CN116903617A - Preparation method of 7-demethoxy tylophorine reference substance - Google Patents
Preparation method of 7-demethoxy tylophorine reference substance Download PDFInfo
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- CN116903617A CN116903617A CN202310871083.0A CN202310871083A CN116903617A CN 116903617 A CN116903617 A CN 116903617A CN 202310871083 A CN202310871083 A CN 202310871083A CN 116903617 A CN116903617 A CN 116903617A
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- Prior art keywords
- tylophorine
- demethoxy
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- eluting
- methanol
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- NCVWJDISIZHFQS-CQSZACIVSA-N (-)-antofine Chemical compound C12=CC(OC)=C(OC)C=C2C2=CC(OC)=CC=C2C2=C1C[C@H]1CCCN1C2 NCVWJDISIZHFQS-CQSZACIVSA-N 0.000 title claims abstract description 139
- NCVWJDISIZHFQS-UHFFFAOYSA-N tylophorine B Natural products C12=CC(OC)=C(OC)C=C2C2=CC(OC)=CC=C2C2=C1CC1CCCN1C2 NCVWJDISIZHFQS-UHFFFAOYSA-N 0.000 title claims abstract description 113
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 239000013558 reference substance Substances 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 68
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000002253 acid Substances 0.000 claims abstract description 35
- 229930008659 antofine Natural products 0.000 claims abstract description 29
- 238000001914 filtration Methods 0.000 claims abstract description 28
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 21
- -1 filtering Substances 0.000 claims abstract description 7
- 238000002791 soaking Methods 0.000 claims abstract description 5
- 239000007787 solid Substances 0.000 claims abstract description 5
- 241000411851 herbal medicine Species 0.000 claims abstract description 4
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 150
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 84
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 43
- 239000012071 phase Substances 0.000 claims description 43
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- 230000002829 reductive effect Effects 0.000 claims description 42
- 239000002904 solvent Substances 0.000 claims description 41
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 32
- 238000001035 drying Methods 0.000 claims description 31
- 239000000741 silica gel Substances 0.000 claims description 29
- 229910002027 silica gel Inorganic materials 0.000 claims description 29
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 25
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 25
- 238000000746 purification Methods 0.000 claims description 25
- 239000010410 layer Substances 0.000 claims description 23
- 239000012046 mixed solvent Substances 0.000 claims description 23
- 239000000284 extract Substances 0.000 claims description 21
- 239000000287 crude extract Substances 0.000 claims description 20
- 238000011068 loading method Methods 0.000 claims description 20
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 17
- 239000007864 aqueous solution Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 12
- 239000008346 aqueous phase Substances 0.000 claims description 11
- 238000011097 chromatography purification Methods 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 9
- 239000000945 filler Substances 0.000 claims description 9
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- 239000000377 silicon dioxide Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 6
- JBTWLSYIZRCDFO-UHFFFAOYSA-N ethyl methyl carbonate Chemical compound CCOC(=O)OC JBTWLSYIZRCDFO-UHFFFAOYSA-N 0.000 claims description 6
- 150000008282 halocarbons Chemical class 0.000 claims description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 6
- SSEUDFYBEOIWGF-AWEZNQCLSA-N Tylophorine Chemical class C1=C(OC)C(OC)=CC2=C(C=C(C(OC)=C3)OC)C3=C(C[C@H]3N(CCC3)C3)C3=C21 SSEUDFYBEOIWGF-AWEZNQCLSA-N 0.000 claims description 4
- UBOXGVDOUJQMTN-UHFFFAOYSA-N 1,1,2-trichloroethane Chemical compound ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 claims description 3
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 3
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 3
- CYTYCFOTNPOANT-UHFFFAOYSA-N Perchloroethylene Chemical group ClC(Cl)=C(Cl)Cl CYTYCFOTNPOANT-UHFFFAOYSA-N 0.000 claims description 3
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 claims description 3
- KXKVLQRXCPHEJC-UHFFFAOYSA-N acetic acid trimethyl ester Natural products COC(C)=O KXKVLQRXCPHEJC-UHFFFAOYSA-N 0.000 claims description 3
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 3
- 239000012044 organic layer Substances 0.000 claims description 3
- BNIXVQGCZULYKV-UHFFFAOYSA-N pentachloroethane Chemical compound ClC(Cl)C(Cl)(Cl)Cl BNIXVQGCZULYKV-UHFFFAOYSA-N 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- 239000011265 semifinished product Substances 0.000 claims description 3
- 229950011008 tetrachloroethylene Drugs 0.000 claims description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 21
- 240000006851 Duhaldea cappa Species 0.000 abstract description 7
- 239000003513 alkali Substances 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract 1
- 238000003810 ethyl acetate extraction Methods 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000001556 precipitation Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 description 27
