCCC, and wherein, (1) is triptolide, (2) NSC-163063.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1:
1) ultrasound-enhanced extraction: take Thunder God rattan and leaf 1.0kg, 80 mesh sieves are crossed after drying and crushing, then take 100 grams of powder and put in 2500mL conical flask, ethanol/water/1-ethyl-3-methylimidazole villaumite (8:2:7 is added by solid-to-liquid ratio 1:5 (W/V), V/V/V) mixture, in ultrasonic power density 100W/cm2, supersonic frequency 100kHz, under the conditions of temperature 45 C, supersound extraction 30min, obtain the extracting solution containing triptolide and NSC-163063 through solid-liquid separation.
2) organic solvent extraction: petroleum ether is joined above-mentioned steps 1) in the extracting solution that obtains, extract 3 times by volume ratio 3:1 of extracting solution with petroleum ether, separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 50 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.The remaining liquid of extraction recycles, and more than this extraction, liquid is mainly composed of ionic liquid I (1-ethyl-3-methylimidazole villaumite).
3) HSCCC is isolated and purified: weigh step 2) triptolide that obtains and the yellow crude 500mg of NSC-163063, select normal hexane-ethyl acetate-alcohol-water-1-pi-allyl-3-Methylimidazole. bromide (2:3:3:2: 0.05, V/V) as HSCCC dicyandiamide solution, HSCCC dicyandiamide solution and the above-mentioned Radix Tripterygii Wilfordii yellow crude of 200mL are separately added in separatory funnel, concussion makes solution be sufficiently mixed, stand overnight, after split-phase balance, separate upper and lower phase.Upper for fixing phase with dicyandiamide solution, lower phase is flowing phase, adjustment rotating speed is 800r/min, column temperature is 30 DEG C, HSCCC serpentine pipe is pumped into 5.0mL/min flow velocity with high-speed counter-current chromatograph (the TBE-1000A type that Shanghai Tongtian Biotechnology Co., Ltd. produces), spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively.
4) vacuum lyophilization: by step 3) the collection liquid that obtains, pre-freezing temperature-20 DEG C, operating pressure 25Pa, sublimation temperature 50 DEG C, resolution temperature 70 DEG C, obtain the purity triptolide more than 95% and NSC-163063.
Detection uses Agilent1100series high performance liquid chromatograph, and chromatographic column is AccQ.TagC18Post (150mm × 3.9mmi.d., 5 μm, Waters, Milford, MA, USA), flowing is 0.05% (v/v) acetic acid-Spirit of Mindererus. (5mmol/L)/acetonitrile/methanol (60:30:10, V/V/V) mutually, and flow velocity is 0.6mL/min, UV-detector detects under 220nm wavelength, and the purity of triptolide and NSC-163063 is all higher than 95%.
Embodiment 2:
1) ultrasound-enhanced extraction: take Thunder God rattan 1.0kg, 60 mesh sieves are crossed after drying and crushing, then take 100 grams of powder and put in 2500mL conical flask, ethanol/water/1-ethyl-3-methylimidazole bromide (4:1:5 is added by solid-to-liquid ratio 1:10 (W/V), V/V/V) mixture, in ultrasonic power density 50W/cm2, supersonic frequency 50kHz, under the conditions of temperature 45 C, supersound extraction 20min, obtain the extracting solution containing triptolide and NSC-163063 through solid-liquid separation.
2) organic solvent extraction: petroleum ether is joined above-mentioned steps 1) in the extracting solution that obtains, extract 3 times by volume ratio 5:1 of extracting solution with petroleum ether, separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 50 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.The remaining liquid of extraction recycles, and more than this extraction, liquid is mainly composed of ionic liquid I (1-ethyl-3-methylimidazole bromide).
