CN108191948B - A method of preparing triptolide and 2- table triptolide - Google Patents
A method of preparing triptolide and 2- table triptolide Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- 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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Abstract
The invention discloses a kind of methods for preparing triptolide and 2- table triptolide.It is very close to be present in triptonide in tripterygium wilfordii, triptolide, triptolide and 2- table triptolide structure, wherein triptolide and 2- table triptolide are a pair of of chiral isomer, and separating difficulty is big.For the prior art when separating these four compounds, nothing is independent of silica gel column chromatography repeatedly, however silica gel column chromatography expends solvent, poor reproducibility repeatedly, is suitable only for laboratory and separates on a small quantity, it is difficult to promote in the industrial production.At present in industrial production, uniquely there was only macroreticular resin in the column chromatography used.Method provided by the invention separates independent of silica gel column chromatography repeatedly and prepares triptonide, triptolide, triptolide and 2- table triptolide.HPLC method provided by the invention can efficiently separate chiral isomer triptolide and 2- table triptolide, at low cost, repeated height based on conventional C18 chromatographic column.
Description
Technical field
The invention belongs to chemical fields, are related to the preparation method and method for separating and analyzing of known compound, and in particular to one
Kind prepare triptonide, triptolide, triptolide and 2- table triptolide method and triptolide
With the HPLC method for separating and analyzing of 2- table triptolide.
Background technique
The study found that the very similar chemical component of four kinds of structures being present in tripterygium wilfordii has excellent antitumor work
Property, it is respectively as follows: triptonide, triptolide, triptolide and 2- table triptolide.Structural formula is as follows:
Chemically structure is it can be found that this four compound structures are very close, wherein triptolide and 2- table Thunder God
Rattan B prime is a pair of of chiral isomer.This causes the separating difficulty of this four compounds very big.The prior art separate this four
When kind compound, nothing is independent of silica gel column chromatography repeatedly, however silica gel column chromatography expends solvent, poor reproducibility repeatedly, only suitable
It is separated on a small quantity together in laboratory, it is difficult to promote in the industrial production.At present in industrial production, uniquely only have in the column chromatography used
Macroreticular resin.
Therefore, a kind of separation method based on macroreticular resin helps to realize the industrialized production system of above-mentioned four kinds of compounds
It is standby.
Summary of the invention
Triptonide, tripterygium wilfordii first are prepared it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of
The HPLC of element, the method and triptolide of triptolide and 2- table triptolide and 2- table triptolide separates analysis
Method.
Above-mentioned purpose of the invention is achieved by following technical solution:
The preparation of triptonide:
A method of triptonide is prepared, is included the following steps:
Step S1 is extracted: the dry root of Celastraceae plant tripterygium wilfordii being crushed, ethanol solution cold soaking extracts, and recycling is extracted
Ethyl alcohol in liquid is simultaneously diluted with water, and filters, and collects filtrate as macroreticular resin sample solution；
Step S2, XDA-1B macroporous adsorbing resin for purification: as separating medium and filling column using XDA-1B macroporous absorbent resin, tree
Rouge diameter height compares for 1:10, mixes sample resin accounts for resin total amount 1/10, wet method dress post mixes resin loading；First use 35% second of 12BV
Alcohol is cleaned with the flow velocity of 12BV/h, and rear 75% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) with 8BV is with 12BV/h
Flow velocity elute and collect 5-8BV eluent；The eluent of collection is concentrated to dryness up to crude product；
The purification of alkali open ring acid closed loop: step S3 crude product obtained by step S2 is dissolved using the buck that pH value is 9.5, filtering
Insoluble matter is removed, filtrate is adjusted with acid pH value to 6.5 again, and precipitate, washing, drying, with 65% second is collected by filtration in low temperature crystallization
Alcohol (triethylamine containing a ten thousandth, volumn concentration) dissolution is used as sample solution；
The separation of step S4, DM130 macroporous absorbent resin: column as separating medium and is filled using DM130 macroporous absorbent resin, resin
Diameter height compares for 1:20, mixes sample resin accounts for resin total amount 1/20, wet method dress post mixes resin loading；(contained with 65% ethyl alcohol of 9BV
A ten thousandth triethylamine, volumn concentration) 8-9BV eluent is eluted and collected with the flow velocity of 4BV/h, 8-9BV eluent is dense
Contracting is drying to obtain triptonide.
