CN113896695B - Method for simultaneously extracting cephalomannine and taxol from Taxus media - Google Patents
Method for simultaneously extracting cephalomannine and taxol from Taxus media Download PDFInfo
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- 241001674343 Taxus x media Species 0.000 title claims abstract description 80
- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 75
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 75
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- DBXFAPJCZABTDR-KUEXGRMWSA-N Cephalomannine Natural products O=C(O[C@@H]1C(C)=C2[C@@H](OC(=O)C)C(=O)[C@]3(C)[C@@H](O)C[C@@H]4[C@](OC(=O)C)([C@H]3[C@H](OC(=O)c3ccccc3)[C@@](O)(C2(C)C)C1)CO4)[C@@H](O)[C@H](NC(=O)/C(=C\C)/C)c1ccccc1 DBXFAPJCZABTDR-KUEXGRMWSA-N 0.000 title claims abstract description 73
- DBXFAPJCZABTDR-WBYYIXQISA-N cephalomannine Chemical compound O([C@@H]1[C@]2(O)C[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]3(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]31)OC(C)=O)C2(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)C(/C)=C/C)C=1C=CC=CC=1)C(=O)C1=CC=CC=C1 DBXFAPJCZABTDR-WBYYIXQISA-N 0.000 title claims abstract description 73
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- 239000001273 butane Substances 0.000 claims description 33
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 claims description 33
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- 238000011068 loading method Methods 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 14
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- 238000003756 stirring Methods 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 10
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
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- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
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- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
Abstract
The invention discloses a method for simultaneously extracting cephalomannine and taxol from Taxus media, which comprises the steps of drying branches and leaves of the Taxus media, crushing, sieving, subcritical extraction, solvent precipitation and a series medium-high pressure column chromatography method, wherein the high-purity cephalomannine and taxol two monomer active ingredients are simultaneously extracted from the Taxus media. The invention has simple process flow, easy operation, green and nontoxic performance, high efficiency and low cost, and is suitable for mass production.
Description
Technical Field
The invention belongs to the technical field of separation and extraction of effective active ingredients from Taxaceae plants, and particularly relates to a method for simultaneously extracting cephalomannine and taxol from Taxus media.
Background
Taxus chinensis is a tree species which integrates ornamental, material and medical functions into a whole and has extremely high economic value, and taxol extracted from bark and branches and leaves of the Taxus chinensis is a world-recognized natural compound with anticancer efficacy, is a novel anticancer drug with taxane diterpene skeleton, and is the only drug which can promote microtubule polymerization and stabilize the polymerized microtubule in the prior art. The traditional Chinese medicine composition has strong inhibition effect on most solid tumors, has no influence on normal cells, and has definite curative effect and small side effect on advanced ovarian cancer, breast cancer, non-small cell lung cancer and Kaposi's sarcoma, so that the traditional Chinese medicine composition becomes the focus of research in the current pharmaceutical industry. With the further development of researches, the anticancer effect of the taxol medicaments is widely applied, and the taxol medicaments become first-line anticancer medicaments and broad-spectrum antitumor medicaments.
Cephalomannine is a natural taxane compound symbiotic with paclitaxel, and paclitaxel differs from paclitaxel only in the amide side chain in chemical structure. Since cephalomannine and paclitaxel are very similar in structure and properties, it is very difficult to separate paclitaxel from cephalomannine, and repeated separation is necessary. In addition, as a biological macromolecular substance, paclitaxel is easily degraded or isomerized to generate other taxane substances under the influence of environmental conditions such as temperature, organic solvents, acids, alkalis and the like. Based on the factors, the research and development of the separation and purification process with simpler, more convenient, higher efficiency and lower cost becomes a focus problem in the study of the taxol.
