CN100427501C - Method for separating and preparing ursolic acid and its derivative from persimmon leaf using counter current chromatography - Google Patents
Method for separating and preparing ursolic acid and its derivative from persimmon leaf using counter current chromatography Download PDFInfo
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- CN100427501C CN100427501C CNB2005100604816A CN200510060481A CN100427501C CN 100427501 C CN100427501 C CN 100427501C CN B2005100604816 A CNB2005100604816 A CN B2005100604816A CN 200510060481 A CN200510060481 A CN 200510060481A CN 100427501 C CN100427501 C CN 100427501C
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Abstract
The present invention discloses a method for separating and preparing ursolic acid and derivatives thereof from persimmon leaves by countercurrent chromatography. The method comprises the following procedures: preparing a two phase solvent system of immobile phase and moving phase; filling a column of a countercurrent chromatograph with immobile phase; making a host machine of the countercurrent chromatograph rotate; pumping the moving phase into the column of the countercurrent chromatograph; feeding raw materials from a sample valve, and obtaining ursolic acid and derivatives thereof according to the detection by detector spectrogram or a thin layer chromatography method; the method is suitable for purifying a persimmon leaf crude extract obtained by various means, separating and preparing ursolic acid, 3 alpha, 19 alpha, 24-trihydroxy ursol-12-alkene-28-acid, 3 beta, 19 alpha, 24-trihydroxy ursol-12-alkene-28-acid and 3 beta, 24-trihydroxy ursol-12-alkene-28-acid by using various types of countercurrent chromatographs; the purity can reach more than 98%; large amounts of crude products or synthetic mixtures can be introduced into countercurrent chromatographs; the separation effect is favorable.
Description
Technical field
The present invention relates to the method for a kind of counter current chromatography separating and preparing ursolic acid and derivative thereof from the persimmon leaf.
Background technology
Ebenaceae (the family Ebenaceae) calamander, the whole world always has kind more than 240, and they mainly are distributed in the torrid zone widely the subtropical zone, and the temperate zone idol has distribution.The west and south is originated in to the southeast in nearly 56 kinds of China, and north is (Hou Kuanzhao compiles, and Chinese spermatophyte section belongs to dictionary, second edition, Beijing: scientific and technological press, 1984,159) to Liaoning.This invents used persimmon leaf, is the dry leave of persimmon, and its bitter, acid, puckery are cool in nature, has removing heat from the lung to relieve cough, cool blood, hypotensive effect.Be mainly used in cough breathe heavily, illness such as pulmonary emphysema, various internal hemorrhage, hypertension.Modern study shows, the persimmon leaf contains triterpenic acids such as abundant flavonoid, tannin, resin, vitamins C, lactones coumarins, volatile oil and ursolic acid, Oleanolic Acid, persimmon leaf all over the world all contains abundant ursolic acid and derivative (Row L.Ramachandra et al thereof, IndianJournal of chemistry, 1969,7:204-206; Mallavadhani U.V.et al, Biochemical Systematics and Ecology, 1998,26:941-942; Mallavadhani U.V.et al, Pharmaceutical Biology, 2001,39 (1): 20-24; Mallavadhani U.V.et al, Biochemical Systematics and Ecology, 1998,26:941-942; Chen Guang etc., Chinese pharmaceutical chemistry magazine, 2000,10 (4): 298-299; Chen Guang etc., 2005,36 (1): 26-28).
