Method for separating and purifying kaempferol from artemisia apiacea byproduct

Abstract

The invention provides a method for separating and purifying kaempferol from an artemisia apiacea byproduct, and belongs to the field of natural product separation engineering. The method for separating and purifying kaempferol from the artemisia apiacea byproduct comprises the following steps: 1, extracting the artemisia apiacea byproduct by adopting an organic solvent 1, filtering, and concentrating filtrate to constant weight to obtain a concentrate 1; the artemisia apiacea byproduct does not contain artemisinin; 2, dissolving the concentrate 1 with an organic solvent 2, extracting with an alkaline solution, and concentrating an organic phase layer to obtain a concentrate 2; 3, carrying out column chromatography separation on the concentrate 2 by using an organic solvent 3 as an eluent, and concentrating the eluent to obtain crude kaempferol; and 4, recrystallizing the crude kaempferol by using an organic solvent 4 to obtain purified kaempferol. According to the method, extraction, column chromatography and recrystallization processes are combined, the process is simple, the artemisia apiacea extract residues obtained after artemisinin is extracted are used as raw materials, the method can be seamlessly connected with a production line for industrially extracting artemisinin, and the cost is saved.

Description

Technical Field

[0001] The invention belongs to the field of natural product separation engineering, relates to a method for separating and purifying kaempferol, and specifically relates to a method for separating and purifying kaempferol from artemisia annua byproducts. Background Art

[0002] Artemisia annua is the above-ground part of the Artemisia annua plant of the Asteraceae family. It has the effect of clearing away heat and detoxifying. It can be used to treat summer heat fever, yin deficiency fever, malaria cold and heat, damp-heat jaundice, etc. It contains active substances such as artemisinin and kaempferol.

[0003] Kaempferol, also known as kaempferol, is a flavonoid compound with multiple effects such as anti-cancer, anti-cancer, anti-viral, anti-bacterial, antioxidant, anti-inflammatory, and anti-ulcer. Kaempferol can scavenge free radicals and protect cells from oxidative damage. It can also inhibit inflammatory responses and reduce the production of inflammatory mediators. Kaempferol has cardiovascular protective effects and helps reduce the risk of cardiovascular disease by improving vascular function and lowering blood pressure. Kaempferol also has a broad spectrum of anti-tumor activity and has an inhibitory effect on the proliferation of liver cancer Huh7 cells, gastric cancer MGC-803 cells, and small cell lung cancer H446 cells. Due to these characteristics, kaempferol has potential application value in food, health products and drug development.

[0004] Although the prior art (Journal: Lijin Duan et al., Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae. Microb Cell Fact Microb Cell, 2017) discloses that kaempferol can be successfully synthesized by engineered microorganisms, its yield is still low. Therefore, in order to obtain a large amount of kaempferol, extraction from plant tissues is a more economical way.

[0005] There are not many extraction processes for kaempferol in the prior art. The invention patent with publication number CN110841019A discloses a method for preparing a kaempferol extract with flavonoids as an indicator, which includes the following processing steps: S1, using kaempferol rhizome as raw material, drying and then crushing into powder; S2, extracting the powder with n-hexane to remove the characteristic odor of kaempferol; S3, extracting the odor-removed kaempferol powder with acetone-water or ethanol-water solution as the extraction system, and then reducing the pressure to recover the extract; S4, adjusting the pH value of the extract after recovering part of the solvent to the range of 2.0-3.0, and continuing to heat and stir; S5, desalting the solution after S4 treatment through a macroporous resin, eluting with an ethanol-water system, and then reducing the pressure to recover the solvent of the obtained ethanol elution component, concentrating, and drying to obtain a kaempferol extract with a high flavonoid content, and the total content of kaempferol and quercetin in the final kaempferol extract is not less than 1.0%. However, the present invention does not extract kaempferol separately.