- 238000010828 elution Methods 0.000 description 14
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- 239000000047 product Substances 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 238000012856 packing Methods 0.000 description 10
- 230000001376 precipitating effect Effects 0.000 description 10
- 239000011260 aqueous acid Substances 0.000 description 8
- 239000002585 base Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 241001673966 Magnolia officinalis Species 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 229930013930 alkaloid Natural products 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 241001571485 Vincetoxicum mongolicum Species 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 150000003797 alkaloid derivatives Chemical class 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
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- 230000006872 improvement Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010052360 Colorectal adenocarcinoma Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 241000132446 Inula Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241001673954 Magnolia sieboldii Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000331439 Tylophora tanakae Species 0.000 description 1
- SSEUDFYBEOIWGF-CQSZACIVSA-N Tylophorine Natural products C1=C(OC)C(OC)=CC2=C(C=C(C(OC)=C3)OC)C3=C(C[C@@H]3N(CCC3)C3)C3=C21 SSEUDFYBEOIWGF-CQSZACIVSA-N 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241001247477 Vincetoxicum hirundinaria Species 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001887 anti-feedant effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000012846 chemical reference substance Substances 0.000 description 1
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- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
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- 201000005202 lung cancer Diseases 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a preparation method of a 7-demethoxy tylophorine reference substance, which comprises the following steps: soaking and extracting Chinese herbal medicine inula cappa with ethanol, filtering, acid regulation, ethyl acetate extraction, alkali regulation, chloroform extraction, silica gel column chromatography, C18 column chromatography and precipitation treatment to obtain 7-demethoxy tylophorine (Antofine) with purity of more than 99%, and through specific pretreatment, the extraction and separation efficiency is effectively improved, and a target object reference substance with high HPLC purity can be obtained in a high throughput manner. In addition, the preparation method of the 7-demethoxy tylophorine solid reference substance has very good industrial production applicability.
Description
Technical Field
The invention relates to a preparation method of 7-demethoxy tylophorine (Antofine), in particular to a preparation method of a solid reference substance which can be used for preparing 7-demethoxy tylophorine applicable to industrialization.
Background
7-demethoxy tylophorine (also known as Antofine, CAS No. 32671-82-2) studied herein is a natural phenanthroindolizidine alkaloid, also known as tylophorine B, with good biological activity. Wang et al report that Antofine has the effects of relieving pain, relieving cough, relieving asthma, eliminating phlegm, relieving diarrhea, reducing fever, immunizing, treating cardiovascular diseases and the like in traditional Chinese medicine. Tripathi has reported in 1990 the insecticidal activity of Antofine and in addition it has antifungal and bacterial biological activity. Antofine has potential cytotoxicity and anticancer activity, and in vitro experiments of the Staerk group in 2002 prove that the drug-sensitive strain KB-3-1 and the multi-drug resistant strain KB-V1 for inhibiting human cervical cancer are cancer cell lines, and Fu in 2007 and the like have the inhibitory activity on the cancer cell lines of human colorectal cancer HCT116, human colorectal adenocarcinoma HT-1080 and lung cancer A549.
Due to the remarkable cytotoxicity and biochemical properties of Antofine, such compounds have received great attention as potential drug leads, and thus there is a great need for Antofine starting compounds. However, at present, only a total synthesis production scheme exists, and no scheme for providing an Antofine raw material compound which is low in cost and easy to implement exists. Research shows that 7-demethoxy tylophorine has the highest content in total alkaloids of the inula root and is likely to be used as a plant source, however, the current situation is that the extraction and separation of the plant source of the 7-demethoxy tylophorine from the plant source are difficult, no existing technology exists, the total content of the 7-demethoxy tylophorine is low, and the 7-demethoxy tylophorine is difficult to crystallize out of the mixture, so that the existing extraction and preparation methods of the 7-demethoxy tylophorine are very limited, and no high-efficiency related industrial extraction and preparation method of the 7-demethoxy tylophorine exists at present. As an important characteristic component, there is an urgent need to develop a preparation method of a 7-demethoxy tylophorine reference substance which has high flux, can provide high-purity 7-demethoxy tylophorine, and is suitable for mass preparation.
