A method for extracting topotecan from sycamore leaves

Abstract

The present invention belongs to the field of biological extraction technology, and particularly relates to a method for extracting topotecan from sycamore leaves. Sycamore leaves are used as raw materials, soaked in a solvent for extraction for a certain period of time, centrifuged, and the supernatant is filtered. The filtrate is filtered using an organic filter membrane to obtain a filtrate; after resin enrichment, column chromatography, evaporation, and purification, topotecan is obtained. The raw materials used are sycamore leaves, and the method provides a method for effectively treating fallen leaf waste, which can effectively alleviate the environmental problems caused by this type of plant waste, realize the transformation of plant waste into valuables, and has broad application prospects. The preparation process is simple, the extract is high in purity, high in safety, strong in availability, and easy to achieve large-scale production.

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, also known as the sycamore and tung hemp, is a large deciduous tree, reaching up to 15 meters tall, with a straight trunk and smooth green bark. Native to China, it is cultivated in provinces throughout northern and southern China. The tree grows quickly, its wood suitable for making musical instruments, its bark for papermaking and rope, its seeds edible or for oil extraction, and its leaves used in medicine and biology. Its smooth trunk and large, elegant leaves make it a popular ornamental tree. However, in autumn, the sycamore leaves fall in large numbers, increasing sanitation efforts while also contributing to environmental pollution.

[0003] Sycamore leaves, the leaves of the sycamore 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 the leaves of the Chinese sycamore tree. Topotecan hydrochloride is commonly used for small cell lung cancer and metastatic ovarian cancer that has failed first-line 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 plays an important role in DNA replication by loosening the DNA helix. Topotecan binds to topoisomerase I and the loosened DNA strands through a covalent complex, thereby preventing the reconnection of broken DNA strands. The subsequent effect of topotecan's inhibition of topoisomerase I on cells is the induction of single-strand breaks in the DNA protein. Traditional topotecan is mostly industrially synthesized, which is costly. 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:

[0007] A method for extracting topotecan from sycamore leaves comprises the following steps:

[0008] (1) Extraction: Using sycamore leaves as raw materials, soaking them in a solvent for a certain period of time, centrifuging them, taking the supernatant and filtering them with an organic filter membrane to obtain filtrate S1;

[0009] (2) Resin enrichment: The filtrate S1 is added to the macroporous adsorption resin. After ultrasonic treatment, the resin is separated from the clear liquid. The macroporous resin is eluted with the eluent D1, and the eluate is collected to obtain S2.

[0010] (3) Column chromatography: S2 is passed through a chromatography column and eluted with mobile phase D2, and the eluate is collected to obtain S3;

[0011] (4) After S3 is evaporated to dryness and purified, topotecan is obtained.

[0012] Preferably, the extraction solvent in step (1) is one or more of alcohols, esters, ethers, water, dichloromethane, and chloroform.

[0013] Preferably, in step (1), the extraction temperature is 20-80° C., the soaking time is 10-48 hours, and the ratio of the sycamore leaves to the solvent is 1 mg: 10-50 mL.

[0014] Preferably, the organic membrane filtration in step (1) is performed using two-stage organic membranes, wherein the first-stage organic membrane has a cutoff value in the range of 15,000-20,000 and the second-stage organic membrane has a cutoff value in the range of 5,000-8,000.

[0015] 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-type macroporous adsorption resin.

[0016] 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 eluent D1 used is 8-12 BV.

[0017] Preferably, the chromatography column in step (3) is an alumina chromatography column.

[0018] Preferably, the mobile phase D2 in step (3) is a mixture of ethanol, acetonitrile and water in a volume ratio of 1:2:2.

[0019] Preferably, the elution rate in step (3) is 1 BV / h, and the amount of eluent used is 8 BV.

[0020] Preferably, the purification is performed by recrystallization in step (4), using a volume ratio of ethanol to water of 1:10-30.

