CN113896695B - Method for simultaneously extracting cephalomannine and paclitaxel from Mandya yew - Google Patents

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

Method for simultaneously extracting cephalomannine and paclitaxel from Mandya yew

Technical field

The invention belongs to the technical field of isolating and extracting effective active ingredients from Taxus plants, and in particular relates to a method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea.

Background technique

Yew is a tree species with extremely high economic value for ornamental, timber and medicinal purposes. Taxol extracted from the bark, branches and leaves of yew is a natural compound recognized worldwide as having anti-cancer effects. A new type of anticancer drug with a taxane diterpene skeleton, it is also the only drug currently known that can promote microtubule polymerization and stabilize polymerized microtubules. It has a strong inhibitory effect on most solid tumors and has basically no effect on normal cells. It is especially effective on advanced ovarian cancer, breast cancer, non-small cell lung cancer and Kaposi's sarcoma with minimal side effects, so it has become a current drug. Industry research focus. With the further deepening of research, the anti-cancer effects of paclitaxel drugs have been more widely used, and now they have become first-line anti-cancer drugs and broad-spectrum anti-tumor drugs.

Cephalomannine is a natural taxane compound that is symbiotic with paclitaxel. Its chemical structure only differs from paclitaxel in the amide side chain. Since cephalomannine and paclitaxel are very similar in structure and properties, the separation of paclitaxel and cephalomannine is very difficult, and repeated separations are necessary. In addition, as a biological macromolecule, paclitaxel is affected by environmental conditions such as temperature, organic solvents, acids, and alkalis, and is prone to degradation or isomerization to generate other taxanes. Based on the above factors, research and development of a simpler, more convenient, efficient, and low-cost separation and purification process has become a focus issue in paclitaxel research.

Although paclitaxel is a natural product extracted primarily from the bark of Pacific yew (Taxus brevifolia), it is also found in other members of the Taxaceae family such as Canada yew (T. canadensis) and Yunnan yew (T. yunnanensis). Paclitaxel was also discovered. Taxol is also present in the aerial parts and roots of other yew species, such as European yew (T. baccata) (the needles of which contain paclitaxel and its analogs), Asian yew (T. wallichiana) and Chinese yew (T.chinensis)) and yew trees cultivated for ornamental purposes. Taxus media is a natural hybrid between Northeastern yew (Taxus cuspidata) and European yew (Taxes baccata). It grows quickly. Not only does the bark contain paclitaxel, but the content of paclitaxel in the branches and leaves is also significantly higher than that of Taxus media. Domestic yew, with a content as high as 0.03% to 0.052%, is the best alternative raw material for yew bark. It not only can effectively protect this resource, but also greatly reduces its production cost due to its high paclitaxel content, so it is most suitable for the production of natural paclitaxel. Since 1995, my country has introduced fine varieties of Mandian yew from Canada and the United States, and has carried out large-scale planting and cultivation.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide a method for simultaneously extracting cephalomannine and paclitaxel from Mandya yew. The process flow is simple, easy to operate, green, non-toxic, efficient, low cost, and suitable for Mass production.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and entrain the powder with ethanol or methanol with a volume concentration of 70 to 95% Subcritical extraction is performed with the agent to obtain the extract and residue of Taxus mandiana; the subcritical extraction conditions are: the solid-liquid ratio of powder to solvent is 1:3~7kg/L, and the volume ratio of entrainer to solvent is 1: 10~50, extraction temperature is 40~90℃, ultrasonic power is 500~800W, extraction pressure is 0.2~1.0MPa, extraction time is 30~100min, and the number of extractions is 1~4 times;

Step S2: Disperse the Mandian yew extract obtained in step S1 with distilled water, and use the first solvent for extraction. The mass ratio of the extract to the first solvent is 1:10 to 30. The residue after extraction is Concentrate under reduced pressure to obtain the extraction residue of Taxus Mandea; the first solvent is petroleum ether, chloroform, n-hexane, methylene chloride, ethyl acetate, or tetrahydrofuran;