Preferably, the solid-to-liquid ratio that step S1 is extracted is 1:5, and 1kg dry root extracts the ethanol solution cold soaking of application 5L.
Preferably, step S1 is extracted with 95% ethyl alcohol cold soaking.
Preferably, step S1 is diluted with water to 0.5g crude drug/mL after recycling the ethyl alcohol in extracting solution, filters, and collects filtrate
As macroreticular resin sample solution.
Preferably, it is that sample solution corresponds to the 1/2 of crude drug quality that step S2, which mixes sample resin quality,.
Preferably, step S3 buck is ammonia spirit.
Preferably, step S3 acid is hydrochloric acid.
Preferably, the temperature of step S3 low temperature crystallization is 4 DEG C.
Preferably, it is molten to be dissolved into 0.5g/mL for step S3 65% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration)
Liquid is as sample solution.
Preferably, step S4 mixes 1/2 that sample resin quality is the corresponding precipitation amount of substance of sample solution.
The preparation of triptolide:
A method of triptolide is prepared, is included the following steps:
Step S1 is extracted: the dry root of Celastraceae plant tripterygium wilfordii being crushed, ethanol solution cold soaking extracts, and recycling is extracted
Ethyl alcohol in liquid is simultaneously diluted with water, and filters, and collects filtrate as macroreticular resin sample solution；
Step S2, XDA-1B macroporous adsorbing resin for purification: as separating medium and filling column using XDA-1B macroporous absorbent resin, tree
Rouge diameter height compares for 1:10, mixes sample resin accounts for resin total amount 1/10, wet method dress post mixes resin loading；First use 35% second of 12BV
Alcohol is cleaned with the flow velocity of 12BV/h, and rear 75% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) with 8BV is with 12BV/h
Flow velocity elute and collect 5-8BV eluent；The eluent of collection is concentrated to dryness up to crude product；
The purification of alkali open ring acid closed loop: step S3 crude product obtained by step S2 is dissolved using the buck that pH value is 9.5, filtering
Insoluble matter is removed, filtrate is adjusted with acid pH value to 6.5 again, and precipitate, washing, drying, with 65% second is collected by filtration in low temperature crystallization
Alcohol (triethylamine containing a ten thousandth, volumn concentration) dissolution is used as sample solution；
The separation of step S4, DM130 macroporous absorbent resin: column as separating medium and is filled using DM130 macroporous absorbent resin, resin
Diameter height compares for 1:20, mixes sample resin accounts for resin total amount 1/20, wet method dress post mixes resin loading；(contained with 65% ethyl alcohol of 12BV
A ten thousandth triethylamine, volumn concentration) 11-12BV eluent, 11-12BV elution are eluted and collected with the flow velocity of 4BV/h
Liquid is concentrated and dried up to triptolide.
Preferably, the solid-to-liquid ratio that step S1 is extracted is 1:5, and 1kg dry root extracts the ethanol solution cold soaking of application 5L.
Preferably, step S1 is extracted with 95% ethyl alcohol cold soaking.
Preferably, step S1 is diluted with water to 0.5g crude drug/mL after recycling the ethyl alcohol in extracting solution, filters, and collects filtrate
As macroreticular resin sample solution.
Preferably, it is that sample solution corresponds to the 1/2 of crude drug quality that step S2, which mixes sample resin quality,.
Preferably, step S3 buck is ammonia spirit.
Preferably, step S3 acid is hydrochloric acid.
Preferably, the temperature of step S3 low temperature crystallization is 4 DEG C.
Preferably, it is molten to be dissolved into 0.5g/mL for step S3 65% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration)
Liquid is as sample solution.
Preferably, step S4 mixes 1/2 that sample resin quality is the corresponding precipitation amount of substance of sample solution.