[0021] Beneficial effects: The raw materials used in the present invention are sycamore leaves, which provides a method for effectively treating fallen leaf garbage, can effectively alleviate the environmental problems caused by this type of plant garbage, realize the transformation of plant garbage into treasure, and has broad application prospects.

[0022] The extraction method of the present invention has low raw material cost, wide raw material sources, simple preparation process, high extract purity, high safety, strong availability, and is easy to achieve large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1is the product topotecan obtained in Example 1 1 H-NMR spectrum,

[0024] Figure 2 13C-NMR and DEPT135 spectra of topotecan obtained in Example 1,

[0025] Figure 3 This is the HMBC chart of topotecan, the product obtained in Example 1. DETAILED DESCRIPTION

[0026] A method for extracting topotecan from sycamore leaves comprises the following steps:

[0027] (1) Extraction: Use sycamore leaves as raw materials, soak them in a solvent for a certain period of time, centrifuge, take the supernatant and filter it, and then filter it with an organic filter membrane to obtain filtrate S1.

[0028] Naturally fallen sycamore leaves were collected, cleaned with distilled water, and air-dried in a cool place. The leaves were then ground into powder using a universal grinder. The powder was then soaked in a specific solvent and extracted for a specific time to allow the active ingredients in the leaves to seep out. After centrifugation, the supernatant was filtered and filtered twice with an organic membrane to obtain filtrate S1.

[0029] The extraction solvent is one or more of alcohols, esters, ethers, water, dichloromethane, and chloroform. An alcohol-water system is particularly recommended.

[0030] The extraction temperature is 20-80°C, optimized to 30-70°C, and recommended to be 35-50°C. The soaking time is 10-48 hours, optimized to 16-35 hours, and recommended to be 18-24 hours.

[0031] The dosage ratio of sycamore leaves to solvent is 1 mg: 10-50 mL.

[0032] 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.

[0033] Filtration using an organic membrane is performed using two-stage organic membranes, with the first-stage organic membrane having a cutoff value ranging from 15,000 to 20,000 and the second-stage organic membrane having a cutoff value ranging from 5,000 to 8,000.

[0034] (2) Resin enrichment: Add the filtrate S1 to the macroporous adsorption resin, and after ultrasonic treatment, separate the resin from the clear liquid. Elute the macroporous resin with eluent D1, collect the eluate, and obtain S2.

[0035] The macroporous adsorption resin is soaked in ethanol aqueous solution, washed, placed in a glass container, added with S1, and ultrasonically vibrated for a certain period of time to enrich the effective components in the resin. The resin is filtered to separate the resin from the clear liquid, and the effective components enriched on the resin are eluted with eluent D1. The eluate is collected to obtain S2.

[0036] The macroporous adsorption resin is a styrene-type non-polar copolymer, a styrene-type weakly polar copolymer, or a weakly acidic cation exchange resin. Preferred are 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, and AB-8 macroporous adsorption resin. D-101 macroporous adsorption resin is recommended.

[0037] The ultrasonic treatment time is 6-24 hours, preferably 7-16 hours, and preferably 8-12 hours.

[0038] The eluent D1 is alcohol, ester, ether or alcohol aqueous solution, preferably alcohol aqueous solution, and ethanol aqueous solution is particularly recommended.

[0039] The elution rate is 1-3 BV / h, and the amount of eluent D1 used is 8-12 BV.

[0040] (3) Column chromatography: S2 is passed through a chromatography column and eluted with mobile phase D2. The eluate is collected to obtain S3.

[0041] The chromatography column adopts an alumina chromatography column.

[0042] The loading rate of S2 in the alumina column is 1-3 BV / h, preferably 2 BV / h.

[0043] The mobile phase D2 was a mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2.

[0044] The elution rate was 1 BV / h, and the amount of eluent used was 8 BV.

[0045] (4) After S3 is evaporated to dryness and purified, topotecan is obtained.

[0046] 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, and particularly 50-60°C. The pressure is preferably controlled at 20-30 mbar.

