CN106831804B - The method that ion exchange and silica gel column chromatography separation prepare Stephania tetrandra first, B prime - Google Patents
The method that ion exchange and silica gel column chromatography separation prepare Stephania tetrandra first, B prime Download PDFInfo
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- CN106831804B CN106831804B CN201710034052.4A CN201710034052A CN106831804B CN 106831804 B CN106831804 B CN 106831804B CN 201710034052 A CN201710034052 A CN 201710034052A CN 106831804 B CN106831804 B CN 106831804B
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 241001369613 Stephania tetrandra Species 0.000 title claims abstract description 14
- 238000000926 separation method Methods 0.000 title claims abstract description 10
- 238000010898 silica gel chromatography Methods 0.000 title claims abstract description 10
- 238000004255 ion exchange chromatography Methods 0.000 title claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 219
- 235000019441 ethanol Nutrition 0.000 claims abstract description 96
- WVTKBKWTSCPRNU-KYJUHHDHSA-N (+)-Tetrandrine Chemical group C([C@H]1C=2C=C(C(=CC=2CCN1C)OC)O1)C(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2C[C@@H]2N(C)CCC3=CC(OC)=C(OC)C1=C23 WVTKBKWTSCPRNU-KYJUHHDHSA-N 0.000 claims abstract description 67
- IIQSJHUEZBTSAT-VMPREFPWSA-N fangchinoline Chemical compound C([C@H]1C=2C=C(C(=CC=2CCN1C)OC)O1)C(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2C[C@@H]2N(C)CCC3=CC(OC)=C(O)C1=C23 IIQSJHUEZBTSAT-VMPREFPWSA-N 0.000 claims abstract description 44
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 13
- 238000004440 column chromatography Methods 0.000 claims abstract description 8
- 230000006837 decompression Effects 0.000 claims abstract description 8
- WVTKBKWTSCPRNU-UHFFFAOYSA-N rac-Tetrandrin Natural products O1C(C(=CC=2CCN3C)OC)=CC=2C3CC(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2CC2N(C)CCC3=CC(OC)=C(OC)C1=C23 WVTKBKWTSCPRNU-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000001953 recrystallisation Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 44
- 239000012043 crude product Substances 0.000 claims description 30
- 229930013930 alkaloid Natural products 0.000 claims description 25
- 238000010828 elution Methods 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000000706 filtrate Substances 0.000 claims description 22
- 238000001914 filtration Methods 0.000 claims description 22
- 150000003797 alkaloid derivatives Chemical class 0.000 claims description 21
- 239000000284 extract Substances 0.000 claims description 21
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 20
- 239000000908 ammonium hydroxide Substances 0.000 claims description 20
- 239000003480 eluent Substances 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- 239000011347 resin Substances 0.000 claims description 18
- 229920005989 resin Polymers 0.000 claims description 18
- 239000000741 silica gel Substances 0.000 claims description 17
- 229910002027 silica gel Inorganic materials 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 15
- 239000003729 cation exchange resin Substances 0.000 claims description 14
- 239000000287 crude extract Substances 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 150000001768 cations Chemical class 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 238000002242 deionisation method Methods 0.000 claims description 6
- 238000004821 distillation Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 5
- 238000005342 ion exchange Methods 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims 2
- 238000010612 desalination reaction Methods 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
- 230000002411 adverse Effects 0.000 claims 1
- 230000001476 alcoholic effect Effects 0.000 claims 1
- 239000003513 alkali Substances 0.000 claims 1
- 238000002425 crystallisation Methods 0.000 claims 1
- 230000008025 crystallization Effects 0.000 claims 1
- 238000005292 vacuum distillation Methods 0.000 claims 1
- 239000003643 water by type Substances 0.000 claims 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 26
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000002360 preparation method Methods 0.000 abstract description 6
- 239000003456 ion exchange resin Substances 0.000 abstract description 4
- 229920003303 ion-exchange polymer Polymers 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 201000010001 Silicosis Diseases 0.000 abstract description 2
- 239000004480 active ingredient Substances 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract description 2
- 230000003288 anthiarrhythmic effect Effects 0.000 abstract description 2
- 230000001093 anti-cancer Effects 0.000 abstract description 2
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 2
- 230000007797 corrosion Effects 0.000 abstract description 2
- 238000005260 corrosion Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 239000000945 filler Substances 0.000 abstract description 2