[0006] The invention patent with the publication number CN101845467A discloses a method for extracting kaempferol, which includes the following steps: (1) alkali extraction: crush the raw material, add alkali solution to dissolve, heat and extract in a boiling water bath, and cool to obtain an extract; (2) enzymatic hydrolysis: adjust the acidity of the extract to weak acidity, filter, add 0.5-1% enzyme of the raw material mass, stir and heat to 40-50°C for hydrolysis for 15-20 hours to obtain a hydrolyzate; (3) extraction: concentrate the above hydrolyzate to 1 / 10-1 / 15 of the original volume, add ethyl acetate and stir to extract, and reduce the pressure of the ethyl acetate phase to recover the ethyl acetate to obtain an extract; (4) recrystallization: the extract is recrystallized with methanol for 3 times to obtain a yellow needle-shaped crystalline product. The raw material refers to one or more of dried kaempferol, calendula, ginkgo leaf or other plants containing kaempferol and its derivatives. This scheme mainly uses enzymatic hydrolysis and recrystallization methods to extract kaempferol from plants. The enzymatic hydrolysis process is relatively slow and requires a long time to complete the hydrolysis, resulting in reduced production efficiency. In addition, the cost of enzymatic hydrolysis is high, and the requirements for reaction conditions such as temperature, pH value, and enzyme type and dosage are relatively strict. Improper control of reaction conditions may affect the quality and yield of the final product.

[0007] The extraction process of kaempferol disclosed in the prior art has strict requirements and high costs, and no scheme for directly extracting kaempferol from the byproducts of Artemisia annua extraction is disclosed. Therefore, in order to meet the needs of the market and industrialization, it is urgent to develop a method for extracting kaempferol from the byproducts of Artemisia annua. Summary of the invention

[0008] The present invention aims at the problems existing in the prior art and provides a method for separating and purifying kaempferol from artemisia annua byproducts. The present invention combines extraction, column chromatography and recrystallization processes, has a simple process, a high extraction rate and purity of kaempferol, uses the residue of artemisia annua extract after extracting artemisinin as a raw material, and can be seamlessly connected with the production line of industrial extraction of artemisinin, saving costs.

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

[0010] In a first aspect, the present invention provides a method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following steps:

[0011] Step 1: extracting the byproduct of Artemisia annua by using organic solvent 1, filtering, and concentrating the filtrate to a constant weight to obtain a concentrate 1; the Artemisia annua byproduct does not contain artemisinin;

[0012] Step 2: dissolving the concentrate 1 with an organic solvent 2, then extracting with an alkaline solution, and concentrating the organic phase layer to obtain a concentrate 2;

[0013] Step 3: The concentrate 2 is separated by column chromatography, the eluent is an organic solvent 3, and the eluate is concentrated to obtain crude kaempferol;

[0014] Step 4: Recrystallize the crude kaempferol using organic solvent 4 to obtain purified kaempferol.

[0015] Specifically, step 1: using organic solvent 1 to extract artemisia annua extraction by-products, the artemisia annua extraction by-products do not contain artemisinin, and are artemisia annua by-products after artemisinin is extracted; the organic solvent 1 is selected from one or more of methanol, ethyl acetate, ethanol, petroleum ether, and dichloromethane, preferably methanol; the mass ratio of organic solvent 1 to artemisia annua by-products is 4-8:1, preferably 5:1; the extraction temperature is 70-90°C, preferably 75°C; preferably multiple extractions are performed, the number of extractions is 2-4 times, preferably 2 times; the time for each extraction is 1-3h, preferably 1.5h; after multiple extractions, filtering, concentrating the filtrate to constant weight, the temperature of the concentrated filtrate is 60-90°C, preferably 70°C, to obtain a concentrate 1.

[0016] In one embodiment, the artemisia annua extract is subjected to column chromatography to separate artemisinin and a mixture not containing artemisinin, and the mixture not containing artemisinin is concentrated to constant weight at a concentration temperature of 60-80° C., preferably 70° C., to obtain an artemisia annua by-product. The specific treatment method of the artemisia annua extract and the column chromatography steps are not limited, and the artemisia annua by-product can be collected.

[0017] In the present invention, the residue of the artemisia annua extract after extracting artemisinin is used as the raw material, which can be seamlessly connected with the production line for industrial extraction of artemisinin, saves the investment of purchasing raw materials, and greatly reduces the cost of raw materials.

