A method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid

Abstract

The present disclosure relates to a method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid, comprising the following steps: S1. ultrasonically extracting a raw material containing artemisinic acid and dihydroartemisinic acid to obtain a first crude extract; S2. extracting the first crude extract with a solvent to obtain a second crude extract; S3. preparing a sample solution of the second crude extract, and separating the components in the sample solution by reversed-phase high-performance liquid chromatography to obtain an artemisinic acid component and a dihydroartemisinic acid component; wherein, the mobile phase is selected from acetonitrile-acetic acid aqueous solution; S4. respectively dissolving, filtering the artemisinic acid component and the dihydroartemisinic acid component, and concentrating the supernatant to obtain purified artemisinic acid monomer and dihydroartemisinic acid monomer. The solution of the present disclosure can quickly remove large-polarity impurities in artemisinic acid and dihydroartemisinic acid, and obtain artemisinic acid and dihydroartemisinic acid with a purity of more than 98%. Using a mobile phase containing acetic acid ensures the stability of artemisinic acid and dihydroartemisinic acid.

Description

Technical Field

[0001] The present disclosure relates to the technical field of separation and purification of plant components, and particularly relates to a method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid. Background Art

[0002] The artemisia annua described in this patent is the aerial part of the artemisia annua L. of the Compositae family, and is the main extraction source of the highly effective antimalarial drug artemisinin. All along, people often ignore the sesquiterpene components such as artemisinic acid, dihydroartemisinic acid, deoxyartemisinin, and artemisinin I in artemisia annua because of the star component artemisinin in artemisia annua. Currently, commercial artemisinin is all extracted, separated, refined and prepared from artemisia annua, but most of the valuable compounds in artemisia annua are discarded, resulting in a great waste of resources. For example, artemisinic acid and dihydroartemisinic acid in artemisia annua have biological activities such as anti-tumor, anti-inflammatory, antibacterial, inhibiting fat generation, and whitening, and their contents in artemisia annua are much higher than that of artemisinin, having great development and utilization value.

[0003] At the same time, the artemisia described in this patent is artemisia caruifolia Buch.-Ham.ex Roxb. of the Compositae family, which also contains artemisinic acid and dihydroartemisinic acid, and its selling price is lower than that of artemisia annua. Using artemisia to prepare artemisinic acid and dihydroartemisinic acid can not only reduce costs, but also improve the utilization value of artemisia.

[0004] Currently, the preparation of artemisinic acid and dihydroartemisinic acid mainly uses artemisia annua or the medicinal residues after extracting artemisinin from artemisia annua for extraction, and then uses methods such as organic solvent-alkali-acid-organic solvent, silica gel column chromatography, preparative liquid phase purification, and recrystallization for separation and purification. For example, in the prior art "Extraction Process of Artemisinic Acid and Dihydroartemisinic Acid in Artemisia annua Mother Liquid" (Chinese Journal of Pharmaceuticals. 2015, 46(2): 149-151), artemisia annua waste liquid was used as the raw material, and ethanol extraction, decolorization, alkali saponification, acidification, and recrystallization were used to obtain artemisinic acid and dihydroartemisinic acid monomers. In addition, artemisinic acid and dihydroartemisinic acid have also been separated and purified by high performance liquid chromatography and other methods.

[0005] Due to the certain instability of the chemical structure of dihydroartemisinic acid, the separation and purification processes will destroy the stability of dihydroartemisinic acid and reduce the yield of dihydroartemisinic acid. And the mobile phase in the separation process by liquid chromatography will also affect the separation effect of artemisinic acid and dihydroartemisinic acid, increasing the instability of artemisinic acid and dihydroartemisinic acid in the separation process. Summary of the Invention

[0006] The purpose of the present disclosure is to improve the stability of artemisinic acid and dihydroartemisinic acid in the separation and purification processes.

