Method for extracting topotecan from phoenix tree leaves

Abstract

The invention belongs to the technical field of biological extraction, and particularly relates to a method for extracting topotecan from phoenix tree leaves. The method comprises the following steps: soaking phoenix tree leaves serving as a raw material in a solvent, extracting for a certain time, centrifuging, taking supernate, filtering, and filtering by using an organic filter membrane to obtain filtrate; and carrying out resin enrichment, column chromatography, drying by distillation and purification to obtain the topotecan. The used raw material is phoenix tree leaves, a method capable of effectively treating the fallen leaf garbage is provided, the environmental problem caused by the plant garbage can be effectively relieved, the plant garbage is turned into wealth, and the application prospect is wide. The preparation process is simple, the extract is high in purity, high in safety and strong in availability, and large-scale production is easy to realize.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extraction, and particularly relates to a method for extracting topotecan from sycamore leaves. Background Art

[0002] The sycamore tree is a plant of the genus sycamore of the family Sterculiaceae. It is also known as the green sycamore and tung hemp. It is also a large deciduous tree, up to 15 meters tall, with a straight trunk and green, smooth bark. It is native to China and is cultivated in provinces in the north and south. The sycamore grows fast, its wood is suitable for making musical instruments, its bark can be used for papermaking and ropes, its seeds can be eaten or pressed for oil, and its leaves can be used in the field of medicine and biology. It is a famous ornamental tree species because of its smooth trunk and large, beautiful leaves. In autumn, sycamore leaves fall in large numbers, which increases sanitation work and easily causes environmental pollution.

[0003] Paulownia leaves, the leaves of the Paulownia tree, have medicinal value. The leaves of the plant are used as medicine, which has the effects of dispelling wind and dampness, detoxifying and reducing swelling, and lowering blood pressure. Some ingredients have the effect of treating cancer.

[0004] Topotecan is an important secondary metabolite that can be extracted from sycamore leaves. Topotecan hydrochloride is often used for small cell lung cancer and metastatic ovarian cancer that has failed first-line chemotherapy or subsequent chemotherapy. Topotecan hydrochloride is a semi-synthetic camptothecin derivative and an anti-tumor drug that inhibits the activity of topoisomerase I. Topoisomerase I can loosen the DNA helix, thus playing an important role in DNA replication. Topotecan binds to the covalent complex formed by topoisomerase I and the loosened DNA chain, thereby hindering the reconnection of the broken DNA single strand. The subsequent effect of topotecan on cells after inhibiting topoisomerase I is to induce the breakage of the DNA protein single strand. Traditional topotecan is mostly industrially synthesized and has high cost. Summary of the invention

[0005] The invention provides a method for extracting topotecan from sycamore leaves.

[0006] The technical solution of this application is as follows: A method for extracting topotecan from sycamore leaves comprises the following steps: (1) Extraction: Using sycamore leaves as raw materials, soaking them in a solvent for a certain period of time, centrifuging them, filtering the supernatant, and filtering them with an organic filter membrane to obtain filtrate S1; (2) Resin enrichment: Add the filtrate S1 to the macroporous adsorption resin, separate the resin from the clear liquid after ultrasonic treatment, elute the macroporous resin with eluent D1, collect the eluent, and obtain S2; (3) Column chromatography: S2 is passed through a chromatography column and eluted with mobile phase D2, and the eluate is collected to obtain S3; (4) After S3 is evaporated to dryness and purified, topotecan is obtained.

[0007] Preferably, the extraction solvent in step (1) is one or more of alcohols, esters, ethers, water, dichloromethane and chloroform.

[0008] Preferably, in step (1), the extraction temperature is 20-80° C., the soaking time is 10-48 hours, and the dosage ratio of the sycamore leaves to the solvent is 1 mg: 10-50 mL.

[0009] Preferably, the filtration using the organic filter membrane in step (1) is performed using two-stage organic filter membranes, wherein the cutoff value of the first stage organic filter membrane is in the range of 15000-20000; and the cutoff value of the second stage organic filter membrane is in the range of 5000-8000.

