Method for isolating and purifying desoxyartemisin from artemisia annua by-products

Abstract

The application discloses a method for separating and purifying desoxyartemisinin from artemisia annua byproducts, which comprises the following steps: step one, column chromatography separation of artemisia annua extract, collection of sesquiterpene mixture without artemisinin, concentration to constant weight; step two, extraction of the sesquiterpene mixture by solvent 1, filtration; step three, concentration to constant weight; step four, saponification of the concentrate, extraction of the product by solvent 2, and neutralization of the extract; step five, concentration of the extract; step six, column chromatography separation and concentration to obtain crude desoxyartemisinin; and step seven, heating and dissolving by solvent 3, cooling and recrystallization to obtain desoxyartemisinin product. The application provides a method for separating and purifying desoxyartemisinin from artemisia annua byproducts by combining column chromatography and solvent extraction, and the desoxyartemisinin obtained has high purity and high yield, and the process is simple, stable and reproducible, and has good popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the field of deoxyartemisinin preparation technology, and in particular to a method for separating and purifying deoxyartemisinin from Artemisia annua byproducts. Background Technology

[0002] Deoxyartemisinin is an orally bioavailable compound isolated from Artemisia annua, possessing anti-inflammatory and anti-ulcer activities. It can inhibit the surface membrane, cell membrane, and mitochondrial membrane of Plasmodium, blocking nutrient absorption and thus achieving a killing effect; it also has antibacterial effects, exhibiting some inhibitory activity against Staphylococcus aureus, Mycobacterium tuberculosis, and Shigella dysenteriae.

[0003] Artemisia annua is the aerial part of Artemisia annua, a plant in the Asteraceae family. It has heat-clearing and detoxifying properties and is used for summer heat fever, yin deficiency fever, malaria with chills and fever, and damp-heat jaundice. Deoxyartemisinin is obtained by separating and purifying deoxyartemisinin from Artemisia annua byproducts through column chromatography; it also has anti-inflammatory and anti-ulcer effects.

[0004] There is a significant market demand for deoxyartemisinin, but existing technologies for its preparation are limited, and methods for directly extracting deoxyartemisinin from Artemisia annua byproducts are not publicly available. Therefore, developing methods for extracting deoxyartemisinin from Artemisia annua byproducts is of great importance to meet market and industrial needs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for separating and purifying deoxyartemisinin from artemisia by-products, addressing the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for separating and purifying deoxyartemisinin from artemisia annua by-products, comprising the following steps: Step 1: Separate the Artemisia annua extract by column chromatography, collect the mixture of sesquiterpenes without artemisinin, and concentrate to constant weight; Step 2: Extract the sesquiterpenoid mixture from Step 1 using solvent 1, and filter after extraction; Step 3: Concentrate the filtrate obtained in Step 2 to constant weight; Step 4: Saponify the concentrate, extract the saponified product with solvent 2, and adjust the extract to neutral. Step 5: Concentrate the extract obtained in Step 4; Step 6: The concentrate obtained in Step 5 is subjected to column chromatography for separation. The separated product is concentrated to obtain crude deoxyartemisinin. Step 7: Dissolve the crude deoxyartemisinin in solvent 3 by heating, filter, cool and recrystallize the filtrate, filter again, discard the filtrate, and obtain the deoxyartemisinin product.

[0007] Preferably, solvent 1 is at least one of methanol, ethyl acetate, and ethanol.

[0008] Preferably, solvent 2 is at least one of ethyl acetate, dichloromethane, petroleum ether, and chloroform.

[0009] Preferably, solvent 3 is at least one of methanol, ethyl acetate, ethanol, and water.

[0010] Preferably, the concentration temperature in step one is 50–90°C, the concentration temperature in step three is 60–90°C, the concentration temperature in step five is 60–80°C, and the concentration temperature in step six is ​​50–90°C.

[0011] Preferably, the extraction temperature in step two is 40–90°C, and the heating temperature in step seven is 50–90°C.

[0012] Preferably, the alkali used for saponification in step four is at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate.

[0013] Preferably, during column chromatography separation in step six, the sample loading volume to silica gel mass ratio is 1:7 to 10.

[0014] Preferably, step six specifically involves mixing the concentrate obtained in step five with 100-200 mesh silica gel at a mass ratio of 1:7 to 10, then packing the mixture into a column, eluting and collecting it with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:1, and concentrating the collected solution at 50-90°C to constant weight to obtain crude deoxyartemisinin.