[0018] Step 2: dissolving the concentrate 1 with an organic solvent 2, wherein the solvent 2 is selected from one or more of chloroform, ethyl acetate, petroleum ether, and dichloromethane, preferably ethyl acetate, and the ratio of the concentrate 1 to the organic solvent 2 is 1kg:1-5L, preferably 1kg:2-5L, and more preferably 1kg:2L; then extracting with an alkaline solution, wherein the alkaline solution is selected from one or more of a 5% mass fraction sodium hydroxide solution, a 10% mass fraction sodium hydroxide solution, and a 10% mass fraction sodium bicarbonate solution, preferably a 5% mass fraction sodium hydroxide solution, and the volume ratio of the organic solvent 2 to the alkaline solution is 1-2:1-2, preferably 1:1, and concentrating the organic phase layer to obtain a concentrate 2.

[0019] Step three: The concentrate 2 is subjected to column chromatography separation, and the eluent is an organic solvent 3, wherein the organic solvent 3 is selected from petroleum ether and ethyl acetate in a volume ratio of 1-5:1, or a volume ratio of 1:3-8 methanol and petroleum ether, preferably a volume ratio of 1-5:1 petroleum ether and ethyl acetate, more preferably a volume ratio of 2-5:1 petroleum ether and ethyl acetate, and most preferably a volume ratio of 3:1 petroleum ether and ethyl acetate; the eluent is concentrated, and the temperature of the concentrated eluent is 60-80°C, preferably 60°C, to obtain crude kaempferol. Preferably, when column chromatography is performed, the mass ratio of silica gel to concentrate 2 is 5-10:1, more preferably 8:1. The silica gel is a type commonly used in the art, including but not limited to 100-200 mesh silica gel.

[0020] Step 4: Recrystallize the crude kaempferol using an organic solvent 4, wherein the organic solvent 4 is selected from one or more of methanol, ethyl acetate, ethanol and water, preferably ethanol, to obtain purified kaempferol. The recrystallization specifically comprises the following steps:

[0021] The crude kaempferol is dissolved by heating with organic solvent 4 at a heating temperature of 50-90° C., preferably 70° C., the mass ratio of crude kaempferol to solvent 4 is 1:3-8, preferably 1:3, and cooled for crystallization at a cooling and crystallization temperature of 0-20° C., preferably 10° C., to obtain purified kaempferol.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] First, the present invention provides for the first time a method for separating and purifying kaempferol from artemisia annua by-products by combining extraction, column chromatography and recrystallization processes. The obtained kaempferol has high purity and high yield, and the process is simple, the process conditions are mild, and the stability and repeatability are good. It has a good prospect for promotion and application. The obtained kaempferol product can be used in various fields such as cosmetics and medicines.

[0024] Secondly, the present invention uses the residual liquid of the artemisia annua extract after extracting artemisinin as raw material, which can be seamlessly connected with the production line of industrial extraction of artemisinin, saving the investment in purchasing raw materials and greatly reducing the cost of raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The liquid chromatogram of 0.1 mg / mL kaempferol reference solution; wherein the abscissa is time (unit: min) and the ordinate is response value (unit: mAU);

[0026] Figure 2 It is the liquid chromatogram of the purified kaempferol in Example 1, wherein the abscissa is time (unit: min) and the ordinate is response value (unit: mAU). DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0028] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0029] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all common commercial products, and their sources are not specifically limited. If the specific conditions are not specified in the following examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0030] In the embodiment of the present invention, the source of the artemisia annua by-product is: extracting artemisia annua with petroleum ether, separating the artemisia annua extract by column chromatography, loading the sample on a silica gel column, eluting with a mixed solution of petroleum ether and ethyl acetate, collecting a solution containing artemisinin, and then collecting a mixed solution without artemisinin, concentrating to a constant weight to obtain the artemisia annua by-product. The content of kaempferol in the mixed solution without artemisinin is 2.08%.

[0031] The above is the general concept of the present invention, and detailed embodiments are provided below to further illustrate the present invention.

[0032] Example 1

[0033] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0034] Step 1: The artemisia annua extract is separated by column chromatography to collect 10 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 9.2 kg at 70° C. to obtain an artemisia annua by-product; the mixture that does not contain artemisinin is extracted twice with 46 kg of methanol (5 times the mass) at 75° C., with each extraction time of 1.5 h. The obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70° C. to obtain 1.02 kg of concentrate 1.