Although paclitaxel is a natural product extracted mainly from the bark of pacific yew (short leaf yew), paclitaxel is also found in other members of the family taxaceae, such as canadian yew (t.canadensis) and yunnanensis (t.yunnanensis). Paclitaxel is also present in aerial parts and roots of other taxus species, such as Taxus baccata (T.baccata) which contains paclitaxel and its analogues in its needle leaves, taxus asiatica (Taxus baccata (T.wallichiana) and Taxus chinensis (T.chinensis)), and Taxus chinensis trees cultivated for ornamental purposes. Taxus media is a natural hybrid of Taxus cuspidata (Taxus cusidata) and Taxus baccata (Taxus baccata), has high growth speed, contains taxol in the bark, has the content of taxol in the branches and leaves obviously higher than that of Taxus chinensis in China, has the content of taxol up to 0.03% -0.052%, is the best substitute raw material for the bark of Taxus chinensis, can effectively protect the resource, and greatly reduces the production cost due to the high content of taxol, so that the Taxus cuspidata is most suitable for producing natural taxol. Canadian and American Taxus media excellent varieties are introduced from 1995 in China, and large-scale planting and cultivation are carried out.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a method for simultaneously extracting cephalomannine and taxol from Taxus media, which has the advantages of simple process flow, easy operation, greenness, no toxicity, high efficiency, low cost and suitability for mass production.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
a method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and ethanol or methanol with the volume concentration of 70-95% as an entrainer, so as to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder material to the solvent is 1:3-7 kg/L, the volume ratio of the entrainer to the solvent is 1:10-50, the extraction temperature is 40-90 ℃, the ultrasonic power is 500-800W, the extraction pressure is 0.2-1.0 MPa, the extraction time is 30-100 min, and the extraction times are 1-4 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using a first solvent, wherein the mass ratio of the extract to the first solvent is 1:10-30, and concentrating the extracted residues under reduced pressure to obtain Taxus media extraction residues; the first solvent is petroleum ether, chloroform, normal hexane, dichloromethane, ethyl acetate, or tetrahydrofuran;
s3, adding the Taxus media extraction residues obtained in the step S2 into a second solvent for dissolution, wherein the feed liquid ratio of the extraction residues to the second solvent is 1:10-20 kg/L; stirring to fully dissolve, standing for 30-100 min, pouring the supernatant into another container, repeating the stirring and standing operation for 3-5 times, and concentrating the supernatant under reduced pressure to be dry; the second solvent is methanol, ethanol or acetonitrile solution with volume concentration of 10-90%;
s4, dissolving the dry matter obtained in the step S3 by adopting a third solvent, wherein the feed liquid ratio of the dry matter to the third solvent is 1:1-5 g/mL, adding active carbon with the mass of 5-20% of the residual matter into the solution, adsorbing for 1-3 hours at the temperature of 60-80 ℃, filtering, collecting filtrate, and concentrating the filtrate to the specific gravity of 2.0-3.0 to obtain Taxus media sample loading liquid; the third solvent is methanol, acetone, ethanol or acetonitrile;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the packing used by the first chromatographic column is MCI, the packing used by the second chromatographic column is C18, acetonitrile or methanol with the volume concentration of 5-30% is adopted to balance the column, the wavelength of an ultraviolet detector is 200-400 nm, after a base line is stable, the sample loading liquid in the step S4 is sampled by adopting a preassembled column, acetonitrile or methanol with the volume concentration of 30-70% is adopted as a mobile phase, an eluent is collected according to UV detection, the detection wavelength of a UV detector is 200-254 nm, the eluent which flows out firstly contains cephalomannine, the eluent which flows out later contains taxol, and the high-purity cephalomannine and taxol are respectively obtained through decompression concentration, filtration and drying, the purity of cephalomannine is 95-99%, and the purity of taxol is 95-99%.
Further, in the step S1, the subcritical extraction conditions are as follows: the feed liquid ratio of the powder material to the butane is 1:3-5 kg/L, the volume ratio of the butane to the ethanol or the methanol is 1:10-35, the extraction temperature is 40-65 ℃, the ultrasonic power is 500-700W, the extraction pressure is 0.2-0.8 MPa, the extraction time is 30-60 min, and the extraction times are 1-3 times.
Further, in the step S1, the subcritical extraction conditions are as follows: the feed liquid ratio of the powder to the butane is 1:4kg/L, the volume ratio of the butane to the ethanol or the methanol is 1:20, the extraction temperature is 40 ℃, the ultrasonic power is 500W, the extraction pressure is 0.8MPa, the extraction time is 30min, and the extraction times are 2 times.
Further, in the step S5, the first chromatographic column has a filler particle size of 10 to 100. Mu.m, a column length of 310 to 920mm and a column inner diameter of 15 to 100mm.
Further, in the step S5, the second column has a packing particle diameter of 5 to 50 μm, a column length of 310 to 920mm, and a column inner diameter of 15 to 100mm.
The invention discloses a method for simultaneously extracting cephalomannine and taxol from Taxus media, wherein the cephalomannine yield is 0.5-1%, and the taxol yield is 0.1-1%.
By adopting the technical scheme, the invention has the following advantages:
the method for simultaneously extracting cephalomannine and taxol from Taxus media has the advantages of flexible and simple operation, mild reaction and treatment conditions, energy conservation, environmental protection and high efficiency, and the subcritical fluid extraction technology can effectively remove pigment and part of gum substances by adopting the first solvent extraction, and can further remove viscous pectin substances contained in the extract by adopting the second solvent and third solvent precipitation method, thereby facilitating the subsequent loading; the activated carbon can not only remove pigment substances in the sample, but also effectively remove some viscous insoluble substances, so that the subsequent column chromatographic packing is better protected, and the next repeated use is convenient; the effective separation effect of cephalomannine and taxol is achieved by a method of connecting the first chromatographic column and the second chromatographic column in series; the whole process adopts a single solvent system, the solvent can be recovered and can be reused, so that the waste of the solvent is avoided, and the environment is polluted; the cephalomannine and taxol can be prepared simultaneously, the purity of the cephalomannine and taxol is high, no toxic organic solvent is used in the preparation process, and the cephalomannine and taxol are prepared green and are suitable for large-scale industrial production.