3) HSCCC is isolated and purified: weigh step 2) triptolide that obtains and the yellow crude 500mg of NSC-163063, select methyl tertiary butyl ether(MTBE)-n-butyl alcohol-acetonitrile-water-1-pi-allyl-3-Methylimidazole. bromide (2:3:3:4: 0.05, V/V) as HSCCC dicyandiamide solution, HSCCC dicyandiamide solution and the above-mentioned Radix Tripterygii Wilfordii yellow crude of 250mL are separately added in separatory funnel, concussion makes solution be sufficiently mixed, stand overnight, after split-phase balance, separate upper and lower phase.Upper for fixing phase with dicyandiamide solution, lower phase is flowing phase, adjustment rotating speed is 750r/min, column temperature is 30 DEG C, HSCCC serpentine pipe is pumped into 5.0mL/min flow velocity with high-speed counter-current chromatograph (the TBE-1000A type that Shanghai Tongtian Biotechnology Co., Ltd. produces), spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively.
4) vacuum lyophilization: by step 3) the collection liquid that obtains, pre-freezing temperature-25 DEG C, operating pressure 25Pa, sublimation temperature 50 DEG C, resolution temperature 65 DEG C, obtain the purity triptolide more than 95% and NSC-163063.
Detection uses Agilent1100series high performance liquid chromatograph, and chromatographic column is AccQ.TagC18Post (150mm × 3.9mmi.d., 5 μm, Waters, Milford, MA, USA), flowing is 0.05% (v/v) acetic acid-Spirit of Mindererus. (5mmol/L)/acetonitrile/methanol (60:30:10, V/V/V) mutually, and flow velocity is 0.6mL/min, UV-detector detects under 220nm wavelength, and the purity of triptolide and NSC-163063 is all higher than 95%.
Embodiment 3:
1) ultrasound-enhanced extraction: take tripterygium leaf 1.0kg, 40 mesh sieves are crossed after drying and crushing, then take 100 grams of powder and put in 2500mL conical flask, ethanol/water/1-butyl-3-Methylimidazole. bromide (5:1:5 is added by solid-to-liquid ratio 1:8 (W/V), V/V/V) mixture, in ultrasonic power density 100W/cm2, supersonic frequency 50kHz, under the conditions of temperature 40 DEG C, supersound extraction 30min, obtain the extracting solution containing triptolide and NSC-163063 through solid-liquid separation.
2) organic solvent extraction: petroleum ether is joined above-mentioned steps 1) in the extracting solution that obtains, extract 3 times by volume ratio 3:1 of extracting solution with petroleum ether, separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 45 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.The remaining liquid of extraction recycles, and more than this extraction, liquid is mainly composed of ionic liquid I (1-ethyl-3-methylimidazole bromide).
3) HSCCC is isolated and purified: weigh step 2) triptolide that obtains and the yellow crude 500mg of NSC-163063, select chloroform-ethanol-water-1-pi-allyl-3-Methylimidazole. bromide (1:2:1: 0.05, v/v) as HSCCC dicyandiamide solution, HSCCC dicyandiamide solution and the above-mentioned Radix Tripterygii Wilfordii yellow crude of 250mL are separately added in separatory funnel, concussion makes solution be sufficiently mixed, stand overnight, after split-phase balance, separate upper and lower phase.Upper for fixing phase with dicyandiamide solution, lower phase is flowing phase, adjustment rotating speed is 700r/min, column temperature is 25 DEG C, HSCCC serpentine pipe is pumped into 5.0mL/min flow velocity with high-speed counter-current chromatograph (the TBE-1000A type that Shanghai Tongtian Biotechnology Co., Ltd. produces), spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively.
4) vacuum lyophilization: by step 3) the collection liquid that obtains, pre-freezing temperature-25 DEG C, operating pressure 25Pa, sublimation temperature 50 DEG C, resolution temperature 60 DEG C, obtain the purity triptolide more than 95% and NSC-163063.
Detection uses Agilent1100series high performance liquid chromatograph, and chromatographic column is AccQ.TagC18Post (150mm × 3.9mmi.d., 5 μm, Waters, Milford, MA, USA), flowing is 0.05% (v/v) acetic acid-Spirit of Mindererus. (5mmol/L)/acetonitrile/methanol (60:30:10, V/V/V) mutually, and flow velocity is 0.6mL/min, UV-detector detects under 220nm wavelength, and the purity of triptolide and NSC-163063 is all higher than 95%.