[0007] To achieve the above object, the present disclosure adopts the following technical solutions:

[0008] A method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid, comprising the following steps:

[0009] S1. Ultrasonically extract the raw material containing artemisinic acid and dihydroartemisinic acid to obtain a first crude extract;

[0010] S2. Extract the first crude extract with a solvent to obtain a second crude extract;

[0011] S3. Prepare an injection solution of the second crude extract, and separate the components in the injection solution by reverse-phase high performance liquid chromatography to obtain an artemisinic acid component and a dihydroartemisinic acid component; wherein, the mobile phase is selected from acetonitrile - acetic acid aqueous solution;

[0012] S4. Dissolve and filter the artemisinic acid component and the dihydroartemisinic acid component respectively, and concentrate the supernatant to obtain purified artemisinic acid monomer and dihydroartemisinic acid monomer.

[0013] Preferably, in step S1, the raw material is soaked with 8 - 20 times the volume of 70 - 100% ethanol and then subjected to the ultrasonic extraction. The number of times of ultrasonic extraction is 2 times, and the extraction time for each time is 30 - 90 minutes.

[0014] More preferably, the raw material is soaked with 8 - 15 times the volume of 95% ethanol and then subjected to the ultrasonic extraction, and the extraction time for each time is 60 - 90 minutes.

[0015] Preferably, in step S2, the solvent is petroleum ether.

[0016] More preferably, step S2 includes:

[0017] S21. Place the first crude extract in water to form a suspension;

[0018] S22. Extract the suspension with the petroleum ether;

[0019] S23. Wash the organic phase with water after combining the organic phases;

[0020] S24. Dry and filter the organic phase to remove the petroleum ether to obtain the second crude extract.

[0021] Preferably, in step S3, the acetic acid aqueous solution contains 0.05% - 0.2% by volume of acetic acid, the flow rate of the mobile phase is 6 - 10 mL / min, the volume ratio of the acetonitrile to the acetic acid aqueous solution is 50 - 90:50 - 10, and the injection volume of the injection solution is 100 - 2000 μL.

[0022] More preferably, the aqueous acetic acid solution contains acetic acid with a volume fraction of 0.1%, the flow rate of the mobile phase is 10 mL / min, the volume ratio of the acetonitrile to the aqueous acetic acid solution is 70:30, and the injection volume of the injection solution is 400 - 2000 μL.

[0023] Preferably, the inner diameter of the reverse-phase column is 20 mm, the length is 250 mm, the column temperature is 40°C, and the detection wavelength is 220 nm.

[0024] More preferably, in step S3, the second crude extract is dissolved in acetonitrile, and the acetonitrile solution of the second crude extract is filtered through a filter paper and then through a 0.45-μm filter membrane to obtain the injection solution.

[0025] More preferably, the raw materials include plants or extracts containing artemisinic acid and dihydroartemisinic acid, including Artemisia annua, Artemisia annua var. annua, and the waste liquid and residue after extracting artemisinin from Artemisia annua var. annua.

[0026] The technical solutions claimed in the present disclosure have achieved the following beneficial effects:

[0027] 1) Extracting the first crude extract obtained by ultrasonic extraction can remove large-polarity and water-soluble impurities in the extract. Redissolving and filtering the artemisinic acid component and dihydroartemisinic acid component separated by reverse-phase high-performance liquid chromatography can further remove large-polarity impurities in artemisinic acid and dihydroartemisinic acid, and finally obtain high-purity artemisinic acid and dihydroartemisinic acid with a product purity of over 98%.

[0028] 2) Adding a mobile phase containing acetic acid in reverse-phase high-performance liquid chromatography can inhibit the dissociation of sample components, increase the retention of components on the stationary phase, and ensure the stability of artemisinic acid and dihydroartemisinic acid while accelerating the separation effect.

[0029] 3) The preparation process has a large sample loading amount and a fast separation speed. The sample loading amount per time can be as high as 2000 μL, and the complete separation of the crude extract can be completed within 30 minutes, and the separated artemisinic acid and dihydroartemisinic acid have high purity. Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only the embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0031] Figure 1 It is a chromatogram for separating dihydroartemisinic acid reference standard and artemisinic acid reference standard by high-performance liquid chromatography.