The preparation of triptolide and 2- table triptolide:
A method of triptolide and 2- table triptolide are prepared, is included the following steps:
Step S1 is extracted: the dry root of Celastraceae plant tripterygium wilfordii being crushed, ethanol solution cold soaking extracts, and recycling is extracted
Ethyl alcohol in liquid is simultaneously diluted with water, and filters, and collects filtrate as macroreticular resin sample solution；
Step S2, XDA-1B macroporous adsorbing resin for purification: as separating medium and filling column using XDA-1B macroporous absorbent resin, tree
Rouge diameter height compares for 1:10, mixes sample resin accounts for resin total amount 1/10, wet method dress post mixes resin loading；First use 35% second of 12BV
Alcohol is cleaned with the flow velocity of 12BV/h, and rear 75% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) with 8BV is with 12BV/h
Flow velocity elute and collect 5-8BV eluent；The eluent of collection is concentrated to dryness up to crude product；
The purification of alkali open ring acid closed loop: step S3 crude product obtained by step S2 is dissolved using the buck that pH value is 9.5, filtering
Insoluble matter is removed, filtrate is adjusted with acid pH value to 6.5 again, and precipitate, washing, drying, with 65% second is collected by filtration in low temperature crystallization
Alcohol (triethylamine containing a ten thousandth, volumn concentration) dissolution is used as sample solution；
The separation of step S4, DM130 macroporous absorbent resin: column as separating medium and is filled using DM130 macroporous absorbent resin, resin
Diameter height compares for 1:20, mixes sample resin accounts for resin total amount 1/20, wet method dress post mixes resin loading；(contained with 65% ethyl alcohol of 6BV
A ten thousandth triethylamine, volumn concentration) 5-6BV eluent is eluted and collected with the flow velocity of 4BV/h, it is concentrated and dried up to thunder
Public rattan B prime and 2- table triptolide mixture；
Step S5, high speed adverse current chromatogram separation: with ethyl acetate-n-butanol-water-glacial acetic acid (4:1:3:0.01) for solvent
System, with thereon mutually for stationary phase, lower phase is mobile phase；Triptolide obtained by step S4 and 2- table triptolide are mixed
The isometric stationary phase of object and flowing phased soln, filtration, as sample solution；Revolving speed is 850r/min, and flow rate of mobile phase is
It is corresponding to collect triptolide, 2- table triptolide chromatographic peak according to chromatogram by 2.5mL/min, Detection wavelength 220nm
Eluent is concentrated and dried up to triptolide, 2- table triptolide.
Preferably, the solid-to-liquid ratio that step S1 is extracted is 1:5, and 1kg dry root extracts the ethanol solution cold soaking of application 5L.
Preferably, step S1 is extracted with 95% ethyl alcohol cold soaking.
Preferably, step S1 is diluted with water to 0.5g crude drug/mL after recycling the ethyl alcohol in extracting solution, filters, and collects filtrate
As macroreticular resin sample solution.
Preferably, it is that sample solution corresponds to the 1/2 of crude drug quality that step S2, which mixes sample resin quality,；Step S4 mixes sample resin quality
The 1/2 of amount of substance is precipitated for sample solution is corresponding.
Preferably, step S3 buck is ammonia spirit.
Preferably, step S3 acid is hydrochloric acid.
Preferably, the temperature of step S3 low temperature crystallization is 4 DEG C.
Preferably, it is molten to be dissolved into 0.5g/mL for step S3 65% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration)
Liquid is as sample solution.
Preferably, the concentration of step S5 sample solution is 10mg/mL.
The HPLC separation method of triptolide and 2- table triptolide:
It is a kind of separate triptolide and 2- table triptolide efficient liquid-phase chromatography method, be with C18 reverse phase silica gel
Stationary phase, with methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72 (volume ratio) for mobile phase.
Preferably, chromatographic column is Ultimate XB-C18.
Preferably, chromatographic column filler partial size is 5 μm.
Preferably, column length 250mm, internal diameter 4.6mm.
Preferably, Detection wavelength 220nm.
Preferably, flow rate of mobile phase 1.0mL/min.
Preferably, sample volume is 10 μ L.
Advantages of the present invention:
Method provided by the invention separates independent of silica gel column chromatography repeatedly and prepares triptonide, tripterygium wilfordii
A prime, triptolide and 2- table triptolide, are suitable for industrialized production.HPLC analysis method provided by the invention is based on
Conventional C18 chromatographic column can efficiently separate chiral isomer triptolide and 2- table triptolide, at low cost, repeated
It is high.
