CN101367862A - Method for quickly and massively separating high purity triptolide from thunder god vine - Google Patents
Method for quickly and massively separating high purity triptolide from thunder god vine Download PDFInfo
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- CN101367862A CN101367862A CNA2008100461638A CN200810046163A CN101367862A CN 101367862 A CN101367862 A CN 101367862A CN A2008100461638 A CNA2008100461638 A CN A2008100461638A CN 200810046163 A CN200810046163 A CN 200810046163A CN 101367862 A CN101367862 A CN 101367862A
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Abstract
The present invention discloses a technique for separating out and purifying triptolide monomer from common threewingnut root. The technique is carried out according to the following technical steps: (A) heating and extraction: after ethanol or methanol is warmly soaked, dregs are removed; (B) solvent extraction: organic solvent is adopted to extract concentrated solution in order to enrich the product; (C) chromatographic column chromatography: with neutral alumina or silica gel as filler, dichloromethane-methanol or chloroform-methanol is used to carry out gradient elution, and cut fraction is collected by stages; (D) high-efficiency preparation and liquid phase separation; (E) crystallization: two-phase solvent crystallization is adopted to use petroleum ether-ethyl acetate, dichloromethane-methanol or methylene dichloride-ethyl acetate to crystallize the cut fraction collected by stages, so that the colorless spiculate crystal product of triptolide, the purity of which reaches over 99.5 percent, can be obtained. The technique, which has the advantages of simplicity, rapidness, little solvent consumption, economy, environment-friendliness and high yield, can prepare a large amount of the product for one time, thus having high economic and academic values.
Description
Technical field
The present invention relates to a kind of from the herbal medicine medicinal material method of isolating active composition, particularly, the present invention relates to a kind of from trypterygine the method for quickly and massively separating high purity triptolide.
Background technology
Triptolide (triptolide) is the highest epoxidation diterpenic lactone of isolated activity from Celastraceae plant trypterygine Tripterygium wilfordiiHook.f., be one of main effective constituent of trypterygine, its relevant potency ratio tripterygium total glycosides is high 100-200 times.Show that through pharmacology and clinical trial it has definite immunosuppression, anti-inflammatory, antifertility and biological activity such as antitumor.At present, Glucosidorum Tripterygll Totorum be main on the market be the preparation of raw material with the trypterygine, be used for rheumatoid arthritis, nephrotic syndrome, the Behcet triad, lepra reaction, autoimmune hepatitis etc., but its toxicity is bigger, effective dose and toxic dose difference are very little, and the security of medicine is difficult to control, and single effective constituent triptolide wherein is because can fine control dosage, also can be used as the precursor of structure of modification, its demand is increasing.Triptolide content in plant is extremely low, and main at present employing normal pressure silica gel column chromatography repeatedly separates, and yield is low, and is not only very big to operator's toxicity, and noxious solvent consumption is also big.
Summary of the invention
The present invention is intended to overcome above-mentioned defective, provide a kind of from trypterygine the method for quickly and massively separating high purity triptolide.Method is simple for this, the consumption solvent is few, yield is high, safety non-toxic, environmental protection.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of from trypterygine the method for quickly and massively separating high purity triptolide, it is characterized in that being undertaken by following processing step:
A. heating is extracted: choosing granularity is 10-60 purpose trypterygine cauline leaf meal, presses the alcoholic solvent that mass ratio adds 5 times of amounts, in 25-65 ℃ of heating 2-5 hours, and slagging-off after filtration, filtrate decompression obtains aqeous suspension after reclaiming and removing alcoholic solvent;
It is 95% ethanol or 100% methyl alcohol that described alcoholic solvent is selected from concentration of volume percent, preferred 95% ethanol.
Preferred 20 orders of the granularity of described trypterygine cauline leaf meal.
Preferred 55 ℃ of the Heating temperature of described trypterygine cauline leaf meal and alcoholic solvent.
B. solvent extraction: with steps A gained aqeous suspension, first petroleum ether extraction with 2 times of volume ratios, water is again with chloroform or dichloromethane extraction, each extracts 3-5 times, each consumption is 3-5 times of aqeous suspension by volume, and petroleum ether layer discards after reclaiming sherwood oil, combined chloroform or dichloromethane layer extraction liquid obtain deep yellow precipitation (density is 1.26) through concentrating under reduced pressure under 45-65 ℃ of temperature.
Described sherwood oil, chloroform or methylene dichloride are analytical pure.
C. column chromatography: carry out the normal pressure column chromatography after the deep yellow precipitation dissolve with methanol dry method of B step gained mixed sample.Wherein, the chromatography column filler is neutral alumina or silica gel, and granularity is 100-400 orders; The chromatographic column specification is decided according to fractional dose, and diameter is 5-50cm.The selected eluent of gradient elution is the mixed solvent of methylene dichloride-methyl alcohol or chloroform-methyl alcohol, by volume from high to low, successively in the eluent ratio of 100-5:1, the Fractional Collections cut.