- 208000031225 myocardial ischemia Diseases 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract 2
- 241000196324 Embryophyta Species 0.000 abstract 1
- 241001330502 Stephania Species 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 231100000004 severe toxicity Toxicity 0.000 abstract 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 27
- 238000011084 recovery Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000012535 impurity Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- -1 methoxyl group Chemical group 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 239000002156 adsorbate Substances 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- JEUXZUSUYIHGNL-UHFFFAOYSA-N n,n-diethylethanamine;hydrate Chemical compound O.CCN(CC)CC JEUXZUSUYIHGNL-UHFFFAOYSA-N 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/18—Bridged systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The method that ion exchange and silica gel column chromatography separation prepare Stephania tetrandra first, B prime.Stephania tetrandra is the dried root of Menispermaceae stephania plant Fourstamen Stephania Root, its active ingredient is tetrandrine, wherein principle active component is hanfangchin A and B prime, and tetrandrine has the effects that resist myocardial ischemia, anti-arrhythmia cordis, decompression, anti-inflammatory, anti-silicosis, anticancer and eases pain.When medicinal, 99% or more is reached to its purity requirement, extraction purification is the medicinal major reason of both limitations.Currently, extraction purification hanfangchin A and B prime technique largely apply to severe toxicity and expensive chloroform and highly basic etc., and chloroform has human and environment prodigious toxic action, in addition strong to the equipment corrosion of amplification production.In addition, sample purity is not high, complex technical process.In this present situation, the present invention develops, with ion exchange resin and C18 bonded silica gel column chromatographies, the hanfangchin A and B prime of high-purity is made in fast separating and purifying, also discloses a kind of method carrying out recrystallization purifying hanfangchin A and B prime using ethyl alcohol.This preparation process does not use any toxic reagent, and column chromatography filler is repeatable to be utilized, ethyl alcohol recoverable used.
Description
Technical field
The present invention relates to the methods that separation prepares active ingredient from Chinese medicine, belong to the field of Chinese medicines.For a kind of utilization
Ion exchange resin and reverse phase C18The method that bonded silica gel column chromatography quickly prepares hanfangchin A and hanfangchin B simultaneously.
Background technology
Hanfangchin A is a kind of alkaloid for extracting separation from the Chinese medicine root of fangji and going out.Pharmacological research in recent years shows
Hanfangchin A has the effects that resist myocardial ischemia, anti-arrhythmia cordis, decompression, anti-inflammatory, anti-silicosis, anticancer and analgesia, at present with
Be widely used in clinic for injection, powder needle, the tablet of raw material production.But hanfangchin A as it is medicinal when, especially make
It is general to require to reach 99% or more for injection in use, must have higher purity, and cannot contain benzene, chloroform etc. its
Its impurity, it is very well sold and in short supply that this allows for high-purity hanfangchin A raw material, far can not meet preparation production needs.It is domestic at present
There are mainly two types of hanfangchin A extraction processes：One is cold benzene method, it is that solvent refluxing carries that basic craft course, which is with ethyl alcohol,
It taking, after extracting solution concentration plus lime precipitation, precipitation are extracted with benzene cold soaking, after recycling benzene, with ethyl alcohol recrystallization, what this method used
Benzene toxicity is very big, is easy residual, harm to the human body is big, does not adapt to produce greatly, it is difficult to obtain the Stephania tetrandra for meeting medicinal requirements
A prime；Another kind is chloroform extraction method, and it is solvent refluxing extraction, acid after extracting solution concentration that basic craft course, which is with ethyl alcohol,
It is heavy, scale is ammoniated after acid liquor filtering, the imitative extraction of lye chlorination uses acetone recrystallization, the chlorine that this method uses after recycling chloroform
Imitative of high cost, toxicity corrodes big, easy leakage greatly, to the sealing ring of equipment, and the chloroform of recycling is difficult to handle, the chloroform in waste liquid
Hardly possible separation, causes serious pollution to the environment, and the Content of Tetrandrine Determined of production is low, color is deep, fusing point is low, of high cost, organic residue is serious.