Detailed description of the invention
Fig. 1 is that the HPLC of XDA-1B macroporous adsorbing resin for purification product detects chromatogram, from this figure, it can be seen that in addition to thunder
Public rattan lactone ketone, triptolide, triptolide and 2- table triptolide, the product also contain more impurity；
Fig. 2 is that the HPLC of alkali open ring acid closed loop refined products detects chromatogram, be can be seen that from the figure, by this step, thunder
Public rattan lactone ketone, triptolide, triptolide and 2- table triptolide are further enriched with, and impurity component significantly subtracts
It is few；
Fig. 3 is the HPLC detection chromatogram that DM130 macroporous absorbent resin 5-6BV eluent is concentrated and dried product, substantially only
Contain triptolide and 2- table triptolide；
Fig. 4 is the HPLC detection chromatogram that DM130 macroporous absorbent resin 8-9BV eluent is concentrated and dried product, substantially only
Contain triptonide；
Fig. 5 is the HPLC detection chromatogram that DM130 macroporous absorbent resin 11-12BV eluent is concentrated and dried product, substantially
Contain only triptolide；
Fig. 6 is to be concentrated to do using the DM130 macroporous absorbent resin 5-6BV eluent for the elution for not adding triethylamine
The HPLC of dry product detects chromatogram, triptonide and triptolide and 2- table triptolide co-elute；
Fig. 7 is to be concentrated to do using the DM130 macroporous absorbent resin 8-9BV eluent for the elution for not adding triethylamine
The HPLC of dry product detects chromatogram, triptonide and triptolide and 2- table triptolide co-elute；
Fig. 8 is HSCCC separation chromatogram, triptolide and 2- table triptolide baseline separation；
Fig. 9 is that HSCCC of the dicyandiamide solution without glacial acetic acid separates spectrogram, and triptolide and 2- table triptolide are washed altogether
It is de-；
Figure 10 is that three kinds of different mobile phases are poor to the separation of triptolide and 2- table triptolide in C18 chromatographic column
It is different.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
One, experimental material
The dry root of tripterygium wilfordii is purchased from Bozhou Chinese Medicinal Materials Markets, is identified as the dry root of Celastraceae plant tripterygium wilfordii；
XDA-1B macroporous absorbent resin is purchased from Zhengzhou Qin Shi Science and Technology Ltd.；
DM130 macroporous absorbent resin is purchased from Anhui Samsung resin Science and Technology Ltd.；
95% ethyl alcohol, triethylamine, ethyl acetate, n-butanol, ammonium hydroxide and hydrochloric acid are purchased from the limited public affairs of Nanjing chemical reagent share
Department；
TBE-300C type high-speed counter-current chromatograph, Shanghai Tongtian Biotechnology Co., Ltd..
Two, experimental method
A method of triptonide, triptolide, triptolide and 2- table triptolide are prepared, is wrapped
It includes:
Step S1 is extracted: the dry root of Celastraceae plant tripterygium wilfordii being crushed, 95% ethyl alcohol cold soaking extracts overnight, solid-liquid
It than 1:5, recycles the ethyl alcohol in extracting solution and is diluted with water to 0.5g crude drug/mL, filter, collect filtrate as macroreticular resin loading
Liquid；
Step S2, XDA-1B macroporous adsorbing resin for purification: as separating medium and filling column using XDA-1B macroporous absorbent resin, tree
Rouge diameter height compares for 1:10, mixes sample resin accounts for resin total amount 1/10, and mixing sample resin quality is that sample solution corresponds to the 1/ of crude drug quality
2, wet method dress post mixes resin loading；It is first cleaned with 35% ethyl alcohol of 12BV with the flow velocity of 12BV/h, rear 75% ethyl alcohol for using 8BV
(triethylamine containing a ten thousandth, volumn concentration) is eluted with the flow velocity of 12BV/h and collects 5-8BV eluent；By washing for collection
De- liquid is concentrated to dryness up to crude product；
Step S3, alkali open ring acid closed loop purification: the ammonia solvent for the use of pH value being 9.5 by crude product obtained by step S2, filtering
Insoluble matter is removed, filtrate uses salt acid for adjusting pH value to 6.5,4 DEG C of low temperature crystallizations again, precipitate, washing, dry, use is collected by filtration
65% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) is dissolved into 0.5g/mL solution as sample solution；