Drawings
FIG. 1 is a process flow diagram of the method of the present invention for simultaneously extracting cephalomannine and paclitaxel from Taxus media;
FIG. 2 is a preparative chromatogram of a method of the present invention for simultaneously extracting cephalomannine and paclitaxel from Taxus media;
FIG. 3a is a mass spectrum of cephalomannine;
FIG. 3b is a nuclear magnetic hydrogen spectrum of cephalomannine;
FIG. 3c is a carbon spectrum of cephalomannine;
FIG. 4a is a mass spectrum of paclitaxel;
FIG. 4b is a nuclear magnetic resonance hydrogen spectrum of paclitaxel;
FIG. 4c is a carbon spectrum of paclitaxel.
Detailed Description
The invention will be described in further detail with reference to the accompanying drawings and the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.
As shown in fig. 1 and 2, a method for simultaneously extracting cephalomannine and paclitaxel from Taxus media comprises the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and ethanol or methanol with the volume concentration of 70-95% as an entrainer, so as to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder material to the solvent is 1:3-7 kg/L, the volume ratio of the entrainer to the solvent is 1:10-50, the extraction temperature is 40-90 ℃, the ultrasonic power is 500-800W, the extraction pressure is 0.2-1.0 MPa, the extraction time is 30-100 min, and the extraction times are 1-4 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using a first solvent, wherein the mass ratio of the extract to the first solvent is 1:10-30, and concentrating the extracted residues under reduced pressure to obtain Taxus media extraction residues; the first solvent is petroleum ether, chloroform, normal hexane, dichloromethane, ethyl acetate, or tetrahydrofuran;
s3, adding the Taxus media extraction residues obtained in the step S2 into a second solvent for dissolution, wherein the feed liquid ratio of the extraction residues to the second solvent is 1:10-20 kg/L; stirring to fully dissolve, standing for 30-100 min, pouring the supernatant into another container, repeating the stirring and standing operation for 3-5 times, and concentrating the supernatant under reduced pressure to be dry; the second solvent is methanol, ethanol or acetonitrile solution with volume concentration of 10-90%;
s4, dissolving the dry matter obtained in the step S3 by adopting a third solvent, wherein the feed liquid ratio of the dry matter to the third solvent is 1:1-5 g/mL, adding active carbon with the mass of 5-20% of the residual matter into the solution, adsorbing for 1-3 hours at the temperature of 60-80 ℃, filtering, collecting filtrate, and concentrating the filtrate to the specific gravity of 2.0-3.0 to obtain Taxus media sample loading liquid; the third solvent is methanol, acetone, ethanol or acetonitrile;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the packing used by the first chromatographic column is MCI, the packing used by the second chromatographic column is C18, acetonitrile or methanol with the volume concentration of 5-30% is adopted to balance the column, the wavelength of an ultraviolet detector is 200-400 nm, after a base line is stable, the sample loading liquid in the step S4 is sampled by adopting a preassembled column, the sample feeding amount is 100-500 mL, the temperature of the column is room temperature, acetonitrile or methanol with the volume concentration of 30-70% is adopted as a mobile phase, the flow rate is 10-50 mL/min, the eluent is collected according to UV detection, the detection wavelength of the UV detector is 200-254 nm, the eluent which flows out firstly contains cephalomannine, the eluent which flows out later contains taxol, the purity of cephalomannine and taxol are respectively obtained through decompression concentration, filtration and drying, the purity of cephalomannine is 95-99%, the purity of taxol is 95-99%, the cephalomannine yield is 0.5-1%, and the taxol yield is 0.1-1%.
In the step S1, the subcritical extraction conditions are preferably as follows: the feed liquid ratio of the powder material to the butane is 1:3-5 kg/L, the volume ratio of the butane to the ethanol or the methanol is 1:10-35, the extraction temperature is 40-65 ℃, the ultrasonic power is 500-700W, the extraction pressure is 0.2-0.8 MPa, the extraction time is 30-60 min, and the extraction times are 1-3 times; more preferably, the subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:4kg/L, the volume ratio of the butane to the ethanol or the methanol is 1:20, the extraction temperature is 40 ℃, the ultrasonic power is 500W, the extraction pressure is 0.8MPa, the extraction time is 30min, and the extraction times are 2 times.
