CN108191948A - A kind of method for preparing triptolide and 2- table triptolides - Google Patents
A kind of method for preparing triptolide and 2- table triptolides Download PDFInfo
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- CN108191948A CN108191948A CN201810117628.8A CN201810117628A CN108191948A CN 108191948 A CN108191948 A CN 108191948A CN 201810117628 A CN201810117628 A CN 201810117628A CN 108191948 A CN108191948 A CN 108191948A
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- Prior art keywords
- triptolide
- resin
- triptolides
- ethyl alcohol
- sample
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- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical compound O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 title claims abstract description 90
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 title claims abstract description 89
- 238000000034 method Methods 0.000 title claims abstract description 43
- 239000011347 resin Substances 0.000 claims abstract description 93
- 229920005989 resin Polymers 0.000 claims abstract description 93
- 241000830536 Tripterygium wilfordii Species 0.000 claims abstract description 18
- 235000015398 thunder god vine Nutrition 0.000 claims abstract description 18
- 239000002904 solvent Substances 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 96
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 90
- 235000019441 ethanol Nutrition 0.000 claims description 47
- 239000012488 sample solution Substances 0.000 claims description 33
- 239000003480 eluent Substances 0.000 claims description 30
- 239000002250 absorbent Substances 0.000 claims description 27
- 230000002745 absorbent Effects 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 25
- 239000002253 acid Substances 0.000 claims description 19
- 238000000605 extraction Methods 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000000926 separation method Methods 0.000 claims description 15
- 239000000706 filtrate Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 13
- 239000012043 crude product Substances 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 229940079593 drug Drugs 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims description 9
- 238000002425 crystallisation Methods 0.000 claims description 9
- 230000008025 crystallization Effects 0.000 claims description 9
- 239000003513 alkali Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 7
- 238000004064 recycling Methods 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 241000208365 Celastraceae Species 0.000 claims description 6
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 230000002411 adverse Effects 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims 1
- SWOVVKGLGOOUKI-ZHGGVEMFSA-N triptonide Chemical compound O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)C(=O)[C@]21[C@H]3O1 SWOVVKGLGOOUKI-ZHGGVEMFSA-N 0.000 abstract description 22
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 21
- 150000001875 compounds Chemical class 0.000 abstract description 7
- 238000010898 silica gel chromatography Methods 0.000 abstract description 6
- 238000009776 industrial production Methods 0.000 abstract description 4
- 238000004440 column chromatography Methods 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- 239000000243 solution Substances 0.000 description 17
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- 241000345998 Calamus manan Species 0.000 description 6
- 238000010262 high-speed countercurrent chromatography Methods 0.000 description 6
- 235000012950 rattan cane Nutrition 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- -1 lactone ketone Chemical class 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a kind of methods for preparing triptolide and 2 table triptolides.It is very close to be present in triptonide in tripterygium wilfordii, triptolide, triptolide and 2 table triptolide structures, wherein triptolide and 2 table triptolides are a pair of of chiral isomer, and separating difficulty is big.For the prior art when detaching 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 detaches 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 triptolides.HPLC methods provided by the invention can efficiently separate chiral isomer triptolide and 2 table triptolides, at low cost, repeated height based on conventional C18 chromatographic columns.
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 prepares the method and triptolide of triptonide, triptolide, triptolide and 2- table triptolides
With the HPLC method for separating and analyzing of 2- table triptolides.
Background technology
The study found that the very similar chemical composition of four kinds of structures being present in tripterygium wilfordii has excellent antitumor work
Property, respectively：Triptonide, triptolide, triptolide and 2- table triptolides.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 Gods
Rattan B prime is a pair of of chiral isomer.This causes the separating difficulty of this four compounds very big.The prior art detach this four
During 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 detached 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.
Invention content
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 separation analyses of element, the method and triptolide of triptolide and 2- table triptolides and 2- table triptolides
Method.
The above-mentioned purpose of the present invention is achieved by following technical solution：
The preparation of triptonide：
A kind of method for preparing triptonide, includes the following steps：
Step S1, extraction：The dry root of Celastraceae plant tripterygium wilfordii is crushed, the extraction of ethanol solution cold soaking, recycling extraction
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：Column as separating medium and is filled using XDA-1B macroporous absorbent resins, is set
Fat blade diameter length ratio is 1:10, sample resin accounts for resin total amount 1/10 is mixed, 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 eluents；The eluent of collection is concentrated to dryness up to crude product；
Step S3, alkali open ring acid closed loop refine：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) dissolving is as sample solution；
Step S4, DM130 macroporous absorbent resin detach：Column as separating medium and is filled using DM130 macroporous absorbent resins, resin
Blade diameter length ratio is 1:20, sample resin accounts for resin total amount 1/20 is mixed, wet method dress post mixes resin loading；(contained with 65% ethyl alcohol of 9BV
A ten thousandth triethylamine, volumn concentration) it is eluted with the flow velocity of 4BV/h and collects 8-9BV eluents, 8-9BV eluents are dense
Contracting is drying to obtain triptonide.