The hanfangchin A of above two method extraction is Extraction solvent with benzene and chloroform, and acetone is used in combination to be recrystallized, such
Extracting method remains the organic solvent being more toxic, and is difficult to detach with hanfangchin B in extraction process, to
Affect the purity of hanfangchin A.It is carried for the hanfangchin A of solvent without benzene and chloroform in addition, yet there are no both at home and abroad at present
Take method, and the report of the hanfangchin A without benzene and chloroform.
Invention content
The ion exchange resin and reverse phase C of easily realizing industrialization production are utilized the present invention provides a kind of18Bonded silica gel system
The method of standby hanfangchin A and hanfangchin B, benzene and chloroform is not used in this preparation method, and at the same time having prepared high-purity
Spend hanfangchin A and hanfangchin B.
Appropriate root of fangji medicinal material is taken, the 80-95% ethyl alcohol of 5-8 times of volume, refluxing extraction 5-8 hours, adjustment pH value to 1- are utilized
7, filtration takes filtrate to be added on cation exchange resin column, is first cleaned with water elution, then uses ethanol elution, uses successively different
The ethanol elution of concentration removes the impurity such as depigmentaton and other non-ionic states finally with anhydrous ethanol elution to colourless；So
Afterwards, first with the ammonium hydroxide of 0.5-1.5 mol/L and 50%-70% ethanol elutions, then with the ammonium hydroxide and 70%- of 1.6-2.5 mol/L
100% ethanol elution collects the elution fractions, through desalting processing.It is concentrated to give liquid extract, is suspended with 50% ethyl alcohol, upper reverse phase C18
Bonded silica gel column is rinsed pillar using 70%-90% ethyl alcohol, is monitored using UV detector, and monitoring wavelength is 230nm, according to color
Spectral peak is received, and is then recrystallized using ethyl alcohol to obtaining Tetrandrine and Fourstamen Stephania Root B prime.Recrystallization is primary, i.e.,
The product of high-purity can be obtained.
Above-mentioned technique detaches the alkalinity of object, hanfangchin A and hanfangchin B molecule knot first based on following principle
There are two the nitrogen-atoms for being in tertiary amine state in structure, alkalinity is stronger.Secondly, detach the lipophilicity of object, hanfangchin A and
Hanfangchin B lipophilicity is stronger, the general dissolubility with Fat-soluble alkaloids.Again, polarity difference between separation object,
Due to the difference of substituent group in the two molecular structure, the former is methoxyl group, and the latter is phenolic hydroxyl group, thus the polarity of hanfangchin A compared with
It is small.
In above-mentioned technical process, extracting solution is adjusted to suitable pH value, so that alkaloid is adequately ionized as cation, and
Nonbasic substances are not ionized.Extracting solution is after cation exchange resin column, biological basic ion and the hydrogen ion on resin column
Ion exchange occurs so that alkaloid is adsorbed on resin column.Here the pH value of extracting solution is unsuitable too high or too low, if mistake
Height, extracting solution is in neutrality or alkalinity, alkaloid cannot be ionized, and also can not just complete the exchange process on resin；If too low,
Hydrogen ion concentration in extracting solution is too high, hinders the hydrogen ion dissociation on resin column, cannot equally favorably accomplish resin friendship
Change process；Thus, it is proper that the pH value for finding to adjust extracting solution in experiment, which is 1-7,.When with water elution resin column, selection
Using deionized water, to ensure not introduce new ion interference.In elution process, those are not by the non-alkaline of resin adsorption
Substance is easy to be washed with water and take off, to come with the alkaloid ion isolation being attracted on resin column.
The exchange elution process of cation has following several, is eluted using basic solvent, and principle is by biological basic ion
It is converted into free compound, is then eluted by organic solvent；In addition, adsorbate is set from resin using the salt of high concentration
It changes, cation can be competitively combined with resin, to which the alkaloid ion exchange being adsorbed on resin column be got off.
Alkaloid can separate out in the salting liquid of high concentration, it is therefore desirable to add suitable acid or alcohol into eluent, exchange is made to get off
Alkaloid dissolving.