In the step S5, the particle size of the filler in the first chromatographic column is 10-100 mu m, the column length is 310-920 mm, and the inner diameter of the column is 15-100 mm; in the second chromatographic column, the particle size of the filler is 5-50 mu m, the column length is 310-920 mm, and the column inner diameter is 15-100 mm.
The invention relates to a method for simultaneously extracting cephalomannine and taxol from Taxus media, wherein the cephalomannine extracted by the method has a chemical structural formula shown in a formula (I), and the taxol has a chemical structural formula shown in a formula (II):
example 1
A method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and ethanol with the volume concentration of 70% as an entrainer to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:3kg/L, the volume ratio of the ethanol to the butane is 1:10, the extraction temperature is 40 ℃, the ultrasonic power is 500W, the extraction pressure is 0.2MPa, the extraction time is 80min, and the extraction times are 2 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using petroleum ether, wherein the mass ratio of the extract to the petroleum ether is 1:10, and concentrating the extracted residues under reduced pressure to obtain the Taxus media extraction residues;
step S3, adding the Taxus media extraction residues obtained in the step S2 into a methanol solution with the volume concentration of 10% for dissolution, stirring to enable the extraction residues to be fully dissolved in the methanol solution at the feed liquid ratio of 1:10kg/L, standing for 30min, pouring the supernatant into another container, repeating the operation for 3 times, and concentrating the supernatant to be dry under reduced pressure;
s4, carrying out thermal dissolution on the dry matter obtained in the step S3 by adopting methanol, wherein the feed liquid ratio of the dry matter to the methanol is 1:1g/mL, adding active carbon accounting for 5% of the mass of the remainder into the dissolution liquid, adsorbing for 1h at 60 ℃, filtering, collecting filtrate, and concentrating the filtrate to a specific gravity of 2.0 to obtain a taxus media loading liquid;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the filler used in the first chromatographic column is MCI, the particle size of the filler is 10 mu m, the column length is 310mm, the inner diameter of the column is 15mm, the filler used in the second chromatographic column is C18, the particle size of the filler is 5 mu m, the column length is 310mm, the inner diameter of the column is 15mm, the acetonitrile with the volume concentration of 5% is adopted to balance the column, the wavelength of an ultraviolet detector is 200nm, after the baseline is stabilized, the sample loading liquid in the step S4 is loaded by adopting the preloaded column, the sample loading amount is 100mL, the column temperature is room temperature, the acetonitrile with the volume concentration of 30% is adopted as a mobile phase, the flow rate is 10mL/min, the eluent is collected according to UV detection, the detection wavelength of a UV detector is 200nm, the eluent obtained firstly contains cephalomannine, the eluent obtained later contains taxol, the yield of cephalomannine is 0.53%, the yield of cephalomannine is 0.26%, the purity of cephalomannine is 96.98% after the HPLC detection, and the purity of the taxol is 96.98%.
With reference to fig. 3a, 3b, 3c, cephalomannine nuclear magnetic data: 1 H-NMR(CDCl 3 ,400MHz)δ:1.15(3H,s,H-17),1.26(3H,s,H-16),1.68(3H,s,H-19),1.76(3H,dd,J＝6.8,1.2Hz,H-4”-CH3),1.80(3H,s,H-18),1.86(1H,ddd,J＝14.1,11.0,2.3Hz,H-6β),2.24,2.35(each 3H,s,2×CH 3 ),2.25(1H,dd,J＝15.1,8.2Hz,H-14β),2.33(1H,dd,J＝15.1,8.2Hz,H-14α),2.52(1H,ddd,J＝14.1,9.9,6.5Hz,H-6α),3.79(1H,d,J＝7.1Hz,H-3),4.20,4.30(each 1H,d,J＝8.1Hz,H-20),4.37(1H,dd,J＝11.1,6.8Hz,H-7),4.71(1H,d,J＝3.1Hz,H-2’),4.94(1H,dd,J＝10.1,2.1Hz,H-5),5.62(1H,dd,J＝8.1,2.3Hz,H-3’),5.68(1H,d,J＝7.1Hz,H-2),6.20(1H,t,J＝8.1Hz,H-13),6.28(1H,s,H-10),6.42(1H,d,J＝7.1Hz,H-3”),7.34(1H,m,Ph-H),7.41(4H,m,Ph-H),7.52(2H,t,J＝8.1Hz,Ph-H),7.63(1H,t,J＝7.1Hz,Ph-H),8.11(2H,d,J＝8.1Hz,Ph-H). 13 C-NMR(CDCl 3 ,100MHz)δ:78.97(C-1),74.95(C-2),45.6(C-3),81.09(C-4),84.38(C-5),35.62(C-6),72.13(C-7),58.57(C-8),203.64(C-9),75.57(C-10),133.10(C-11),142.05(C-12),72.28(C-13),35.50(C-14),43.18(C-15),20.84(C-16),26.83(C-17),14.78(C-18),9.55(C-19),77.20(C-20),172.78(C-1’),73.28(C-2’),54.83(C-3’),170.31,22.57(4-OAc),171.23,20.84(10-OAc),166.94(2-OBz,C＝O),129.15,130.18,128.22(2-OBz,Ph),131.29,126.96,128.80,128.94(4’-OBz,Ph),169.60(C-5’),138.10(C-6’),131.89(C-7’),13.97(C-8’),12.41(C-9’)。