[0032] Figure 2 1H NMR spectrum of artemisinic acid 1

[0033] Figure 3 13C NMR spectrum of artemisinic acid 13

[0034] Figure 4 1H NMR spectrum of dihydroartemisinic acid 1

[0035] Figure 5 13C NMR spectrum of dihydroartemisinic acid 13

[0036] Figure 6 HPLC chromatogram of methanol

[0037] Figure 7 HPLC chromatogram of artemisinic acid sample

[0038] Figure 8 HPLC chromatogram of dihydroartemisinic acid sample

[0039] Figure 9 Chromatogram with mixed chromatographic peaks of Artemisia annua extract sample in Comparative Example 1 Specific Embodiments

[0040] To make the objectives, technical solutions and beneficial effects of the embodiments in the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present disclosure. Obviously, the described embodiments are a part rather than all of the embodiments of the present disclosure. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present disclosure without creative efforts shall fall within the scope of protection of the present disclosure.

[0041] Embodiments

[0042] This embodiment provides a method for simultaneously obtaining high-purity artemisinic acid and dihydroartemisinic acid, which specifically includes the following steps:

[0043] (1) Extraction of artemisinic acid and dihydroartemisinic acid: The raw material containing artemisinic acid and dihydroartemisinic acid is ultrasonically extracted twice with 8-20 times the volume of 80-95% ethanol, and the extraction time for each extraction is 30-90 min. The extract is concentrated to dryness to obtain a first crude extract. Among them, the raw material used in this embodiment is a plant or extract containing artemisinic acid and dihydroartemisinic acid, including Artemisia annua, Artemisia annua L., and the waste residue and waste liquid after extracting artemisinin from Artemisia annua L.

[0044] ​​​​(2) Preliminary impurity removal: The first crude extract is placed in water to form a suspension, which is extracted with petroleum ether. The organic phases are combined, dried over anhydrous sodium sulfate, and filtered. The petroleum ether is removed by rotary evaporation to obtain the second crude extract. This step can remove some of the large-polarity and water-soluble impurities in the artemisinic acid and dihydroartemisinin extracts.

[0045] (3) Preparation of injection solution: The second crude extract sample obtained in step (2) is dissolved in acetonitrile, filtered through a filter paper, and then passed through a 0.45 μm filter membrane to obtain the injection solution.

[0046] (4) Liquid-phase purification: The artemisinic acid and dihydroartemisinin in the injection solution are separated by reverse-phase high-performance liquid chromatography.

[0047] The separation conditions are as follows:

[0048] Chromatographic column: Shimadzu PRC-ODS C18 reverse-phase column, with an inner diameter of 20 mm and a length of 250 mm;

[0049] Chromatographic conditions: The mobile phase is an aqueous solution of acetonitrile - acetic acid with a volume fraction of 0.05% - 0.2%, with a volume ratio of 50 - 90:50 - 10, a flow rate of 6 - 10 mL / min, a detection wavelength of 220 nm, a column temperature of 40 °C, and an injection volume of 100 - 2000 μL.

[0050] After separation, it is vacuum concentrated until the solvent evaporates completely to obtain the artemisinic acid component and the dihydroartemisinin component;

[0051] (5) Filtration: To further remove the large-polarity impurities in artemisinic acid and dihydroartemisinin and improve the purity of artemisinic acid and dihydroartemisinin, the two components obtained in step (4) are separately dissolved in petroleum ether and filtered. The supernatant is vacuum concentrated to evaporate the petroleum ether completely, and high-purity artemisinic acid and dihydroartemisinin monomers are obtained respectively.

