CN115536665A - Separation method of high-purity cepharanthine - Google Patents
Separation method of high-purity cepharanthine Download PDFInfo
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- CN115536665A CN115536665A CN202211117274.XA CN202211117274A CN115536665A CN 115536665 A CN115536665 A CN 115536665A CN 202211117274 A CN202211117274 A CN 202211117274A CN 115536665 A CN115536665 A CN 115536665A
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- cepharanthine
- purity
- silica gel
- methanol
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- VQAWRQZAAIQXHM-UHFFFAOYSA-N Cepharanthine Natural products O1C(C=C2)=CC=C2CC(C=23)N(C)CCC3=CC=3OCOC=3C=2OC(=CC=23)C(OC)=CC=2CCN(C)C3CC2=CC=C(O)C1=C2 VQAWRQZAAIQXHM-UHFFFAOYSA-N 0.000 title claims abstract description 64
- YVPXVXANRNDGTA-WDYNHAJCSA-N cepharanthine Chemical compound C1C(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2C[C@H](C2=C3)N(C)CCC2=CC(OC)=C3OC2=C(OCO3)C3=CC3=C2[C@H]1N(C)CC3 YVPXVXANRNDGTA-WDYNHAJCSA-N 0.000 title claims abstract description 64
- 238000000926 separation method Methods 0.000 title claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 102
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000000463 material Substances 0.000 claims abstract description 28
- 239000000843 powder Substances 0.000 claims abstract description 21
- 239000000741 silica gel Substances 0.000 claims abstract description 21
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 21
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 15
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 15
- 241001598357 Stephania delavayi Species 0.000 claims abstract description 10
- 239000012043 crude product Substances 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 238000004262 preparative liquid chromatography Methods 0.000 claims abstract description 9
- 239000007787 solid Substances 0.000 claims abstract description 9
- 241000218164 Menispermaceae Species 0.000 claims abstract description 4
- 239000003480 eluent Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000004570 mortar (masonry) Substances 0.000 claims description 14
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 238000001179 sorption measurement Methods 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000002137 ultrasound extraction Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000004215 Carbon black (E152) Substances 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 229930195733 hydrocarbon Natural products 0.000 claims description 6
- 125000001183 hydrocarbyl group Chemical group 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 239000003607 modifier Substances 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 4
- 239000000499 gel Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 2
- 239000001099 ammonium carbonate Substances 0.000 claims description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 2
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 2
- 125000004185 ester group Chemical group 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000006069 physical mixture Substances 0.000 claims description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 238000010025 steaming Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 9
- 239000012528 membrane Substances 0.000 abstract description 8
- 239000000047 product Substances 0.000 abstract description 8
- 238000009776 industrial production Methods 0.000 abstract description 4
- 238000010298 pulverizing process Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 18
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 8
- 238000012856 packing Methods 0.000 description 6
- 238000000825 ultraviolet detection Methods 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 238000001816 cooling Methods 0.000 description 5
- 238000011049 filling Methods 0.000 description 5
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 5
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 241000270708 Testudinidae Species 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 229930192392 Mitomycin Natural products 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 241001369613 Stephania tetrandra Species 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 201000002364 leukopenia Diseases 0.000 description 2
- 231100001022 leukopenia Toxicity 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010018687 Granulocytopenia Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- QOSMNYMQXIVWKY-UHFFFAOYSA-N Propyl levulinate Chemical group CCCOC(=O)CCC(C)=O QOSMNYMQXIVWKY-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000004442 acylamino group Chemical group 0.000 description 1
- 150000001335 aliphatic alkanes Chemical group 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000005360 mashing Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- UEAPAHNNFSZHMW-UHFFFAOYSA-N stepahnine Natural products COC1=CC=CC(C2=C34)=C1CC3N(C)CCC4=CC1=C2OCO1 UEAPAHNNFSZHMW-UHFFFAOYSA-N 0.000 description 1
- UEAPAHNNFSZHMW-CQSZACIVSA-N stephanine Chemical compound CN([C@@H]1CC2=C(C3=C11)C=CC=C2OC)CCC1=CC1=C3OCO1 UEAPAHNNFSZHMW-CQSZACIVSA-N 0.000 description 1
Images
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
- C07D491/22—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 four or more hetero rings
Abstract
The invention belongs to the technical field of separation and purification, and provides a separation method of high-purity cepharanthine, which comprises the following steps: A. pulverizing sliced Stephania delavayi Diels, radix Stephaniae Japonicae or Menispermaceae Chinese medicinal materials containing cepharanthine into powder, adding extractant, ultrasonic extracting for 20min, and concentrating under reduced pressure to remove extractant; B. b, carrying out chromatography on the extracting solution obtained in the step A through a silica gel column, and eluting to obtain a cepharanthine crude product; C. purifying the crude cepharanthine product by preparative liquid chromatography after passing through a 0.45 μm membrane; D. and D, performing reduced pressure rotary evaporation on the fraction obtained in the step C at the temperature of 35 ℃ to remove methanol, and performing freeze drying to obtain a white solid, namely the purified cepharanthine. The method of the invention has the advantages of high cepharanthine purity, simple and controllable separation process, and easy automation of industrial production.