Preferably, the solid-to-liquid ratio of step S1 extractions is 1:5,1kg dry roots extract 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 drugs/mL after recycling the ethyl alcohol in extracting solution, filters, and collects filtrate
As macroreticular resin sample solution.
Preferably, step S2 mixes sample resin quality and corresponds to the 1/2 of crude drug quality for sample solution.
Preferably, step S3 bucks are 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 sample resin quality and is precipitated the 1/2 of amount of substance for sample solution correspondence.
The preparation of triptolide：
A kind of method for preparing triptolide, includes the following steps：
Step S1, extraction：The dry root of Celastraceae plant tripterygium wilfordii is crushed, the extraction of ethanol solution cold soaking, recycling extraction
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：Column as separating medium and is filled using XDA-1B macroporous absorbent resins, is set
Fat blade diameter length ratio is 1:10, sample resin accounts for resin total amount 1/10 is mixed, 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 eluents；The eluent of collection is concentrated to dryness up to crude product；
Step S3, alkali open ring acid closed loop refine：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) dissolving is as sample solution；
Step S4, DM130 macroporous absorbent resin detach：Column as separating medium and is filled using DM130 macroporous absorbent resins, resin
Blade diameter length ratio is 1:20, sample resin accounts for resin total amount 1/20 is mixed, wet method dress post mixes resin loading；(contained with 65% ethyl alcohol of 12BV
A ten thousandth triethylamine, volumn concentration) it is eluted with the flow velocity of 4BV/h and collects 11-12BV eluents, 11-12BV elutions
Liquid is concentrated and dried up to triptolide.
Preferably, the solid-to-liquid ratio of step S1 extractions is 1:5,1kg dry roots extract 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 drugs/mL after recycling the ethyl alcohol in extracting solution, filters, and collects filtrate
As macroreticular resin sample solution.
Preferably, step S2 mixes sample resin quality and corresponds to the 1/2 of crude drug quality for sample solution.
Preferably, step S3 bucks are 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 sample resin quality and is precipitated the 1/2 of amount of substance for sample solution correspondence.
The preparation of triptolide and 2- table triptolides：
A kind of method for preparing triptolide and 2- table triptolides, includes the following steps：
Step S1, extraction：The dry root of Celastraceae plant tripterygium wilfordii is crushed, the extraction of ethanol solution cold soaking, recycling extraction
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：Column as separating medium and is filled using XDA-1B macroporous absorbent resins, is set
Fat blade diameter length ratio is 1:10, sample resin accounts for resin total amount 1/10 is mixed, 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 eluents；The eluent of collection is concentrated to dryness up to crude product；
Step S3, alkali open ring acid closed loop refine：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) dissolving is as sample solution；
Step S4, DM130 macroporous absorbent resin detach：Column as separating medium and is filled using DM130 macroporous absorbent resins, resin
Blade diameter length ratio is 1:20, sample resin accounts for resin total amount 1/20 is mixed, wet method dress post mixes resin loading；(contained with 65% ethyl alcohol of 6BV
A ten thousandth triethylamine, volumn concentration) it is eluted with the flow velocity of 4BV/h and collects 5-6BV eluents, it is concentrated and dried up to thunder
Public rattan B prime and 2- table triptolide mixtures；
Step S5, high speed adverse current chromatogram separation：With ethyl acetate-n-butanol-water-glacial acetic acid (4:1:3:0.01) it is solvent
System, with thereon mutually for stationary phase, lower phase is mobile phase；Triptolide obtained by step S4 and 2- tables triptolide are mixed
The isometric stationary phase of object and flowing phased soln, filtration, as sample solution；Rotating speed is 850r/min, and flow rate of mobile phase is
It is corresponding to collect triptolide, 2- table triptolide chromatographic peaks according to chromatogram by 2.5mL/min, Detection wavelength 220nm
Eluent is concentrated and dried up to triptolide, 2- table triptolides.
Preferably, the solid-to-liquid ratio of step S1 extractions is 1:5,1kg dry roots extract 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 drugs/mL after recycling the ethyl alcohol in extracting solution, filters, and collects filtrate
As macroreticular resin sample solution.