[0052] In step (4) above, the elution peak times of artemisinic acid and dihydroartemisinin are determined by high-performance liquid chromatography. When separating for the first time, the fractions of each chromatographic peak are collected. 10 μL of each fraction is injected into an analytical high-performance liquid phase, and analyzed under the above chromatographic conditions (the chromatographic column is ZORBAX SB-C18 (4.6 mm * 250 mm, 5 μm)). At the same time, artemisinic acid and dihydroartemisinin reference standards are prepared into solutions, and the elution peak times (retention times) of artemisinic acid and dihydroartemisinin reference standards are determined by high-performance liquid chromatography under the same chromatographic conditions above to judge the fractions where artemisinic acid and dihydroartemisinin are located, and the area normalization method is used to judge the purity. For subsequent separations, only the fractions corresponding to the corresponding time are collected.

[0053] The following further clearly describes the solutions in the examples in combination with specific application examples.

[0054] Application Example 1

[0055] This application example provides a method for simultaneously obtaining high-purity artemisinic acid and dihydroartemisinin, which specifically includes the following steps:

[0056] S1. Weigh 100 g of Artemisia annua, add 800 mL of 95% ethanol, extract ultrasonically twice, each time for 1 h, combine the extracts, filter, and recover the solvent under vacuum to obtain 4.6 g of the first crude extract;

[0057] S2. Add 20 mL of water to the crude extract obtained in step S1 to make a suspension, extract successively with 20 mL of petroleum ether, combine the organic phases, wash the organic phases with 20 mL of water, dry the organic phases with anhydrous sodium sulfate, and recover the solvent under vacuum to obtain 3 g of the second crude extract after preliminary impurity removal;

[0058] S3. Dissolve the second crude extract obtained in step S2 with 4 mL of acetonitrile, filter through a 0.45 μm microporous membrane, and load it onto a preparative high-performance liquid chromatography. The following preparative chromatography conditions are used: Shimadzu PRC-ODS, reverse-phase column, inner diameter 20 mm, length 250 mm; mobile phase: acetonitrile / 0.1% water (volume ratio 70:30); flow rate 10 mL / min; detection wavelength 220 nm; column temperature 40°C; injection volume 400 μL.

[0059] When separating for the first time, collect the fractions of each chromatographic peak. Take 10 μL of each fraction and inject it into an analytical high-performance liquid chromatography, and analyze it under the above chromatographic conditions (the chromatographic column is ZORBAX SB-C18 (4.6 mm * 250 mm, 5 μm)). At the same time, prepare solutions of artemisinic acid and dihydroartemisinin standards, and determine the peak time / retention time Figure 1 () of artemisinic acid and dihydroartemisinin standards by high-performance liquid chromatography under the same above chromatographic conditions to judge the fractions where artemisinic acid and dihydroartemisinin are located, and use the area normalization method to judge the purity. For subsequent separations, only collect the fractions within the corresponding time. At this time, the purities of artemisinic acid and dihydroartemisinin are 89% and 70% respectively.

[0060] For subsequent separations, only collect the fractions where artemisinic acid and dihydroartemisinin are located, combine the fractions of dihydroartemisinin and artemisinic acid, recover the solvent under vacuum, dissolve with 5 mL of petroleum ether, filter, take the supernatant and concentrate and dry it to obtain pure products of artemisinic acid and dihydroartemisinin. The weights of the products are 0.21 g and 0.07 g respectively, and the purities are 99.6% and 99.8% respectively.

[0061] S4. Identify the structures of artemisinic acid and dihydroartemisinin obtained in step S3:

[0062] Artemisinic acid minus hydrogen [C 15 H 22 O2-H]- The theoretical exact mass number is 233.1542. The exact mass number of the hydrogen-depleted artemisinic acid obtained in step S3 of this example measured by high-resolution mass spectrometry is 233.1546, and the two data are basically the same; for dihydroartemisinic acid minus hydrogen 15 H 24 O2-H] - the theoretical exact mass number is 235.1698, and the exact mass number of the hydrogen-depleted dihydroartemisinic acid obtained in step S3 of this example measured by high-resolution mass spectrometry is 235.1700, and the two data are basically the same.