Embodiment 4:
1) ultrasound-enhanced extraction: take Radix et Rhizoma Tripterygii 1.0kg, 80 mesh sieves are crossed after drying and crushing, then take 100 grams of powder and put in 2500mL conical flask, ethanol/water/1-octyl group-3-Methylimidazole. villaumite (10:1:5 is added by solid-to-liquid ratio 1:50 (W/V), V/V/V) mixture, in ultrasonic power density 20W/cm2, supersonic frequency 70kHz, under the conditions of temperature 65 DEG C, supersound extraction 90min, obtain the extracting solution containing triptolide and NSC-163063 through solid-liquid separation.
2) organic solvent extraction: petroleum ether is joined above-mentioned steps 1) in the extracting solution that obtains, extract 2 times by volume ratio 4:1 of extracting solution with petroleum ether, separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 50 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.The remaining liquid of extraction recycles, and more than this extraction, liquid is mainly composed of ionic liquid I (1-octyl group-3-Methylimidazole. villaumite).
3) HSCCC is isolated and purified: weigh step 2) triptolide that obtains and the yellow crude 500mg of NSC-163063, select chloroform-ethanol-water-1-ethyl-3-methylimidazole hexafluorophosphate (1:2:1: 0.05, v/v) as HSCCC dicyandiamide solution, HSCCC dicyandiamide solution and the above-mentioned Radix Tripterygii Wilfordii yellow crude of 250mL are separately added in separatory funnel, concussion makes solution be sufficiently mixed, stand overnight, after split-phase balance, separate upper and lower phase.Upper for fixing phase with dicyandiamide solution, lower phase is flowing phase, adjustment rotating speed is 800r/min, column temperature is 20 DEG C, HSCCC serpentine pipe is pumped into 4.0mL/min flow velocity with high-speed counter-current chromatograph (the TBE-1000A type that Shanghai Tongtian Biotechnology Co., Ltd. produces), spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively.
4) vacuum lyophilization: by step 3) the collection liquid that obtains, pre-freezing temperature-20 DEG C, operating pressure 30Pa, sublimation temperature 55 DEG C, resolution temperature 75 DEG C, obtain the purity triptolide more than 95% and NSC-163063.
Detection uses Agilent1100series high performance liquid chromatograph, and chromatographic column is AccQ.TagC18Post (150mm × 3.9mmi.d., 5 μm, Waters, Milford, MA, USA), flowing is 0.05% (v/v) acetic acid-Spirit of Mindererus. (5mmol/L)/acetonitrile/methanol (60:30:10, V/V/V) mutually, and flow velocity is 0.6mL/min, UV-detector detects under 220nm wavelength, and the purity of triptolide and NSC-163063 is all higher than 95%.
Embodiment 5:
1) ultrasound-enhanced extraction: take Thunder God rattan 1.0kg, 60 mesh sieves are crossed after drying and crushing, then take 100 grams of powder and put in 2500mL conical flask, ethanol/water/1-pi-allyl-3-Methylimidazole. villaumite (3:1:3 is added by solid-to-liquid ratio 1:70 (W/V), V/V/V) mixture, in ultrasonic power density 50W/cm2, supersonic frequency 200kHz, under the conditions of temperature 35 DEG C, supersound extraction 70min, obtain the extracting solution containing triptolide and NSC-163063 through solid-liquid separation.
2) organic solvent extraction: petroleum ether is joined above-mentioned steps 1) in the extracting solution that obtains, extract 3 times by volume ratio 3:1 of extracting solution with petroleum ether, separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 55 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.The remaining liquid of extraction recycles, and more than this extraction, liquid is mainly composed of ionic liquid I (1-pi-allyl-3-Methylimidazole. villaumite).