[0010] Preferably, the macroporous adsorption resin in step (2) is a styrene-type non-polar copolymer, a styrene-type weakly polar copolymer or a weakly acidic cation exchange resin macroporous adsorption resin.

[0011] Preferably, the ultrasonic treatment time in step (2) is 6-24 hours, the eluent D1 is an alcohol, ester, ether or alcohol aqueous solution, the elution rate is 1-3 BV / h, and the amount of the eluent D1 used is 8-12 BV.

[0012] Preferably, the chromatography column in step (3) is an alumina chromatography column.

[0013] Preferably, the mobile phase D2 in step (3) is a mixture of ethanol, acetonitrile and water in a volume ratio of 1:2:2.

[0014] Preferably, the elution rate in step (3) is 1 BV / h, and the amount of eluent used is 8 BV.

[0015] Preferably, in step (4), the purification is carried out by recrystallization, and the volume ratio of ethanol to water is 1:10-30.

[0016] Beneficial effects: The raw material used in the present invention is sycamore leaves, which provides a method for effectively treating fallen leaf garbage, can effectively reduce the environmental problems caused by this type of plant garbage, realize the transformation of plant garbage into treasure, and has broad application prospects.

[0017] The extraction method of the invention has low raw material cost, wide raw material sources, simple preparation process, high extract purity, high safety, strong availability, and is easy to realize large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the product topotecan obtained in Example 1 1 H-NMR spectrum, Figure 2 13C-NMR and DEPT135 spectra of topotecan obtained in Example 1, Figure 3 This is the HMBC chart of topotecan, the product obtained in Example 1. DETAILED DESCRIPTION

[0019] A method for extracting topotecan from sycamore leaves comprises the following steps: (1) Extraction: Using sycamore leaves as raw materials, soaking them in a solvent for a certain period of time, centrifuging them, filtering the supernatant, and filtering them with an organic filter membrane to obtain filtrate S1.

[0020] Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Use a universal grinder to grind the sycamore leaves into powder, soak them in a certain solvent, and extract them for a certain period of time to make the effective ingredients in the sycamore leaves seep out. After centrifugation, take the supernatant and filter it. After filtering twice with an organic filter membrane, obtain the filtrate S1.

[0021] The solvent for extraction is one or more of alcohols, esters, ethers, water, dichloromethane, and chloroform. An alcohol-water system is particularly recommended.

[0022] The extraction temperature is 20-80°C, optimized to 30-70°C, recommended to 35-50°C, and the soaking time is 10-48 hours, optimized to 16-35 hours, and recommended to 18-24 hours.

[0023] The dosage ratio of sycamore leaf to solvent is 1 mg: 10-50 mL.

[0024] The rotation speed of the centrifugal process is 3000-10000 r / min, optimized to 5000-8000 r / min, and the centrifugal time is 5-10 minutes.

[0025] The filtration using organic filter membrane is to use two-stage organic filter membrane for filtration, the first stage organic filter membrane cutoff value range is 15000-20000; the second stage organic filter membrane cutoff value range is 5000-8000.

[0026] (2) Resin enrichment: Add the filtrate S1 to the macroporous adsorption resin. After ultrasonic treatment, separate the resin from the clear liquid. Elute the macroporous resin with eluent D1 and collect the eluent to obtain S2.

[0027] The macroporous adsorption resin is soaked in ethanol aqueous solution, washed, placed in a glass container, S1 is added, ultrasonic vibration is performed for a certain period of time to enrich the effective components in the resin, and the resin is separated from the clear liquid by suction filtration. The effective components enriched on the resin are eluted with eluent D1, and the eluent is collected to obtain S2.

[0028] The macroporous adsorption resin is a styrene-type non-polar copolymer, a styrene-type weakly polar copolymer or a weakly acidic cation exchange resin macroporous adsorption resin, preferably D-101 macroporous adsorption resin, D-101B macroporous adsorption resin, XDA-1 macroporous adsorption resin, XDA-1B macroporous adsorption resin, DK-110A macroporous adsorption resin, D-401 macroporous adsorption resin, AB-8 macroporous adsorption resin, and D-101 macroporous adsorption resin is recommended.

