A method for extracting and separating artemisinic acid from artemisia annua byproducts

Abstract

This invention belongs to the field of medicinal chemistry technology, specifically relating to a method for extracting and separating artemisinin acid from artemisia annua byproducts. The method includes the following steps: first, dissolving the artemisia annua byproducts in solvent 1, filtering, and collecting the filtrate; extracting the filtrate with solvent 2, separating the layers, and adjusting the extraction layer to acidity; extracting the acidic extraction layer to obtain an insoluble substance; then mixing the insoluble substance with solvent 3 to obtain a crude product; and finally mixing the crude product with solvent 4 to obtain the final product. Solvent 2 is selected from one or more of sodium hydroxide, sodium bicarbonate, and potassium hydroxide. This invention provides, for the first time, a method for extracting and separating artemisinin acid from artemisinin byproducts. The obtained artemisinin acid has a high content and high yield, and the process is simple, uses few types of solvents, and exhibits good stability and reproducibility. It transforms artemisinin byproducts into a valuable resource and has promising application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry, and particularly relates to a method for extracting and separating artemisinic acid from artemisia annua byproducts. Background Art

[0002] Artemisinic acid is one of the sesquiterpenoid components in Artemisia annua L., and is the most important chiral synthetic monomer and precursor for the synthesis and metabolism of artemisinin and its derivatives. Artemisinic acid has a suitable chemical conformation and has important medicinal and development value, such as anti-malarial, anti-inflammatory and anti-tumor activities, especially known for its anti-malarial activity. The structural formula of artemisinic acid is shown in Formula 1.

[0003] .

[0004] There are not many preparation processes for artemisinic acid in the prior art. For example, Chinese invention patent application CN103570739A discloses a method for separating artemisinic acid from artemisia annua essential oil, comprising the following steps: (1) extraction: extracting the artemisinin essential oil with an organic solvent under room temperature for 2-4 times, filtering, combining the filtrate, and concentrating under reduced pressure to obtain a thick extract; (2) acidification: adding dilute hydrochloric acid to the thick extract, stirring and dispersing, filtering, and obtaining an acidic filtrate; (3) extraction: adding an equal volume of organic solvent to the acidic filtrate for extraction, separating the organic layer, and washing with water until neutral; (4) decolorization: adding a decolorizer to the organic layer, heating and refluxing for 1-3 h, filtering, and obtaining a filtrate; (5) concentration and crystallization: concentrating the filtrate under reduced pressure, cooling and crystallizing, filtering, and obtaining a crude artemisinic acid; (6) recrystallization and purification: dissolving the crude artemisinic acid with an organic solvent by heating, cooling and crystallizing.

[0005] Another Chinese invention patent application CN107011157A discloses a method for extracting artemisinic acid from an extract produced by artemisinin production, which is carried out according to the following steps: a. collecting the petroleum ether-ethyl acetate eluate in the artemisinin production process; b. concentrating the collected petroleum ether-ethyl acetate eluate and separating it by chromatography, and then eluting it with methanol, ethanol, acetone or ethyl acetate solvent to collect the eluate rich in artemisinic acid; c. concentrating the collected eluate rich in artemisinic acid and standing it for crystallization to obtain crude crystals; d. dissolving the crude artemisinic acid crystals with petroleum ether and recrystallizing it to obtain the finished artemisinic acid product.

[0006] The above patent only uses simple acidification and alkalization treatment, which has complex process, cumbersome operation, uses many types of solvents, and has high cost. In addition, the yield and content of artemisinic acid are not high, which is not conducive to the large-scale production of artemisinic acid.

[0007] Extracting artemisinin from Artemisia annua produces Artemisia annua byproducts with a high content, while the Artemisia annua byproducts contain a large amount of artemisinic acid that has not been effectively extracted and utilized. In view of this, the art urgently needs a method for obtaining artemisinic acid with a higher yield and content from Artemisia annua byproducts, which is simple in process and suitable for industrial production. Summary of the invention

[0008] The present invention aims at the problems existing in the prior art and provides a method for extracting and separating artemisinic acid from artemisia annua byproducts.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] A method for extracting and separating artemisinic acid from artemisia annua byproducts, comprising the following steps:

[0011] (1) First, dissolve the Artemisia annua byproduct with solvent 1, filter, and collect the filtrate;

[0012] (2) extracting the filtrate with solvent 2, separating the layers, and adjusting the extraction layer to be acidic;

[0013] (3) extracting the acidic extraction layer to obtain insoluble matter;

[0014] (4) The insoluble matter is then mixed with solvent 3 to obtain a crude product; the crude product is then mixed with solvent 4 to obtain,

[0015] Wherein, solvent 2 is selected from one or more of sodium hydroxide, sodium bicarbonate and potassium hydroxide.