Non-patent document 1: staerk, d., lykkeberg, A.K, christensen, j., et al In vitro cytotoxic activityof
phenanthroindolizidine alkaloids from Cynanchumvincetoxicum and Tylophoratanakae against drug-sensitive and multidrug-resistant cancer cells.J Nat Prod，2002，65(9):1299-1302
Non-patent document 2: wang, Q.G, xie, L, zhai, J.Tylophorine B benzonate, acteCrystalline graphic section C-Crystal Structure Communications,2000, 56:197-198
Non-patent document 3: tripathi, A.K., singh, D., persistency of Tylophorine as an Insect Antifeedant againstSpilosoma-obliquawalker, phytopherer Res,1990,4:144-147
Non-patent document 4: fu, Y, lee, S.K., min, H.Y., et al Synthesis and structure-activity studies of antofineanalogues as potential anticancer agents.bioorganic & Medicinal Chemistry Letters,2007, 17 (1): 97-100)
Non-patent document 5: study of chemical composition and biological Activity of Cynanchum Komarovii university of agriculture and forestry, northwest, 2004.
Disclosure of Invention
The invention aims to provide a preparation method of a 7-demethoxy tylophorine reference substance, which has high flux, can provide high-purity 7-demethoxy tylophorine and is suitable for mass preparation. In the present invention, 7-demethoxy tylophorine is sometimes referred to as an isolation target. The invention greatly improves the separation and purification of 7-demethoxy tylophorine, introduces a unique and easy-to-operate combination method of column adsorption and solvation treatment, makes the whole extraction method suitable for extracting 7-demethoxy tylophorine components from a medicinal material of the inula cappa, and further finds a cost-controllable extraction and separation means through deep exploration, thereby realizing a industrialized target compound purification method, and greatly improving the amount and purity of the compound, thereby completing the invention.
The invention can separate and obtain the 7-demethoxy tylophorine with high purity through a simple and efficient operation flow. The preparation and separation method sequentially comprises the following steps:
normal phase silica gel chromatography purification procedure: loading the crude extract solution containing 7-demethoxy tylophorine to a normal phase chromatographic column, eluting with ethyl acetate solvent, eluting for 8-15 chromatographic column volumes, eluting with alcohol solvent containing 0.5-1.5wt% ammonia, collecting target part, concentrating the eluate, and drying to obtain crude extract I of 7-demethoxy tylophorine;
solvent treatment and purification steps: adding methanol/ethanol into the crude extract I of 7-demethoxy tylophorine obtained in the normal phase silica gel chromatographic purification process, performing ultrasonic treatment, filtering to obtain precipitate, and drying to obtain a crude extract II of 7-demethoxy tylophorine;
reversed phase chromatography purification procedure: loading the 7-demethoxy tylophorine crude extract II onto a reversed phase chromatographic column, eluting with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water, collecting a target, concentrating under reduced pressure to obtain a 7-demethoxy tylophorine (Antofine) semi-finished product, detecting and collecting an eluent according to TLC or HPLC, concentrating the eluent under reduced pressure, and drying to obtain a 7-demethoxy tylophorine reference substance with the purity of 99%.
In a preferred embodiment of the present invention, the method for preparing the total extract solution of the magnolia bark herb material comprises pulverizing dried magnolia bark herb, adding 5-15 times of alcohol solvent, soaking at room temperature for 2-5 days, filtering the soaking solution, concentrating under reduced pressure to thick extract, adding aqueous acid for dissolving, filtering, extracting aqueous acid layer with organic solvent, adjusting pH of aqueous acid phase with concentrated ammonia water to 9-10, extracting with organic solvent, combining organic layers, concentrating under reduced pressure to obtain crude extract solution containing 7-demethoxy tylophorine.
In a preferred embodiment of the present invention, the alcohol-based aqueous solution in the preparation method of the total extract solution of the magnolia bark herb is 60% -90% aqueous methanol solution or 60% -90% aqueous ethanol solution.
In a preferred embodiment of the present invention, in the normal phase chromatography purification step, the packing material of the normal phase chromatography column is silica gel having a particle diameter of 100 to 300 meshes, and elution is performed with an alcoholic solvent containing 0.9 to 1.1wt% of ammonia.
In a preferred embodiment of the present invention, in the reversed phase chromatography purification step, the elution is performed under a pressure of 0.1 to 0.3Mpa, and the elution is performed with an acetonitrile/water mixture solvent of 48% v/v to 70% v/v or a methanol/water mixture solvent of 70% v/v to 95% v/v, and an octadecylsilane chemically bonded silica filler of 40 to 60 μm is selected.
In a preferred embodiment of the present invention, in the acid-base treatment step, the aqueous acid solution is 0.06 to 0.10M hydrochloric acid.
In a preferred embodiment of the present invention, in the acid-base treatment step, the organic solvent used for extraction is an ester solvent or a halogenated hydrocarbon solvent.