Ursolic acid and derivative thereof have purposes widely in the persimmon leaf, as are used for medicine, makeup etc.Ursolic acid can hypertension, atherosclerosis, effect (Somova L.I.et al such as anti-oxidant, Journalof Ethnopharmacology, 2003,84:299-305), and it has cell toxicant, induces differentiation and anti-angiogenic formation effect multiple carcinogenic cells, its anti-cancer and kill cancer action draws attention, become new focus (the Chiang Lien-Chai et al of modern tumour medicine research, The American Journal of ChineseMedicine, 2003,31 (1): 37-46; Xia Guohao etc., foreign medical oncology credit volume, 2002,29 (6): 420-422), ursolic acid also might become anti-HIV potential drug (Qu é r é Luc et al, Biochemical and Biophysical Research Communications1996,227:484-488).The derivative of persimmon leaf ursolic acid also has anti-inflammatory (Kusakari Takeshi et al, Japanese Patent, JP 2004010531 A2 20040115) (Maria del Carmen Recio et al, PlantaMedica, 1995,61:9-12.) and can suppress the generation of superoxide in the neutrophilic granulocyte in the human body, make it to have potential pharmaceutical use (Chen Guang et al, Clinica Chimica Acta2002,320:11-16), or the like.Existing bibliographical information adopts the method for column chromatography and high performance liquid chromatography to separate, but purity is not high, and the rate of recovery is very low.High speed adverse current chromatogram (High-speed countercurrentchromatography, HSCCC) be that a kind of successive that grew up in recent years need not the efficient of any solid support, liquid liquid divides color matching to say isolation technique fast, it avoided variety of issue that solid state adhesion body or carrier bring as: sample easily is adsorbed, loss and sex change, when using other liquid phase chromatography amount of being prepared to separate, its allocative efficiency can obviously reduce, solvent-oil ratio is big, HSCCC guarantees higher peak shape resolution, fractional dose is big, the sample free of losses, loss yield height, isolating environment relaxes, and saves solvent.Counter current chromatograph can directly advance slightly get sample in a large number product or synthetic mixture, and separating resulting can reach quite high purity, has been widely used in the preparation separation and the purifying of field chemical substances such as biology, medicine, environmental protection.
Summary of the invention
The method that the purpose of this invention is to provide a kind of counter current chromatography separating and preparing ursolic acid and derivative thereof from the persimmon leaf.
The step of method is as follows:
1) adopt alkane: fatty ester: Fatty Alcohol(C12-C14 and C12-C18): the volume ratio of water is the solvent systems of 1-10: 1-20: 1-10: 1-10; The extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material;
2) place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time;
3) use the phase that fixes mutually, make down moving phase mutually or with time fixing phase mutually, on make moving phase mutually; Use as stationary phase, be full of the chromatogram pillar in the whole counter current chromatograph, adjust main frame and just change, rotating speed is 200-2000rmp, will descend phase moving phase to pump into from the head end of pillar with the flow velocity of 1.0-10.0mL/min and carry out wash-out in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; As stationary phase, be full of whole adverse current chromatogram pillar with down, the adjustment main frame is just changeing, and rotating speed is 200-2000rmp, will go up phase moving phase with the flow velocity of 1.0-10.0mL/min and pump into from the tail end of pillar and carry out wash-out in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction;
4) spectrogram or the thin-layer chromatography according to detector detects, and obtains ursolic acid and derivative (3 α, 19 α respectively through recrystallization, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).
Described alkane is one or more mixture of normal hexane, isohexane, Skellysolve A, iso-pentane, normal heptane, isoheptane, octane, octane-iso or sherwood oil.Fatty ester is one or more a mixture of ethyl acetate, propyl acetate, isopropyl acetate or n-butyl acetate.Fatty Alcohol(C12-C14 and C12-C18) is one or both a mixture of ethanol or methyl alcohol.
The ursolic acid derivative is 3 α, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, acid of 24-trihydroxy-ursol-12-alkene-28-or 3 β, in 24-trihydroxy-ursol-12-alkene-28-acid any or several.
Pump into that to carry out wash-out in the post be the constant gradient wash-out or change proportioning gradually and carry out gradient elution.Thin-layer chromatography detects and adopts the purification on normal-phase silica gel thin layer chromatography board, and the developping agent system is a normal hexane: ethyl acetate: the volume ratio of acetone is 4-8: 2-6: 1-3, toluene: ethyl acetate: the volume ratio of acetate is 12: 4: 0.5, benzene: acetoacetic ester: the volume ratio of acetate is 12: 4: 0.5.Counter current chromatograph is analysis mode, half preparation type or preparation type.