[0063] Take 20 mg of artemisinic acid and dihydroartemisinic acid obtained in step S3 respectively in a nuclear magnetic tube, add 0.6 mL of deuterated chloroform for dissolution, and perform 1 H, 13 C NMR scans. The 1 H, 13 C NMR spectra of artemisinic acid are as shown in Figure 2 , Figure 3 shown, and the 1 H, 13 C NMR spectra of dihydroartemisinic acid are as shown in Figure 4 , Figure 5 shown.

[0064] Artemisinic acid 1 1H NMR(CDCl3): δ(ppm) 6.46(s, 1H, H-13), 5.56(s, 1H, H-13), 4.98(s, 1H, H-5), 2.70(dt, J = 12, 3.6 Hz, 1H, H-7), 2.61(s, 1H, H-6), 1.93(m, 1H, H-2), 1.87(dd, J = 12.0, 4.2 Hz, 1H, H-3), 1.77(dd, J = 16.8, 5.4 Hz, 1H, H-3), 1.71(dd, J = 12.6, 3.0 Hz, 1H, H-9), 1.59(s, 3H, H-15), 1.54(m, 1H, H-2), 1.41(m, 3H, H-10, H-8, H-1), 1.35(m, 1H, H-8), 1.07(m, 1H, H-9), 0.90(d, J = 6.0 Hz, 3H, H-14); 1313C NMR δ (ppm): 172.8 (COOH), 142.6 (C-11), 135.0 (C-4), 126.7 (C-13), 120.2 (C-5), 42.0 (C-7), 41.4 (C-1), 37.9 (C-6), 35.2 (C-9), 27.6 (C-10), 26.4 (C-3), 26.0 (C-8), 25.6 (C-2), 23.7 (C-15), 19.8 (C-14);

[0065] Dihydroartemisinic acid 1 1H NMR (CDCl3): δ (ppm) 5.12 (s, 1H, H-5), 2.50 (m, 2H, H-11 and H-6), 1.95 (m, 2H, H-9 and H-8), 1.81 (m, 1H, H-8), 1.64 (s, 3H, H-15), 1.61 (m, 2H, H-2 and H-1), 1.43 (m, 1H, H-10), 1.27 (m, 1H, H-7), 1.19 (d, J = 6.6 Hz, 3H, H-13), 1.11 (m, 1H, H-3), 0.97 (m, 1H, H-2), 0.87 (d, J = 6.6 Hz, 3H, H-14); 13 13C NMR δ (ppm): 183.9 (COOH), 136.0 (C-4), 119.2 (C-5), 43.6 (C-1), 42.3 (C6), 41.7 (C-7), 36.3 (C-11), 35.2 (C-2), 27.7 (C-10), 27.4 (C-3), 26.6 (C-8), 25.8 (C-9), 23.8 (C-15), 19.6 (C-14), 15.1 (C-13).

[0066] The above structural identification results indicate that the separation and purification method provided in this example can simultaneously obtain artemisinic acid and dihydroartemisinic acid with high purity and stable structure.

[0067] Application Example 2

[0068] This application example analyzes the purity of artemisinic acid and dihydroartemisinic acid obtained in Example 1.

[0069] Preparation method of test sample: Accurately weigh 2.0 mg of the pure artemisinic acid and dihydroartemisinic acid obtained in Example 1 respectively and place them in a 5 mL volumetric flask, add methanol to dissolve and make up the volume to 5 mL, and filter through a 0.45 μm filter membrane to obtain the test sample solution for the purity detection of artemisinic acid and dihydroartemisinic acid.

[0070] Chromatographic conditions: Agilent 1260 high performance liquid chromatograph, DAD diode array detector, chromatographic column: ZORBAX SB-C18 (4.6 mm * 250 mm, 5 μm); mobile phase: acetonitrile: 0.1% acetic acid-water (72:28 v / v); flow rate: 1.0 mL / min; column temperature: 30 °C; detection wavelength: 204 nm; injection volume: 10 μL;

[0071] Purified water (Wahaha), acetonitrile (chromatographic grade, Fisher).