with reference to fig. 4a, 4b, 4c, paclitaxel nuclear magnetic data: 1 H-NMR(CDCl 3 ,400MHz)δ:1.14(3H,s,H-17),1.24(3H,s,H-16),1.68(3H,s,H-19),1.80(3H,s,H-18),1.88(1H,m,H-6β),2.23(3H,s,10-OAc),2.33(2H,m,H-14),2.38(3H,s,4-OAc),2.54(1H,ddd,J＝15.1,9.9,6.3Hz,H-6α),3.79(1H,d,J＝7.1Hz,H-3),4.19(1H,d,J＝8.1Hz,H-20b),4.31(1H,d,J＝8.1Hz,H-20a),4.40(1H,dd,J＝11.1,6.5Hz,H-7),4.79(1H,br.s,H-2’),4.95(1H,dd,J＝9.1,2.2Hz,H-5),5.67(1H,d,J＝7.1Hz,H-2),5.78(1H,d,J＝9.1Hz,H-3’),6.23(1H,t,J＝8.8Hz,H-13),6.27(1H,s,H-10),6.95(1H,d,J＝8.4Hz,3’-NH),7.42(2H,o.t,4'-OBz,Ph-m),7.47(1H,o.t,4'-OBz Ph-p),7.75(2H,d,4'-OBz,Ph-o),7.50-7.35(5H,3’-Ph),7.49(2H,o.t,2-OBz,Ph-m),7.60(1H,t,2-OBz,Ph-p),8.13(2H,d,2-OBz,Ph-o). 13 C-NMR(CDCl 3 ,400MHz)δ:78.99(C-1),75.19(C-2),45.58(C-3),81.15(C-4),84.41(C-5),35.66(C-6),72.13(C-7),58.59(C-8),203.6(C-9),75.65(C-10),132.73(C-11),142.76(C-12),72.81(C-13),35.53(C-14),43.16(C-15),20.84(C-16),26.84(C-17),14.82(C-18),9.5(C-19),77.19(C-20),170.35,22.62(4-OAc),172.5,22.61(10-OAc),167.47(2-OBz,C＝O),129.72,128.4,133.61(2-OBz,Ph),172.71(C-1’),73.1(C-2’),55.02(C-3’),128.40,126.91(3’-Ph),166.90(4’-OBz,C＝O),126.91,128.75,130.19(4’-OBz,Ph)。
example 2
A method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and ethanol with the volume concentration of 75% as an entrainer to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:3.5kg/L, the volume ratio of the ethanol to the butane is 1:15, the extraction temperature is 55 ℃, the ultrasonic power is 500W, the extraction pressure is 0.5MPa, the extraction time is 45min, and the extraction times are 3 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using petroleum ether, wherein the mass ratio of the extract to the petroleum ether is 1:15, and concentrating the extracted residues under reduced pressure to obtain the Taxus media extraction residues;
step S3, adding 30% methanol solution to dissolve the Taxus media extraction residues obtained in the step S2, stirring to fully dissolve the extraction residues and the methanol at a feed liquid ratio of 1:13kg/L, standing for 40min, pouring the supernatant into another container, repeating the operation for 4 times, and concentrating the supernatant under reduced pressure to be dry;
s4, dissolving the dry matter obtained in the step S3 by adopting acetone, wherein the feed liquid ratio of the dry matter to the acetone is 1:2g/mL, adding active carbon with the mass of 10% of the residual matter into the solution, adsorbing for 2 hours at 70 ℃, filtering, collecting filtrate, and concentrating the filtrate to a specific gravity of 2.5 to obtain a Taxus media sample loading liquid;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the filler used in the first chromatographic column is MCI, the particle size of the filler is 30 mu m, the column length is 310mm, the inner diameter of the column is 30mm, the filler used in the second chromatographic column is C18, the particle size of the filler is 10 mu m, the column length is 380mm, the inner diameter of the column is 35mm, the acetonitrile with the volume concentration of 5% is adopted to balance the column, the wavelength of an ultraviolet detector is 227nm, after the baseline is stabilized, the sample loading liquid in the step S4 is loaded by adopting the preloaded column, the sample loading amount is 150mL, the column temperature is room temperature, the acetonitrile with the volume concentration of 38% is adopted as a mobile phase, the flow rate is 30mL/min, the eluent is collected according to UV detection, the detection wavelength of a UV detector is 200nm, the eluent obtained firstly contains cephalomannine, the eluent obtained later contains taxol, the yield of cephalomannine is 0.65%, the yield of cephalomannine is 0.55%, the purity of cephalomannine is 98.8%, and the purity of the taxol is 98.99%.