[0016] Preferably, the solvent 2 is sodium hydroxide or potassium hydroxide.

[0017] Preferably, the solvent 1 in step (1) is selected from one or more of petroleum ether, ethyl acetate and dichloromethane.

[0018] More preferably, the solvent 1 is petroleum ether or ethyl acetate.

[0019] Preferably, the mass volume ratio of the Artemisia annua by-product to the solvent 1 is 1 g:1-5 mL.

[0020] Preferably, the mass percentage concentration of the solvent 2 in step (2) is 5-15%.

[0021] Preferably, the volume ratio of the filtrate to the solvent 2 is 1:1-5.

[0022] Preferably, the acidic solvent adjusted to acidity in step (2) is selected from one or more of hydrochloric acid, phosphoric acid and acetic acid.

[0023] Preferably, the concentration of the acidic solvent is 1-5 mol / L.

[0024] Preferably, the pH of the acidic extraction layer in step (3) is 4-5.

[0025] Preferably, the extractant used for extraction in step (3) is selected from one or more of petroleum ether, ethyl acetate and dichloromethane.

[0026] More preferably, the extractant is ethyl acetate or dichloromethane.

[0027] Preferably, after the extraction in step (3), the extraction layer is collected and concentrated, and the concentration temperature is 40-80°C.

[0028] Preferably, the solvent 3 and the solvent 4 in step (4) are each independently selected from at least one of acetonitrile, methanol, ethanol and ethyl acetate.

[0029] More preferably, the solvent 3 and the solvent 4 are both ethyl acetate.

[0030] Preferably, the mixture in step (4) needs to be heated to 60-90°C.

[0031] Preferably, the mass volume ratio of the insoluble matter to the solvent 3 is 1 kg:2-5 L. After the insoluble matter and the solvent 3 are mixed, they need to be cooled, crystallized and dried.

[0032] Preferably, the mass volume ratio of the crude product to the solvent 4 is 1 kg:2-5 L. After the crude product and the solvent 4 are mixed, they need to be cooled, recrystallized and dried.

[0033] Preferably, the artemisia annua by-product in step (1) is obtained by separating artemisinin from artemisia annua, collecting the by-product containing artemisinin, and concentrating it to a constant weight.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention provides for the first time a method for extracting and separating artemisinic acid from artemisinin by-products. The obtained artemisinic acid has high content and high yield, and the process is simple, a small number of solvents are used, and the stability and repeatability are good. The artemisinin by-products are turned into treasure, and the method has good application prospects. The obtained artemisinic acid can be widely used in various fields such as cosmetics and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the liquid chromatography spectrum of artemisinic acid reference substance.

[0037] Figure 2 This is the liquid phase spectrum of artemisinic acid prepared in Example 3. DETAILED DESCRIPTION

[0038] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which hydrochloric acid, with a specification of 36.5%, was purchased from Guangdong Xiaoda Chemical Co., Ltd.; artemisinic acid reference substance was purchased from Shanghai Myrel Biochemical Technology Co., Ltd.; methanol, with a specification of 99%, was purchased from Zhongan United Coal Chemical Co., Ltd.

[0039] Example 1

[0040] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0041] (1) extracting and separating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, and concentrating them at 60°C to a constant weight to obtain 10 kg of Artemisia annua byproducts, dissolving the obtained Artemisia annua byproducts in 10 L of ethyl acetate, filtering, and collecting the filtrate;

[0042] (2) The filtrate obtained in step (1) was extracted with 5% by mass concentration of sodium hydroxide (the volume ratio of the filtrate to sodium hydroxide was 1:2), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 5 with 1 mol / L hydrochloric acid;

[0043] (3) extracting the aqueous layer obtained in step (2) with dichloromethane, collecting the dichloromethane layer and concentrating it at 40° C. to constant weight to obtain 1.2 kg of insoluble matter;

[0044] (4) The insoluble matter obtained in step (3) was dissolved with 3.6 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 0.71 kg of crude product. Finally, the crude product was dissolved with 2.1 L of ethyl acetate at 70°C, cooled to below 10°C for recrystallization, and dried at 50°C to obtain 0.52 kg of artemisinic acid.

[0045] Example 2

[0046] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0047] (1) extracting and separating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, and concentrating them at 60°C to a constant weight to obtain 100 kg of Artemisia annua byproducts, dissolving the obtained Artemisia annua byproducts in 100 L of ethyl acetate, filtering, and collecting the filtrate;

[0048] (2) The filtrate obtained in step (1) was extracted with 5% by mass concentration of sodium hydroxide (the volume ratio of the filtrate to sodium hydroxide was 1:2), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 4 with 1 mol / L hydrochloric acid;

[0049] (3) extracting the aqueous layer obtained in step (2) with dichloromethane, collecting the dichloromethane layer and concentrating it at 60° C. to constant weight to obtain 12.5 kg of insoluble matter;

[0050] (4) The insoluble matter obtained in step (3) was dissolved with 37.5 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 8.50 kg of crude product. Finally, the crude product was dissolved with 24 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 5.61 kg of artemisinic acid.