[0072] First, measure the chromatographic peaks of the solvent (methanol) used, then measure the chromatographic peaks of the artemisinic acid and dihydroartemisinic acid sample solutions, and perform area normalization quantification on the sample chromatographic peaks after deducting the solvent chromatographic peaks. The chromatographic conditions should ensure that all impurities in the sample can be separated from the dihydroartemisinic acid peak, and the retention time of the peak is appropriate. The sample running time is at least 3 times the retention time of the chromatographic peak.

[0073] The HPLC chromatogram of the solvent (methanol) used is as Figure 6 shown; the HPLC chromatogram of the artemisinic acid sample is as Figure 7 shown, and the peak table is shown in Table 1; the HPLC chromatogram of the dihydroartemisinic acid sample is as Figure 8 shown, and the peak table is shown in Table 2.

[0074] Table 1: Peak table of artemisinic acid sample detected by HPLC

[0075]

[0076] Table 2: Peak table of dihydroartemisinic acid sample detected by HPLC

[0077]

[0078] The above results show that the separation and purification method in Application Example 1 can obtain high-purity artemisinic acid and dihydroartemisinic acid simultaneously.

[0079] Application Example 3

[0080] This application example provides a method for simultaneously obtaining high-purity artemisinic acid and dihydroartemisinic acid, which specifically includes the following steps:

[0081] S1. Weigh 100 g of Artemisia annua, add 1500 mL of 95% ethanol, extract by ultrasound 2 times, 1.5 h each time, combine the extracts, filter, and recover the solvent under vacuum to obtain 5.7 g of the first crude extract;

[0082] S2. Suspend the first crude extract obtained in step S2 in 30 mL of water, extract successively with 30 mL of petroleum ether, combine the organic phases, wash the organic phases with 30 mL of water, dry the organic phases with anhydrous sodium sulfate, and recover the solvent under vacuum to obtain 4.3 g of the second crude extract after preliminary impurity removal;

[0083] S4. Dissolve the second crude extract obtained in step S3 in 8 mL of acetonitrile, filter through a 0.45 μm microporous membrane, and load onto a preparative high performance liquid chromatography. The following preparative chromatography conditions are adopted: use Shimadzu PRC-ODS, a reverse phase column with an inner diameter of 20 mm and a length of 250 mm; the mobile phase is acetonitrile / 0.1% water (volume ratio 70:30); the flow rate is 10 mL / min; the detection wavelength is 220 nm; the column temperature is 40 °C; the injection volume is 1500 μL.

[0084] The method for judging the fractions and purities of artemisinic acid and dihydroartemisinic acid is the same as that in Example 2. At this time, the purities of artemisinic acid and dihydroartemisinic acid are 91% and 67% respectively; combine the fractions of dihydroartemisinic acid and artemisinic acid, recover the solvent under vacuum, dissolve in 5 mL of petroleum ether, filter, take the supernatant and concentrate and dry to obtain pure products of artemisinic acid and dihydroartemisinic acid. The product weights are 0.22 g and 0.05 g respectively, and the purities are 99.3% and 99.4% respectively. The structural identification of the prepared compound is the same as that in Example 1.

[0085] Application Example 4

[0086] This application example provides a method for simultaneously obtaining high-purity artemisinic acid and dihydroartemisinic acid, which specifically includes the following steps:

[0087] S1. Weigh 200 g of Artemisia annua, add 4000 mL of 95% ethanol, extract ultrasonically 2 times, 30 min each time, combine the extracts, filter, and recover the solvent under vacuum to obtain 13.4 g of the first crude extract;

[0088] S2. Suspend the first crude extract obtained in step S2 in 70 mL of water, extract successively with 70 mL of petroleum ether, combine the organic phases, wash the organic phases with 60 mL of water, dry the organic phases with anhydrous sodium sulfate, and recover the solvent under vacuum to obtain 11.5 g of the second crude extract after preliminary impurity removal;