Example 3
A method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and methanol with the volume concentration of 78% as an entrainer to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:4kg/L, the volume ratio of the methanol to the butane is 1:20, the extraction temperature is 60 ℃, the ultrasonic power is 550W, the extraction pressure is 0.6MPa, the extraction time is 60min, and the extraction times are 3 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by adopting normal hexane, wherein the mass ratio of the extract to the normal hexane is 1:18, and concentrating the extracted residues under reduced pressure to obtain the Taxus media extraction residues;
s3, adding the Taxus media extraction residues obtained in the step S2 into an ethanol solution with the volume concentration of 50% for dissolution, stirring to enable the extraction residues to be fully dissolved, standing for 50min, pouring the supernatant into another container, repeating the operation for 5 times, and concentrating the supernatant to be dry under reduced pressure;
s4, dissolving the dry matter obtained in the step S3 by adopting acetone, wherein the feed liquid ratio of the dry matter to the acetone is 1:3g/mL, adding active carbon with the mass of the rest being 15% into the solution, adsorbing for 3 hours at 80 ℃, filtering, collecting filtrate, and concentrating the filtrate to a specific gravity of 3.0 to obtain a Taxus media sample loading liquid;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the filler used in the first chromatographic column is MCI, the particle size of the filler is 60 mu m, the column length is 500mm, the inner diameter of the column is 50mm, the filler used in the second chromatographic column is C18, the particle size of the filler is 20 mu m, the column length is 460mm, the inner diameter of the column is 50mm, the methanol with the volume concentration of 15% is adopted to balance the column, the wavelength of an ultraviolet detector is 300nm, after the base line is stabilized, the sample loading liquid in the step S4 is loaded by adopting the preloaded column, the sample loading amount is 200mL, the column temperature is room temperature, the methanol with the volume concentration of 50% is adopted as a mobile phase, the flow rate is 35mL/min, the eluent is collected according to UV detection, the detection wavelength of a UV detector is 234nm, the eluent obtained firstly contains cephalomannine, the eluent obtained later contains taxol, the yield of the cephalomannine is 0.81%, the yield of the cephalomannine is 0.49%, the purity of the cephalomannine is 97.98% after the HPLC detection, and the purity of the cephalomannine is 97.98%.
Example 4
A method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and methanol with the volume concentration of 82% as an entrainer to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:5kg/L, the volume ratio of the methanol to the butane is 1:30, the extraction temperature is 70 ℃, the ultrasonic power is 650W, the extraction pressure is 0.7MPa, the extraction time is 50min, and the extraction times are 3 times;
s2, scattering the Taxus media extract obtained in the step S1 with distilled water, extracting with ethyl acetate, wherein the mass ratio of the extract to the ethyl acetate is 1:20, and concentrating the extracted residues under reduced pressure to obtain Taxus media extraction residues;
step S3, adding the Taxus media extraction residues obtained in the step S2 into an ethanol solution with the volume concentration of 55% for dissolution, stirring to enable the extraction residues to be fully dissolved, standing for 60min, pouring the supernatant into another container, repeating the operation for 3 times, and concentrating the supernatant to be dry under reduced pressure;
s4, dissolving the dry matter obtained in the step S3 by adopting acetone, wherein the feed liquid ratio of the dry matter to the acetone is 1:3.5g/mL, adding 17% of active carbon of the mass of the rest into the dissolving liquid, adsorbing for 2 hours at 80 ℃, filtering, collecting filtrate, and concentrating the filtrate to a specific gravity of 2.6 to obtain a taxus media loading liquid;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the filler used by the first chromatographic column is MCI, the particle size of the filler is 60 mu m, the column length is 500mm, the inner diameter of the column is 50mm, the filler used by the second chromatographic column is C18, the particle size of the filler is 20 mu m, the column length is 460mm, the inner diameter of the column is 50mm, the methanol with the volume concentration of 18% is adopted to balance the column, the wavelength of an ultraviolet detector is 350nm, after the baseline is stabilized, the sample loading liquid in the step S4 is loaded by adopting the preloaded column, the sample loading amount is 300mL, the column temperature is room temperature, the methanol with the volume concentration of 55% is adopted as a mobile phase, the flow rate is 40mL/min, the eluent is collected according to UV detection, the detection wavelength of a UV detector is 229nm, the eluent obtained firstly contains cephalomannine, the eluent obtained later contains taxol, the yield of cephalomannine is 0.81%, the yield of cephalomannine is 0.49%, the purity of cephalomannine is 97.98% after decompression concentration, filtration and drying are respectively obtained.