Step S3: Add the yew extraction residue obtained in step S2 to the second solvent for dissolution. The solid-liquid ratio of the extraction residue and the second solvent is 1:10~20kg/L; stir to fully dissolve. Let stand for 30 to 100 minutes, pour the supernatant into another container, repeat the above stirring and standing operations 3 to 5 times, and finally concentrate the supernatant under reduced pressure to dryness; the second solvent has a volume concentration of 10 to 90% methanol, ethanol or acetonitrile solution;

Step S4: Dissolve the dry substance obtained in Step S3 with a third solvent. The solid-liquid ratio of the dry substance to the third solvent is 1:1~5g/mL. Add the mass of the remaining substance to the above dissolved solution. 5 to 20% activated carbon, adsorb at 60 to 80°C for 1 to 3 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.0 to 3.0 to obtain the Mandia yew sample solution; the third solvent is methanol, acetone, ethanol or Acetonitrile;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatography column and the second chromatography column in series. The filler used in the first chromatography column is MCI, and the filler used in the second chromatography column is C18. Use acetonitrile or acetonitrile with a volume concentration of 5 to 30%. Methanol equilibrium column, the wavelength of the UV detector is 200~400nm. After the baseline is stable, use the prepacked column to load the sample solution in step S4. Use acetonitrile or methanol with a volume concentration of 30~70% as the mobile phase. According to UV detection collects the eluate. The detection wavelength of the UV detector is 200~254nm. The eluate that flows out first contains cephalomannine, and the eluate that flows out later contains paclitaxel. They are concentrated under reduced pressure, filtered and dried, respectively. High-purity cephalomannine and paclitaxel are obtained. The purity of cephalomannine is 95% to 99%, and the purity of paclitaxel is 95% to 99%.

Further, in the above-mentioned step S1, the subcritical extraction conditions are: the material-liquid ratio of powder to butane is 1:3~5kg/L, the volume ratio of butane to ethanol or methanol is 1:10~35, and the extraction The temperature is 40~65℃, the ultrasonic power is 500~700W, the extraction pressure is 0.2~0.8MPa, the extraction time is 30~60min, and the number of extractions is 1~3 times.

Furthermore, in the above-mentioned step S1, the subcritical extraction conditions are: the solid-liquid ratio of powder to butane is 1:4kg/L, the volume ratio of butane to ethanol or methanol is 1:20, and the extraction temperature is 40 ℃, the ultrasonic power is 500W, the extraction pressure is 0.8MPa, the extraction time is 30min, and the number of extractions is 2 times.

Further, in the above step S5, in the first chromatographic column, the filler particle size is 10-100 μm, the column length is 310-920 mm, and the column inner diameter is 15-100 mm.

Further, in the above step S5, in the second chromatographic column, the filler particle size is 5-50 μm, the column length is 310-920 mm, and the column inner diameter is 15-100 mm.

The present invention uses a method for simultaneously extracting cephalomannine and paclitaxel from Mandian yew. The yield rate of cephalomannine is 0.5-1% and the yield rate of paclitaxel is 0.1-1%.

Due to the adoption of the technical solution as described above, the present invention has the following advantages:

The method of the present invention for simultaneously extracting cephalomannine and paclitaxel from Mandian yew has flexible and simple operation, mild reaction and treatment conditions, and the subcritical fluid extraction technology has the advantages of energy saving, environmental protection and high efficiency. It adopts the first Solvent extraction can effectively remove pigments and some gum substances. The use of second solvent and third solvent precipitation methods can further remove the sticky pectin substances contained in the extract to facilitate subsequent sample loading; using activated carbon can not only remove samples The pigment substances in the column can also effectively remove some viscous insoluble matter, better protect the subsequent column chromatography packing, and facilitate reuse next time; through the method of connecting the first chromatographic column and the second chromatographic column in series, the tri-tip is achieved Effective separation effect of cephalomannine and paclitaxel; a single solvent system is used in the entire process, the solvent can be recovered and reused, avoiding solvent waste and environmental pollution; cephalomannine and paclitaxel can be prepared at the same time, and the three The purity of apomanine and paclitaxel is high, and no toxic organic solvents are used in the preparation process. The preparation is green and suitable for large-scale industrial production.