The separation of step S4, DM130 macroporous absorbent resin: column as separating medium and is filled using DM130 macroporous absorbent resin, resin
Diameter height compares for 1:20, mixes sample resin accounts for resin total amount 1/20, and mixing sample resin quality, to be that sample solution is corresponding be precipitated the 1/ of amount of substance
2, wet method dress post mixes resin loading；With 65% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) of 12BV with 4BV/h
Flow velocity elute and collect 5-6BV, 8-9BV, 11-12BV eluent；5-6BV eluent be concentrated and dried up to triptolide and
2- table triptolide mixture, 8-9BV eluent are concentrated and dried up to triptonide, and the concentration of 11-12BV eluent is dry
Dry triptolide to obtain the final product；
Step S5, high speed adverse current chromatogram (HSCCC) separation: with ethyl acetate-n-butanol-water-glacial acetic acid (4:1:3:
It 0.01) is dicyandiamide solution, with thereon mutually for stationary phase, lower phase is mobile phase；By triptolide obtained by step S4 and 2- table thunder
Public rattan B prime mixture 10mL stationary phase and 10mL mobile phase ultrasonic dissolution, filtration, as sample solution；Revolving speed is 850r/
Min, flow rate of mobile phase 2.5mL/min, Detection wavelength 220nm collect triptolide, 2- table tripterygium wilfordii according to chromatogram
The corresponding eluent of B prime chromatographic peak is concentrated and dried up to triptolide, 2- table triptolide.
The specific method is as follows for high speed adverse current chromatogram:
It is placed in separatory funnel, is fullyd shake with ethyl acetate-n-butanol-water-glacial acetic acid (4:1:3:0.01) mixed liquor
Stratification afterwards takes phase up and down, ultrasonic degassing 20min respectively.Upper phase is stationary phase, and lower phase is mobile phase.By stationary phase with
The volume flow of 20mL/min adjusts in the upper phase injection HSCCC separating pipe of dicyandiamide solution after upper phase is full of entire pipeline
Engine speed is 850r/min, then injects mobile phase with the volume flow of 2.5mL/min, and it is flat that two-phase reaches dynamic in separating pipe
After weighing apparatus, 20mL sample liquid (10mg/mL) is injected by injection port, 25 DEG C of temperature, Detection wavelength 220nm obtains HSCCC separation figure.
The HPLC analysis method parameter of each sample:
Chromatographic column: moon rising sun Ultimate XB-C18 (5 μm, 4.6 × 250mm)
Mobile phase: methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72 (volume ratio) isocratic elution
Detection wavelength: 220nm
Flow velocity: 1.0mL/min.
Three, experimental result
1, XDA-1B macroporous adsorbing resin for purification result
The effect of XDA-1B macroporous absorbent resin is the triptonide being enriched in tripterygium wilfordii, triptolide, thunder
Public rattan B prime and 2- table triptolide.The HPLC testing result of the crude product of enrichment as shown in Figure 1, it will be seen from figure 1 that in addition to
Triptonide, triptolide, triptolide and 2- table triptolide also contain more impurity in the crude product.
2, alkali open ring acid closed loop upgrading result
The step of alkali open ring acid closed loop refines is using under lactone compound under alkaline condition open loop, acid condition
The principle of closed loop is further enriched with triptonide, triptolide, triptolide and 2- table triptolide.By excellent
The pH value for changing alkaline condition and acid condition can obtain target lactone constituents to the maximum extent.The step refined products
HPLC testing result is as shown in Fig. 2, figure it is seen that by this step, triptonide, triptolide, tripterygium wilfordii
B prime and 2- table triptolide are further enriched with, and impurity component substantially reduces.
3, DM130 macroporous absorbent resin separating resulting
The effect of DM130 macroporous absorbent resin is to separate triptonide, triptolide, Thunder God in tripterygium wilfordii
Rattan B prime and 2- table triptolide.5-6BV eluent is concentrated and dried to obtain the relatively large triptolide of polarity and 2- table
Triptolide mixture, HPLC testing result is as shown in figure 3, the two total content reaches 93.3%；The concentration of 8-9BV eluent is dry
Dry to obtain the relatively medium triptonide of polarity, HPLC testing result is as shown in figure 4, content reaches 95.5%；11-12BV
Eluent is concentrated and dried to obtain the relatively small triptolide of polarity, and HPLC testing result is as shown in figure 5, content reaches
97.1%.