Example 5
A method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and methanol with the volume concentration of 90% as an entrainer to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:6kg/L, the volume ratio of the methanol to the butane is 1:40, the extraction temperature is 80 ℃, the ultrasonic power is 700W, the extraction pressure is 0.8MPa, the extraction time is 60min, and the extraction times are 4 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using petroleum ether, wherein the mass ratio of the extract to the petroleum ether is 1:25, and concentrating the extracted residues under reduced pressure to obtain the Taxus media extraction residues;
step S3, adding the Taxus media extraction residues obtained in the step S2 into an acetonitrile solution with the volume concentration of 70% for dissolution, stirring to enable the extraction residues to be fully dissolved, standing for 80min, pouring the supernatant into another container, repeating the operation for 5 times, and concentrating the supernatant to be dry under reduced pressure;
s4, dissolving the dry matter obtained in the step S3 by adopting methanol, wherein the feed liquid ratio of the dry matter to the methanol is 1:4g/mL, adding active carbon with the mass of 20% of the residual matter into the solution, adsorbing for 1.5 hours at 75 ℃, filtering, collecting filtrate, and concentrating the filtrate to a specific gravity of 3.0 to obtain a Taxus media sample loading liquid;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the filler used in the first chromatographic column is MCI, the particle size of the filler is 80 mu m, the column length is 460mm, the inner diameter of the column is 50mm, the filler used in the second chromatographic column is C18, the particle size of the filler is 50 mu m, the column length is 800mm, the inner diameter of the column is 100mm, the acetonitrile with the volume concentration of 30% is adopted to balance the column, the wavelength of an ultraviolet detector is 350nm, after the base line is stabilized, the sample loading liquid in the step S4 is loaded by adopting the preloaded column, the sample loading amount is 400mL, the column temperature is room temperature, the acetonitrile with the volume concentration of 60% is adopted as a mobile phase, the flow rate is 50mL/min, the eluent is collected according to UV detection, the detection wavelength of a UV detector is 254nm, the eluent obtained firstly contains cephalomannine, the eluent obtained later contains taxol, the yield of the cephalomannine is 0.84%, the yield of the cephalomannine is 0.95%, the purity of the cephalomannine is 98.5%, and the purity of the taxol is 2.99% after the HPLC detection.
Example 6
A method for simultaneously extracting cephalomannine and paclitaxel from Taxus media, comprising the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and methanol with the volume concentration of 95% as an entrainer to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder to the butane is 1:7kg/L, the volume ratio of the methanol to the butane is 1:50, the extraction temperature is 90 ℃, the ultrasonic power is 800W, the extraction pressure is 1.0MPa, the extraction time is 100min, and the extraction times are 4 times;
step S2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using tetrahydrofuran, wherein the mass ratio of the extract to the tetrahydrofuran is 1:30, and concentrating the extracted residues under reduced pressure to obtain the Taxus media extraction residues;
step S3, adding the Taxus media extraction residues obtained in the step S2 into a methanol solution with the volume concentration of 90% for dissolution, stirring to enable the extraction residues to be fully dissolved, standing for 50min, pouring the supernatant into another container, repeating the operation for 5 times, and concentrating the supernatant to be dry under reduced pressure;
s4, dissolving the dry matter obtained in the step S3 by adopting acetone, wherein the feed liquid ratio of the dry matter to the acetone is 1:5g/mL, adding active carbon with the mass of 10% of the residual matter into the solution, adsorbing for 2 hours at 60 ℃, filtering, collecting filtrate, and concentrating the filtrate to a specific gravity of 2.2 to obtain a Taxus media sample loading liquid;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, the filler used by the first chromatographic column is MCI, the filler particle diameter is 100 mu m, the column length is 920mm, the filler used by the second chromatographic column with the inner diameter of 100mm is C18, the filler particle diameter is 50 mu m, the column length is 920mm, the column inner diameter is 100mm, the methanol with the volume concentration of 30% is adopted to balance the column, the ultraviolet detector wavelength is 400nm, after the baseline is stabilized, the sample loading liquid in the step S4 is sampled by adopting the preassembled column, the sample loading amount is 500mL, the column temperature is room temperature, the methanol with the volume concentration of 70% is adopted as the mobile phase, the flow rate is 50mL/min, the eluent is collected according to UV detection, the detection wavelength of a UV detector is 254nm, the first-out eluent contains cephalomannine, the later eluent contains taxol, the yield of cephalomannine with high purity and taxol is 0.82% through decompression concentration, filtration and drying, the yield of cephalomannine is 0.78% and the taxol with the purity of 99.98% by HPLC detection, and the purity of cephalomannine is obtained respectively.