Description of the drawings

Figure 1 is a process flow chart of the method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandria according to the present invention;

Figure 2 is a preparation chromatogram of the method for simultaneously extracting cephalomannine and paclitaxel from Mandya yew according to the present invention;

Figure 3a is the mass spectrum of cephalomannine;

Figure 3b is the H NMR spectrum of cephalomannine;

Figure 3c is the carbon spectrum of cephalomannine;

Figure 4a is the mass spectrum of paclitaxel;

Figure 4b is the hydrogen nuclear magnetic spectrum of paclitaxel;

Figure 4c is the carbon spectrum of paclitaxel.

Detailed ways

The present invention can be further described in detail with reference to the accompanying drawings and the following examples; however, the following examples are only illustrations, and the present invention is not limited to these examples.

As shown in Figures 1 and 2, a method for simultaneously extracting cephalomannine and paclitaxel from Mandya yew includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and entrain the powder with ethanol or methanol with a volume concentration of 70 to 95% Subcritical extraction is performed with the agent to obtain the extract and residue of Taxus mandiana; the subcritical extraction conditions are: the solid-liquid ratio of powder to solvent is 1:3~7kg/L, and the volume ratio of entrainer to solvent is 1: 10~50, extraction temperature is 40~90℃, ultrasonic power is 500~800W, extraction pressure is 0.2~1.0MPa, extraction time is 30~100min, and the number of extractions is 1~4 times;

Step S2: Disperse the Mandian yew extract obtained in step S1 with distilled water, and use the first solvent for extraction. The mass ratio of the extract to the first solvent is 1:10 to 30. The residue after extraction is Concentrate under reduced pressure to obtain the extraction residue of Taxus Mandea; the first solvent is petroleum ether, chloroform, n-hexane, methylene chloride, ethyl acetate, or tetrahydrofuran;

Step S3: Add the yew extraction residue obtained in step S2 to the second solvent for dissolution. The solid-liquid ratio of the extraction residue and the second solvent is 1:10~20kg/L; stir to fully dissolve. Let stand for 30 to 100 minutes, pour the supernatant into another container, repeat the above stirring and standing operations 3 to 5 times, and finally concentrate the supernatant under reduced pressure to dryness; the second solvent has a volume concentration of 10 to 90% methanol, ethanol or acetonitrile solution;

Step S4: Dissolve the dry substance obtained in Step S3 with a third solvent. The solid-liquid ratio of the dry substance to the third solvent is 1:1~5g/mL. Add the mass of the remaining substance to the above dissolved solution. 5 to 20% activated carbon, adsorb at 60 to 80°C for 1 to 3 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.0 to 3.0 to obtain the Mandia yew sample solution; the third solvent is methanol, acetone, ethanol or Acetonitrile;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatography column and the second chromatography column in series. The filler used in the first chromatography column is MCI, and the filler used in the second chromatography column is C18. Use acetonitrile or acetonitrile with a volume concentration of 5 to 30%. Methanol equilibrium column, the wavelength of the UV detector is 200~400nm. After the baseline is stable, use the prepacked column to load the sample solution in step S4. The injection volume is 100~500mL, the column temperature is room temperature, and the volume concentration is used 30-70% acetonitrile or methanol is used as the mobile phase, the flow rate is 10-50mL/min, and the eluate is collected according to UV detection. The detection wavelength of the UV detector is 200-254nm. The eluate that flows out first contains cephalosporin. Alkali, and the eluate flowing out later contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalomannine and paclitaxel are obtained respectively. The purity of cephalomannine is 95% to 99%, and the purity of paclitaxel is 95% ~ 99%, the yield of cephalomannine is 0.5 ~ 1%, and the yield of paclitaxel is 0.1 ~ 1%.

In the above step S1, the preferred subcritical extraction conditions are: the material-to-liquid ratio of powder to butane is 1:3~5kg/L, the volume ratio of butane to ethanol or methanol is 1:10~35, and the extraction The temperature is 40~65°C, the ultrasonic power is 500~700W, the extraction pressure is 0.2~0.8MPa, the extraction time is 30~60min, and the number of extractions is 1~3 times; more preferably, the subcritical extraction conditions are: powder and The material-to-liquid ratio of butane is 1:4kg/L, the volume ratio of butane to ethanol or methanol is 1:20, the extraction temperature is 40°C, the ultrasonic power is 500W, the extraction pressure is 0.8MPa, the extraction time is 30min, extraction The number of times is 2 times.