By the optimization of a variety of different types of macroporous absorbent resins and eluting solvent, triptolide and 2- table tripterygium wilfordii
B prime is difficult to separate since polarity is too similar.Triptonide under this condition can be with triptolide and 2- table tripterygium wilfordii
B prime separation is also just to realize by optimization repeatedly, if not adding triethylamine, 5-6BV, 8-9BV eluent than eluting solvent
The HPLC testing result of products therefrom is as shown in Figure 6,7, and triptonide and triptolide and 2- table triptolide are total to
Elution.
4, high speed adverse current chromatogram (HSCCC) separating resulting
High speed adverse current chromatogram is different from the separation principle of macroporous absorbent resin, by optimizing repeatedly, triptolide and 2-
Table triptolide is separated under the above conditions, and chromatogram is as shown in Figure 8.Fig. 9 is not add glacial acetic acid in dicyandiamide solution
Separating effect figure, triptolide and 2- table triptolide co-elute.
5, the optimization process and result of HPLC analysis method
Triptolide and 2- table triptolide are a pair of of chiral isomer, and compound polarity is very much like.For this
Kind chiral isomer, generallys use chiral chromatographic column and is separated, the conventional reverse-phase chromatographic column separating difficulty based on C18 filler
Greatly.But chiral chromatographic column is extremely expensive, and especially enervated, the impurity component of sample to be analysed cannot be more, elution requirement
Cannot too acutely, most cannot be neglected defect is poor repeatability (poor repeatability especially between different experiments room).Use hand
Property chromatographic column exploitation triptolide and 2- table triptolide method belong to analysis method exploitation be easy, it is poor using stability,
It is at high cost；Triptolide is developed using reverse-phase chromatographic column and 2- table triptolide method belongs to analysis method and develops hardly possible, is answered
It is good, at low cost with stability.By optimizing repeatedly, above-mentioned HPLC separation parameter is obtained, it may be assumed that
Chromatographic column: moon rising sun Ultimate XB-C18 (5 μm, 4.6 × 250mm)
Mobile phase: methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72 (volume ratio) isocratic elution
Detection wavelength: 220nm
Flow velocity: 1.0mL/min；
Sample volume: 10 μ L；
Liquid chromatograph: Agilent 1260.
Mobile phase is mixed using three kinds of organic solvents, as seen in Figure 10 the advantage of above-mentioned flow visualizing.
Sample solution is 30% methanol solution containing 0.25mg/mL triptolide, 0.25mg/mL 2- table triptolide.Figure
In 10 the mobile phase of A be methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72, B mobile phase be methanol/
Acetonitrile/triethylamine/water=20/10/0.1/70, C mobile phase is methanol/acetonitrile/triethylamine/water=14/14/0.1/72.Figure
The mobile phase polarity that A, B, C chromatogram use in 10 is close, but for triptolide and 2- table triptolide in C18 chromatography
Reserve capability influences not identical, methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72 discrimination on column
Good, triptolide and 2- table triptolide reach baseline separation in C18 chromatographic column, which can be used for separating analysis thunder
Public rattan B prime and 2- table triptolide.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (10)
1. a kind of method for preparing triptolide and 2- table triptolide, which comprises the steps of:
Step S1 is extracted: the dry root of Celastraceae plant tripterygium wilfordii being crushed, ethanol solution cold soaking extracts, and recycles in extracting solution
Ethyl alcohol and be diluted with water, filter, collect filtrate as macroreticular resin sample solution；
Step S2, XDA-1B macroporous adsorbing resin for purification: column as separating medium and is filled using XDA-1B macroporous absorbent resin, resin path
Height mixes sample resin accounts for resin total amount 1/10, wet method dress post mixes resin loading than being 1:10；First with 35% ethyl alcohol of 12BV with
The flow velocity of 12BV/h cleans, after with 8BV contain 75% ethyl alcohol of the triethylamine that volumn concentration is a ten thousandth with 12BV/h's
Flow velocity elutes and collects 5-8BV eluent；The eluent of collection is concentrated to dryness up to crude product；