The present invention obtains 001 × 4 by including that gel resin and macroreticular resin screen to different kinds of ions exchanger resin
Belong to macroreticular resin with 001 × 7 cation exchange resin, the micropore with perpetuity, the advantages that surface area is big, and exchange velocity is fast.
Alkaloid is more advantageous to by being eluted on resin.
The present invention is using the alkaloid on Alkaline Elution agent elution resin, and under alkaline environment, and alkaloid is mostly with free
The form of state exists, and the solubility of alkaloid in water will substantially reduce, and elution efficiency is caused to reduce.It is of the invention based on this
The ethyl alcohol for having screened various concentration improves elution efficiency as cosolvent to improve solubility of the alkaloid in eluant, eluent.Together
When some impurity due to same resin-bonded ability difference, and its dissolubility is preferable in ethanol, the present invention devises several differences
Concentration ethanol and ammonium hydroxide are eluted, and removal of impurities is played the role of, to improve containing for hanfangchin A and B prime in ammonia alcohol eluen
Amount.
Technical scheme is as follows：
1, it takes 1 parts by weight of crushed of the root of fangji at coarse powder, the 80-95% ethyl alcohol of 6-10 parts by weight is added to carry out circumfluence distillation, extraction
Liquid filters, and it is spare that filtrate decompression is concentrated into no alcohol taste thick paste；
2, with the thick paste in the deionized water suspending step 1 of 5 times of volumes 10- is stood then with hydrochloric acid tune pH value to 1-7
24 hours, filtration, filtrate repeated 2-4 all over upper cation exchange resin；
3, it is eluted, is cleaned using deionization pure water and 10%-90% ethanol solutions successively；
3, following procedure is purified by flash again：First with the ethanol containing ammonia of 0.5-1.5 mol/L（Concentration of alcohol is
50%-70%）Elution, then with the ethanol containing ammonia of 1.6-2.5 mol/L（Concentration of alcohol is 70%-100%）Elution, collecting should
Elution fractions（Each eluent 5BV to 10BV）, through desalting processing；
4, by above-mentioned eluent, ethyl alcohol is recovered under reduced pressure, is suspended with water, filters, filter residue is washed with deionized water only, obtains Stephania tetrandra
A prime and hanfangchin B crude extract；
5, above-mentioned 1 parts by weight of total alkaloid crude extract, strutting chromatography reverse phase C18 bonded silica gel 0.5-2 parts by weight is taken to stir
It mixes uniformly, sets and fill in 5-15 parts by weight column chromatography reverse phase C18 bonded silica gel chromatographic columns, with 0.01%-0.03% triethylamine water
Solution and ethanol elution, the two ratio are 1:3 to 1:4, using UV detector, at 230nm, hanfangchin A is collected by peak
And hanfangchin B；
6, above-mentioned hanfangchin A and hanfangchin B crude product 10-30 times of ethanol solution for accounting for crude product weight are dissolved,
Filtering, filtrate are concentrated to 4-10 times of former crude product weight, and standing, which is let cool, is precipitated hanfangchin A and B prime；Filtering, drying, i.e.,
Hanfangchin A and hanfangchin B are obtained respectively.
The present invention, using cation exchange resin and reverse phase C18 reversed-phase bonded silicas, the hanfangchin A and the Chinese prepared
The recovery rate of root of fangji B prime, hanfangchin A and hanfangchin B reaches 70% or more, and purity is up to 99.0% ~ 99.8%, fusing point
216 ~ 221 DEG C, meet drug standards requirement.Compared to hanfangchin A extraction process instantly, advantages of the present invention has；
1, chloroform is not used in preparation process of the present invention, and chloroform is huge to production equipment corrosion, to production equipment in addition, chlorine
Imitating cannot be metabolized and be accumulated in body, there is strong carcinogenicity；
2, the organic solvent ethyl alcohol that preparation process of the present invention uses is recycled；
3, the ion exchange resin of preparation process of the present invention and reversed phase column chromatography filler can be recycled；
4, the present invention is relative to current production technology, it is only necessary to which primary recrystallization can be obtained the product of high-purity；
5, the present invention is recrystallized by hot ethanol solution instead of acetone solution sample, is on the one hand reduced and is produced into
This, on the other hand avoids the use of toxic solvent.