Description
Technical Field
The invention belongs to the technical field of separation and purification, and particularly relates to a separation method of high-purity cepharanthine.
Background
Cepharanthine (Cepharanthine), a leukocyte increasing drug, can promote the proliferation of bone marrow tissue, thereby increasing leukocytes. Can be used for treating leukopenia. Can increase peripheral blood white blood cells, and animal experiments show that the compound can obviously prevent white blood cell reduction caused by mitomycin, but does not inhibit the anticancer effect of the mitomycin. The action mechanism is to promote the proliferation of bone marrow tissue, thereby generating the function of increasing leucocytes. Can be used for treating granulocytopenia caused by tumor chemotherapy and radiotherapy and leukopenia caused by other reasons. In the prior art, the separation and purification method of cepharanthine is complex in process and is not easy to realize automation of industrial production.
Disclosure of Invention
The invention provides a separation method of high-purity cepharanthine aiming at the problems, which is characterized in that stephania tetrandra Chinese medicinal materials of stephaniaceae, stephania root slices and stephanine are taken as raw materials, an extracting agent is used for extraction, the extracting solution is subjected to silica gel column chromatography and elution, and liquid chromatography separation is prepared to obtain purified cepharanthine;
the chromatography material is filled in the adsorption column, and is organic hydrocarbon bonded silica gel or organic hydrocarbon bonded silica gel containing polar organic functional groups; the adsorption column material is a physical mixture of more than two bonded silica gels or more than two organic hydrocarbon bonded silica gels;
the polar organic functional group is selected from ester group, amino group, amide group, and urea group.
Preferably, the method comprises the following steps:
A. mashing the Chinese medicinal material SHANCUI slice into powder with mortar, adding extractant, performing ultrasonic extraction, and performing reduced pressure rotary evaporation to concentrate to remove the extractant;
B. b, carrying out chromatography on the extracting solution obtained in the step A through a silica gel column, and eluting to obtain a cepharanthine crude product;
C. filtering the crude cepharanthine product, and purifying by preparative liquid chromatography;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain a white solid, namely the purified cepharanthine.
Preferably, the extractant is one or more of methanol, ethanol, n-propanol and isopropanol.
Preferably, the particle size of the chromatographic material is 5-100um, and the pore diameter is
Preferably, in the step B, the elution flow rate is 5 to 30 column volumes/hour.
Preferably, in the step B, the eluent is selected from any one of organic solvents of methanol, acetonitrile, acetone, tetrahydrofuran, ethanol, isopropanol and dioxane, or a mixture of one or more organic solvents is mixed with water, or a mixture of one or more organic solvents is mixed with an aqueous solution added with a modifier.
Preferably, the ratio of water or aqueous solution viscosity in the eluent is 10% -60%.
Preferably, the modifier is one or more of formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, phosphoric acid, ammonium bicarbonate, ammonia water, triethylamine, diethylamine, ammonium acetate and ammonium formate.
The invention has the beneficial effects that:
the invention discloses a separation method of high-purity cepharanthine, which comprises the steps of extraction of cepharanthine, chromatographic separation, collection and chromatographic purity detection. Pulverizing Stephania delavayi Diels, stephania delavayi Diels or Menispermaceae Chinese medicinal materials containing cepharanthine, extracting with methanol, passing through bonded silica gel chromatography column, concentrating the obtained fraction, and crystallizing to obtain pure cepharanthine with purity of 98%. The method of the invention has the advantages of high cepharanthine purity, simple and controllable separation process, and easy automation of industrial production.