[0047] The product is purified by recrystallization, wherein the volume ratio of ethanol to water is 1:10-30.

[0048] Example 1: A method for extracting topotecan from sycamore leaves, comprising the following steps:

[0049] (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder using a universal grinder and soak them in 80℃ water for 48 hours at a ratio of 1mg / 50ml to allow the active ingredients in the sycamore leaves to seep out. After centrifugation at 3000 r / min for 10 minutes, collect the supernatant and filter it first with a filter membrane with a cutoff value of 20000 and then with a filter membrane with a cutoff value of 8000 to obtain filtrate S1.

[0050] (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 active ingredients in the resin. Filter it to separate the resin from the clear liquid, and elute the active ingredients enriched on the resin with 160 ml of ethyl acetate at an elution rate of 1 BV / h. Collect the eluate to obtain S2.

[0051] (3) The sample was loaded at a rate of 1 BV / h, and S2 was passed through the chromatography column. A mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2 was used for elution at a rate of 1 BV / h. The amount of the elution machine used was 8 BV. The product was further purified to obtain S3.

[0052] (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 with 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 diagram. See Figure 1 、 Figure 2 、 Figure 3 .

[0053] Example 2: A method for extracting topotecan from sycamore leaves, comprising the following steps:

[0054] (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 weight to ethanol dosage ratio of 1 mg / 10 ml, so that the effective components in the sycamore leaves can seep out. Centrifuge at 10,000 r / min for 5 minutes, take the supernatant, filter it with a filter membrane with a cutoff value of 15,000 first, and then filter it with a filter membrane with a cutoff value of 5,000 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 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 ethanol at an elution rate of 3 BV / h. Collect the eluate to obtain S2.

[0055] (3) The sample was loaded at a rate of 1 BV / h, and S2 was passed through the chromatography column. A mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2 was used for elution at a rate of 1 BV / h. The amount of the elution machine used was 8 BV. The product was further purified to obtain S3.

[0056] (4) The S3 solution was subjected to rotary evaporation at 40°C and 20 mbar to obtain crude topotecan. The resulting product was recrystallized using an ethanol / water antisolvent system with a volume ratio of 1:30 to obtain the desired product. The extraction yield of the obtained topotecan was 0.0336% and the purity was 91.38%.

[0057] Example 3: A method for extracting topotecan from sycamore leaves, comprising the following steps:

[0058] (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 ratio of 1mg / 30ml of sycamore leaf weight to 60% ethanol, so that the effective components in the sycamore leaves can seep out. Centrifuge at 5000 r / min for 8 minutes, take the supernatant, filter it with a filter membrane with a cutoff value of 15000 first, 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 components in the resin. Filter it to separate the resin from the clear liquid, and elute the effective components enriched on the resin with 200 ml of 95% ethanol solution at an elution rate of 2BV / h to obtain S2.

[0059] (3) Load the sample at a rate of 1 BV / h, allowing S2 to flow through the chromatography column, and elute it with ethanol, acetonitrile, and water in a ratio of 1:2:2 at a rate of 1 BV / h. The amount of the elution machine used is 8 BV. The product is further purified to obtain S3.

[0060] (4) The S3 solution was subjected to rotary evaporation at 60°C and 25 mbar to obtain crude topotecan. The resulting product was recrystallized using an ethanol / water antisolvent system with a volume ratio of 1:20 to obtain the target product. The extraction yield of the obtained topotecan was 0.0581% and the purity was 98.89%.

[0061] Example 4: A method for extracting topotecan from sycamore leaves, comprising the following steps:

[0062] (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and dry them in a cool place. Grind them into powder using a universal grinder. Soak them in a 50% methanol aqueous solution at 50°C for 16 h at a ratio of 1 mg / 20 ml to allow the active ingredients in the sycamore leaves to seep out. After centrifugation at 3000 r / min for 10 minutes, collect the supernatant and filter it first with a filter membrane with a cutoff value of 15000 and then with a filter membrane with a cutoff value of 5000 to obtain filtrate S1.