Present invention process, flow is simple, efficient, and low energy consumption, and pollutant is few, is especially beneficial amplification production.
Specific implementation mode
Embodiment 1
1, it takes 1 kg of the root of fangji to be ground into coarse powder, 85% ethyl alcohol of 4L is added to carry out circumfluence distillation, extracting solution filtering, filtrate decompression
It is concentrated into no alcohol taste, it is spare to obtain liquid extract；
2, step 1 medicinal extract 9L 0.5mol/L hydrochloric acid solutions are dissolved, it is 3 to adjust pH value, stands 24 hours, is filtered, filter
Liquid crosses the cation handled well and exchanges 001 × 7（Cation exchange resin dosage volume：Medicinal extract weight=30 milliliter：1g）, repeatedly
Loading 3 times；
3,5BV deionizations pure water, 60% ethyl alcohol of 5BV, 0.5 mol/L ammonium hydroxide of 5BV, 75% ethanol solution, 5BV are used successively
2.0 mol/L ammonium hydroxide, 90% ethanol solution collects 2.0 mol/L ammonium hydroxide, 90% ethanol solution eluent；
4, by above-mentioned eluent in step 3, ethyl alcohol being recovered under reduced pressure, is suspended with water, filter, filter residue is washed with deionized water only,
Obtain alkaloid crude extract；
5, above-mentioned total alkaloid crude extract 20g, strutting chromatography reverse phase C are taken18Bonded silica gel 20g, stirs evenly, and sets and fills
400g column chromatographies reverse phase C18In bonded silica gel chromatographic column, with 0.02% triethylamine aqueous solution and ethanol elution, the two ratio is 1:
3, using UV detector, at 230nm, hanfangchin A and hanfangchin B is collected by peak, obtains hanfangchin A crude product
7.1g, hanfangchin B crude product 4.7g；
6, above-mentioned hanfangchin A crude product 200ml 85% is dissolved, is heated to 60 DEG C, filtering, filtrate is concentrated to 50ml
Left and right, standing, which is let cool, is precipitated hanfangchin A, filters, and drying, weigh to obtain 6.3g.Hanfangchin A recovery rate is 71.5%, profit
It is 99.2% to measure purity with HPLC；
7, above-mentioned hanfangchin B 150ml 78% to be dissolved, is heated to 65 DEG C, filtering, filtrate is concentrated to 30ml or so,
Standing lets cool and hanfangchin B is precipitated, and filters, and drying, weigh to obtain 4.1g.Hanfangchin B recovery rate is 71.9%, is utilized
It is 99.1% that HPLC, which measures purity,.
Embodiment 2
1, it takes 1 kg of the root of fangji to be ground into coarse powder, 85% ethyl alcohol of 4L is added to carry out circumfluence distillation, extracting solution filtering, filtrate decompression
It is concentrated into no alcohol taste, it is spare to obtain liquid extract；
2, step 1 medicinal extract 9L 0.5mol/L hydrochloric acid solutions are dissolved, it is 3 to adjust pH value, stands 24 hours, is filtered, filter
Liquid crosses the cation handled well and exchanges 001 × 4（Cation exchange resin dosage volume：Medicinal extract weight=30 milliliter：1g）, repeatedly
Loading 3 times；
3,5BV deionizations pure water, 60% ethyl alcohol of 5BV, 0.5 mol/L ammonium hydroxide of 5BV, 75% ethanol solution, 5BV are used successively
2.0 mol/L ammonium hydroxide, 90% ethanol solution collects 2.0 mol/L ammonium hydroxide, 90% ethanol solution eluent；
4, by above-mentioned eluent in step 3, ethyl alcohol being recovered under reduced pressure, is suspended with water, filter, filter residue is washed with deionized water only,
Obtain alkaloid crude extract；
5, above-mentioned total alkaloid crude extract 20g, strutting chromatography reverse phase C are taken18Bonded silica gel 20g, stirs evenly, and sets and fills
400g column chromatographies reverse phase C18In bonded silica gel chromatographic column, with 0.02% triethylamine aqueous solution and ethanol elution, the two ratio is 1:
3, using UV detector, at 230nm, hanfangchin A and hanfangchin B is collected by peak, obtains hanfangchin A crude product
7.1g hanfangchin B crude product 4.7g；
6, above-mentioned hanfangchin A crude product 200ml 85% is dissolved, is heated to 60 DEG C, filtering, filtrate is concentrated to the left sides 50ml
The right side, standing, which is let cool, is precipitated hanfangchin A, filters, and drying, weigh to obtain 6.3g.Hanfangchin A recovery rate is 71.5%, is utilized
It is 99.2% that HPLC, which measures purity,；
7, above-mentioned hanfangchin B 150ml78% is dissolved, is heated to 65 DEG C, filtering, filtrate is concentrated to 30ml or so, quiet
Cold i.e. precipitation hanfangchin B is put, is filtered, drying, weigh to obtain 4.1g.Hanfangchin B recovery rate is 71.9%, utilizes HPLC
It is 99.1% to measure purity.