Drawings
FIG. 1 is a typical cepharanthine extract chromatographic separation online detection chart;
FIG. 2 is a mass spectrometry detection chart of cepharanthine extract;
FIG. 3 is a liquid chromatography-mass spectrometry detection chart of purified cepharanthine.
Detailed Description
In order to make the aforementioned features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below, but the present invention is not limited thereto.
In the description of the present invention, reference to "one embodiment" means that a particular feature, structure, or parameter, step, or the like described in the embodiment is included in at least one embodiment according to the present invention. Thus, appearances of the phrases such as "in one embodiment," "in one embodiment," and the like in this specification are not necessarily all referring to the same embodiment, nor are other phrases such as "in another embodiment," "in a different embodiment," and the like. Those of skill in the art will understand that the particular features, structures or parameters, steps, etc. disclosed in one or more embodiments of the present specification can be combined in any suitable manner.
Example 1
A. Smashing the Chinese medicinal material Stephania delavayi Diels into powder by using a mortar, smashing the powder by using the mortar, weighing 50g of the powder into a conical flask with a plug, adding 1000ml of methanol, carrying out ultrasonic extraction for 20min, standing, cooling, decompressing, carrying out rotary evaporation and concentration to remove an extractant;
B. b, performing chromatography on the extracting solution obtained in the step A through a bonded silica gel column, and eluting to obtain a cepharanthine crude product;
wherein the chromatography material is octadecyl bonded silica gel (organic alkane bonded silica gel) filled in an adsorption column; the eluent is methanol;
C. purifying the crude cepharanthine product by preparative liquid chromatography after passing through a 0.45 μm membrane;
wherein, the filling material: bioseps-FlashC18,20-45 μm,
packing specification: 30 column volumes/hour;
sample introduction volume: 500ml of
Eluent: 1) Methanol: water =85 (15V/V) held for 30min, 2) for 25min, the ratio is adjusted linearly to methanol: water =95 (V/V); 3) Keeping the proportion of the step 2) for 5min.
Elution flow rate: 6 column volumes/hour;
the collection mode is as follows: ultraviolet detection is carried out for 282nm;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain 37.5mg of white solid, namely the purified cepharanthine.
And (3) chromatographic analysis: diluting the extract with methanol 2 times, passing through 0.45um membrane, and performing chromatography analysis in a mass spectrometer under the following chromatographic conditions:
a chromatographic column: biosepsAQC18, 2.1X 100mm,3um,
mobile phase: methanol: 0.1 The TEA =80 (V/V)
Detection wavelength: 282nm
Flow rate: 0.3ml/min;
column temperature: 35 deg.C
Sample injection amount: 1ul
Mass spectrum voltage: 3000V, a scanning range of 150-1500 m/z and a Pos-TIC mode
And (4) performing purification detection, wherein the purity of the purified cepharanthine sample is 98.5% through LCMS detection.
The purified cepharanthine is detected by liquid chromatography-mass spectrometry, the liquid chromatography column is octadecyl bonded silica gel matrix, 2.1 × 100mm, and the pore diameter is
The particle size is 3 μm, the mobile phase is methanol and 0.1 triethylamine phosphate aqueous solution, and the ratio of 70:30 volume ratio of the mixed solution, the detection wavelength is 282nm, and the mass spectrum adopts a positive mode full scanning mode to detect a peak corresponding to an excimer ion peak of 607.7, namely the cepharanthine.
Example 2
A. Smashing the Chinese medicinal material Stephania delavayi Diels into powder by using a mortar, smashing the powder by using the mortar, weighing 50g of the powder into a conical flask with a plug, adding 1000ml of methanol, carrying out ultrasonic extraction for 20min, standing, cooling, decompressing, carrying out rotary evaporation and concentration to remove an extractant;
B. b, performing chromatography on the extracting solution obtained in the step A through a bonded silica gel column, and eluting to obtain a cepharanthine crude product;
wherein, the chromatographic material is octyl bonded silica gel filled in the adsorption column; the eluent is methanol;
C. passing the crude cepharanthine product through 0.45 μm membrane, and purifying by preparative liquid chromatography;
wherein, the filling material: bioseps-FlashC8,20-45 μm,
packing specification: 50 x 250mm;
sample injection volume: 500ml of
Eluent: tetrahydrofuran (tetrahydrofuran): water =78 (V/V)
Elution flow rate: 10 column volumes/hour;
the collection mode is as follows: ultraviolet detection is carried out for 282nm;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain 37.4mg of white solid, namely the purified cepharanthine.