[0035] Step 2: The concentrate 1 was dissolved with 2.04 L ethyl acetate (2 times), and then dissolved with 2.04 L 5% sodium hydroxide (2 times the volume), stirred, extracted and separated, and the ethyl acetate layer was concentrated to obtain 0.82 kg of concentrate 2.

[0036] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate at 60°C to constant weight to obtain 0.25 kg of crude kaempferol.

[0037] Step 4: Dissolve the crude kaempferol with 0.75 kg of ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 0.18 kg of purified kaempferol.

[0038] The structural formula of kaempferol is as follows:

[0039]

[0040] Example 2

[0041] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0042] Step 1: separate the Artemisia annua extract by column chromatography, collect 100 kg of a mixed solution that does not contain artemisinin, and concentrate it to a constant weight of 91.5 kg at 70°C; obtain Artemisia annua by-products; extract the mixture that does not contain artemisinin with 457.5 kg of methanol (5 times the mass) at 75°C twice, each extraction time is 1.5 hours, the obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; concentrate the filtrate to a constant weight at 70°C to obtain 10.5 kg of concentrate 1.

[0043] Step 2: The concentrate 1 was dissolved with 21 L of ethyl acetate (2 times), and then dissolved with 21 L of 5% sodium hydroxide (2 times the volume), stirred, extracted and separated, and the ethyl acetate layer was concentrated to obtain 8.12 kg of concentrate 2.

[0044] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate at 60°C to constant weight to obtain 2.8 kg of crude kaempferol.

[0045] Step 4: Dissolve the crude kaempferol with 8.4 kg of ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 1.81 kg of purified kaempferol.

[0046] Example 3

[0047] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0048] Step 1: The artemisia annua extract is separated by column chromatography to collect 1000 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 925 kg at 70°C to obtain an artemisia annua by-product; the mixture that does not contain artemisinin is extracted twice with 4625 kg of methanol (5 times the mass) at 75°C, each extraction time is 1.5 hours, the obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70°C to obtain 105 kg of concentrate 1.

[0049] Step 2: The concentrate 1 was dissolved with 210 L of ethyl acetate (2 times), and then dissolved with 210 L of 5% sodium hydroxide (2 times the volume), stirred, extracted and layered, and the ethyl acetate layer was concentrated to obtain 81.2 kg of concentrate 2.

[0050] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate at 60°C to constant weight to obtain 23.35 kg of crude kaempferol.

[0051] Step 4: Dissolve the crude kaempferol with 70.05 kg of ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 17.95 kg of purified kaempferol.

[0052] Comparative Example 1

[0053] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0054] Step 1: The artemisia annua extract is separated by column chromatography to collect 10 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 9.1 kg at 70°C to obtain an artemisinin by-product; the mixture that does not contain artemisinin is extracted once with 27.3 kg of methanol (3 times the mass) at 75°C, and each extraction time is 1.5 h. The obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70°C to obtain 0.75 kg of concentrate 1.

[0055] Step 2: The concentrate 1 was dissolved with 1.5 L of ethyl acetate (2 times), and then dissolved with 1.5 L of 5% sodium hydroxide solution (2 times the volume), stirred, extracted and separated, and the ethyl acetate layer was concentrated to obtain 0.52 kg of concentrate 2.

[0056] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate the collected liquid at 60°C to constant weight to obtain 0.15 kg of crude kaempferol.

[0057] Step 4: Dissolve the crude kaempferol with 0.45 kg ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 0.09 kg purified kaempferol.

[0058] Comparative Example 2

[0059] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0060] Step 1: The artemisia annua extract is separated by column chromatography to collect 10 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 9.2 kg at 70° C. to obtain an artemisia annua by-product; the mixture that does not contain artemisinin is extracted twice with 46 kg of methanol (5 times the mass) at 75° C., each extraction time is 1.5 h, the obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70° C. to obtain 1.08 kg of concentrate 1.

[0061] Step 2: The concentrate 1 was dissolved with 1.08 L ethyl acetate (1 times), and then dissolved with 1.08 L 5% sodium hydroxide (1 times the volume), stirred, extracted and separated, and the ethyl acetate layer was concentrated to obtain 0.39 kg of concentrate 2.

[0062] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate at 60°C to constant weight to obtain 0.21 kg of crude kaempferol.