Embodiment 3
1, it takes 1 kg of the root of fangji to be ground into coarse powder, 85% ethyl alcohol of 4L is added to carry out circumfluence distillation, extracting solution filtering, filtrate decompression
It is concentrated into no alcohol taste, it is spare to obtain liquid extract；
2, step 1 medicinal extract 9L 0.5mol/L hydrochloric acid solutions are dissolved, it is 3 to adjust pH value, stands 24 hours, is filtered, filter
Liquid crosses the cation handled well and exchanges D001（Cation exchange resin dosage volume:Medicinal extract weight=30 milliliter：1g）, repeatedly on
Sample 3 times；
3, successively use 5BV deionizations pure water, 60% ethyl alcohol of 5BV, 0.5 mol/L ammonium hydroxide of 5BV, 75% ethyl alcohol, solution,
2.0 mol/L ammonium hydroxide of 5BV, 90% ethanol solution collects 2.0 mol/L ammonium hydroxide, 90% ethanol solution eluent；
4, by above-mentioned eluent in step 3, ethyl alcohol being recovered under reduced pressure, is suspended with water, filter, filter residue is washed with deionized water only,
Obtain alkaloid crude extract；
5, above-mentioned total alkaloid crude extract 20g, strutting chromatography reverse phase C are taken18Bonded silica gel 20g, stirs evenly, and sets and fills
400g column chromatographies reverse phase C18In bonded silica gel chromatographic column, with 0.02% triethylamine aqueous solution and ethanol elution, the two ratio is 1:
3, using UV detector, at 230nm, hanfangchin A and hanfangchin B is collected by peak, obtains hanfangchin A crude product
4.1g, hanfangchin B crude product 2.7g；
6, above-mentioned hanfangchin A crude product 200ml 85% is dissolved, is heated to 60 DEG C, filtering, filtrate is concentrated to the left sides 50ml
The right side, standing, which is let cool, is precipitated hanfangchin A, filters, and drying, weigh to obtain 2.3g.The recovery rate of hanfangchin A is 26%, is utilized
It is 88.9% that HPLC, which measures purity,；
7, above-mentioned hanfangchin B 150ml 78% to be dissolved, is heated to 65 DEG C, filtering, filtrate is concentrated to 30ml or so,
Standing lets cool and hanfangchin B is precipitated, and filters, and drying, weigh to obtain 1.1g.The recovery rate of hanfangchin B is 19%, is utilized
It is 90.1% that HPLC, which measures purity,.