[0015] Preferably, the method for separating and purifying deoxyartemisinin from artemisia byproducts includes the following steps: Step 1: Separate the Artemisia annua extract by column chromatography, collect the mixture of sesquiterpenes without artemisinin, concentrate to constant weight, and concentrate at 50-90℃. Step 2: Extract the sesquiterpene mixture treated in Step 1 using a mixed solvent of methanol and ethyl acetate at a volume ratio of 1:2 at 40-90℃. Cool the resulting extract and filter it. Step 3: Concentrate the filtrate obtained in Step 2 to constant weight at a temperature of 60–90°C. Step 4: Saponify the concentrate, extract the saponification product with dichloromethane, and adjust the extract to neutral. Step 5: Concentrate the extract obtained in Step 4 at a concentration temperature of 60–80°C; Step 6: Mix the concentrate obtained in Step 5 with 100-200 mesh silica gel at a mass ratio of 1:7 to 10, then pack the mixture into a column. Elute with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:1. Concentrate the collected solution at 50-90°C to constant weight to obtain crude deoxyartemisinin. Step 7: Dissolve crude deoxyartemisinin in ethanol at 50-90°C, filter, cool the filtrate to recrystallize, filter again, discard the filtrate, and obtain the deoxyartemisinin product.

[0016] The beneficial effects of this invention are: This invention provides for the first time a method for separating and purifying deoxyartemisinin from artemisia byproducts using a combination of column chromatography and solvent extraction. The resulting deoxyartemisinin has high purity and high yield, and the process is simple, stable, and reproducible, showing great promise for widespread application. The obtained deoxyartemisinin product can be used in various fields such as cosmetics and pharmaceuticals. Attached Figure Description

[0017] Figure 1 This is a chromatogram of deoxyartemisinin from Example 1. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0019] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0021] This invention provides a method for isolating and purifying deoxyartemisinin from artemisia annua byproducts, comprising the following steps: Step 1: Separate the Artemisia annua extract by column chromatography, collect the mixture of sesquiterpenes without artemisinin, and concentrate to constant weight; in the preferred embodiment, the concentration temperature is 50-90℃.

[0022] Step 2: Extract the sesquiterpenoid mixture from Step 1 using solvent 1, and filter after extraction; In a preferred embodiment, solvent 1 is at least one of ethyl acetate, dichloromethane, petroleum ether, and chloroform; more preferably, solvent 1 is a mixed solvent of methanol and ethyl acetate in a volume ratio of 1:2; and the extraction temperature is 40–90°C.

[0023] Step 3: Concentrate the filtrate obtained in Step 2 to a constant weight; in a preferred embodiment, the concentration temperature is 60-90°C.

[0024] Step 4: Saponify the concentrate, extract the saponified product with solvent 2, and adjust the extract to neutral with acid; In a preferred embodiment, the alkali used for saponification is at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate; and the solvent 2 is at least one of ethyl acetate, dichloromethane, petroleum ether, and chloroform.

[0025] Step 5: Concentrate the extract obtained in Step 4; in a preferred embodiment, the concentration temperature is 60-80°C.

[0026] Step 6: Mix the concentrate obtained in Step 5 with 100-200 mesh silica gel at a mass ratio of 1:7 to 10, then pack the mixture into a column. Elute with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:1. Concentrate the collected solution at 50-90°C to constant weight to obtain crude deoxyartemisinin.

[0027] Step 7: Dissolve the crude deoxyartemisinin in solvent 3 by heating, filter, cool and recrystallize the filtrate, filter again, discard the filtrate, and obtain the deoxyartemisinin product.

[0028] In a preferred embodiment, solvent 3 is at least one of methanol, ethyl acetate, ethanol, and water; the heating temperature is 50–90°C.

[0029] The above is the overall concept of the present invention. The following detailed embodiments are provided to further illustrate the present invention. Example 1

[0030] A method for isolating and purifying deoxyartemisinin from artemisia annua byproducts, comprising the following steps: Step 1: The artemisia extract was separated by column chromatography, and 10 kg of a mixture of sesquiterpenes without artemisinin was collected and concentrated to a constant weight of 8.3 kg at 70 °C. Step 2: Extract the sesquiterpene mixture treated in Step 1 twice at 75°C using a mixed solvent of 5 times the mass of methanol and ethyl acetate in a volume ratio of 1:2. Cool the resulting extracts, filter them, and combine the filtrates from both extractions. Step 3: Concentrate the filtrate obtained in Step 2 at 70℃ to constant weight, yielding 5.8 kg of concentrate; Step 4: Mix the concentrate obtained in Step 3 with 10L of 0.1wt% sodium hydroxide aqueous solution and stir at 50°C for 2 hours to carry out saponification. After the reaction is completed, cool and let stand to separate the layers. Discard the water layer and extract the oil layer twice with 10L of dichloromethane. Combine the extracts and adjust to neutral with 0.1wt% hydrochloric acid aqueous solution. Step 5: Concentrate the extract obtained in Step 4 at 60℃ to constant weight; Step 6: Mix the concentrate obtained in Step 5 with 100-200 mesh silica gel at a mass ratio of 1:8, then pack the mixture into a column. Elute and collect the product with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:1. Start collecting from the second column volume. Concentrate the collected solution at 60°C to constant weight to obtain 0.86 kg of crude deoxyartemisinin. Step 7: Dissolve the crude deoxyartemisinin in 15 times its weight of ethanol at 70°C, filter, cool the filtrate to recrystallize, filter again, discard the filtrate, and obtain 0.65 kg of refined deoxyartemisinin, which is the final product. Example 2