In a preferred embodiment of the invention, the ester solvent is selected from ethyl acetate, butyl acetate, methyl acetate,
the halogenated hydrocarbon solvent is selected from dichloromethane, chloroform, carbon tetrachloride, 1-dichloroethane, 1, 2-dichloroethane, 1-trichloroethane, 1, 2-trichloroethane, and mixtures thereof 1, 2-tetrachloroethane, 1, 2-tetrachloroethane, pentachloroethane, 1-dichloroethylene, 1, 2-dichloroethylene, trichloroethylene, and tetrachloroethylene.
The invention also provides a solid reference substance of 7-demethoxy tylophorine with the purity of more than 99 percent, which is prepared by the preparation method.
The invention has the following characteristics:
the invention has simple and efficient operation, can amplify the process and separate to obtain a large amount of 7-demethoxy tylophorine monomers; the method for preparing and separating the high-purity 7-demethoxy tylophorine from the Chinese herbal medicine inula cappa has the advantages that the special simplification is obtained by utilizing the special chemical property of the 7-demethoxy tylophorine, the requirements of the raw materials of the traditional Chinese medicine chemical reference substances are met, and the method can be used as the reference substances. The method has the advantages of simple process, convenient preparation and separation operation, low equipment cost, easy production, large single-batch processing capacity, stable and reliable process, good reproducibility and high yield, and can be used for large-scale industrialized production. Meanwhile, the method is also the first patent for preparing and separating the high-purity 7-demethoxy tylophorine (Antofine), and fills the industry blank. The invention separates more than 7g of 7-demethoxy tylophorine from 50Kg of medicinal materials of the inula cappa, the purity is more than 99 percent, the yield is about 0.014 percent, and the 7-demethoxy tylophorine can be used as a reference substance for detection and is the first time in the patent report of the literature. Can be scaled up to hundred grams according to the requirements, thus having great improvements in terms of preparation method and throughput.
Drawings
FIG. 1 is a typical HPLC profile of examples 1-4;
FIG. 2 is an HPLC chart of comparative example 1;
FIG. 3 is an HPLC chart of comparative example 2;
FIG. 4 is an HPLC chart of comparative example 3;
FIG. 5 is an HPLC chromatogram of comparative example 4.
Detailed Description
The following describes specific embodiments of the present invention.
The percentage of the purity and content of the target in the present invention is the content in terms of mass ratio, unless otherwise specified. The percentage of liquid to liquid content ratios in the present invention, e.g., the ratio of formic acid content in aqueous acetonitrile, is measured in volume fractions, unless otherwise specified.
The existing method for separating the target 7-demethoxy tylophorine mainly utilizes the combination of normal phase and reverse phase chromatography, uses solvent gradient elution to gradually and slowly separate the target through the difference of the polarity of the target and impurities, generally, according to the polarity of antofine, for example, extract concentrate of crushed material of the magnolia sieboldii herbal medicine can be loaded onto a reverse phase chromatographic column, eluent containing the target is obtained through gradient elution, and the eluent is collected, concentrated and freeze-dried to obtain the solid target. However, the inventor of the invention discovers that other secondary metabolites in the inula cappa can greatly influence the purification of 7-demethoxy tylophorine, and the purity of the 7-demethoxy tylophorine obtained by directly utilizing reverse phase chromatography to separate the compounds contained in the inula cappa herb raw materials is very similar to the polarity of a target object, so that the final purity is difficult to improve the reverse phase chromatography separation efficiency and is not high, and the purity of the 7-demethoxy tylophorine can not be always improved. Although the concept of continuous purity improvement by using a reverse phase chromatographic column with higher pressure and better separation degree can gradually improve the purity of 7-demethoxy tylophorine, the yield is low and the solvent is expensive in the separation process.
The inventor of the present invention has conducted intensive studies to determine that the preparation and separation method of the present invention sequentially comprises the following steps in order to be suitable for industrial mass production, in order to solve the problems of yield and separation efficiency: normal phase silica gel chromatography purification procedure: loading the crude extract solution containing 7-demethoxy tylophorine to a normal phase chromatographic column, eluting with ethyl acetate solvent, eluting for 8-15 chromatographic column volumes, eluting with alcohol solvent containing 0.5-1.5wt% ammonia, collecting target part, concentrating the eluate, and drying to obtain crude extract I of 7-demethoxy tylophorine;
solvent treatment and purification steps: adding methanol/ethanol into the crude extract I of 7-demethoxy tylophorine obtained in the normal phase silica gel chromatographic purification process, performing ultrasonic treatment, filtering to obtain precipitate, and drying to obtain a crude extract II of 7-demethoxy tylophorine;
reversed phase chromatography purification procedure: loading the 7-demethoxy tylophorine crude extract II to a reversed phase chromatographic column, eluting with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water, collecting a target, concentrating under reduced pressure to obtain a 7-demethoxy tylophorine (Antofine) semi-finished product, detecting and collecting an eluent according to TLC or HPLC, concentrating the eluent under reduced pressure, and drying to obtain the 7-demethoxy tylophorine reference substance with the purity of 99%.