[0029] The ultrasonic treatment time is 6-24 hours, preferably 7-16 hours, and preferably 8-12 hours.

[0030] The eluent D1 is alcohol, ester, ether or alcohol aqueous solution, preferably alcohol aqueous solution, and ethanol aqueous solution is particularly recommended.

[0031] The elution rate is 1-3 BV / h, and the amount of eluent D1 used is 8-12 BV.

[0032] (3) Column chromatography: Pass S2 through a chromatography column, elute it with mobile phase D2, and collect the eluate to obtain S3.

[0033] The chromatography column adopts an alumina chromatography column.

[0034] The loading rate of S2 in the alumina column is 1-3 BV / h, preferably 2 BV / h.

[0035] The mobile phase D2 was a mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2.

[0036] The elution rate was 1 BV / h and the amount of eluent used was 8 BV.

[0037] (4) After S3 is evaporated to dryness and purified, topotecan is obtained.

[0038] The S3 solution is subjected to rotary evaporation to obtain crude topotecan. The rotary evaporation temperature is controlled at 40-70° C., preferably 50-70° C., particularly 50-60° C., and the pressure is preferably controlled at 20-30 mbar.

[0039] The product is purified by recrystallization, wherein the volume ratio of ethanol to water is 1:10-30.

[0040] Example 1: A method for extracting topotecan from sycamore leaves, comprising the following steps: (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder with a universal grinder, soak them in 80℃ water for 48 hours at a ratio of 1mg / 50ml of sycamore leaf mass to water to allow the effective ingredients in the sycamore leaves to seep out. Centrifuge at 3000 r / min for 10 minutes, take the supernatant, first filter it with a filter membrane with a cutoff value of 20000, and then filter it with a filter membrane with a cutoff value of 8000 to obtain filtrate S1.

[0041] (2) Take 20 mL of D-110A macroporous resin, soak it in 90% ethanol aqueous solution, wash it, put it in a glass container, add S1, and ultrasonically vibrate it for 24 hours to enrich the effective components in the resin. Filter it to separate the resin from the clear liquid, and elute the effective components enriched on the resin with 160 ml of ethyl acetate at an elution rate of 1 BV / h. Collect the eluate to obtain S2.

[0042] (3) S2 was loaded at a rate of 1 BV / h to flow through the chromatography column and eluted with a mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2 at a rate of 1 BV / h. The amount of the eluting machine used was 8 BV. The product was further purified to obtain S3.

[0043] (4) Rotary evaporation: Rotary evaporation was performed on the S3 solution at a temperature of 70°C and a pressure of 20 mbar to obtain crude topotecan. The obtained product was recrystallized using an ethanol-water anti-solvent system with a volume ratio of 1:10 to obtain topotecan. The extraction rate of the obtained topotecan was 0.0286% and the purity was 88.24%. 1 H-NMR spectrum, 13C-NMR and DEPT135 spectrum, HMBC chart. See Figure 1 , Figure 2 , Figure 3 .

[0044] Example 2: A method for extracting topotecan from sycamore leaves, comprising the following steps: (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder with a universal grinder, soak them in ethanol at 20℃ for 10 hours according to the sycamore leaf mass and ethanol dosage ratio of 1mg / 10ml, so that the effective ingredients in the sycamore leaves can seep out. Centrifuge at 10000 r / min for 5 minutes, take the supernatant, first filter it with a filter membrane with a cutoff value of 15000, and then filter it with a filter membrane with a cutoff value of 5000 to obtain filtrate S1. (2) Take 20 mL of D-AB-8 macroporous resin, soak it with 95% ethanol aqueous solution, wash it, put it in a glass container, add S1, and ultrasonically vibrate it for 6 hours to enrich the effective ingredients in the resin. Filter it to separate the resin from the clear liquid, and elute the effective ingredients enriched on the resin with 240ml ethanol at an elution rate of 3BV / h. Collect the eluate to obtain S2.