D. efficient production liquid phase separation: after the cut of C step Fractional Collections merged, add methyl alcohol, obtain the triptolide work in-process through the efficient production liquid phase separation again to just dissolving.
Described efficient production liquid phase filler is octadecylsilane chemically bonded silica (C18), and the post specification is 25cm*10cm or 25cm*5cm, chromatographic condition: detect wavelength 215nm, moving phase is methyl alcohol-water or acetonitrile-water, and content all can reach 90%.
The concentration of volume percent of methyl alcohol is 20-80% in the described moving phase.
The concentration of volume percent of acetonitrile is 15-45% in the described moving phase.
.E. crystallization:, adopt the two-phase solvent crystallization process to be further purified to obtain purity to reach 99.5% colourless needle crystal product with D step gained triptolide work in-process.Used recrystallisation solvent be in the combination of sherwood oil-ethyl acetate, methylene dichloride-methyl alcohol or methylene dichloride-ethyl acetate any one or several.
The volume ratio of described sherwood oil-ethyl acetate is 5-1:1.
The volume ratio of described methylene dichloride-methyl alcohol is 20-10:1.
The volume ratio of described methylene dichloride-ethyl acetate is 20-5:1.
The invention has the advantages that:
1, step is few, weak point consuming time, and the yield height, easy and simple to handle, be easy to suitability for industrialized production.
2, solvent consumption is few, and the solvent in the steps such as extraction, extraction, column chromatography, preparation liquid phase separation is all recyclable to be utilized again.
3, solvent for use toxicity is less, used sherwood oil, ethyl acetate, acetone etc. in the chromatography process, the chloroform that alternative common column chromatography adopts, methyl alcohol etc., safety non-toxic, environmental protection.
4, good in economic efficiency.Can carry out scale operation, and operation is easy, only comprises 5 steps, be easy to control, the separation efficiency height.
5, has high academic value.Domestic bibliographical information about the triptolide separation and purification all is conventional column chromatography at present, and the separation and purification that high performance preparative liquid chromatography is used for triptolide also belongs to first.The present invention provides pharmacology raw material, theory and practice support for the further development and use of triptolide.
Description of drawings
Fig. 1 detects collection of illustrative plates for the described HPLC of B step of the present invention
Fig. 2 is online surveillance map spectrum for the described preparation of D step of the present invention separates
Fig. 3 detects collection of illustrative plates for the described HPLC of D step of the present invention
Fig. 4 detects collection of illustrative plates for the described HPLC of E step of the present invention
Embodiment
Embodiment 1
A kind of from trypterygine the method for quickly and massively separating high purity triptolide, it is characterized in that being undertaken by following processing step:
A. heating is extracted: choosing granularity is 10 purpose trypterygine cauline leaf meal 30kg, and the mass ratio of pressing 1:5 adds methyl alcohol, heated 3 hours down at 50 ℃, and slagging-off after filtration, filtrate decompression obtains aqeous suspension after reclaiming and removing methyl alcohol;
B. solvent extraction: get steps A gained aqeous suspension 10L, use the 20L petroleum ether extraction earlier, water is used the 30L dichloromethane extraction again, and each extracts 3 times.Petroleum ether layer discards after reclaiming sherwood oil, and dichloromethane extraction liquid merges back 50 ℃ and concentrates, and obtains deep yellow precipitation (density is 1.26) 150g.
C. column chromatography: get B step deep yellow precipitation 20g, the dissolve with methanol dry method is carried out the normal pressure column chromatography after mixing sample.The chromatography column filler is a silica gel, and granularity is 300-400 orders.The chromatographic column specification is diameter 5cm.Gradient elution, eluent are the mixed solvent of methylene dichloride-methyl alcohol, from high to low, press 100:1 successively, 50:1,20:1 and 5:1, Fractional Collections cut.Cut stage casing triptolide purity reaches 50%.
D. efficient production liquid phase separation: after the cut of C step Fractional Collections merged, add methyl alcohol, obtain purity through the efficient production liquid phase separation again and reach 90% triptolide work in-process to just dissolving.
Described preparation liquid phase filler is octadecylsilane chemically bonded silica (C18), and the post specification is 25cm*5cm, chromatographic condition: detect wavelength 215nm, moving phase is acetonitrile-water (volume percent of acetonitrile is 19%).
E. crystallization:, adopt the two-phase solvent crystallization process to be further purified to obtain purity to reach 99.5% colourless needle crystal product 1g with D step gained triptolide work in-process.Used recrystallisation solvent is sherwood oil-ethyl acetate, and its volume ratio is 5:1.