[0031] A method for isolating and purifying deoxyartemisinin from artemisia annua byproducts, comprising the following steps: Step 1: The Artemisia annua extract was separated by column chromatography, and 50 kg of a mixture of sesquiterpenes without artemisinin was collected and concentrated to a constant weight of 42.3 kg at 70 °C. Step 2: Extract the sesquiterpene mixture treated in Step 1 twice at 75°C using a mixed solvent of 5 times the mass of methanol and ethyl acetate in a volume ratio of 1:2. Cool the resulting extracts, filter them, and combine the filtrates from both extractions. Step 3: Concentrate the filtrate obtained in Step 2 at 70℃ to constant weight, yielding 30.2 kg of concentrate; Step 4: Mix the concentrate obtained in Step 3 with 50L of 0.1wt% sodium hydroxide aqueous solution and stir at 50℃ for 2h to carry out saponification. After the reaction is completed, cool and let stand to separate the layers. Discard the water layer and extract the oil layer twice with 50L of dichloromethane. Combine the extracts and adjust to neutral with 0.1wt% hydrochloric acid aqueous solution. Step 5: Concentrate the extract obtained in Step 4 at 60℃ to constant weight; Step 6: Mix the concentrate obtained in Step 5 with 100-200 mesh silica gel at a mass ratio of 1:8, then pack the mixture into a column. Elute and collect the product with a mixed eluent of petroleum ether:ethyl acetate at a volume ratio of 50:1. Start collecting from the second column volume. Concentrate the collected solution at 60°C to constant weight to obtain 4.7 kg of crude deoxyartemisinin. Step 7: Dissolve the crude deoxyartemisinin in 15 times its weight of ethanol at 70°C, filter, cool the filtrate to recrystallize, filter again, discard the filtrate, and obtain 3.1 kg of refined deoxyartemisinin, which is the final product. Example 3

[0032] A method for isolating and purifying deoxyartemisinin from artemisia annua byproducts, comprising the following steps: Step 1: The Artemisia annua extract was separated by column chromatography, and 500 kg of a mixture of sesquiterpenes without artemisinin was collected. The mixture was then concentrated to a constant weight of 410 kg at 70 °C. Step 2: Extract the sesquiterpene mixture treated in Step 1 twice at 75°C using a mixed solvent of 5 times the mass of methanol and ethyl acetate in a volume ratio of 1:2. Cool the resulting extracts, filter them, and combine the filtrates from both extractions. Step 3: Concentrate the filtrate obtained in Step 2 at 70℃ to constant weight, yielding 310 kg of concentrate; Step 4: Mix the concentrate obtained in Step 3 with 500L of 0.1wt% sodium hydroxide aqueous solution and stir at 50°C for 2 hours to carry out saponification. After the reaction is completed, cool and let stand to separate the layers. Discard the water layer and extract the oil layer twice with 500L of dichloromethane. Combine the extracts and adjust to neutral with 0.1wt% hydrochloric acid aqueous solution. Step 5: Concentrate the extract obtained in Step 4 at 60℃ to constant weight; Step 6: Mix the concentrate obtained in Step 5 with 100-200 mesh silica gel at a mass ratio of 1:8, then pack the mixture into a column. Elute and collect the product with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:1. Start collecting from the second column volume. Concentrate the collected solution at 60°C to constant weight to obtain 49.2 kg of crude deoxyartemisinin. Step 7: Dissolve the crude deoxyartemisinin in 15 times its weight of ethanol at 70°C, filter, cool the filtrate to recrystallize, filter again, discard the filtrate, and obtain 30.9 kg, which is the final product.