The purpose of the normal phase chromatographic purification step is generally to distinguish impurities with larger polarity differences from the target, and the normal phase chromatographic column packing used in the normal phase chromatographic purification step may be known normal phase packing, i.e., packing with a polarity of the stationary phase greater than that of the mobile phase, and if the mobile phase is an organic solvent, the usual packing is silica gel (specifically, siO 2 Silicon dioxide), al 2 O 3 Polar-bonded phase fillers, and the like. However, the use of silica gel is desirable in the present inventionThe normal phase chromatographic purification procedure of the invention is not very similar to the procedure, and the method does not use a gradient elution method for fine separation, but still has good target enrichment effect. The key point of the invention is to find that the normal phase silica gel chromatographic column is particularly favorable for removing non-antofine components in the inula cappa, and the reversibility of adsorption and desorption of the antofine components on the normal phase silica gel chromatographic column is good. Based on the characteristics, the ethyl acetate solvent can be used for elution after loading, the elution is basically non-Antofine components, and generally, the elution of 8-15 chromatographic column volumes can remove most of components except 7-demethoxy tylophorine (Antofine), and the components are often similar in polarity to 7-demethoxy tylophorine in reverse phase chromatography and are difficult to separate, so that the normal phase silica gel chromatographic purification process has a crucial effect on efficiently separating target components although the operation is simple. It should be noted that, in the methanol used in the final elution, alkalinity needs to be increased, and in theory, both organic alkali and inorganic alkali can help to increase desorption rate, but it has been found through research that adding ammonia water can simply and effectively desorb the target compound from the normal phase silica gel column, and impurities are less. In the preferred production method of the present invention, the size of the silica gel is not particularly limited, and the filler of the first normal phase column is silica gel having a particle size of 100 to 300 mesh in the purification step of the first normal phase column, in view of efficiency and availability of the filler. In order to obtain a better separation efficiency, and from the viewpoints of the degree of separation and adsorption loss, silica gel is preferably used, and silica gel of 100 to 300 mesh is more preferably used. In a preferred embodiment of the present invention, in the normal phase chromatography purification step, the packing material of the normal phase chromatography column is silica gel having a particle diameter of 100 to 300 meshes, and elution is performed with an alcoholic solvent containing 0.9 to 1.1wt% of ammonia. The method of loading the crude raw material in the present invention is not particularly limited, and the crude raw material may be loaded by dissolving in a good solvent, or may be loaded by stirring with a filler, and in order to reduce the volume of the loaded sample, the loading with a stirring filler is preferable.
The starting material of the separation method can be crude extract of any type of medicinal materials of the Cynanchum komarovii, and can be purchased raw materials or self-extracted raw materials. Because the target product is an alkaloid, the target product can be further enriched if the crude extract is subjected to acid-base treatment, the extraction efficiency can be improved, and the separation effect can be greatly improved if the acid-base treatment process is added, but the separation effect is not required. Therefore, the preparation method of the preferred starting material in the invention is to crush the dried Cynanchum komarovii, add 5-15 times of alcohol solvent, soak for 2-5 days at room temperature, filter and decompress the soak solution to thick extract, add aqueous acid solution for dissolution, filter, extract the aqueous acid layer with organic solvent, adjust pH of the aqueous acid phase with concentrated ammonia water to 9-10, extract with organic solvent, combine the organic layers and decompress and concentrate. In a preferred embodiment of the present invention, in the acid-base treatment step, the organic solvent used for extraction is an ester solvent or a halogenated hydrocarbon solvent. In a preferred embodiment of the present invention, the alcohol-based aqueous solution in the preparation method of the total extract solution of the magnolia bark herb is 60% -90% aqueous methanol solution or 60% -90% aqueous ethanol solution. In a preferred embodiment of the present invention, the aqueous solution of the alcohol system is 60% to 90% aqueous methanol solution or 60% to 90% aqueous ethanol solution, and from the viewpoint of safety of the flow path, it is preferable to use 60% to 90% aqueous ethanol solution. In a preferred embodiment of the invention, the ester solvent is selected from ethyl acetate, butyl acetate, methyl acetate, the halogenated hydrocarbon solvent is selected from dichloromethane, chloroform, carbon tetrachloride, 1-dichloroethane, 1, 2-dichloroethane, 1-trichloroethane, 1, 2-trichloroethane, and mixtures thereof 1, 2-tetrachloroethane, 1, 2-tetrachloroethane, pentachloroethane, 1-dichloroethylene, 1, 2-dichloroethylene, trichloroethylene, and tetrachloroethylene. In a preferred embodiment of the present invention, in the acid-base treatment step, the aqueous acid solution is 0.06 to 0.10M hydrochloric acid; the base may be an organic base or an inorganic base, and sodium bicarbonate is preferably used.