In the above step S5, in the first chromatographic column, the filler particle size is 10-100 μm, the column length is 310-920 mm, and the column inner diameter is 15-100 mm; in the second chromatography column, the filler particle size is 5-50 μm, and the column length is The diameter is 310~920mm, and the inner diameter of the column is 15~100mm.

The present invention is a method for simultaneously extracting cephalomannine and paclitaxel from Mandian yew. The extracted cephalomannine has a chemical structural formula as shown in formula (I), and paclitaxel has a chemical structural formula as shown in formula (II). The chemical structural formula shown:

Example 1

A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; subcritically conduct the powder with butane as the solvent and ethanol with a volume concentration of 70% as the entrainer. After extraction, the extract and residue of Taxus Mandea are obtained; the subcritical extraction conditions are: the material-liquid ratio of powder to butane is 1:3kg/L, the volume ratio of ethanol to butane is 1:10, and the extraction temperature is 40℃, ultrasonic power is 500W, extraction pressure is 0.2MPa, extraction time is 80min, and the number of extractions is 2 times;

Step S2: Disperse the Taxus Mandya extract obtained in Step S1 with distilled water, and extract it with petroleum ether. The mass ratio of the extract to petroleum ether is 1:10. The residue after extraction is concentrated under reduced pressure. Obtain the extraction residue of Taxus Mandya;

Step S3: Add the yew extraction residue obtained in step S2 to a methanol solution with a volume concentration of 10% to dissolve. The solid-liquid ratio of the extraction residue to the methanol solution is 1:10kg/L. Stir to fully dissolve it. , let it stand for 30 minutes, pour the supernatant into another container, repeat the above operation 3 times, and finally concentrate the supernatant under reduced pressure until it is dry;

Step S4: The dry matter obtained in step S3 is thermally dissolved with methanol. The solid-liquid ratio of the dry matter to methanol is 1:1g/mL. 5% of the mass of the remaining activated carbon is added to the above solution. Adsorb at 60°C for 1 hour, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.0 to obtain the Mandian yew sample solution;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series. The filler used in the first chromatographic column is MCI, the filler particle size is 10 μm, the column length is 310mm, and the column inner diameter is 15mm. The packing used in the column is C18, the packing particle size is 5 μm, the column length is 310 mm, and the column inner diameter is 15 mm. An acetonitrile with a volume concentration of 5% is used to balance the column. The wavelength of the UV detector is 200 nm. After the baseline is stable, change the upper column in step S4. The sample solution is loaded on a prepacked column. The injection volume is 100 mL. The column temperature is room temperature. Acetonitrile with a volume concentration of 30% is used as the mobile phase. The flow rate is 10 mL/min. The eluate is collected according to UV detection. The UV detector The detection wavelength is 200nm. The first eluent contains cephalomannine and the later eluate contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalomannine and paclitaxel are obtained respectively. HPLC detection , the yield of cephalomannine was 0.53%, the yield of paclitaxel was 0.26%, the purity of cephalomannine was 96.5%, and the purity of paclitaxel was 98.7%.

Combining Figure 3a, Figure 3b, and Figure 3c, the NMR data of cephalomannine: 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＝7.1 Hz, Ph-H), 8.11 (2H, d, J = 8.1 Hz, 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').