[0063] Step 4: Dissolve the crude kaempferol with 0.63 kg of ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 0.12 kg of purified kaempferol.

[0064] Comparative Example 3

[0065] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0066] Step 1: The artemisia annua extract is separated by column chromatography to collect 10 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 9.1 kg at 70° C. to obtain an artemisia annua by-product; the mixture that does not contain artemisinin is extracted twice with 45.5 kg of methanol (5 times the mass) at 75° C., each extraction time is 1.5 h, the obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70° C. to obtain 1.02 kg of concentrate 1.

[0067] Step 2: The concentrate 1 was dissolved with 2.04 L ethyl acetate (2 times), and then dissolved with 2.04 L 5% sodium hydroxide (2 times the volume), stirred, extracted and separated, and the ethyl acetate layer was concentrated to obtain 0.82 kg of concentrate 2.

[0068] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 6:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:2. Collect from the second column volume, and concentrate at 60°C to constant weight to obtain 0.31 kg of crude kaempferol.

[0069] Step 4: Dissolve the crude kaempferol with 0.93 kg of ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 0.19 kg of purified kaempferol.

[0070] Comparative Example 4

[0071] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0072] Step 1: The Artemisia annua extract is separated by column chromatography to collect 10 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 9.1 kg at 70°C to obtain an Artemisia annua by-product; the mixture that does not contain artemisinin is extracted twice with 46 kg of methanol (5 times the mass) at 75°C, each extraction time is 1.5 h, the obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70°C to obtain 1.01 kg of concentrate 1.

[0073] Step 2: The concentrate 1 was dissolved with 2.02 L of ethyl acetate (2 times), and then dissolved with 2.02 L of 5% by mass sodium bicarbonate solution (2 times the volume), stirred, extracted and layered, and the ethyl acetate layer was concentrated to obtain 0.62 kg of concentrate 2.

[0074] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate the collected liquid at 60°C to constant weight to obtain 0.16 kg of crude kaempferol.

[0075] Step 4: Dissolve the crude kaempferol with 0.48 kg ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 0.10 kg of purified kaempferol.

[0076] Comparative Example 5

[0077] A method for separating and purifying kaempferol from artemisia annua byproducts, comprising the following specific steps:

[0078] Step 1: The Artemisia annua extract is separated by column chromatography to collect 10 kg of a mixed solution that does not contain artemisinin, and concentrated to a constant weight of 9.1 kg at 70°C to obtain an Artemisia annua by-product; the mixture that does not contain artemisinin is extracted twice with 46 kg of methanol (5 times the mass) at 75°C, each extraction time is 1.5 h, the obtained extract is cooled, filtered, and the filtrates obtained from the two extractions are combined; the filtrate is concentrated to a constant weight at 70°C to obtain 1.01 kg of concentrate 1.

[0079] Step 2: The concentrate 1 was dissolved with 2.02 L of ethyl acetate (2 times), and then dissolved with 2.02 L of 10% mass fraction sodium hydroxide solution (2 times volume), stirred, extracted and layered, and the ethyl acetate layer was concentrated to obtain 0.62 kg of concentrate 2.

[0080] Step 3: Mix the concentrate 2 with 100-200 mesh silica gel, and then load it into a column. The mass ratio of silica gel to concentrate 2 is 8:1. Elution is performed. The eluent is petroleum ether and ethyl acetate in a volume ratio of 3:1. Collect from the second column volume, and concentrate the collected liquid at 60°C to constant weight to obtain 0.16 kg of crude kaempferol.

[0081] Step 4: Dissolve the crude kaempferol with 0.48 kg ethanol (3 times the mass) at 70° C., filter, cool to 10° C. for crystallization, and filter to obtain 0.11 kg of purified kaempferol.

[0082] Test example

[0083] The kaempferol content (i.e., the content of kaempferol in kaempferol) prepared in Examples 1-3 and Comparative Examples 1-5 was detected by the following method:

[0084] Determined according to high performance liquid chromatography (General Rule 0512);

[0085] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.1% glacial acetic acid solution (65:35) is used as mobile phase; detection wavelength is 240nm, temperature is 30℃, detection time is 40min, and the number of theoretical plates calculated by peak should be no less than 2000;

[0086] Preparation of reference solution: accurately weigh an appropriate amount of kaempferol reference substance and add methanol to make a solution containing 0.1 mg per 1 ml.