[0045] (3) S2 was loaded at a rate of 1 BV / h to flow through the chromatography column and eluted with a mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2 at a rate of 1 BV / h. The amount of the eluting machine used was 8 BV. The product was further purified to obtain S3.

[0046] (4) The S3 solution was subjected to rotary evaporation at a temperature of 40°C and a pressure of 20 mbar to obtain a crude topotecan. The obtained product was recrystallized using an ethanol-water anti-solvent system with a volume ratio of 1:30 to obtain the target product. The extraction rate of the obtained topotecan was 0.0336% and the purity was 91.38%.

[0047] Example 3: A method for extracting topotecan from sycamore leaves, comprising the following steps: (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder with a universal grinder, soak them in 45℃ water for 24 hours according to the sycamore leaf weight and 60% ethanol dosage ratio of 1mg / 30ml, so that the effective ingredients in the sycamore leaves can seep out. Centrifuge at 5000 r / min for 8 minutes, take the supernatant, first filter it with a filter membrane with a cutoff value of 15000, and then filter it with a filter membrane with a cutoff value of 5000 to obtain filtrate S1. (2) Take 20 mL of D-101A macroporous resin, soak it with 90% ethanol aqueous solution, wash it, put it in a glass container, add S1, and ultrasonically vibrate it for 12 hours to enrich the effective ingredients in the resin. Filter it to separate the resin from the clear liquid, and elute the effective ingredients enriched on the resin with 200ml 95% ethanol solution at a rate of 2BV / h to obtain S2.

[0048] (3) S2 was loaded at a rate of 1 BV / h to flow through the chromatography column and eluted with ethanol, acetonitrile and water in a ratio of 1:2:2 at a rate of 1 BV / h. The amount of the eluting machine used was 8 BV. The product was further purified to obtain S3.

[0049] (4) The S3 solution was subjected to rotary evaporation at a temperature of 60°C and a pressure of 25 mbar to obtain crude topotecan. The obtained product was recrystallized using an ethanol-water anti-solvent system with a volume ratio of 1:20 to obtain the target product. The extraction rate of the obtained topotecan was 0.0581% and the purity was 98.89%.

[0050] Example 4: A method for extracting topotecan from sycamore leaves, comprising the following steps: (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder with a universal grinder, soak them in 50% methanol aqueous solution at 50℃ for 16 hours according to the ratio of sycamore leaf weight to 50% methanol aqueous solution of 1mg / 20ml, so that the effective ingredients in the sycamore leaves can seep out. After centrifugation at 3000 r / min for 10 minutes, take the supernatant, first filter it with a filter membrane with a retention value of 15000, and then filter it with a filter membrane with a retention value of 5000 to obtain filtrate S1.

[0051] (2) Take 20 mL of D101-B macroporous resin, soak it in 90% ethanol aqueous solution, wash it, put it in a glass container, add S1, and ultrasonically vibrate it for 12 hours to enrich the effective components in the resin. Filter it to separate the resin from the clear liquid, and elute the effective components enriched on the resin with 160 ml of 80% methanol aqueous solution at a rate of 3 BV / h to obtain S2.

[0052] (3) S2 was loaded at a rate of 1 BV / h to flow through the chromatography column and eluted with ethanol, acetonitrile and water in a ratio of 1:2:2 at a rate of 1 BV / h. The amount of the eluting machine used was 8 BV. The product was further purified to obtain S3.

[0053] (4) The S3 solution was subjected to rotary evaporation at a temperature of 50°C and a pressure of 20 mbar to obtain a crude topotecan. The obtained product was recrystallized using an ethanol-water anti-solvent system with a volume ratio of 1:10 to obtain the target product. The extraction rate of the obtained topotecan was 0.0372% and the purity was 94.52%.