Advantage of the present invention is: used persimmon leaf is widely distributed in China, often be taken as waste material and throw away or burn, so persimmon leaf raw material of the present invention is easy to get; Used various chemical reagent is all very common, easily mass purchase; This separation process can be carried out continuously, and is easy and simple to handle, the efficient height, and ursolic acid and derivative purity thereof are greater than 98%; Compare with column chromatography isochromatic spectrum method, it need not any solid-state upholder or carrier, therefore the sample absorption loss that does not have solid-state carrier and caused, stain and problem such as sex change, be a kind of economy from persimmon leaf crude extract, separate the method for preparing high-purity ursolic acid and derivative thereof; Can directly advance a large amount of persimmon leaf crude extract or synthetic mixture; The counter current chromatograph separating and preparing ursolic acid and the derivative thereof that are suitable for various models.
Embodiment
Embodiment 1:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=3: 6: 4: the solvent systems of 2 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material, (extracting method of the ethyl acetate extract of persimmon leaf sees that number of patent application is the application for a patent for invention of CN200410025770.8); On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier as stationary phase, be full of whole pillar, adjust main frame and just change, rotating speed is 450rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 2:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=3: 6: 5: the solvent systems of 2 (v/v/v/v); The dichloromethane extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On half preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, earlier with down as stationary phase, be full of whole pillar, the adjustment main frame is just changeing, rotating speed is 750rmp, will go up phase moving phase with the flow velocity of 3.0mL/min and pump in the post from the tail end of pillar; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a toluene: ethyl acetate: acetate=1 2: 4: 0.5) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition:
Chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=7: 3 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 3:
Adopt sherwood oil: ethyl acetate: methyl alcohol: water=3: 6: 4: the solvent systems of 2 (v/v/v/v); The acetone extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On analysis mode HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 2000rmp, with the flow velocity of 1.0mL/min the head end of moving phase from pillar is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a benzene: ethyl acetate: acetate=12: 4: 0.5) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=9: 1 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 4:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=4: 7: 4: the solvent systems of 3 (v/v/v/v); The dichloromethane extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 500rmp, with the flow velocity of 5.0mL/min the head end of moving phase from pillar is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=8: 6: 3) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=7: 3 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 5:
Adopt normal heptane: ethyl acetate: methyl alcohol: water=3: 7: 5: the solvent systems of 2 (v/v/v/v); The chloroform extraction thing of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 550rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=8: 6: 3) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 6:
Adopt normal hexane: ethyl acetate: ethanol: water=3: 5: 4: the solvent systems of 2 (v/v/v/v); The ethylene dichloride extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 400rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 2: 1) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 7:
Adopt hexanaphthene: propyl acetate: methyl alcohol: water=5: 6: 4: the solvent systems of 2 (v/v/v/v); The tetrachloromethane extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 450rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 8:
Adopt normal hexane: ethyl acetate: ethanol: water=1: 1: 2: the solvent systems of 1 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 550rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 4.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=7.8: 2.2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 9:
Adopt octane: ethyl acetate: methyl alcohol: water=3: 6: 4: the solvent systems of 2 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 450rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 3.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 1 50mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 10:
Adopt normal hexane: butylacetate: methyl alcohol: water=3: 7: 4: the solvent systems of 2 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, earlier with being filled with whole pillar for stationary phase mutually down, adjusting main frame and just changeing, rotating speed is 600rmp, will go up phase moving phase with the flow velocity of 10.0mL/min and pump in the post from the tail end of pillar; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=5: 3: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 11:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=1: 1: 2: the solvent systems of 1 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 550rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=5: 2: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 12:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=1: 1: 1: the solvent systems of 1 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 550rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=5: 2: 2) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 13:
Adopting normal hexane and 1: 1 mixture of normal heptane, ethyl acetate, methyl alcohol and alcoholic acid 1: 1 mixture, water volume ratio is 3: 6: 4: the solvent systems of 2 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, earlier with being filled with whole pillar for stationary phase mutually down, adjusting main frame and just changeing, rotating speed is 200rmp, will go up phase moving phase with the flow velocity of 2.0mL/min and pump in the post from the tail end of pillar; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 1) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18HPLC posts.