[0051] Example 3

[0052] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0053] (1) extracting and separating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, concentrating at 60°C to constant weight to obtain 500 kg of Artemisia annua byproducts, dissolving the obtained Artemisia annua byproducts in 500 L of ethyl acetate, filtering, and collecting the filtrate;

[0054] (2) The filtrate obtained in step (1) was extracted with 5% by mass concentration of sodium hydroxide (the volume ratio of the filtrate to sodium hydroxide was 1:2), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 4 with 1 mol / L hydrochloric acid;

[0055] (3) extracting the aqueous layer obtained in step (2) with dichloromethane, collecting the dichloromethane layer and concentrating it at 80° C. to constant weight to obtain 63.1 kg of insoluble matter;

[0056] (4) The insoluble matter obtained in step (3) was dissolved with 187.5 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 40.5 kg of crude product. Finally, the crude product was dissolved with 121.5 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 27.23 kg of artemisinic acid.

[0057] Example 4

[0058] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0059] (1) extracting and isolating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, and concentrating them at 60°C to a constant weight to obtain 10 kg of Artemisia annua byproducts, dissolving the obtained Artemisia annua byproducts with 10 L of petroleum ether, filtering, and collecting the filtrate;

[0060] (2) The filtrate obtained in step (1) was extracted with sodium bicarbonate having a mass percentage concentration of 10% (the volume ratio of the filtrate to sodium bicarbonate was 1:3), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 5 with 2 mol / L hydrochloric acid;

[0061] (3) extracting the aqueous layer obtained in step (2) with petroleum ether, collecting the petroleum ether layer and concentrating it at 60° C. to constant weight to obtain 0.8 kg of insoluble matter;

[0062] (4) The insoluble matter obtained in step (3) was dissolved with 4 L of ethyl acetate at 60°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 0.52 kg of crude product. Finally, the crude product was dissolved with 2.4 L of ethyl acetate at 60°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 0.48 kg of artemisinic acid.

[0063] Comparative Example 1

[0064] The same as Example 3, the only difference is that solvent 1 is n-hexane.

[0065] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0066] (1) Extracting and separating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, and concentrating them at 60°C to a constant weight to obtain 500 kg of Artemisia annua byproducts. Dissolving the obtained Artemisia annua byproducts with 500 L of n-hexane, filtering, and collecting the filtrate;

[0067] (2) The filtrate obtained in step (1) was extracted with 5% by mass concentration of sodium hydroxide (the volume ratio of the filtrate to sodium hydroxide was 1:2), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 4 with 1 mol / L hydrochloric acid;

[0068] (3) extracting the aqueous layer obtained in step (2) with dichloromethane, collecting the dichloromethane layer and concentrating it at 80° C. to constant weight to obtain 30.1 kg of insoluble matter;

[0069] (4) The insoluble matter obtained in step (3) was dissolved in 90 L of ethyl acetate by heating at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 20.5 kg of crude product. Finally, the crude product was dissolved in 60 L of ethyl acetate by heating at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 18.5 kg of artemisinic acid.

[0070] Comparative Example 2

[0071] The same as Example 3, the only difference is that solvent 2 is calcium hydroxide with a mass percentage concentration of 5%.

[0072] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0073] (1) extracting and separating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, concentrating at 60°C to constant weight to obtain 500 kg of Artemisia annua byproducts, dissolving the obtained Artemisia annua byproducts in 500 L of ethyl acetate, filtering, and collecting the filtrate;

[0074] (2) The filtrate obtained in step (1) was extracted with calcium hydroxide having a mass percentage concentration of 5% (the volume ratio of the filtrate to calcium hydroxide was 1:2), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 4 with 1 mol / L hydrochloric acid;

[0075] (3) extracting the aqueous layer obtained in step (2) with dichloromethane, collecting the dichloromethane layer and concentrating it at 80° C. to constant weight to obtain 34.2 kg of insoluble matter;

[0076] (4) The insoluble matter obtained in step (3) was dissolved in 102 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 20.4 kg of crude product. Finally, the crude product was dissolved in 60 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 19.2 kg of artemisinic acid.

[0077] Comparative Example 3

[0078] The same as Example 3, the only difference is that the extractant in step (3) is n-hexane.