Combining Figure 4a, Figure 4b, and Figure 4c, the NMR data of paclitaxel: 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,ot,4'-OBz,Ph-m),7.47(1H,ot,4'-OBz Ph-p),7.75(2H,d,4'-OBz ,Ph-o),7.50-7.35(5H,3'-Ph),7.49(2H,ot,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 Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and ethanol with a volume concentration of 75% as the entrainer for subcritical processing of the powder. Extraction is performed to obtain the extract and residue of Taxus Mandea; the subcritical extraction conditions are: the material-to-liquid ratio of powder to butane is 1:3.5kg/L, the volume ratio of ethanol to butane is 1:15, and the extraction temperature The temperature is 55℃, the ultrasonic power is 500W, the extraction pressure is 0.5MPa, the extraction time is 45min, and the number of extractions is 3 times;

Step S2: Disperse the Taxus Mandiana extract obtained in Step S1 with distilled water, and extract it with petroleum ether. The mass ratio of the extract to petroleum ether is 1:15. The residue after extraction is concentrated under reduced pressure. Obtain the extraction residue of Taxus Mandya;

Step S3: Add the yew extraction residue obtained in step S2 to a methanol solution with a volume concentration of 30% to dissolve. The solid-liquid ratio of the extraction residue to methanol is 1:13kg/L. Stir to fully dissolve it. Let it stand for 40 minutes, pour the supernatant into another container, repeat the above operation 4 times, and finally concentrate the supernatant under reduced pressure until it is dry;

Step S4: Dissolve the dry matter obtained in step S3 with acetone. The solid-liquid ratio of the dry matter to acetone is 1:2g/mL. Add 10% of the remaining mass of activated carbon to the above dissolved liquid, 70 °C for 2 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.5 to obtain a Mandian yew loading solution;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series. The filler used in the first chromatographic column is MCI, the filler particle size is 30 μm, the column length is 310mm, and the column inner diameter is 30mm. The packing used in the column is C18, the packing particle size is 10 μm, the column length is 380 mm, and the column inner diameter is 35 mm. The column is balanced with acetonitrile with a volume concentration of 5%. The wavelength of the ultraviolet detector is 227 nm. After the baseline is stable, remove the upper column in step S4. The sample solution is loaded on a prepacked column. The injection volume is 150 mL. The column temperature is room temperature. Acetonitrile with a volume concentration of 38% is used as the mobile phase. The flow rate is 30 mL/min. The eluate is collected according to UV detection. The UV detector The detection wavelength is 200nm. The first eluent contains cephalomannine and the later eluate contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalomannine and paclitaxel are obtained respectively. HPLC detection , the yield of cephalomannine was 0.65%, the yield of paclitaxel was 0.55%, the purity of cephalomannine was 98.8%, and the purity of paclitaxel was 99.4%.

Example 3

A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and methanol with a volume concentration of 78% as the entrainer for subcritical processing After extraction, the extract and residue of Taxus Mandea are obtained; the subcritical extraction conditions are: the material-to-liquid ratio of powder to butane is 1:4kg/L, the volume ratio of methanol to butane is 1:20, and the extraction temperature is 60℃, ultrasonic power is 550W, extraction pressure is 0.6MPa, extraction time is 60min, and the number of extractions is 3 times;

Step S2: Disperse the Taxus Mandya extract obtained in Step S1 with distilled water, and extract it with n-hexane. The mass ratio of the extract to n-hexane is 1:18. The residue after extraction is concentrated under reduced pressure. Obtain the extraction residue of Taxus Mandya;

Step S3: Add the yew extraction residue obtained in step S2 to an ethanol solution with a volume concentration of 50% to dissolve. The solid-liquid ratio of the extraction residue to the ethanol solution is 1:15kg/L. Stir to fully dissolve it. Let it stand for 50 minutes, pour the supernatant into another container, repeat the above operation 5 times, and finally concentrate the supernatant under reduced pressure until it is dry;

Step S4: Dissolve the dry matter obtained in step S3 with acetone. The solid-liquid ratio of the dry matter to acetone is 1:3g/mL. Add 15% of the remaining mass of activated carbon to the above dissolved liquid, 80 °C for 3 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 3.0 to obtain the Mandian yew sample solution;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series. The filler used in the first chromatographic column is MCI, the filler particle size is 60 μm, the column length is 500mm, and the column inner diameter is 50mm. The filler used in the column is C18, the filler particle size is 20 μm, the column length is 460 mm, and the column inner diameter is 50 mm. Methanol with a volume concentration of 15% is used to balance the column. The wavelength of the UV detector is 300 nm. After the baseline is stable, turn the upper column in step S4 The sample solution is loaded on a prepacked column. The injection volume is 200 mL. The column temperature is room temperature. Methanol with a volume concentration of 50% is used as the mobile phase. The flow rate is 35 mL/min. The eluate is collected according to UV detection. The UV detector The detection wavelength is 234nm. The first eluate contains cephalomannine and the later eluate contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalomannine and paclitaxel are obtained respectively. HPLC detection , the yield of cephalomannine was 0.81%, the yield of paclitaxel was 0.49%, the purity of cephalomannine was 97.8%, and the purity of paclitaxel was 98.6%.