The solvent treatment purification process in the present invention is also a critical step, and the inventors found that the substances eluted from the normal phase column have a large difference in solubility in methanol and/or ethanol, and that the target substance is not dissolved in methanol and/or ethanol, but a large amount of impurities are dissolved in methanol and/or ethanol. Through this solvent treatment purification procedure, it has been possible to increase the purity of 7-demethoxylated tylophorine to a very high degree. More importantly, most of impurities which are difficult to separate from 7-demethoxy tylophorine in the reversed phase chromatography purification can be removed through the step, so that the difficulty in the reversed phase chromatography purification is reduced.
The sequence of the steps in the invention greatly influences the extraction and separation efficiency, and generally, solvent purification treatment such as recrystallization and the like is carried out finally. The order of steps is also an important feature of the present invention.
And loading the 7-demethoxy tylophorine crude extract II to a reversed phase chromatographic column, eluting with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water, and obtaining a 7-demethoxy tylophorine reference substance with very high purity. In the present invention, the reverse phase chromatography purification step is used to separate other components having a polarity different from that of the target.
In a preferred embodiment of the present invention, the reverse phase chromatography purification step is performed in a medium-low pressure reverse phase chromatography purification step, and high pressure reverse phase chromatography purification is not required, so that a common preparative liquid phase, flash column, or the like can be used. The term "medium-low pressure" and "high pressure" are not strictly defined, and in the present invention, the medium-low pressure reverse phase chromatography purification step is considered to be used for separating other components having a polarity different from that of the target, and the equilibrium between resolution and velocity can be achieved by using an elution pressure of 0.05 to 1Mpa, and in the medium-low pressure column separation step, this pressure is particularly preferable because the separation effect and separation velocity can be balanced. The reversed-phase packing of the reversed-phase column used in the column preparation step of the present invention may be silica gel having a known nonpolar, bonded functional group of alkane (for example, C18 (ODS), C8, C4, etc.). Preferably, a C18 (ODS) silica gel column, namely octadecylsilane chemically bonded silica, is preferably used as a 40 to 60 μm packing in the so-called medium-low pressure reverse phase chromatography purification step, and a reasonable particle size is advantageous in maintaining an appropriate column pressure and resolution, and octadecylsilane chemically bonded silica is easily commercially available. In the preferred preparation method of the present invention, the packing of the reverse phase chromatography column is octadecylsilane chemically bonded silica having a particle size of 40 to 60 μm from the viewpoints of efficiency and availability of the packing. In the reverse phase chromatography purification step, the elution is preferably performed under a pressure of 0.1 to 0.3MPa, and the elution is performed with a 48% v/v to 70% v/v acetonitrile/water mixed solvent or a 70% v/v to 95% v/v methanol/water mixed solvent, more preferably with a 48% v/v to 70% v/v acetonitrile/water mixed solvent, and an octadecylsilane chemically bonded silica filler of 40 to 60 μm is selected. When eluting with an eluent of too high polarity, the product obtained is of lower purity.
Examples
The typical extraction method of the present invention will be described in further detail by examples. The following experimental protocol is merely exemplary and not limiting of the invention. Any alterations will occur to those skilled in the art without departing from the principles and spirit of the invention.
Example 1
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing the solvent from the target part, adding methanol for ultrasonic treatment, filtering, precipitating and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting with acetonitrile (48-70%v/v), collecting a target, concentrating, and drying under reduced pressure to obtain 7.084g of 7-demethoxy tylophorine (Antofine) pure product, wherein the HPLC purity is more than 99%, and the yield is 35.42%.
Comparative example 1
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing the solvent from the target part, adding methanol for ultrasonic treatment, filtering, precipitating and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting with acetonitrile (45%v/v), collecting a target, concentrating, and drying under reduced pressure to obtain a pure 7-demethoxy tylophorine (Antofine) product, wherein the HPLC purity is more than 94.0%.