3) HSCCC is isolated and purified: weigh step 2) triptolide that obtains and the yellow crude 500mg of NSC-163063, select chloroform-ethanol-water-1-ethyl-3-methylimidazole tetrafluoroborate (1:2:1: 0.05, v/v) as HSCCC dicyandiamide solution, HSCCC dicyandiamide solution and the above-mentioned Radix Tripterygii Wilfordii yellow crude of 400mL are separately added in separatory funnel, concussion makes solution be sufficiently mixed, stand overnight, after split-phase balance, separate upper and lower phase.Upper for fixing phase with dicyandiamide solution, lower phase is flowing phase, adjustment rotating speed is 750r/min, column temperature is 25 DEG C, HSCCC serpentine pipe is pumped into 4.5mL/min flow velocity with high-speed counter-current chromatograph (the TBE-1000A type that Shanghai Tongtian Biotechnology Co., Ltd. produces), spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively.
4) vacuum lyophilization: by step 3) the collection liquid that obtains, pre-freezing temperature-25 DEG C, operating pressure 20Pa, sublimation temperature 55 DEG C, resolution temperature 75 DEG C, obtain the purity triptolide more than 95% and NSC-163063.
Detection uses Agilent1100series high performance liquid chromatograph, and chromatographic column is AccQ.TagC18Post (150mm × 3.9mmi.d., 5 μm, Waters, Milford, MA, USA), flowing is 0.05% (v/v) acetic acid-Spirit of Mindererus. (5mmol/L)/acetonitrile/methanol (60:30:10, V/V/V) mutually, and flow velocity is 0.6mL/min, UV-detector detects under 220nm wavelength, and the purity of triptolide and NSC-163063 is all higher than 95%.
Embodiment 6:
1) ultrasound-enhanced extraction: take tripterygium leaf 1.0kg, 40 mesh sieves are crossed after drying and crushing, then take 100 grams of powder and put in 2500mL conical flask, ethanol/water/1-butyl-3-Methylimidazole. bromide (5:1:4 is added by solid-to-liquid ratio 1:80 (W/V), V/V/V) mixture, in ultrasonic power density 200W/cm2, supersonic frequency 100kHz, under the conditions of temperature 55 DEG C, supersound extraction 15min, obtain the extracting solution containing triptolide and NSC-163063 through solid-liquid separation.
2) organic solvent extraction: petroleum ether is joined above-mentioned steps 1) in the extracting solution that obtains, extract 3 times by volume ratio 3:1 of extracting solution with petroleum ether, separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 55 DEG C near dry, obtains the yellow crude containing triptolide and NSC-163063.The remaining liquid of extraction recycles, and more than this extraction, liquid is mainly composed of ionic liquid I (1-butyl-3-Methylimidazole. bromide).
3) HSCCC is isolated and purified: weigh step 2) triptolide that obtains and the yellow crude 500mg of NSC-163063, select chloroform-ethanol-water--butyl-3-methyl imidazolium tetrafluoroborate (1:2:1: 0.05, v/v) as HSCCC dicyandiamide solution, HSCCC dicyandiamide solution and the above-mentioned Radix Tripterygii Wilfordii yellow crude of 500mL are separately added in separatory funnel, concussion makes solution be sufficiently mixed, stand overnight, after split-phase balance, separate upper and lower phase.Upper for fixing phase with dicyandiamide solution, lower phase is flowing phase, adjustment rotating speed is 750r/min, column temperature is 25 DEG C, HSCCC serpentine pipe is pumped into 4.5mL/min flow velocity with high-speed counter-current chromatograph (the TBE-1000A type that Shanghai Tongtian Biotechnology Co., Ltd. produces), spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively.
4) vacuum lyophilization: by step 3) the collection liquid that obtains, pre-freezing temperature-25 DEG C, operating pressure 25Pa, sublimation temperature 55 DEG C, resolution temperature 60 DEG C, obtain the purity triptolide more than 95% and NSC-163063.
Detection uses Agilent1100series high performance liquid chromatograph, and chromatographic column is AccQ.TagC18Post (150mm × 3.9mmi.d., 5 μm, Waters, Milford, MA, USA), flowing is 0.05% (V/V) acetic acid-Spirit of Mindererus. (5mmol/L)/acetonitrile/methanol (60:30:10, V/V/V) mutually, and flow velocity is 0.6mL/min, UV-detector detects under 220nm wavelength, and the purity of triptolide and NSC-163063 is all higher than 95%.