Preferably, step S2 mixes sample resin quality and corresponds to the 1/2 of crude drug quality for sample solution；Step S4 mixes sample resin quality
It is corresponded to for sample solution and is precipitated the 1/2 of amount of substance.
Preferably, step S3 bucks are 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, a concentration of 10mg/mL of step S5 sample solutions.
The HPLC separation methods of triptolide and 2- table triptolides：
A kind of efficient liquid-phase chromatography method for detaching triptolide and 2- table triptolides, using C18 reverse phase silica gels as
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 grain 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 size 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 triptolides, are suitable for industrialized production.HPLC analysis methods provided by the invention are based on
Conventional C18 chromatographic columns can efficiently separate chiral isomer triptolide and 2- table triptolides, at low cost, repeated
It is high.
Description of the drawings
Fig. 1 is that the HPLC of XDA-1B macroporous adsorbing resin for purification products detects chromatogram, from this figure, it can be seen that in addition to thunder
Public rattan lactone ketone, triptolide, triptolide and 2- table triptolides, 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 triptolides are further enriched with, and impurity component significantly subtracts
It is few；
Fig. 3 is the HPLC detection chromatograms that DM130 macroporous absorbent resin 5-6BV eluents are concentrated and dried product, substantially only
Contain triptolide and 2- table triptolides；
Fig. 4 is the HPLC detection chromatograms that DM130 macroporous absorbent resin 8-9BV eluents are concentrated and dried product, substantially only
Contain triptonide；
Fig. 5 is the HPLC detection chromatograms that DM130 macroporous absorbent resin 11-12BV eluents are concentrated and dried product, substantially
Contain only triptolide；
Fig. 6 is dry using the DM130 macroporous absorbent resin 5-6BV eluents concentration for the elution for not adding triethylamine
The HPLC detection chromatograms of dry product, triptonide and triptolide and 2- table triptolide co-elutes；
Fig. 7 is dry using the DM130 macroporous absorbent resin 8-9BV eluents concentration for the elution for not adding triethylamine
The HPLC detection chromatograms of dry product, triptonide and triptolide and 2- table triptolide co-elutes；
Fig. 8 is HSCCC separation chromatography figures, triptolide and 2- table triptolide baseline separations；
Fig. 9 is that HSCCC of the dicyandiamide solution without glacial acetic acid detaches spectrogram, and triptolide and 2- table triptolides are washed altogether
It is de-；
Figure 10 is poor to the separation of triptolide and 2- tables triptolide in C18 chromatographic columns for three kinds of different mobile phases
It is different.
Specific embodiment
The specific guarantor for introducing essentiality content of the present invention, but the present invention not being limited with this with reference to the accompanying drawings and examples
Protect range.
First, experiment 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 resins are purchased from Zhengzhou Qin Shi Science and Technology Ltd.s；
DM130 macroporous absorbent resins are 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 chromatographs, Shanghai Tongtian Biotechnology Co., Ltd..
2nd, experimental method
A kind of method for preparing triptonide, triptolide, triptolide and 2- table triptolides, packet
It includes：
Step S1, extraction：The dry root of Celastraceae plant tripterygium wilfordii is crushed, 95% ethyl alcohol cold soaking extracts overnight, solid-liquid
Than 1:5, it recycles the ethyl alcohol in extracting solution and is diluted with water to 0.5g crude drugs/mL, filter, collect filtrate as macroreticular resin loading
Liquid；
Step S2, XDA-1B macroporous adsorbing resin for purification：Column as separating medium and is filled using XDA-1B macroporous absorbent resins, is set
Fat blade diameter length ratio is 1:10, sample resin accounts for resin total amount 1/10 is mixed, sample resin quality is mixed and corresponds to the 1/ of crude drug quality for sample solution
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 eluents；By washing for collection
De- liquid is concentrated to dryness up to crude product；
Step S3, alkali open ring acid closed loop refine：Crude product obtained by step S2 is used into the ammonia solvent that pH value is 9.5, filtering
Insoluble matter is removed, with salt acid for adjusting pH value to 6.5,4 DEG C of low temperature crystallizations, precipitate, washing, dry, use is collected by filtration again in filtrate
65% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) is dissolved into 0.5g/mL solution as sample solution；
Step S4, DM130 macroporous absorbent resin detach：Column as separating medium and is filled using DM130 macroporous absorbent resins, resin
Blade diameter length ratio is 1:20, sample resin accounts for resin total amount 1/20 is mixed, mixes sample resin quality corresponds to precipitation amount of substance for sample solution 1/
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 eluents be concentrated and dried up to triptolide and
2- table triptolide mixtures, 8-9BV eluents are concentrated and dried up to triptonide, and the concentration of 11-12BV eluents is done
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:
0.01) it is dicyandiamide solution, with thereon mutually for stationary phase, lower phase is mobile phase；By triptolide obtained by step S4 and 2- table thunders
Public rattan B prime mixture 10mL stationary phases and 10mL mobile phase ultrasonic dissolutions, filtration, as sample solution；Rotating speed is 850r/
Min, flow rate of mobile phase 2.5mL/min, Detection wavelength 220nm collect triptolide, 2- table tripterygium wilfordiis according to chromatogram
The corresponding eluent of B prime chromatographic peak is concentrated and dried up to triptolide, 2- table triptolides.