Embodiment 2
A kind of from trypterygine the method for quickly and massively separating high purity triptolide, it is characterized in that being undertaken by following processing step:
A. heating is extracted: choosing granularity is 20 purpose trypterygine cauline leaf meal 500kg, and the mass ratio of pressing 1:5 adds 95% ethanol, heated 3 hours down at 55 ℃, and slagging-off after filtration, filtrate decompression obtains aqeous suspension after reclaiming and removing ethanol;
B. solvent extraction: get steps A gained aqeous suspension 500L, use the 1000L petroleum ether extraction earlier, water is used the 1500L dichloromethane extraction again, and each extracts 5 times.Petroleum ether layer discards after reclaiming sherwood oil, and dichloromethane extraction liquid merges back 55 ℃ and concentrates, and obtains deep yellow precipitation 9000g (density is 1.26).
C. column chromatography: get and carry out the normal pressure column chromatography after B step deep yellow precipitation 9000g dissolve with methanol dry method is mixed sample.The chromatography column filler is a silica gel, and granularity is 100-200 orders.The chromatographic column specification is diameter 50cm.Gradient elution, eluent are the mixed solvent of chloroform-methyl alcohol, from high to low, press 100:1 successively, 50:1,20:1 and 5:1, Fractional Collections cut.Cut stage casing triptolide purity reaches more than 50%, triptolide purity is 10%-30% in leading portion and the back segment.
D. efficient production liquid phase separation: after the cut of C step Fractional Collections merged, add methyl alcohol, obtain the triptolide work in-process through the efficient production liquid phase separation again to just dissolving.
Described preparation liquid phase filler is octadecylsilane chemically bonded silica (C18), and the post specification is 25cm*20cm, chromatographic condition: detect wavelength 215nm, moving phase is methyl alcohol-water (concentration of volume percent of methyl alcohol is 40%).
E. crystallization:, adopt the two-phase solvent crystallization process to be further purified to obtain purity to reach 99.5% colourless needle crystal product 20g with D step gained triptolide work in-process.Used recrystallisation solvent is selected methylene dichloride-ethyl acetate for use, and its volume ratio is 4:1.
Claims (9)
1. the method for a separating high-purity triptolide from trypterygine is characterized in that being undertaken by following processing step:
A. heating is extracted: choosing granularity is 10-60 purpose trypterygine cauline leaf powder, presses the alcoholic solvent that mass ratio adds 5 times of amounts, in 25-65 ℃ of heating 2-5 hours, and slagging-off after filtration, filtrate decompression obtains aqeous suspension after reclaiming and removing alcoholic solvent;
B. solvent extraction: with steps A gained aqeous suspension, first petroleum ether extraction with 2 times of volume ratios, water is again with chloroform or dichloromethane extraction, each extracts 3-5 times, each consumption is 3-5 times of aqeous suspension by volume, and petroleum ether layer discards after reclaiming sherwood oil, combined chloroform or dichloromethane layer extraction liquid, obtaining density through concentrating under reduced pressure under 45-65 ℃ of temperature is 1.26 deep yellows precipitations;
C. column chromatography: carry out the normal pressure column chromatography after the deep yellow precipitation dissolve with methanol dry method of B step gained mixed sample, wherein, the chromatography column filler is aluminum oxide or silica gel, and granularity is 100-400 orders, and the chromatogram diameter is 5-50cm; The selected eluent of gradient elution is the mixed solvent of methylene dichloride-methyl alcohol or chloroform-methyl alcohol, by volume from high to low, successively in the eluent ratio of 100-5:1, the Fractional Collections cut;
D. efficient production liquid phase separation: after the cut of C step Fractional Collections merged, add methyl alcohol, obtain the triptolide work in-process through the efficient production liquid phase separation again to just dissolving;
E. crystallization: with D step gained triptolide work in-process, adopt the two-phase solvent crystallization process to be further purified to obtain purity to reach 99.5% colourless needle crystal product, used recrystallisation solvent be in sherwood oil-ethyl acetate, methylene dichloride-methyl alcohol or methylene dichloride-ethyl acetate combination any one or several.
2. according to claim 1 from trypterygine the method for separating high-purity triptolide, it is characterized in that it is 95% ethanol or 100% methyl alcohol that the described alcoholic solvent of steps A is selected from concentration of volume percent.
3. according to claim 1 from trypterygine the method for separating high-purity triptolide, the Heating temperature that it is characterized in that steps A described trypterygine cauline leaf powder and alcoholic solvent is 55 ℃.
4. according to claim 1 from trypterygine the method for separating high-purity triptolide, it is characterized in that the described efficient production liquid phase of step D filler is an octadecylsilane chemically bonded silica, the post specification is 25cm*10cm or 25cm*5cm, chromatographic condition: detect wavelength 215nm, moving phase is methyl alcohol-water or acetonitrile-water.