Example 4

A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and methanol with a volume concentration of 82% as the entrainer for subcritical processing. Extraction is performed to obtain the extract and residue of Taxus Mandea; the subcritical extraction conditions are: the material-to-liquid ratio of powder to butane is 1:5kg/L, the volume ratio of methanol to butane is 1:30, and the extraction temperature is 70℃, ultrasonic power is 650W, extraction pressure is 0.7MPa, extraction time is 50min, and the number of extractions is 3 times;

Step S2: Disperse the Mandian yew extract obtained in step S1 with distilled water, and extract it with ethyl acetate. The mass ratio of the extract to ethyl acetate is 1:20. The residue after extraction is decompressed. Concentrate to obtain the remainder of the Mandian yew extract;

Step S3: Add the yew extract residue obtained in step S2 to an ethanol solution with a volume concentration of 55% for dissolution. The solid-liquid ratio of the extraction residue to the ethanol solution is 1:16kg/L, and stir to fully dissolve. Let it stand for 60 minutes, pour the supernatant into another container, repeat the above operation three times, and finally concentrate the supernatant under reduced pressure until it is dry;

Step S4: Dissolve the dry matter obtained in step S3 with acetone. The solid-liquid ratio of the dry matter to acetone is 1:3.5g/mL. Add 17% of the remaining mass of activated carbon to the above dissolved solution. Adsorb at 80°C for 2 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.6 to obtain the Mandian yew sample solution;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series. The filler used in the first chromatographic column is MCI, the filler particle size is 60 μm, the column length is 500mm, and the column inner diameter is 50mm. The packing used in the column is C18, the packing particle size is 20 μm, the column length is 460 mm, and the column inner diameter is 50 mm. Methanol with a volume concentration of 18% is used to balance the column. The wavelength of the ultraviolet detector is 350 nm. After the baseline is stable, change the upper column in step S4. The sample solution is loaded on a prepacked column. The injection volume is 300 mL. The column temperature is room temperature. Methanol with a volume concentration of 55% is used as the mobile phase. The flow rate is 40 mL/min. The eluate is collected according to UV detection. The UV detector The detection wavelength is 229nm. The first eluate contains cephalomannine and the later eluate contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalomannine and paclitaxel are obtained respectively. HPLC detection , the yield of cephalomannine was 0.81%, the yield of paclitaxel was 0.49%, the purity of cephalomannine was 97.8%, and the purity of paclitaxel was 98.6%.

Example 5

A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and methanol with a volume concentration of 90% as the entrainer for subcritical processing After extraction, the extract and residue of Taxus Mandea are obtained; the subcritical extraction conditions are: the material-to-liquid ratio of powder to butane is 1:6kg/L, the volume ratio of methanol to butane is 1:40, and the extraction temperature is 80℃, ultrasonic power is 700W, extraction pressure is 0.8MPa, extraction time is 60min, and the number of extractions is 4 times;

Step S2: Disperse the Taxus Mandiana extract obtained in Step S1 with distilled water, and extract it with petroleum ether. The mass ratio of the extract to petroleum ether is 1:25. The residue after extraction is concentrated under reduced pressure. Obtain the extraction residue of Taxus Mandya;

Step S3: Add the yew extraction residue obtained in step S2 to an acetonitrile solution with a volume concentration of 70% to dissolve. The solid-liquid ratio of the extraction residue to the acetonitrile solution is 1:18kg/L. Stir to fully dissolve it. , let it stand for 80 minutes, pour the supernatant into another container, repeat the above operation 5 times, and finally concentrate the supernatant under reduced pressure until it is dry;