[0087] Preparation of the test solution: Take 2 portions of kaempferol from Examples 1-3 and Comparative Examples 1-5 (precisely weighed, placed in a stoppered conical flask, accurately add 10 ml of methanol, weigh the weight, ultrasonically treat (power 250 W, frequency 25 kHz) for 15 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and obtain;

[0088] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0089] The liquid chromatogram of 0.1 mg / mL kaempferol reference solution is as follows: Figure 1 As shown, the liquid chromatogram of the purified kaempferol in Example 1 is as shown in Figure 2 The purity of the purified kaempferol and the yield of the purified kaempferol are shown in Table 1 below.

[0090] Table 1

[0091]

[0092]

[0093] The results in Table 1 show that the determination results are relatively stable, the kaempferol in Examples 1-3 has high purity and high yield, and the kaempferol is separated and purified from 10-1000 kg of Artemisia annua by-products, with high process stability and good repeatability.

[0094] 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 separating and purifying kaempferol from artemisia annua byproducts, characterized in that: The following steps are involved: Step 1: extracting the byproduct of Artemisia annua by using organic solvent 1, filtering, and concentrating the filtrate to a constant weight to obtain a concentrate 1; the Artemisia annua byproduct does not contain artemisinin; Step 2: dissolving the concentrate 1 with an organic solvent 2, then extracting with an alkaline solution, and concentrating the organic phase layer to obtain a concentrate 2; Step 3: The concentrate 2 is separated by column chromatography, the eluent is an organic solvent 3, and the eluate is concentrated to obtain crude kaempferol; Step 4: Recrystallize the crude kaempferol using organic solvent 4 to obtain purified kaempferol.

2. The method according to claim 1, characterized in that: In the step 1, the organic solvent 1 is selected from one or more of methanol, ethyl acetate, ethanol, petroleum ether, and dichloromethane; the mass ratio of the organic solvent 1 to the Artemisia annua by-product is 4-8:1; the number of extractions is 2-4 times; and the temperature of the concentrated filtrate is 60-90°C.

3. The method according to claim 1, characterized in that: In the step 2, the organic solvent 2 is selected from one or more of chloroform, ethyl acetate, petroleum ether, and dichloromethane; the alkaline solution is selected from one or more of 5% by mass sodium hydroxide solution, 10% by mass sodium hydroxide solution, and 10% by mass sodium bicarbonate solution; the ratio of the concentrate 1 to the organic solvent 2 is 1kg:1-5L; the volume ratio of the organic solvent 2 to the alkaline solution is 1-2:1-2.

4. The method according to claim 3, characterized in that: In the step 2, the organic solvent 2 is ethyl acetate; the alkaline solution is selected from a 5% mass fraction sodium hydroxide solution; the ratio of the concentrate 1 to the organic solvent 2 is 1kg:2-5L; the volume ratio of the organic solvent 2 to the alkaline solution is 1-2:1-2.

5. The method according to claim 1, characterized in that: In the step 3, the organic solvent 3 is selected from petroleum ether and ethyl acetate in a volume ratio of 1-5:1, or methanol and petroleum ether in a volume ratio of 1:3-8; the temperature of the concentrated eluent is 60-80°C.

6. The method according to claim 5, characterized in that: The organic solvent 3 is petroleum ether and ethyl acetate in a volume ratio of 1-5:

1.

7. The method according to claim 6, characterized in that: The organic solvent 3 is petroleum ether and ethyl acetate in a volume ratio of 2-5:

1.

8. The method according to claim 1, characterized in that: The organic solvent 4 is selected from one or more of methanol, ethyl acetate, ethanol and water.

9. The method according to claim 1, characterized in that: When column chromatography separation is performed in step 3, the mass ratio of silica gel to concentrate 2 is 5-10:

1.

10. The method according to claim 1, characterized in that: In the step 4, the recrystallization specifically comprises the following steps: The crude kaempferol is dissolved by heating with organic solvent 4 at a heating temperature of 50-90° C., the mass ratio of the crude kaempferol to solvent 4 is 1:3-8, and the mixture is cooled and crystallized at a cooling and crystallization temperature of 0-20° C. to obtain purified kaempferol.

Images