Claims (3)

1. the method utilizing ionic liquid extract triptolide and NSC-163063, it is characterised in that comprise the following steps:
1) ultrasound-enhanced extraction: Radix et Rhizoma Tripterygii, stem or leaf sample drying cross 40～80 mesh sieves after pulverizing, adding volume ratio by solid-to-liquid ratio 1:5～100 is the ethanol of 1:0.1～2.0:0.5～2.0, water and glyoxaline ion liquid I mixed liquor, in ultrasonic power density 10～200W/cm2And under supersonic frequency 10～100kHz, 20～95 DEG C of supersound extraction 5～90min, solid-liquid separation must contain the extracting solution of triptolide and NSC-163063；
2) organic solvent extraction: petroleum ether is joined described step 1) in the extracting solution that obtains, extract 1～5 time by the volume ratio 1～5:1 of extracting solution with petroleum ether, then aqueous phase chloroform aqueous phase by volume: chloroform=1～5:1 extract 1～3 time；Separate organic facies and obtain triptolide and NSC-163063 extraction phase, then the organic extraction obtained is evaporated at 40～60 DEG C near dry, obtain the yellow crude containing triptolide and NSC-163063, the remaining liquid of extraction recycles, and more than this extraction, liquid Main Ingredients and Appearance is above-mentioned glyoxaline ion liquid I；
3) HSCCC is isolated and purified: select HSCCC dicyandiamide solution, the yellow crude of HSCCC dicyandiamide solution and above-mentioned triptolide and NSC-163063 is joined in separatory funnel by by volume/weight ratio 0.2～2:1 (mL/mg), concussion, stratification, after balance, by upper and lower phase separately, upper for fixing phase with dicyandiamide solution, the lower phase of dicyandiamide solution is flowing phase, adjust rotating speed, flowing phase is pumped into certain flow rate, spectrogram Fraction collection object according to detector, obtains triptolide and the solution of NSC-163063 respectively；
4) vacuum lyophilization: by above-mentioned each triptolide and NSC-163063 solution, vacuum lyophilization under proper temperature, drying pressure, reclaim organic extractant, respectively obtains the purity triptolide more than 95% and NSC-163063；
Wherein, the ion of described glyoxaline ion liquid I is at least one salt in 1-ethyl-3-methylimidazole villaumite, 1-ethyl-3-methylimidazole bromide, 1-butyl-3-Methylimidazole. bromide, 1-octyl group-3-Methylimidazole. villaumite, 1-pi-allyl-3-Methylimidazole. villaumite；
Wherein, described HSCCC dicyandiamide solution be volume ratio be the HSCCC dicyandiamide solution of normal hexane-ethyl acetate-alcohol-water-ionic liquid II preparation of 2:3:3:2: 0.05；Volume ratio is any one in the HSCCC dicyandiamide solution of methyl tertiary butyl ether(MTBE)-n-butyl alcohol-acetonitrile-water-ionic liquid II preparation of 2:3:3:4: 0.05 or HSCCC dicyandiamide solution that volume ratio is 1:2:1: 0.05 chloroform-ethanol-water-ionic liquid II preparation；Wherein, at least one during the ion in described glyoxaline ion liquid II is 1-ethyl-3-methylimidazole tetrafluoroborate, 1-ethyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methyl imidazolium tetrafluoroborate, 1-pi-allyl-3-Methylimidazole. bromide.
The most the method for claim 1, wherein in step 2) in also include removing organic facies and obtaining the remaining liquid of extraction, the method that more than this extraction, liquid is recycled in step 1 obtains containing triptolide and the extracting solution of NSC-163063.
3. method as claimed in claim 2, wherein, more than this extraction, liquid Main Ingredients and Appearance is volume ratio 1:0.1～the ethanol of 2.0:0.5～2.0, water and glyoxaline ion liquid I mixed liquor.
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