Claims (3)
1. the method that ion exchange and silica gel column chromatography separation prepare Stephania tetrandra first, B prime, it is characterised in that processing step：
1. extraction：The root of dry Fourstamen Stephania Root is taken, is crushed, the 80-95% alcohol reflux 5-8h of 6-10 times of volume, vacuum distillation are utilized
Ethyl alcohol is recycled, liquid extract is obtained；
2. crossing cation exchange resin：The liquid extract obtained by hydrochloric acid suspending step 1, and pH value is adjusted to 1-7, filtering takes filter
Liquid is added on cation exchange resin column after progress loading, is first washed with 5-10BV deionized waters and 10%-90% ethanol solutions
Ethanol elution that is miscellaneous, then using various concentration successively is removed, specially first with the ammonium hydroxide second of 5-10BV 0.5-1.5 mol/L
Alcoholic solution elutes, and wherein concentration of alcohol is 50%-70%, then is eluted with the ethanol containing ammonia of 5-10BV 1.6-2.5 mol/L,
Wherein concentration of alcohol is 70%-100%, finally with anhydrous ethanol elution to colourless, collection alkaline ethanol eluent, after desalination, mistake
Filter, is washed with deionized water, obtains tetrandrine crude extract；Wherein, cation exchange resin used is 001 × 7,001 × 4；It is mixed
The hydrochloric acid solution of outstanding liquid extract, a concentration of 0.5 mol/L-1.5 mol/L；The amount of the cation exchange resin used is, sun from
The volume (mL) of sub-exchange resin:Liquid extract weight (g)=30-50：1；Filtrate sample loading mode is to adsorb repeatedly, using adverse current side
Formula loading repeatedly for three times；Alkaline ethanol eluent is the ethanol containing ammonia of 1.6-2.5 mol/L, and wherein concentration of alcohol is
The eluent of 70%-100%；Desalination mode is to be adjusted to pH value to 6-7 with 0.5-2.0 mol/L ammonium hydroxide, then filter；
3.RP-C18Bonded silica gel column chromatography：With 2 gained tetrandrine crude product of ethyl alcohol dissolving step, it is suspended, uses RP-C18Silica gel fills
Divide and mix thoroughly, RP-C is added18In bonded silica gel column, eluent is monitored using UV detector, and monitoring wavelength is 230nm, is pressed
It is received according to chromatographic peak, obtains hanfangchin A and hanfangchin B crude product；Wherein, ethyl alcohol is absolute ethyl alcohol, and dosage is anti-for the Chinese
1-1.5 times of own alkali crude product weight；Mix the RP-C of sample18Silica gel is 0.5-2 times of medicinal extract weight；RP-C18Bonded silica gel column chromatography
Eluant, eluent is the triethylamine aqueous solution and ethyl alcohol of volume fraction 0.01%-0.03%, and the two ratio is 1:3 to 1:4；
4. recrystallization：The hanfangchin A and hanfangchin B crude product that in step 3, are received according to chromatographic peak are separated；Respectively
Solvent is recovered under reduced pressure in the two, respectively by each crude product, is dissolved with fixed proportion ethyl alcohol, is heated to proper temperature, keeps sample abundant
Dissolving, filtering, filtrate is concentrated, standing, is waited that crystallization is precipitated, is cleaned, drying obtains the Chinese that purity is 99% or more respectively
Root of fangji A prime and hanfangchin B；Wherein, it is each crude product weight to dissolve hanfangchin A and the crude product ethanol solution dosage of B prime
10-30 times；Dissolve the fixed proportion ethanol solution of hanfangchin A crude product, concentration of alcohol 70%-100%；Dissolve Stephania tetrandra
The fixed proportion ethanol solution of B prime crude product, concentration of alcohol 70%-100%；The temperature of hanfangchin A crude product is dissolved using ethyl alcohol
Degree can be 30 DEG C -75 DEG C；The temperature that hanfangchin B crude product is dissolved using ethyl alcohol is 30 DEG C -75 DEG C；Concentrate the filtrate to former crude product
4-10 times of quality.