Example 2
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; a target portion; removing the solvent from the target part, adding methanol for ultrasonic treatment, filtering, precipitating and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting methanol (70-95% v/v), collecting a target, concentrating, and drying under reduced pressure to obtain 7.026g of 7-demethoxy tylophorine (Antofine) pure product, wherein the HPLC purity is more than 99% and the yield is 35.13%.
Comparative example 2
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing the solvent from the target part, adding methanol for ultrasonic treatment, filtering, precipitating and drying; dissolving and loading a small amount of mixed solvent of dichloromethane and methanol into a C18 column for high-efficiency preparation, eluting with methanol (65%v/v), collecting the target, concentrating, and drying under reduced pressure to obtain 7.399 g of 7-demethoxy tylophorine (Antofine) pure product with HPLC purity of 91.5%
Example 3
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing solvent from target part, adding ethanol, ultrasonic treating, filtering, precipitating, and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting with acetonitrile (48-70%v/v), collecting a target, concentrating, and drying under reduced pressure to obtain 7.580g of 7-demethoxy tylophorine (Antofine) pure product, wherein the HPLC purity is more than 99% and the yield is 37.90%.
Comparative example 3
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing solvent from target part, adding ethanol, ultrasonic treating, filtering, precipitating, and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting with acetonitrile (75%v/v), collecting a target, concentrating, and drying under reduced pressure to obtain the 7-demethoxy tylophorine (Antofine) with the HPLC purity of 98.4% at maximum, the weight of 4.55g and the yield of 22.75%.
Example 4
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing solvent from target part, adding ethanol, ultrasonic treating, filtering, precipitating, and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting with methanol (70-95% v/v), collecting a target, concentrating, and drying under reduced pressure to obtain 7.030g of 7-demethoxy tylophorine (Antofine) pure product, wherein the HPLC purity is more than 99%, and the yield is 35.15%.
Comparative example 4
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing solvent from target part, adding ethanol, ultrasonic treating, filtering, precipitating, and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting with methanol (100%v/v), collecting a target, concentrating, and drying under reduced pressure to obtain the 7-demethoxy tylophorine (Antofine) with the HPLC purity of 96.4% at maximum.
Comparative example 5 (without introducing acid-base treatment Process)
50kg of the officinal magnolia bark herb is added with 10 times of 90 percent ethanol, soaked for 3 days at room temperature, filtered and decompressed and concentrated into thick extract, and mixed with silica gel; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing solvent from target part, adding ethanol, ultrasonic treating, filtering, precipitating, and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting acetonitrile (48-70% v/v), collecting a target, concentrating, and drying under reduced pressure to obtain 6.89g of 7-demethoxy tylophorine (Antofine) pure product with the HPLC purity of 89%.
Comparative example 6 (without introducing solvent treatment)
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; removing the solvent from the target portion, and drying; and (3) dissolving and loading a small amount of mixed solvent of dichloromethane and methanol to a C18 column for efficient preparation, eluting methanol (70-95% v/v), collecting a target, concentrating, and drying under reduced pressure to obtain 7.12g of 7-demethoxy tylophorine (Antofine) pure product with an HPLC purity of 95.0%.
Comparative example 7 (solvent treatment sequence adjustment)
50kg of the cortex magnoliae officinalis herb material is added with 90% ethanol with the amount of 10 times that of the cortex magnoliae officinalis herb material, soaked for 3 days at room temperature, the soaked solution is filtered and concentrated to thick extract under reduced pressure, 5% of acid aqueous solution is added for dissolution and filtration, the acid aqueous layer is extracted by ethyl acetate, the pH of the acid aqueous phase is adjusted to 9-10 by ammonia water, chloroform is used for extraction, a chloroform layer is combined, and the mixture is mixed with silica gel after reduced pressure concentration; eluting with ethyl acetate for 10 column volumes, eluting with methanol (containing 1% ammonia water), and collecting target part; concentrating the target part, performing C18 high-pressure preparation, eluting with methanol (70-95% v/v), collecting the target, concentrating, drying under reduced pressure, adding ethanol, performing ultrasonic treatment, filtering, precipitating, and drying to obtain 4.030g of 7-demethoxy tylophorine (Antofine) pure product, wherein the HPLC purity is greater than 99%, the yield (20.15%) is far lower than that of the examples 1-4, and the construction period is 5 times that before the exchange sequence.
Analysis of detection conditions
Chromatographic conditions:
1.0mg of sample is taken and 1mL of methanol is added for dissolution to prepare 1.0mg/mL, and the sample solution is filtered by a 0.22 mu m filter membrane.