[0079] A method for extracting and separating artemisinic acid from artemisinin byproducts, comprising the following steps:

[0080] (1) extracting and separating artemisinin from Artemisia annua, collecting byproducts containing artemisinin, concentrating at 60°C to constant weight to obtain 500 kg of Artemisia annua byproducts, dissolving the obtained Artemisia annua byproducts in 500 L of ethyl acetate, filtering, and collecting the filtrate;

[0081] (2) The filtrate obtained in step (1) was extracted with 5% by mass concentration of sodium hydroxide (the volume ratio of the filtrate to sodium hydroxide was 1:2), the layers were separated, the aqueous layer was collected, and the pH of the aqueous layer was adjusted to 4 with 1 mol / L hydrochloric acid;

[0082] (3) extracting the aqueous layer obtained in step (2) with n-hexane, collecting the n-hexane layer and concentrating it at 80° C. to constant weight to obtain 45.1 kg of insoluble matter;

[0083] (4) The insoluble matter obtained in step (3) was dissolved with 135 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 30.2 kg of crude product. Finally, the crude product was dissolved with 90 L of ethyl acetate at 70°C, cooled to below 10°C for crystallization, and dried at 50°C to obtain 21.5 kg of artemisinic acid.

[0084] Test Case

[0085] The content of artemisinic acid prepared in Examples 1-4 and Comparative Examples 1-3 was tested respectively according to the high performance liquid chromatography method (General Rule 0512). The chromatographic conditions and system suitability test were as follows:

[0086] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0087] Mobile phase: acetonitrile-0.1% phosphoric acid solution (35:65, v:v);

[0088] Detection wavelength: 348 nm;

[0089] Temperature: 30℃;

[0090] Detection time: 40 min;

[0091] The number of theoretical plates calculated based on peaks should not be less than 2000.

[0092] Preparation of reference solution: Accurately weigh an appropriate amount of artemisinic acid reference substance and add methanol to prepare a solution containing 0.1 mg per 1 mL.

[0093] Preparation of test solution: Accurately weigh 10.0 mg of artemisinic acid prepared in Example 1-4 and Comparative Example 1-3 respectively, place in a stoppered conical flask, accurately add 10 mL of methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 25 kHz) for 15 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and obtain.

[0094] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0095] The liquid phase spectrum of artemisinic acid reference substance is as follows Figure 1 As shown, the liquid phase spectrum of artemisinic acid prepared in Example 3 is as follows Figure 2 The content detection results are shown in Table 1.

[0096] Table 1 Test results

[0097]

[0098] According to the sample content and yield results of Examples 1-4 and Comparative Examples 1-3, the determination results are relatively stable. Compared with Comparative Examples 1-3, the artemisinic acid product of Examples 1-4 has high content, high yield, high process stability and good repeatability.

[0099] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for extracting and separating artemisinic acid from artemisia annua byproducts, characterized in that: The steps include: (1) First, dissolve the Artemisia annua byproduct with solvent 1, filter, and collect the filtrate; (2) extracting the filtrate with solvent 2, separating the layers, and adjusting the extraction layer to be acidic; (3) extracting the acidic extraction layer to obtain insoluble matter; (4) The insoluble matter is then mixed with solvent 3 to obtain a crude product; the crude product is then mixed with solvent 4 to obtain, Wherein, the solvent 2 in step (2) is selected from one or more of sodium hydroxide, sodium bicarbonate and potassium hydroxide, the mass percentage concentration of the solvent 2 is 5-15%, and the volume ratio of the filtrate to the solvent 2 is 1:1-5; The artemisia annua byproduct in step (1) is obtained by collecting the byproduct containing artemisinin after separating artemisinin from artemisia annua and concentrating it to a constant weight, wherein the solvent 1 is selected from one or more of petroleum ether, ethyl acetate and dichloromethane; The extraction agent used in step (3) is ethyl acetate or dichloromethane, and after the extraction is completed, the extraction layer is collected and concentrated, and the concentration temperature is 40-80°C; The mixture in step (4) needs to be heated to 60-90°C, the mass volume ratio of the insoluble matter to the solvent 3 is 1 kg:2-5 L, and after the insoluble matter and the solvent 3 are mixed, they need to be cooled, crystallized and dried. The mass volume ratio of the crude product to the solvent 4 is 1 kg:2-5 L, and after the crude product and the solvent 4 are mixed, they need to be cooled, recrystallized and dried.

2. The method according to claim 1, characterized in that: The mass volume ratio of the artemisia annua byproduct to the solvent 1 is 1 g: 1-5 mL.

3. The method according to claim 1, characterized in that The acidic solvent adjusted to acidity in step (2) is selected from one or more of hydrochloric acid, phosphoric acid and acetic acid.

4. The method according to claim 3, characterized in that The concentration of the acidic solvent is 1-5 mol / L.

5. The method according to claim 1, characterized in that The pH of the acidic extraction layer in step (3) is 4-5.

6. The method according to claim 1, characterized in that In step (4), the solvent 3 and the solvent 4 are each independently selected from at least one of acetonitrile, methanol, ethanol and ethyl acetate.

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