[0033] The content of deoxyartemisinin in the products prepared in Examples 1-3 (i.e., the content of deoxyartemisinin in the high-quality deoxyartemisinin products) was detected using the following methods: Determined according to high performance liquid chromatography (General Rule 0512); Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.1% glacial acetic acid solution (55:45) was used as the mobile phase; the detection wavelength was 200 nm, the temperature was 30 °C, the detection time was 40 min, and the theoretical plate number calculated based on the peak should not be less than 2000. Preparation of reference solution: Accurately weigh an appropriate amount of deoxyartemisinin reference standard, add methanol to prepare a solution containing 0.1 mg per ml; Preparation of test solution: Take 2 portions of each of the above examples 1-3, deoxyartemisinin (accurately weighed, placed in a stoppered conical flask, accurately add 10 ml of methanol, weigh, sonicate (power 250W, frequency 25kHz) for 15 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and the test solution is obtained. Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0034] Reference Figure 1 The chromatogram of deoxyartemisinin in Example 1 is shown, wherein the retention time of deoxyartemisinin is 9.717 min.

[0035] The content detection results and the yield of deoxyartemisinin product are shown in Table 1 below: Table 1 Example 1 98.2% 87.5% Example 2 98.6% 86.9% Example 3 97.9% 87.2% Based on the sample content and yield results of the above three examples, the test results are relatively stable. The deoxyartemisinin products of Examples 1-3 have high purity, high yield, high process stability, and good repeatability. The refined product yield refers to the ratio of the deoxyartemisinin content in the refined deoxyartemisinin product to the total deoxyartemisinin content in the medicinal material.

[0036] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for isolating and purifying deoxyartemisinin from Artemisia annua byproducts, characterized in that, Includes the following steps: Step 1: Separate the Artemisia annua extract by column chromatography, collect the mixture of sesquiterpenes without artemisinin, and concentrate to constant weight; Step 2: Extract the sesquiterpenoid mixture from Step 1 using solvent 1, and filter after extraction; Step 3: Concentrate the filtrate obtained in Step 2 to constant weight; Step 4: Saponify the concentrate, extract the saponified product with solvent 2, and adjust the extract to neutral. Step 5: Concentrate the extract obtained in Step 4; Step 6: The concentrate obtained in Step 5 is subjected to column chromatography for separation. The separated product is concentrated to obtain crude deoxyartemisinin. Step 7: Dissolve crude deoxyartemisinin in solvent 3 by heating, filter, cool and recrystallize the filtrate, filter again, discard the filtrate, and obtain the deoxyartemisinin product. Solvent 1 is at least one of methanol, ethyl acetate, and ethanol; Solvent 2 is at least one of ethyl acetate, dichloromethane, petroleum ether, and chloroform; Solvent 3 is at least one of methanol, ethyl acetate, and ethanol; The concentration temperature in step one is 50-90℃, the concentration temperature in step three is 60-90℃, the concentration temperature in step five is 60-80℃, and the concentration temperature in step six is ​​50-90℃. The extraction temperature in step two is 40–90°C, and the heating temperature in step seven is 50–90°C. The alkali used for saponification in step four is at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate.

2. The method for separating and purifying deoxyartemisinin from Artemisia annua byproducts according to claim 1, characterized in that, In step six, when performing column chromatography separation, the sample loading volume to silica gel mass ratio is 1:7 to 10.

3. The method for separating and purifying deoxyartemisinin from Artemisia annua byproducts according to claim 2, characterized in that, Step six specifically involves mixing the concentrate obtained in step five with 100-200 mesh silica gel at a mass ratio of 1:7 to 10, then packing the mixture into a column, eluting and collecting it with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:1, and concentrating the collected solution at 50-90°C to constant weight to obtain crude deoxyartemisinin.

4. The method for separating and purifying deoxyartemisinin from Artemisia annua byproducts according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Separate the Artemisia annua extract by column chromatography, collect the mixture of sesquiterpenes without artemisinin, concentrate to constant weight, and concentrate at 50-90℃. Step 2: Extract the sesquiterpene mixture treated in Step 1 using a mixed solvent of methanol and ethyl acetate at a volume ratio of 1:2 at 40-90℃. Cool the resulting extract and filter it. Step 3: Concentrate the filtrate obtained in Step 2 to constant weight at a temperature of 60–90°C. Step 4: Saponify the concentrate, extract the saponification product with dichloromethane, and adjust the extract to neutral. Step 5: Concentrate the extract obtained in Step 4 at a concentration temperature of 60–80°C; Step 6: Mix the concentrate obtained in Step 5 with 100-200 mesh silica gel at a mass ratio of 1:7 to 10, then pack the mixture into a column. Elute with a mixed eluent of petroleum ether and ethyl acetate at a volume ratio of 50:

1. Concentrate the collected solution at 50-90°C to constant weight to obtain crude deoxyartemisinin. Step 7: Dissolve crude deoxyartemisinin in ethanol at 50-90°C, filter, cool the filtrate to recrystallize, filter again, discard the filtrate, and obtain the deoxyartemisinin product.

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