[0054] Example 5: A method for extracting topotecan from sycamore leaves, comprising the following steps: (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder with a universal grinder, soak them in 75% ethanol water at 35°C for 35 hours at a ratio of 1 mg / 40 ml of sycamore leaf mass to ethanol water solution, so that the effective ingredients in the sycamore leaves can seep out. After centrifugation at 8000 r / min for 5 minutes, take the supernatant, first filter it with a filter membrane with a cutoff value of 20000, and then filter it with a filter membrane with a cutoff value of 5000 to obtain filtrate S1.

[0055] (2) Take 20 mL of AB-8 macroporous resin, soak it in 90% ethanol aqueous solution, wash it, put it in a glass container, add S1, and ultrasonically vibrate it for 8 hours to enrich the effective components in the resin. Filter it to separate the resin from the clear liquid, and elute the effective components enriched on the resin with 240 ml of 80% ethanol aqueous solution at an elution rate of 1 BV / h to obtain S2.

[0056] (3) S2 was loaded at a rate of 1 BV / h to flow through the chromatography column and eluted with ethanol, acetonitrile and water in a ratio of 1:2:2 at a rate of 1 BV / h. The amount of the eluting machine used was 8 BV. The product was further purified to obtain S3.

[0057] (4) The S3 solution was subjected to rotary evaporation at a temperature of 60°C and a pressure of 30 mbar to obtain crude topotecan. The obtained product was recrystallized using an ethanol-water anti-solvent system with a volume ratio of 1:30 to obtain the target product. The extraction rate of the obtained topotecan was 0.0458% and the purity was 96.13%.

[0058] The above is only a specific implementation of the present invention. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working process of the system, module and unit described above can refer to the corresponding process in the aforementioned method embodiment, and will not be repeated here. It should be understood that the protection scope of the present invention is not limited to this. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be covered within the protection scope of the present invention.

Claims

1. A method for extracting topotecan from sycamore leaves, characterized in that The following steps are involved: (1) Extraction: Using sycamore leaves as raw materials, soaking them in a solvent for a certain period of time, centrifuging them, filtering the supernatant, and filtering them with an organic filter membrane to obtain filtrate S1; (2) Resin enrichment: Add the filtrate S1 to the macroporous adsorption resin, separate the resin from the clear liquid after ultrasonic treatment, elute the macroporous resin with eluent D1, collect the eluent, and obtain S2; (3) Column chromatography: S2 is passed through a chromatography column and eluted with mobile phase D2, and the eluate is collected to obtain S3; (4) After S3 is evaporated to dryness and purified, topotecan is obtained.

2. The method according to claim 1, characterized in that The extraction solvent in step (1) is one or more of alcohols, esters, ethers, water, dichloromethane and chloroform.

3. The method according to claim 1, characterized in that In step (1), the extraction temperature is 20-80° C., the soaking time is 10-48 hours, and the dosage ratio of the sycamore leaves to the solvent is 1 mg: 10-50 mL.

4. The method according to claim 1, characterized in that The filtration using organic membrane in step (1) is to use two-stage organic membrane for filtration, the first stage organic membrane has a cutoff value in the range of 15000-20000; the second stage organic membrane has a cutoff value in the range of 5000-8000.

5. The method according to claim 1, characterized in that In step (2), the macroporous adsorption resin is a styrene-type non-polar copolymer, a styrene-type weakly polar copolymer or a weakly acidic cation exchange resin macroporous adsorption resin.

6. The method according to claim 1, characterized in that In step (2), the ultrasonic treatment time is 6-24 hours, the eluent D1 is an alcohol, ester, ether or alcohol aqueous solution, the elution rate is 1-3 BV / h, and the amount of the eluent D1 used is 8-12 BV.

7. The method according to claim 1, characterized in that In step (3), the chromatography column is an alumina chromatography column.

8. The method according to claim 1, characterized in that In step (3), the mobile phase D2 is a mixture of ethanol, acetonitrile and water in a volume ratio of 1:2:

2.

9. The method according to claim 1, characterized in that In step (3), the elution rate is 1 BV / h and the amount of eluent used is 8 BV.

10. The method according to claim 1, characterized in that In step (4), the product is purified by recrystallization, using ethanol and water in a volume ratio of 1:10-30.

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