5. according to claim 4 from trypterygine the method for separating high-purity triptolide, the concentration of volume percent that it is characterized in that methyl alcohol in the described moving phase is 20-80%.
6. according to claim 4 from trypterygine the method for separating high-purity triptolide, the concentration of volume percent that it is characterized in that acetonitrile in the described moving phase is 15-45%.
7. according to claim 1 from trypterygine the method for separating high-purity triptolide, the volume ratio that it is characterized in that the described sherwood oil-ethyl acetate of step e is 5-1:1.
8. according to claim 1 from trypterygine the method for separating high-purity triptolide, the volume ratio that it is characterized in that the described methylene dichloride-methyl alcohol of step e is 20-10:1.
9. according to claim 1 from trypterygine the method for separating high-purity triptolide, the volume ratio that it is characterized in that the described methylene dichloride-ethyl acetate of step e is 20-5:1.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101747406B (en) * | 2010-01-06 | 2012-10-24 | 福建省汉堂生物制药股份有限公司 | Technology for producing triptodiolide with leaf of tripterygium |
| CN103159822A (en) * | 2013-03-04 | 2013-06-19 | 华侨大学 | Preparation method of triptolide and ramification composition thereof |
| CN103242414A (en) * | 2013-05-17 | 2013-08-14 | 成都彼斯特生物科技有限公司 | Method for separating and purifying tripterine from medicinal material celastrus orbiculatus |
| CN103610727A (en) * | 2013-11-26 | 2014-03-05 | 浙江得恩德制药有限公司 | Method for reducing organic solvent residue in tripterygium glycoside |
| CN104231032A (en) * | 2013-06-13 | 2014-12-24 | 宁波工程学院 | Method for separating tripdiolide from Tripterygium Wilfordii Hook F leaves |
| CN104788532A (en) * | 2015-04-30 | 2015-07-22 | 成都健腾生物技术有限公司 | Extraction preparation method of triptolide |
| CN106153794A (en) * | 2015-04-10 | 2016-11-23 | 浙江海洋学院 | The method of triptolide in a kind of microwave radiation exaraction/HPLC detection tripterygium leaf |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1431336A (en) * | 1973-04-03 | 1976-04-07 | Kupchan S M | Diterpenoid triepoxides their isolation and use |
| AU5594794A (en) * | 1993-11-08 | 1995-05-29 | Pharmagenesis, Inc. | Immunotherapy composition and method |
| WO2005077008A2 (en) * | 2004-02-09 | 2005-08-25 | Pharmagenesis, Inc. | Methods for isolation of triptolide compounds from tripterygium wilfordii |
| CN1327775C (en) * | 2004-02-20 | 2007-07-25 | 浙江医药股份有限公司新昌制药厂 | Preparation method of thunder godvine extract |
-
2008
- 2008-09-25 CN CN2008100461638A patent/CN101367862B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101747406B (en) * | 2010-01-06 | 2012-10-24 | 福建省汉堂生物制药股份有限公司 | Technology for producing triptodiolide with leaf of tripterygium |
| CN103159822A (en) * | 2013-03-04 | 2013-06-19 | 华侨大学 | Preparation method of triptolide and ramification composition thereof |
| CN103159822B (en) * | 2013-03-04 | 2016-05-18 | 华侨大学 | The preparation method of triptolide and derivative composition thereof |
| CN103242414A (en) * | 2013-05-17 | 2013-08-14 | 成都彼斯特生物科技有限公司 | Method for separating and purifying tripterine from medicinal material celastrus orbiculatus |
| CN103242414B (en) * | 2013-05-17 | 2015-09-30 | 成都彼斯特生物科技有限公司 | A kind of method from Stem of Oriental Bittersweet medicinal material separation and purification Tripterine |
| CN104231032A (en) * | 2013-06-13 | 2014-12-24 | 宁波工程学院 | Method for separating tripdiolide from Tripterygium Wilfordii Hook F leaves |
| CN103610727A (en) * | 2013-11-26 | 2014-03-05 | 浙江得恩德制药有限公司 | Method for reducing organic solvent residue in tripterygium glycoside |
| CN103610727B (en) * | 2013-11-26 | 2015-12-30 | 浙江得恩德制药有限公司 | A kind of method reducing organic solvent residual in tripterygium glycosides |
| CN106153794A (en) * | 2015-04-10 | 2016-11-23 | 浙江海洋学院 | The method of triptolide in a kind of microwave radiation exaraction/HPLC detection tripterygium leaf |
| CN104788532A (en) * | 2015-04-30 | 2015-07-22 | 成都健腾生物技术有限公司 | Extraction preparation method of triptolide |
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