2. the method that ion exchange according to claim 1 and silica gel column chromatography separation prepare Stephania tetrandra first, B prime,
It is characterized in that：
1, it takes 1 kg of the root of fangji to be ground into coarse powder, 85% ethyl alcohol of 4L is added to carry out circumfluence distillation, extracting solution filtering, filtrate decompression concentration
To no alcohol taste, it is spare to obtain liquid extract；
2, step 1 medicinal extract 9L 0.5mol/L hydrochloric acid solutions are dissolved, it is 3 to adjust pH value, stands 24 hours, filtration, filtrate mistake
The cation handled well exchanges 001 × 7, wherein cation exchange resin dosage volume：Medicinal extract weight=30 milliliter：1g, repeatedly on
Sample 3 times；
3,5BV deionizations pure water, 60% ethyl alcohol of 5BV, 0.5 mol/L ammonium hydroxide of 5BV, 75% ethanol solution, 5BV 2.0 are used successively
90% ethanol solution of mol/L ammonium hydroxide collects 2.0 mol/L ammonium hydroxide, 90% ethanol solution eluent；
4, by above-mentioned eluent in step 3, ethyl alcohol is recovered under reduced pressure, is suspended with water, filter, filter residue is washed with deionized water only, obtains life
Alkaloids crude extract；
5, above-mentioned total alkaloid crude extract 20g, strutting chromatography reverse phase C are taken18Bonded silica gel 20g, stirs evenly, and sets and fills 400g
Column chromatography reverse phase C18In bonded silica gel chromatographic column, with 0.02% triethylamine aqueous solution and ethanol elution, the two ratio is 1:3, profit
Hanfangchin A and hanfangchin B are collected by peak, obtains hanfangchin A crude product, Stephania tetrandra at 230nm with UV detector
B prime crude product；
6, the above-mentioned hanfangchin A crude product ethyl alcohol that 200ml volume fractions are 85% is dissolved, is heated to 60 DEG C, filters, filter
Liquid is concentrated to 50ml or so, and standing, which is let cool, is precipitated hanfangchin A, filters, drying, obtains the Stephania tetrandra first that purity is 99% or more
Element；
7, the ethyl alcohol that above-mentioned hanfangchin B 150ml volume fractions are 78% is dissolved, is heated to 65 DEG C, filtering, filtrate concentrates
30ml or so is arrived, standing, which is let cool, is precipitated hanfangchin B, filters, drying, obtains the hanfangchin B that purity is 99% or more.
3. the method that ion exchange according to claim 1 and silica gel column chromatography separation prepare Stephania tetrandra first, B prime,
It is characterized in that：
1, it takes 1 kg of the root of fangji to be ground into coarse powder, 85% ethyl alcohol of 4L is added to carry out circumfluence distillation, extracting solution filtering, filtrate decompression concentration
To no alcohol taste, it is spare to obtain liquid extract；
2, step 1 medicinal extract 9L 0.5mol/L hydrochloric acid solutions are dissolved, it is 3 to adjust pH value, stands 24 hours, filtration, filtrate mistake
The cation handled well exchanges 001 × 4, wherein cation exchange resin dosage volume：Medicinal extract weight=30 milliliter：1g, repeatedly on
Sample 3 times；
3,5BV deionizations pure water, 60% ethyl alcohol of 5BV, 0.5 mol/L ammonium hydroxide of 5BV, 75% ethanol solution, 5BV 2.0 are used successively
90% ethanol solution of mol/L ammonium hydroxide collects 2.0 mol/L ammonium hydroxide, 90% ethanol solution eluent；
4, by above-mentioned eluent in step 3, ethyl alcohol is recovered under reduced pressure, is suspended with water, filter, filter residue is washed with deionized water only, obtains life
Alkaloids crude extract；
5, above-mentioned total alkaloid crude extract 20g, strutting chromatography reverse phase C are taken18Bonded silica gel 20g, stirs evenly, and sets and fills 400g
Column chromatography reverse phase C18In bonded silica gel chromatographic column, with 0.02% triethylamine aqueous solution and ethanol elution, the two ratio is 1:3, profit
Hanfangchin A and hanfangchin B are collected by peak, obtains hanfangchin A crude product, Stephania tetrandra at 230nm with UV detector
B prime crude product；
6, the above-mentioned hanfangchin A crude product ethyl alcohol that 200ml volume fractions are 85% is dissolved, is heated to 60 DEG C, filters, filter
Liquid is concentrated to 50ml or so, and standing, which is let cool, is precipitated hanfangchin A, filters, drying, obtains the Stephania tetrandra first that purity is 99% or more
Element；
7, the ethyl alcohol that above-mentioned hanfangchin B 150ml volume fractions are 78% is dissolved, is heated to 65 DEG C, filtering, filtrate is concentrated to
30ml or so, standing, which is let cool, is precipitated hanfangchin B, filters, drying, obtains the hanfangchin B that purity is 99% or more.
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