Embodiment 14:
Adopting isohexane and 1: 1 mixture of octane, ethyl acetate and 1: 1 mixture of n-butyl acetate, ethanol, water volume ratio is 3: 7: 5: the solvent systems of 2 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 550rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 5.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 1) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100RP-18 HPLC post.
Embodiment 15: adopting iso-pentane and 1: 1 mixture of octane-iso, propyl acetate and 1: 1 mixture of isopropyl acetate, ethanol, water volume ratio is 3: 6: 5: the solvent systems of 2 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On analysis mode HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, earlier with being filled with whole pillar for stationary phase mutually down, adjusting main frame and just changeing, rotating speed is 2000rmp, will go up phase moving phase with the flow velocity of 5.0mL/min and pump in the post from the tail end of pillar; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 1) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 16:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=10: 20: 9: the solvent systems of 6 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On preparation type HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 200rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 2.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 1) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Embodiment 17:
Adopt normal hexane: ethyl acetate: methyl alcohol: water=10: 19: 10: the solvent systems of 10 (v/v/v/v); The ethyl acetate extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material; On analysis mode HSCCC, carry out separating and preparing ursolic acid and derivative thereof.At first place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time.Then, use earlier mutually to stationary phase is filled with whole pillar, adjust main frame and just change, rotating speed is 2000rmp, will descend phase moving phase to pump in the post from the head end of pillar with the flow velocity of 1.0mL/min; After treating that whole system is set up running balance, by the sampling valve sample introduction; (the developping agent system is a normal hexane: ethyl acetate: acetone=4: 3: 1) detect according to the spectrogram of detector and thin-layer chromatography method then, obtain ursolic acid and derivative (3 α thereof respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β, 24-trihydroxy-ursol-12-alkene-28-acid).HPLC check and analysis condition: chromatographic column is
(Merk), 25 ℃ of column temperatures, elutriant are methyl alcohol for 150mm * 4.6mm, 5 μ m: phosphate aqueous solution=8: 2 (v/v), flow velocity 1.0mL/min detects wavelength 210nm to 100 RP-18 HPLC posts.
Claims (10)
1, the method for a kind of counter current chromatography separating and preparing ursolic acid and derivative thereof from the persimmon leaf is characterized in that the step of method is as follows:
1) adopt alkane: fatty ester: Fatty Alcohol(C12-C14 and C12-C18): the volume ratio of water is the solvent systems of 1-10: 1-20: 1-10: 1-10; The extract of persimmon leaf is after most of ursolic acid is removed in refining, purification and crystallization, and filtrate evaporate to dryness gained solid is a raw material;
2) place separating funnel by above-mentioned volume ratio preparation solvent systems, after shaking up, standing demix, isoequilibrium separated mutually with upper and lower after for some time;
3) use the phase that fixes mutually, make down moving phase mutually or with time fixing phase mutually, on make moving phase mutually; Use as stationary phase, be full of the chromatogram pillar in the whole counter current chromatograph, adjust main frame and just change, phase moving phase is down pumped into from the head end of pillar carry out wash-out in the post, treat that whole system is set up running balance after, by the sampling valve sample introduction; As stationary phase, be full of whole adverse current chromatogram pillar with down, the adjustment main frame is just changeing, and last phase moving phase is pumped into from the tail end of pillar carry out wash-out in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction;
4) spectrogram or the thin-layer chromatography according to detector detects, obtain i.e. 3 α of ursolic acid and derivative thereof, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β respectively through recrystallization, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid and 3 β or 24-trihydroxy-ursol-12-alkene-28-acid.
2, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that described alkane is one or more mixture of normal hexane, isohexane, Skellysolve A, iso-pentane, normal heptane, isoheptane, octane, octane-iso or sherwood oil.
3, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that described fatty ester is one or more a mixture of ethyl acetate, propyl acetate, isopropyl acetate or n-butyl acetate.
4, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf is characterized in that, described Fatty Alcohol(C12-C14 and C12-C18) is one or both a mixture of ethanol or methyl alcohol.
5, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that described ursolic acid derivative is 3 α, 19 α, 24-trihydroxy-ursol-12-alkene-28-acid, 3 β, 19 α, acid of 24-trihydroxy-ursol-12-alkene-28-or 3 β, in 24-trihydroxy-ursol-12-alkene-28-acid any or several.
6, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf is characterized in that, describedly pumps into that to carry out wash-out in the post be the constant gradient wash-out or changes proportioning gradually and carry out gradient elution.
7, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that: described thin-layer chromatography detects and adopts the purification on normal-phase silica gel thin layer chromatography board, and the developping agent system is a normal hexane: ethyl acetate: the volume ratio of acetone is 4-8: 2-6: 1-3, toluene: ethyl acetate: the volume ratio of acetate is 12: 4: 0.5, benzene: acetoacetic ester: the volume ratio of acetate is 12: 4: 0.5.
8, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that: described counter current chromatograph is analysis mode, half preparation type or preparation type.
9, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that: described engine speed is 200-2000rmp.
10, the method for a kind of counter current chromatography according to claim 1 separating and preparing ursolic acid and derivative thereof from the persimmon leaf, it is characterized in that: the flow velocity of described moving phase is 1.0-10.0mL/min.
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| US8748658B2 (en) | 2010-03-22 | 2014-06-10 | Council Of Scientific And Industrial Research | Fast isolation method for the natural scaffold ursolic acid from Diospyros melanoxylon |
| CN107200767A (en) * | 2017-03-02 | 2017-09-26 | 长沙博海生物科技有限公司 | A kind of preparation method of blood-sugar decreasing active Corosolic acid |
| CN110508031A (en) * | 2019-07-30 | 2019-11-29 | 浙江工业大学 | A method of separating ursolic acid and oleanolic acid from plant |
| CN110559688A (en) * | 2019-08-30 | 2019-12-13 | 浙江工业大学 | Method for separating isomers of oleanolic acid and ursolic acid |
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| CN1136024C (en) * | 1999-06-14 | 2004-01-28 | 北京市新技术应用研究所 | Highspeed counter-current chromatographic separation process for preparing high purity catechin |
| CN1186350C (en) * | 2003-05-19 | 2005-01-26 | 西安交通大学 | Process of extracting and separating ursolic acid from plant |
| CN1594356A (en) * | 2004-06-29 | 2005-03-16 | 浙江大学 | Process for extracting active component ursolic acid from persimmon leaf |
| CN1210567C (en) * | 2002-12-10 | 2005-07-13 | 中国农业科学院茶叶研究所 | Countercurrent chromatographic separation process for reducing fixed phase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1136024C (en) * | 1999-06-14 | 2004-01-28 | 北京市新技术应用研究所 | Highspeed counter-current chromatographic separation process for preparing high purity catechin |
| CN1210567C (en) * | 2002-12-10 | 2005-07-13 | 中国农业科学院茶叶研究所 | Countercurrent chromatographic separation process for reducing fixed phase |
| CN1186350C (en) * | 2003-05-19 | 2005-01-26 | 西安交通大学 | Process of extracting and separating ursolic acid from plant |
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