The purification of alkali open ring acid closed loop: step S3 crude product obtained by step S2 is dissolved using the buck that pH value is 9.5, filtering removal
Insoluble matter, filtrate are adjusted with acid pH value to 6.5 again, low temperature crystallization, are collected by filtration precipitate, washing, dry, with containing volume hundred
The 65% ethyl alcohol dissolution that content is the triethylamine of a ten thousandth is divided to be used as sample solution；
The separation of step S4, DM130 macroporous absorbent resin: as separating medium and filling column using DM130 macroporous absorbent resin, and resin path is high
Than mixing sample resin accounts for resin total amount 1/20, wet method dress post mixes resin loading for 1:20；Containing volumn concentration with 6BV is
65% ethyl alcohol of the triethylamine of a ten thousandth elutes with the flow velocity of 4BV/h and collects 5-6BV eluent, is concentrated and dried up to Thunder God
Rattan B prime and 2- table triptolide mixture；
Step S5, high speed adverse current chromatogram separate: being for ethyl acetate-n-butanol-water-glacial acetic acid of 4:1:3:0.01 with volume ratio
Dicyandiamide solution, with thereon mutually for stationary phase, lower phase is mobile phase；By triptolide obtained by step S4 and 2- table triptolide
The isometric stationary phase of mixture and flowing phased soln, filtration, as sample solution；Revolving speed is 850r/min, flow rate of mobile phase
For 2.5mL/min, Detection wavelength 220nm, it is corresponding that triptolide, 2- table triptolide chromatographic peak are collected according to chromatogram
Eluent, be concentrated and dried up to triptolide, 2- table triptolide.
2. according to the method described in claim 1, it is characterized by: the solid-to-liquid ratio that step S1 is extracted is 1:5,1kg dry root pair
It is extracted using the ethanol solution cold soaking of 5L.
3. method according to claim 1 or 2, it is characterised in that: step S1 is extracted with 95% ethyl alcohol cold soaking.
4. according to the method described in claim 1, it is characterized by: step S1 is diluted with water to after recycling the ethyl alcohol in extracting solution
0.5g crude drug/mL, filtering collect filtrate as macroreticular resin sample solution.
5. according to the method described in claim 1, it is characterized by: it is that sample solution corresponds to crude drug matter that step S2, which mixes sample resin quality,
The 1/2 of amount；Step S4 mixes 1/2 that sample resin quality is the corresponding precipitation amount of substance of sample solution.
6. according to the method described in claim 1, it is characterized by: step S3 buck is ammonia spirit.
7. according to the method described in claim 1, it is characterized by: step S3 acid is hydrochloric acid.
8. according to the method described in claim 1, it is characterized by: the temperature of step S3 low temperature crystallization is 4 DEG C.
9. according to the method described in claim 1, it is characterized by: step S3 with containing volumn concentration is a ten thousandth
65% ethyl alcohol of triethylamine is dissolved into 0.5g/mL solution as sample solution.
10. according to the method described in claim 1, it is characterized by: the concentration of step S5 sample solution is 10mg/mL.
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| CN101445545A (en) * | 2008-12-29 | 2009-06-03 | 浙江大学 | Method for separating triptolide from thunder god vine leaves |
| CN103524591A (en) * | 2012-07-05 | 2014-01-22 | 广西大学 | Preparation method of effective components of triperygium wilfordii |
| CN104231032A (en) * | 2013-06-13 | 2014-12-24 | 宁波工程学院 | Method for separating tripdiolide from Tripterygium Wilfordii Hook F leaves |
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| CN1911932A (en) * | 2006-08-25 | 2007-02-14 | 浙江大学 | Method for separating preparing tripterygium wilfordii monomer from tripterygium wilfordii by countercurrent flow chromatography |
| CN101445545A (en) * | 2008-12-29 | 2009-06-03 | 浙江大学 | Method for separating triptolide from thunder god vine leaves |
| CN103524591A (en) * | 2012-07-05 | 2014-01-22 | 广西大学 | Preparation method of effective components of triperygium wilfordii |
| CN104231032A (en) * | 2013-06-13 | 2014-12-24 | 宁波工程学院 | Method for separating tripdiolide from Tripterygium Wilfordii Hook F leaves |
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