High speed adverse current chromatogram specific method is as follows：
With ethyl acetate-n-butanol-water-glacial acetic acid (4:1:3:0.01) mixed liquor is placed in separatory funnel, fully shaking
Stratification afterwards takes phase up and down, ultrasound degassing 20min respectively.Upper phase is stationary phase, and lower phase is mobile phase.By stationary phase with
The volume flow of 20mL/min is adjusted in the upper phase injection HSCCC separating pipes 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, by injection port injection 20mL sample liquids (10mg/mL), 25 DEG C, Detection wavelength 220nm of temperature obtains HSCCC separation figures.
The HPLC analysis method parameters 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.
3rd, experimental result
1st, XDA-1B macroporous adsorbing resin for purification result
The effect of XDA-1B macroporous absorbent resins is the triptonide, triptolide, thunder being enriched in tripterygium wilfordii
Public rattan B prime and 2- table triptolides.The HPLC testing results 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 triptolides also contain more impurity in the crude product.
2nd, alkali open ring acid closed loop upgrading result
The step that 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 triptolides.By excellent
Target lactone constituents can be obtained to the maximum extent by changing the pH value of alkaline condition and acid condition.The step refined products
HPLC testing results are as shown in Fig. 2, figure it is seen that by this step, triptonide, triptolide, tripterygium wilfordii
B prime and 2- table triptolides are further enriched with, and impurity component substantially reduces.
3rd, DM130 macroporous absorbent resins separating resulting
The effect of DM130 macroporous absorbent resins is to detach triptonide, triptolide, Thunder God in tripterygium wilfordii
Rattan B prime and 2- table triptolides.5-6BV eluents are concentrated and dried to obtain the relatively large triptolide of polarity and 2- tables
Triptolide mixture, HPLC testing results are as shown in figure 3, the two total content reaches 93.3%；The concentration of 8-9BV eluents is dry
Dry to obtain the relatively medium triptonide of polarity, HPLC testing results are 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 results are 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 wilfordiis
B prime is difficult to detach since polarity is too similar.Triptonide under this condition can be with triptolide and 2- table tripterygium wilfordiis
B prime separation is also just to be realized by optimization repeatedly, if not adding triethylamine, 5-6BV, 8-9BV eluent than eluting solvent
As shown in Figure 6,7, triptonide is total to the HPLC testing results of products therefrom with triptolide and 2- table triptolides
Elution.
4th, 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 detached under these 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-elutes.
5th, the optimization process and result of HPLC analysis methods
Triptolide and 2- tables triptolide are a pair of of chiral isomer, and compound polarity is very much like.For this
Kind chiral isomer, generally use chiral chromatographic column are detached, the conventional reverse-phase chromatographic column separating difficulty based on C18 fillers
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, the defects of most can not be ignored is poor repeatability (poor repeatability especially between different experiments room).Use hand
Property chromatographic column exploitation triptolide and 2- table triptolide methods to belong to analysis method exploitation easy, using stability it is poor,
It is of high cost；Triptolide is developed using reverse-phase chromatographic column and 2- table triptolide methods belong to analysis method exploitation hardly possible, it should
It is good, at low cost with stability.By optimizing repeatedly, above-mentioned HPLC separation parameters are obtained, i.e.,：
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 size：10μL；
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 triptolides, 0.25mg/mL 2- table triptolides.Figure
In 10 the mobile phase of A be the mobile phase of methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72, B be methanol/
The mobile phase of acetonitrile/triethylamine/water=20/10/0.1/70, C 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- tables triptolide in C18 chromatographies
Reserve capability is influenced and is differed on column, the discrimination of methanol/acetonitrile/tetrahydrofuran/triethylamine/water=15/10/3/0.1/72
Good, triptolide and 2- tables triptolide can be used for separation to analyze thunder in C18 chromatographic columns up to baseline separation, the mobile phase
Public rattan B prime and 2- table triptolides.