Instrument: waters 2695HPLC chromatograph;
chromatographic column: agilent Plus C18, 4.6x150mm, 5 μm;
column temperature: 30 ℃;
sample injection amount: 10. Mu.L;
detection wavelength: 280nm;
gradient set-up of mobile phase (A: acetonitrile, B: water)
The HPLC spectrum of the final target can be referred to fig. 1, and it can be seen that the present invention can stably provide the target compound with high purity.
The technical features disclosed above are not limited to the combination with other features disclosed, and other combinations of the technical features can be performed by those skilled in the art according to the purpose of the invention to achieve the purpose of the invention, and various modifications made by those skilled in the art to the technical solution of the invention should fall within the scope of protection defined by the claims of the invention without departing from the design spirit of the invention.
Claims (9)
1. The preparation method of the 7-demethoxy tylophorine reference substance is characterized by comprising the following steps in sequence:
normal phase silica gel chromatography purification procedure: loading the crude extract solution containing 7-demethoxy tylophorine to a normal phase chromatographic column, eluting with ethyl acetate solvent, eluting with 8-15 chromatographic column volumes, eluting with alcohol solvent containing 0.5-1.5wt% ammonia, collecting target part, concentrating the eluate, and drying to obtain crude extract I of 7-demethoxy tylophorine;
solvent treatment and purification steps: adding methanol/ethanol into the crude extract I of 7-demethoxy tylophorine obtained in the normal phase silica gel chromatographic purification process, performing ultrasonic treatment, filtering to obtain precipitate, and drying to obtain a crude extract II of 7-demethoxy tylophorine;
reversed phase chromatography purification procedure: loading the 7-demethoxy tylophorine crude extract II onto a reversed phase chromatographic column, eluting with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water, collecting a target, concentrating under reduced pressure to obtain a 7-demethoxy tylophorine (Antofine) semi-finished product, detecting and collecting an eluent according to TLC or HPLC, concentrating the eluent under reduced pressure, and drying to obtain a 7-demethoxy tylophorine reference substance with the purity of 99%.
2. The method for preparing the 7-demethoxytylophorine control according to claim 1, characterized in that,
the preparation method of the total extract solution of the cortex magnoliae officinalis herbal medicine comprises the steps of crushing dried cortex magnoliae officinalis, adding 5-15 times of alcohol solvent, soaking for 2-5 days at room temperature, filtering and concentrating the soaking solution under reduced pressure to obtain thick extract, adding an acid aqueous solution for dissolving, filtering, extracting an acid aqueous layer by using an organic solvent, adjusting the pH of the acid aqueous phase to 9-10 by using concentrated ammonia water, extracting by using the organic solvent, merging the organic layers, and concentrating under reduced pressure to obtain the crude extract solution containing 7-demethoxy tylophorine.
3. The method for preparing the 7-demethoxy tylophorine control according to claim 2, wherein the aqueous solution of the alcohol system is 60% -90% aqueous methanol solution or 60% -90% aqueous ethanol solution.
4. The method for preparing the 7-demethoxytylophorine control according to claim 1, characterized in that,
in the normal phase chromatographic purification step, the filler of the normal phase chromatographic column is silica gel with the particle size of 100-300 meshes, and methanol containing 0.9-1.1 wt% of ammonia is used for eluting.
5. The method for preparing the 7-demethoxytylophorine control according to claim 1, characterized in that,
in the reverse phase chromatographic purification process, the pressure of 0.1-0.3 MPa is used for eluting, 48-70% v/v acetonitrile water mixed solvent or 70-95% v/v methanol water mixed solvent is used for eluting, and 40-60 mu m octadecylsilane chemically bonded silica filler is selected.
6. The method for preparing a 7-demethoxy tylophorine control according to claim 1, wherein in the acid-base treatment process, the acid aqueous solution is 0.06-0.10M hydrochloric acid.
7. The method for preparing a 7-demethoxylated tylophorine control according to claim 2, wherein the organic solvent used for extraction in the acid-base treatment process is an ester solvent or a halogenated hydrocarbon solvent.
8. The method for preparing a 7-demethoxylated tylophorine control according to claim 7, wherein the ester solvent is selected from ethyl acetate, butyl acetate, methyl acetate,
the halogenated hydrocarbon solvent is selected from dichloromethane, chloroform, carbon tetrachloride, 1-dichloroethane, 1, 2-dichloroethane, 1-trichloroethane, 1, 2-trichloroethane, and mixtures thereof 1, 2-tetrachloroethane, 1, 2-tetrachloroethane, pentachloroethane, 1-dichloroethylene, 1, 2-dichloroethylene, trichloroethylene, and tetrachloroethylene.
9. A solid control of 7-demethoxy tylophorine having a purity of 99% or more, prepared by the method of claim 1.
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