And (4) performing purification detection, wherein the purity of the purified cepharanthine sample is 98.3% through LCMS detection.
Example 3
A. Smashing the Chinese medicinal material Stephania delavayi Diels into powder by using a mortar, smashing the powder by using the mortar, weighing 50g of the powder into a conical flask with a plug, adding 1000ml of methanol, carrying out ultrasonic extraction for 20min, standing, cooling, decompressing, carrying out rotary evaporation and concentration to remove an extractant;
B. b, performing chromatography on the extracting solution obtained in the step A through a bonded silica gel column, and eluting to obtain a cepharanthine crude product;
wherein the chromatography material is NH 2 -modified octadecyl bonded silica gel is packed in an adsorption column; the eluent is methanol;
C. passing the crude cepharanthine product through 0.45 μm membrane, and purifying by preparative liquid chromatography;
wherein, the filling material: bioseps-FlashC18-N,20-45 μm,
packing specification: 50 x 250mm;
sample introduction volume: 500ml of
Eluent: acetonitrile: water =85 (V/V)
Elution flow rate: 15 column volumes/hour;
the collection mode is as follows: ultraviolet detection is carried out for 282nm;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain 37.5mg of white solid, namely the purified cepharanthine.
And (4) performing purification detection, wherein the purity of the purified cepharanthine sample is 97.1% by LCMS detection.
Example 4
A. Smashing the Chinese medicinal herb tortoise slices into powder by using a mortar, smashing into powder by using the mortar, weighing 50g of the powder into a conical flask with a plug, adding 1000ml of an extracting agent (acetone/methanol mixed solution with the volume ratio of 20;
B. b, performing chromatography on the extracting solution obtained in the step A through a bonded silica gel column, and eluting to obtain a cepharanthine crude product;
wherein, the chromatographic material is octadecyl bonded silica gel modified by acylamino and filled in an adsorption column; the eluent is an acetone/methanol mixed solution with the volume ratio of 20;
C. purifying the crude cepharanthine product by preparative liquid chromatography after passing through a 0.45 μm membrane;
wherein, the filling material: bioseps-FlashC18-NC,20-45 μm,
packing specification: 50 x 250mm;
sample injection volume: 500ml of
Eluent: acetone: methanol: water (containing 0.05% trifluoroacetic acid and 0.01% triethylamine) =10
Elution flow rate: 25 column volumes/hour;
the collection mode comprises the following steps: ultraviolet detection is carried out for 282nm;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain 37.2mg of white solid, namely the purified cepharanthine.
And (4) performing purification detection, wherein the purity of the purified cepharanthine sample is 97.8% through LCMS detection.
Example 5
A. Smashing a Chinese medicinal material, namely the tortoise slices into powder by using a mortar, smashing into powder by using the mortar, weighing 50g of the powder into a conical bottle with a plug, adding 1000ml of methanol, carrying out ultrasonic extraction for 20min, standing, cooling, decompressing, carrying out rotary evaporation and concentration to remove an extracting agent;
B. b, performing chromatography on the extracting solution obtained in the step A through a bonded silica gel column, and eluting to obtain a cepharanthine crude product;
wherein the chromatographic material is NH 2 -modified octadecyl bonded silica gel is packed in an adsorption column; the eluent is methanol: water =85:15 (V/V)
C. Purifying the crude cepharanthine product by preparative liquid chromatography after passing through a 0.45 μm membrane;
wherein the filler: bioseps-FlashC18-N,20-45 μm,
packing specification: 50 x 250mm;
sample injection volume: 500ml of
Eluent: methanol: ethanol: water (with 0.1% formic acid) =68
Elution flow rate: 5 column volumes/hour;
the collection mode is as follows: ultraviolet detection is carried out for 282nm;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain 37.3mg of white solid, namely the purified cepharanthine.
And (4) performing purification detection, wherein the purity of the purified cepharanthine sample is 98.1% through LCMS detection.