The effect of above-described embodiment is specifically to introduce the essentiality content of the present 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)
- A kind of 1. method for preparing triptolide and 2- table triptolides, which is characterized in that include the following steps：Step S1, extraction：The dry root of Celastraceae plant tripterygium wilfordii is crushed, the extraction of ethanol solution cold soaking is recycled 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 resins, resin path Height is than being 1:10, sample resin accounts for resin total amount 1/10 is mixed, wet method dress post mixes resin loading；First with 35% ethyl alcohol of 12BV with The flow velocity cleaning of 12BV/h, rear 75% ethyl alcohol (triethylamine containing a ten thousandth, volumn concentration) with 8BV is with the stream of 12BV/h Speed elutes and collects 5-8BV eluents；The eluent of collection is concentrated to dryness up to crude product；Step S3, alkali open ring acid closed loop refine：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, and precipitate, washing, drying, with 65% ethyl alcohol is collected by filtration in low temperature crystallization (triethylamine containing a ten thousandth, volumn concentration) dissolving is as sample solution；Step S4, DM130 macroporous absorbent resin detach：Column as separating medium and is filled using DM130 macroporous absorbent resins, resin path is high Than being 1:20, sample resin accounts for resin total amount 1/20 is mixed, wet method dress post mixes resin loading；With 65% ethyl alcohol of 6BV (containing very much One of triethylamine, volumn concentration) eluted with the flow velocity of 4BV/h and collect 5-6BV eluents, be concentrated and dried up to tripterygium wilfordii B prime and 2- table triptolide mixtures；Step S5, high speed adverse current chromatogram separation：With ethyl acetate-n-butanol-water-glacial acetic acid (4:1:3:0.01) it is solvent body System, with thereon mutually for stationary phase, lower phase is mobile phase；By triptolide obtained by step S4 and 2- table triptolide mixtures With isometric stationary phase and flowing phased soln, filtration, as sample solution；Rotating speed is 850r/min, and flow rate of mobile phase is It is corresponding to collect triptolide, 2- table triptolide chromatographic peaks according to chromatogram by 2.5mL/min, Detection wavelength 220nm Eluent is concentrated and dried up to triptolide, 2- table triptolides.
- 2. according to the method described in claim 1, it is characterized in that：The solid-to-liquid ratio of step S1 extractions is 1:5,1kg dry roots 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 in that：It is diluted with water to after ethyl alcohol in step S1 recycling extracting solutions 0.5g crude drugs/mL, filtering collect filtrate as macroreticular resin sample solution.
- 5. according to the method described in claim 1, it is characterized in that：Step S2 mixes sample resin quality and corresponds to crude drug matter for sample solution The 1/2 of amount；Step S4 mixes 1/2 that sample resin quality corresponds to precipitation amount of substance for sample solution.
- 6. according to the method described in claim 1, it is characterized in that：Step S3 bucks are ammonia spirit.
- 7. according to the method described in claim 1, it is characterized in that：Step S3 acid is hydrochloric acid.
- 8. according to the method described in claim 1, it is characterized in that：The temperature of step S3 low temperature crystallizations is 4 DEG C.
- 9. according to the method described in claim 1, it is characterized in that：Step S3 65% ethyl alcohol (triethylamine containing a ten thousandth, body Product percentage composition) 0.5g/mL solution is dissolved into as sample solution.
- 10. according to the method described in claim 1, it is characterized in that：A concentration of 10mg/mL of step S5 sample solutions.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108329292A (en) * | 2018-04-16 | 2018-07-27 | 黄榜昇 | A method of preparing former haematoxylin B |
| CN108546260A (en) * | 2018-04-16 | 2018-09-18 | 黄榜昇 | A method of preparing former haematoxylin C |
| CN108546261A (en) * | 2018-04-16 | 2018-09-18 | 黄榜昇 | A method of preparing protosappanin A |
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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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| 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108329292A (en) * | 2018-04-16 | 2018-07-27 | 黄榜昇 | A method of preparing former haematoxylin B |
| CN108546260A (en) * | 2018-04-16 | 2018-09-18 | 黄榜昇 | A method of preparing former haematoxylin C |
| CN108546261A (en) * | 2018-04-16 | 2018-09-18 | 黄榜昇 | A method of preparing protosappanin A |
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