[0063] (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 active ingredients in the resin. Filter it to separate the resin from the clear liquid, and elute the active ingredients enriched on the resin with 160 ml of 80% methanol aqueous solution at an elution rate of 3 BV / h to obtain S2.

[0064] (3) Load the sample at a rate of 1 BV / h, allowing S2 to flow through the chromatography column, and elute it with ethanol, acetonitrile, and water in a ratio of 1:2:2 at a rate of 1 BV / h. The amount of the elution machine used is 8 BV. The product is further purified to obtain S3.

[0065] (4) The S3 solution was subjected to rotary evaporation at 50°C and 20 mbar to obtain crude topotecan. The resulting product was recrystallized using an ethanol / water antisolvent system with a volume ratio of 1:10 to obtain the target product. The extraction yield of the obtained topotecan was 0.0372%, and the purity was 94.52%.

[0066] Example 5: A method for extracting topotecan from sycamore leaves, comprising the following steps:

[0067] (1) Collect naturally fallen sycamore leaves, clean them with distilled water, and air-dry them in a cool place. Grind them into powder using a universal grinder and soak them in 75% ethanol at 35°C for 35 h at a ratio of 1 mg / 40 ml of ethanol-water solution to allow the active ingredients in the leaves to seep out. After centrifugation at 8000 r / min for 5 minutes, collect the supernatant and filter it first with a filter membrane with a cutoff value of 20,000 and then with a filter membrane with a cutoff value of 5000 to obtain filtrate S1.

[0068] (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 active ingredients in the resin. Filter it to separate the resin from the clear liquid, and elute the active ingredients enriched on the resin with 240 ml of 80% ethanol aqueous solution at an elution rate of 1 BV / h to obtain S2.

[0069] (3) Load the sample at a rate of 1 BV / h, allowing S2 to flow through the chromatography column, and elute it with ethanol, acetonitrile, and water in a ratio of 1:2:2 at a rate of 1 BV / h. The amount of the elution machine used is 8 BV. The product is further purified to obtain S3.

[0070] (4) The S3 solution was subjected to rotary evaporation at 60°C and 30 mbar to obtain crude topotecan. The resulting product was recrystallized using an ethanol / water antisolvent system with a volume ratio of 1:30 to obtain the target product. The extraction yield of the obtained topotecan was 0.0458%, and the purity was 96.13%.

[0071] The above description is only a specific embodiment of the present invention. Those skilled in the art will clearly understand that for the convenience and brevity of description, the specific working processes of the systems, modules and units described above can refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here. It should be understood that the protection scope of the present invention is not limited to this. Any person skilled in the art 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 included in 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, taking the supernatant and filtering, and filtering through an organic filter membrane to obtain a filtrate S1, wherein the extraction solvent is one or more of alcohols, esters, ethers, water, dichloromethane, and chloroform; the filtration using the organic filter membrane is performed using a two-stage organic filter membrane, the first stage organic filter membrane has a cutoff value range of 15000-20000; the second stage organic filter membrane has a cutoff value range of 5000-8000; (2) Resin enrichment: The filtrate S1 is added to a macroporous adsorption resin, and after ultrasonic treatment, the resin is separated from the clear liquid, and the macroporous resin is eluted with an eluent D1. The eluent is collected to obtain S2. 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; 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 eluent D1 used is 8-12 BV; (3) Column chromatography: S2 was passed through a chromatography column and eluted with mobile phase D2, and the eluate was collected to obtain S3; the mobile phase D2 was a mixture of ethanol, acetonitrile, and water in a volume ratio of 1:2:2, the elution rate was 1 BV / h, and the amount of eluent used was 8 BV; (4) After S3 is evaporated to dryness and purified, topotecan is obtained; it is purified by recrystallization, using a volume ratio of ethanol to water of 1:10-30.

2. 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.

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

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