Example 6
A. Smashing a Chinese medicinal material, namely the tortoise slices into powder by using a mortar, smashing into powder by using the mortar, weighing 50g of the powder into a conical bottle with a plug, adding 1000ml of methanol, carrying out ultrasonic extraction for 20min, standing, cooling, decompressing, carrying out rotary evaporation and concentration to remove an extracting agent;
B. b, carrying out chromatography on the extracting solution obtained in the step A through a silica gel column, and eluting to obtain a cepharanthine crude product;
wherein, the chromatographic material is octadecyl bonding silica gel modified by propyl ester group and filled in an adsorption column; the eluent is methanol and aqueous solution added with formic acid, wherein the volume ratio of the aqueous solution added with formic acid is 10%.
C. Purifying the crude cepharanthine product by preparative liquid chromatography after passing through a 0.45 μm membrane;
wherein, the filling material: bioseps-FlashC18,20-45 μm,
packing specification: 50 x 250mm;
sample introduction volume: 500ml of
Eluent: methanol: water =85 (V/V)
Elution flow rate: 20 column volumes/hour;
the collection mode is as follows: ultraviolet detection is carried out for 282nm;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain 37.4mg of white solid, namely the purified cepharanthine.
And (4) performing purification detection, wherein the purity of the purified cepharanthine sample is 98.5% through LCMS detection.
In conclusion, the cepharanthine separated by the method has high purity, the separation process is simple and controllable, and the method is very easy for automation of industrial production.
Claims (8)
1. A method for separating high-purity cepharanthine is characterized in that Stephania delavayi Diels slice, stephania delavayi Diels or Menispermaceae traditional Chinese medicinal material containing cepharanthine are used as raw materials, extraction is carried out by an extractant, the extract is processed by silica gel column chromatography and elution, and liquid chromatography separation is carried out to obtain purified cepharanthine;
the chromatography material is filled in the adsorption column, and is organic hydrocarbon bonded silica gel or organic hydrocarbon bonded silica gel containing polar organic functional groups; the adsorption column material is a physical mixture of more than two bonded silica gels or more than two organic hydrocarbon bonded silica gels;
the polar organic functional group is selected from ester group, amino group, amide group, and urea group.
2. The method for separating high-purity cepharanthine according to claim 1, comprising the following steps:
A. crushing the raw materials of Stephania delavayi Diels slice, stephania delavayi Diels slice or Menispermaceae Chinese medicinal material containing cepharanthine into powder by using a mortar, adding an extracting agent, performing ultrasonic extraction, and performing reduced pressure rotary steaming and concentration to remove the extracting agent;
B. b, carrying out chromatography on the extracting solution obtained in the step A through a silica gel column, and eluting to obtain a cepharanthine crude product;
C. filtering crude cepharanthine, and purifying by preparative liquid chromatography;
D. and D, carrying out reduced pressure rotary evaporation on the fraction obtained in the step C to remove methanol, and carrying out freeze drying to obtain a white solid, namely the purified cepharanthine.
3. The method of claim 1, wherein the extractant is one or more of methanol, ethanol, n-propanol, and isopropanol.
4. The method for separating high-purity cepharanthine according to claim 1, wherein the chromatography material has a particle size of 5-100um and a pore size of 5-100um
5. The method for separating highly pure cepharanthine according to claim 2, wherein in step B, the elution flow rate is 5-30 column volumes/hour.
6. The method according to claim 2, wherein in the step B, the eluent is selected from any one of organic solvents of methanol, acetonitrile, acetone, tetrahydrofuran, ethanol, isopropanol and dioxane, or a mixture of more than one organic solvents is mixed with water, or a mixture of more than one organic solvents is mixed with an aqueous solution added with a modifier.
7. The method for separating highly pure cepharanthine according to claim 6, wherein the ratio of water or aqueous solution with modifier added in the eluent is 10% -60%.
8. The method for separating highly pure cepharanthine according to claim 6, wherein the modifier is one or more of formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, phosphoric acid, ammonium bicarbonate, ammonia, triethylamine, diethylamine, ammonium acetate, or ammonium formate.
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| PCT/CN2022/139271 WO2024055459A1 (en) | 2022-09-14 | 2022-12-15 | Method for separating high-purity cepharanthine |
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