[0089] S4. Dissolve the second crude extract obtained in step S3 in 20 mL of acetonitrile, filter through a 0.45 μm microporous membrane, and load onto a preparative high performance liquid chromatography. The following preparative chromatography conditions are adopted: use Shimadzu PRC-ODS, a reverse phase column with an inner diameter of 20 mm and a length of 250 mm; the mobile phase is acetonitrile / 0.1% water (volume ratio 70:30); the flow rate is 10 mL / min; the detection wavelength is 220 nm; the column temperature is 40 °C; the injection volume is 2000 μL.

[0090] The judgment method for the fractions and purities of artemisinic acid and dihydroartemisinic acid is the same as that in Example 2. At this time, the purities of artemisinic acid and dihydroartemisinic acid are 87% and 63% respectively. The fractions of dihydroartemisinic acid and artemisinic acid are combined, the solvent is recovered under vacuum, dissolved in 10 mL of petroleum ether, filtered, the supernatant is taken and concentrated and dried to obtain pure products of artemisinic acid and dihydroartemisinic acid. The weights of the products are 0.54 g and 0.12 g respectively, and the purities are 98.6% and 99.1% respectively. The structural identification of the obtained compound is the same as that in Example 1.

[0091] Comparative Example 1

[0092] S1. Weigh 100 g of dry Artemisia annua samples, add 800 mL of petroleum ether, reflux and extract at 60 °C for 3 times, 1 h each time. Combine the extracts, filter, and recover the petroleum ether under vacuum to obtain 4.3 g of crude extract.

[0093] S2. Resuspend 1.5 mL of acetonitrile in the crude extract obtained in step S1, filter by vacuum, pass the filtrate through a 0.45 μm microporous filter membrane, and load it onto a preparative high-performance liquid chromatography. The following preparative chromatography conditions are used: Shimadzu PRC-ODS, reverse-phase column, inner diameter 20 mm, length 250 mm; the mobile phase is acetonitrile / water (volume ratio 60:40); the flow rate is 10 mL / min; the detection wavelength is 192 nm; the column temperature is 40 °C; the injection volume is 400 μL. The chromatographic peaks of the samples in this comparative example are mixed (as Figure 9 shown), and artemisinic acid and dihydroartemisinic acid are not separated.

[0094] Comparative Example 2

[0095] S1. Weigh 20 g of dry Artemisia annua samples, add 40 mL of 95% ethanol, reflux and extract at a hot temperature for 3 times, 1 h each time. Combine the extracts, filter, concentrate and recover the solvent, and dissolve it in petroleum ether.

[0096] S2. Add an equal volume of 5% NaOH solution to the petroleum ether solution in step S1 and extract 3 times, let it stand for layer separation, separate, and combine the aqueous phases.

[0097] S3. Add dilute hydrochloric acid to the aqueous phase obtained in step S2 to adjust the pH to 3, add ethyl acetate and extract 3 times, let it stand for layer separation, separate, and combine the organic phases.

[0098] S4. Add anhydrous sodium sulfate to dry the organic phase, concentrate and recover the solvent to obtain the refined Artemisia annua after pretreatment.

[0099] S5. The Artemisia annua L. refined product was added to a pre-treated silica gel column, and eluted with an eluent of petroleum ether:ethyl acetate at 10:1 (volume ratio). TLC (thin layer chromatography) detection was carried out, the eluates of artemisinic acid and dihydroartemisinin were collected and dried. The contents and purities of artemisinic acid and dihydroartemisinin in the extracts obtained in each extraction step during the preparation process were determined, and the determination method was the same as that in Example 2. The determination results are shown in Table 3.

[0100] Table 3: Determination results of artemisinic acid and dihydroartemisinin in each extraction step in Comparative Example 2

[0101]

[0102] As can be seen from Table 3, the addition of alkali saponification and hydrochloric acid acidification during the separation and purification process resulted in a large loss of artemisinic acid and dihydroartemisinin, and high-purity dihydroartemisinin could not be obtained by silica gel column chromatography.