In the above examples 2 to 6, the method of the present invention for simultaneously extracting cephalomannine and paclitaxel from Taxus media was carried out, and the nuclear magnetic data of cephalomannine and paclitaxel obtained by the extraction was the same as that of example 1.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (3)
1. A method for simultaneously extracting cephalomannine and taxol from Taxus media is characterized by comprising the following steps: which comprises the following steps:
s1, drying branches and leaves of Taxus media, crushing and sieving to obtain powder with the size of 0.1-2.5 mm; subcritical extraction is carried out on the powder material by taking butane as a solvent and ethanol or methanol with the volume concentration of 70-95% as an entrainer, so as to obtain Taxus media extract and residues; subcritical extraction conditions are: the feed liquid ratio of the powder material to the solvent is 1:3-7 kg/L, the volume ratio of the entrainer to the solvent is 1:10-50, the extraction temperature is 40-90 ℃, the ultrasonic power is 500-800W, the extraction pressure is 0.2-1.0 MPa, the extraction time is 30-100 min, and the extraction times are 1-4 times;
s2, scattering the Taxus media extract obtained in the step S1 by using distilled water, extracting by using a first solvent, wherein the mass ratio of the extract to the first solvent is 1:10-30, and concentrating the extracted residues under reduced pressure to obtain Taxus media extraction residues; the first solvent is petroleum ether, chloroform, normal hexane, dichloromethane, ethyl acetate, or tetrahydrofuran;
s3, adding the Taxus media extraction residues obtained in the step S2 into a second solvent for dissolution, wherein the feed liquid ratio of the extraction residues to the second solvent is 1:10-20 kg/L; stirring to fully dissolve, standing for 30-100 min, pouring the supernatant into another container, repeating the stirring and standing operation for 3-5 times, and concentrating the supernatant under reduced pressure to be dry; the second solvent is methanol, ethanol or acetonitrile solution with volume concentration of 10-90%;
s4, dissolving the dry matter obtained in the step S3 by adopting a third solvent, wherein the feed liquid ratio of the dry matter to the third solvent is 1:1-5 g/mL, adding active carbon with the mass of 5-20% of the residual matter into the solution, adsorbing for 1-3 hours at the temperature of 60-80 ℃, filtering, collecting filtrate, and concentrating the filtrate to the specific gravity of 2.0-3.0 to obtain Taxus media sample loading liquid; the third solvent is methanol, acetone, ethanol or acetonitrile;
step S5, separating by medium-high pressure liquid chromatography: the first chromatographic column and the second chromatographic column are connected in series, wherein the filler used for the first chromatographic column is MCI, the particle size of the filler is 10-100 mu m, the column length is 310-920 mm, and the inner diameter of the column is 15-100 mm; the filler used for the second chromatographic column is C18, the particle size of the filler is 5-50 mu m, the column length is 310-920 mm, and the inner diameter of the column is 15-100 mm; and (3) adopting acetonitrile or methanol with volume concentration of 5-30% to balance a column, stabilizing the wavelength of an ultraviolet detector at 200-400 nm, loading the sample solution in the step S4 by adopting a preassembled column after the baseline is stabilized, adopting acetonitrile or methanol with volume concentration of 30-70% as a mobile phase, collecting eluent according to UV detection, wherein the detection wavelength of the UV detector is 200-254 nm, the eluent which flows out firstly contains cephalomannine, the eluent which flows out later contains taxol, concentrating under reduced pressure, filtering and drying to respectively obtain high-purity cephalomannine and taxol, wherein the purity of cephalomannine is 95-99%, the purity of taxol is 95-99%, the yield of cephalomannine is 0.5-1%, and the yield of taxol is 0.1-1%.
2. The method for simultaneously extracting cephalomannine and paclitaxel from Taxus media of claim 1, wherein the method comprises the following steps: in the step S1, subcritical extraction conditions are as follows: the feed liquid ratio of the powder material to the butane is 1:3-5 kg/L, the volume ratio of the butane to the ethanol or the methanol is 1:10-35, the extraction temperature is 40-65 ℃, the ultrasonic power is 500-700W, the extraction pressure is 0.2-0.8 MPa, the extraction time is 30-60 min, and the extraction times are 1-3 times.
3. The method for simultaneously extracting cephalomannine and paclitaxel from Taxus media of claim 2, wherein the method comprises the following steps: in the step S1, subcritical extraction conditions are as follows: the feed liquid ratio of the powder to the butane is 1:4kg/L, the volume ratio of the butane to the ethanol or the methanol is 1:20, the extraction temperature is 40 ℃, the ultrasonic power is 500W, the extraction pressure is 0.8MPa, the extraction time is 30min, and the extraction times are 2 times.
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