Step S4: Dissolve the dry substance obtained in step S3 with methanol. The solid-liquid ratio of the dry substance to methanol is 1:4g/mL. Add 20% of the remaining mass of activated carbon to the above dissolved solution at 75°C. Adsorb for 1.5 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 3.0 to obtain the Mandian yew sample solution;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series. The filler used in the first chromatographic column is MCI, the filler particle size is 80 μm, the column length is 460mm, and the column inner diameter is 50mm. The packing used in the column is C18, the packing particle size is 50 μm, the column length is 800 mm, and the column inner diameter is 100 mm. An acetonitrile equilibrium column with a volume concentration of 30% is used. The UV detector wavelength is 350 nm. After the baseline is stable, change the upper column in step S4. The sample solution is loaded on a prepacked column. The injection volume is 400 mL. The column temperature is room temperature. Acetonitrile with a volume concentration of 60% is used as the mobile phase. The flow rate is 50 mL/min. The eluate is collected according to UV detection. The UV detector The detection wavelength is 254nm. The first eluate contains cephalomannine and the later eluate contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalomannine and paclitaxel are obtained respectively. HPLC detection , the yield of cephalomannine was 0.84%, the yield of paclitaxel was 0.95%, the purity of cephalomannine was 98.5%, and the purity of paclitaxel was 99.2%.

Example 6

A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, which includes the following steps:

Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and methanol with a volume concentration of 95% as the entrainer for subcritical processing. Extraction is performed to obtain the extract and residue of Taxus Mandea; the subcritical extraction conditions are: the material-to-liquid ratio of powder to butane is 1:7kg/L, the volume ratio of methanol to butane is 1:50, and the extraction temperature is 90℃, ultrasonic power is 800W, extraction pressure is 1.0MPa, extraction time is 100min, and the number of extractions is 4 times;

Step S2: Disperse the Taxus mandia extract obtained in Step S1 with distilled water, and extract it with tetrahydrofuran. The mass ratio of the extract to tetrahydrofuran is 1:30. The residue after extraction is concentrated under reduced pressure to obtain Mandia yew extract. Taxus yew extract residue;

Step S3: Add the yew extraction residue obtained in step S2 to a methanol solution with a volume concentration of 90% to dissolve. The material-liquid ratio of the extraction residue to the methanol solution is 1:20kg/L. Stir to fully dissolve it. , let it stand for 50 minutes, pour the supernatant into another container, repeat the above operation 5 times, and finally concentrate the supernatant under reduced pressure until it is dry;

Step S4: Dissolve the dry matter obtained in step S3 with acetone. The solid-liquid ratio of the dry matter to acetone is 1:5g/mL. Add 10% of the remaining mass of activated carbon to the above dissolved solution at 60°C. Adsorb for 2 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.2 to obtain the Mandia yew loading solution;

Step S5, medium and high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series. The filler used in the first chromatographic column is MCI, the filler particle size is 100 μm, the column length is 920mm, and the column inner diameter is 100mm. The second chromatographic column The filler used is C18, the filler particle size is 50 μm, the column length is 920 mm, and the column inner diameter is 100 mm. Methanol with a volume concentration of 30% is used to balance the column. The UV detector wavelength is 400 nm. After the baseline is stable, load the sample in step S4. The liquid is loaded on a prepacked column. The injection volume is 500mL. The column temperature is room temperature. Methanol with a volume concentration of 70% is used as the mobile phase. The flow rate is 50mL/min. The eluate is collected according to UV detection. The UV detector detects The wavelength is 254nm. The first eluate contains cephalomannine and the later eluate contains paclitaxel. After concentration, filtration and drying under reduced pressure, high purity cephalomannine and paclitaxel are obtained respectively. HPLC detection. The yield of cephalomannine was 0.82%, the yield of paclitaxel was 0.78%, the purity of cephalomannine was 99.3%, and the purity of paclitaxel was 98.2%.