[0103] The extraction scheme provided by the present disclosure performs extraction on the first crude extract obtained by ultrasonic extraction to preliminarily remove large-polarity and water-soluble impurities in the extract. Then, the artemisinic acid component and dihydroartemisinin component separated by reversed-phase high-performance liquid chromatography are redissolved and filtered to further remove large-polarity impurities in artemisinic acid and dihydroartemisinin, and finally high-purity artemisinic acid and dihydroartemisinin are obtained, with the product purity above 98%. Moreover, since a mobile phase containing acetic acid is used in reversed-phase high-performance liquid chromatography, it can inhibit the dissociation of sample components, increase the retention of components on the stationary phase, and ensure the stability of artemisinic acid and dihydroartemisinin while accelerating the separation effect.

[0104] The above-described embodiments and application examples are only exemplary descriptions of the present disclosure, and do not limit the scope of the present disclosure. Without departing from the design spirit of the present disclosure, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present disclosure shall fall within the protection scope determined by the present disclosure.

Claims

1. A method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid, characterized in that, It includes the following steps: S1. Ultrasonically extract the raw material containing artemisinic acid and dihydroartemisinic acid to obtain a first crude extract; S2. Extract the first crude extract with petroleum ether to obtain a second crude extract; S3. Prepare the sample solution of the second crude extract, separate the components in the sample solution by reverse-phase high performance liquid chromatography to obtain artemisinic acid component and dihydroartemisinic acid component; wherein, the mobile phase is acetonitrile - acetic acid aqueous solution; dissolve the second crude extract with acetonitrile, filter the acetonitrile solution of the second crude extract with filter paper and then filter it through a 0.45 μm filter membrane to obtain the sample solution; S4. Dissolve and filter the artemisinic acid component and the dihydroartemisinic acid component respectively, and concentrate the supernatant to obtain purified artemisinic acid monomer and dihydroartemisinic acid monomer.

2. The method according to claim 1, characterized in that, In step S1, soak the raw material with 8 - 20 times the volume of 70 - 100% ethanol and then carry out the ultrasonic extraction. The number of times of ultrasonic extraction is 2 times, and the extraction time for each time is 30 - 90 minutes.

3. The method according to claim 2, wherein Soak the raw material with 8 - 15 times the volume of 95% ethanol and then carry out the ultrasonic extraction. The extraction time for each time is 60 - 90 minutes.

4. The method according to claim 1, wherein Step S2 includes: S21. Place the first crude extract in water to form a suspension; S22. Extract the suspension with the petroleum ether; S23. Wash the organic phase with water after combining the organic phases; S24. Dry and filter the organic phase to remove the petroleum ether to obtain the second crude extract.

5. The method according to claim 1, wherein In step S3, the aqueous acetic acid solution contains acetic acid with a volume fraction of 0.05% to 0.2%, the flow rate of the mobile phase is 6 to 10 mL / min, the volume ratio of acetonitrile to the aqueous acetic acid solution is 50 to 90:50 to 10, and the injection volume of the injection solution is 100 to 2000 .

6. The method according to claim 5, wherein The aqueous acetic acid solution contains acetic acid with a volume fraction of 0.1%. The flow rate of the mobile phase is 10 mL / min. The volume ratio of acetonitrile to the aqueous acetic acid solution is 70:

30. The injection volume of the injection solution is 400 - 2000 .

7. The method according to claim 1, wherein The inner diameter of the reversed-phase column is 20 mm, the length is 250 mm, the column temperature is 40 °C, and the detection wavelength is 220 nm.

8. The method according to claim 1, wherein The raw material includes plants or extracts containing artemisinic acid and dihydroartemisinic acid, including Artemisia annua, Artemisia annua var. artemisiifolia, and the waste liquid and residue after extracting artemisinin from Artemisia annua var. artemisiifolia.

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