In the above-mentioned Examples 2 to 6, the present invention uses a method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandria. The NMR data of the extracted cephalomannine and paclitaxel are the same as those in Example 1.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (3)

1. A method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandea, characterized by: it includes the following steps: Step S1: Dry the raw materials Mandian yew branches and leaves in the shade, crush and sieve to obtain a powder of 0.1 to 2.5 mm; use butane as the solvent and entrain the powder with ethanol or methanol with a volume concentration of 70 to 95% Subcritical extraction is performed with the agent to obtain the extract and residue of Taxus mandiana; the subcritical extraction conditions are: the solid-liquid ratio of powder to solvent is 1:3~7kg/L, and the volume ratio of entrainer to solvent is 1: 10~50, extraction temperature is 40~90℃, ultrasonic power is 500~800W, extraction pressure is 0.2~1.0MPa, extraction time is 30~100min, and the number of extractions is 1~4 times; Step S2: Disperse the Mandian yew extract obtained in step S1 with distilled water, and use the first solvent for extraction. The mass ratio of the extract to the first solvent is 1:10 to 30. The residue after extraction is Concentrate under reduced pressure to obtain the extraction residue of Taxus Mandea; the first solvent is petroleum ether, chloroform, n-hexane, methylene chloride, ethyl acetate, or tetrahydrofuran; Step S3: Add the yew extraction residue obtained in step S2 to the second solvent for dissolution. The solid-liquid ratio of the extraction residue and the second solvent is 1:10~20kg/L; stir to fully dissolve. Let stand for 30 to 100 minutes, pour the supernatant into another container, repeat the above stirring and standing operations 3 to 5 times, and finally concentrate the supernatant under reduced pressure to dryness; the second solvent has a volume concentration of 10 to 90% methanol, ethanol or acetonitrile solution; Step S4: Dissolve the dry substance obtained in Step S3 with a third solvent. The solid-liquid ratio of the dry substance to the third solvent is 1:1~5g/mL. Add the mass of the remaining substance to the above dissolved solution. 5 to 20% activated carbon, adsorb at 60 to 80°C for 1 to 3 hours, filter, collect the filtrate, and concentrate the filtrate to a specific gravity of 2.0 to 3.0 to obtain the Mandia yew sample solution; the third solvent is methanol, acetone, ethanol or Acetonitrile; Step S5, medium and high pressure liquid chromatography separation: connect the first chromatography column and the second chromatography column in series. The filler used in the first chromatography column is MCI, the filler particle size is 10-100 μm, the column length is 310-920 mm, and the column inner diameter is 15 ~100mm; the filler used in the second chromatographic column is C18, the particle size of the filler is 5~50μm, the column length is 310~920mm, and the column inner diameter is 15~100mm; the column is balanced with acetonitrile or methanol with a volume concentration of 5~30%, and UV detection The wavelength of the instrument is 200-400nm. After the baseline is stable, load the sample solution in step S4 with a pre-packed column. Use acetonitrile or methanol with a volume concentration of 30-70% as the mobile phase, and collect the eluent according to UV detection. , the detection wavelength of the UV detector is 200~254nm. The eluate flowing out first contains cephalomannine, and the eluate flowing out later contains paclitaxel. After concentration, filtration and drying under reduced pressure, high-purity cephalosporin is obtained respectively. The purity of cephalomannine and paclitaxel is 95% ~ 99%, the purity of paclitaxel is 95% ~ 99%, the yield of cephalomannine is 0.5 ~ 1%, and the yield of paclitaxel is 0.1 ~ 1% . 2. The method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandria according to claim 1, characterized in that: in step S1, the subcritical extraction conditions are: a mixture of powder and butane. The liquid ratio is 1:3~5kg/L, the volume ratio of butane to ethanol or methanol is 1:10~35, the extraction temperature is 40~65℃, the ultrasonic power is 500~700W, and the extraction pressure is 0.2~0.8MPa. The extraction time is 30 to 60 minutes, and the number of extractions is 1 to 3 times. 3. The method for simultaneously extracting cephalomannine and paclitaxel from Taxus Mandria according to claim 2, characterized in that: in step S1, the subcritical extraction conditions are: a mixture of powder and butane. The liquid ratio is 1:4kg/L, the volume ratio of butane to ethanol or methanol is 1:20, the extraction temperature is 40°C, the ultrasonic power is 500W, the extraction pressure is 0.8MPa, the extraction time is 30min, and the number of extractions is 2 times .