Method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid

Abstract

The invention relates to a method for simultaneously extracting high-purity artemisinic acid and dihydroartemisinic acid, which comprises the following steps: S1, carrying out ultrasonic extraction on 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 introduction solution of the second crude extract, and separating components in the sample introduction solution by using reversed-phase high-performance liquid chromatography to obtain an arteannuic acid component and a dihydroarteannuic acid component; wherein an acetonitrile-acetic acid aqueous solution is selected as a mobile phase; and S4, respectively dissolving the arteannuic acid component and the dihydroarteannuic acid component, filtering, and concentrating the supernatant to obtain the purified arteannuic acid monomer and dihydroarteannuic acid monomer. According to the scheme, large-polarity impurities in arteannuic acid and dihydroarteannuic acid can be rapidly removed, arteannuic acid and dihydroarteannuic acid with the purity being 98% or above are obtained, the mobile phase containing acetic acid is used, and the stability of arteannuic acid and dihydroarteannuic acid is guaranteed.

Description

Technical Field

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

[0002] The Artemisia annua L. described in this patent is the above-ground part of the Asteraceae plant Artemisia annua L., which is the main extraction source of the highly effective antimalarial drug artemisinin. For a long time, people often ignore the sesquiterpenoid components such as artemisinic acid, dihydroartemisinic acid, deoxyartemisinin, and artemisinin I because of the star ingredient artemisinin in Artemisia annua L. At present, commercialized artemisinins are extracted, separated and refined from Artemisia annua L., but most of the high-value compounds in Artemisia annua L. are discarded, resulting in a great waste of resources. For example, artemisinic acid and dihydroartemisinic acid in Artemisia annua L. have biological activities such as anti-tumor, anti-inflammatory, antibacterial, lipogenesis inhibition, and whitening, and their content in Artemisia annua L. is much higher than artemisinin, and has great development and utilization value.

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

[0004] At present, the preparation of artemisinic acid and dihydroartemisinic acid mainly uses the residues after extracting artemisinin from Artemisia annua or Artemisia annua to extract, and then uses organic solvent-alkali-acid-organic solvent, silica gel column chromatography, preparative liquid phase purification, recrystallization and other methods for separation and purification. For example, in the prior art "Extraction process of artemisinic acid and dihydroartemisinic acid from artemisia annua mother liquor" (China Journal of Pharmaceutical Industry. 2015, 46 (2): 149-151), artemisinic acid and dihydroartemisinic acid monomers are obtained by ethanol extraction, decolorization, alkaline saponification, acidification and recrystallization using artemisinic acid waste liquid as raw material. In addition, there are also methods for separating and purifying artemisinic acid and dihydroartemisinic acid by high performance liquid chromatography and the like.

[0005] Since the chemical structure of dihydroartemisinic acid is somewhat unstable, the separation and purification process will destroy the stability of dihydroartemisinic acid and reduce the yield of dihydroartemisinic acid. The mobile phase in the liquid chromatography separation process will also affect the separation effect of artemisinic acid and dihydroartemisinic acid, increasing the instability of artemisinic acid and dihydroartemisinic acid during the separation process. Summary of the invention

[0006] The object of the present disclosure is to improve the stability of artemisinic acid and dihydroartemisinic acid during separation and purification.

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

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

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

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

[0011] S3. preparing an injection solution of the second crude extract, and separating 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 an 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 ultrasonic extraction is performed after the raw material is soaked in 8 to 20 times the volume of 70 to 100% ethanol, the ultrasonic extraction is performed twice, and the extraction time for each time is 30 to 90 minutes.

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

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

[0016] More preferably, step S2 comprises:

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

[0018] S22. The suspension is extracted with the petroleum ether;

[0019] S23. After combining the organic phases, washing the organic phases with water;

[0020] S24. Drying and filtering 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% to 0.2% volume fraction of acetic acid, the flow rate of the mobile phase is 6 to 10 mL / min, the volume ratio of the acetonitrile to the acetic acid aqueous solution is 50 to 90:50 to 10, and the injection volume of the injection solution is 100 to 2000 μL.

[0022] More preferably, the acetic acid aqueous solution contains 0.1% volume fraction of acetic acid, the flow rate of the mobile phase is 10 mL / min, the volume ratio of the acetonitrile and the acetic acid aqueous 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, the acetonitrile solution of the second crude extract is filtered through filter paper and then filtered 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, and waste liquid and waste residue after artemisinin is extracted from Artemisia annua.

[0026] The technical solution claimed in the present disclosure has achieved the following beneficial effects:

[0027] 1) Extracting the first crude extract obtained by ultrasonic extraction can remove highly polar and water-soluble impurities in the extract, and re-dissolving and filtering the artemisinic acid component and dihydroartemisinic acid component separated by reversed-phase high performance liquid chromatography can further remove highly polar impurities in artemisinic acid and dihydroartemisinic acid, and finally obtain high-purity artemisinic acid and dihydroartemisinic acid, and the product purity is above 98%.

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

[0029] 3) The preparation process has a large sample loading capacity and a fast separation speed. The sample loading capacity can be as high as 2000 μL at a time. The crude extract can be completely separated within 30 minutes, and the separated artemisinic acid and dihydroartemisinic acid are of high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are merely embodiments of the present disclosure, and a person of ordinary skill in the art can obtain other drawings based on the provided drawings without paying any creative work.

[0031] Figure 1 The chromatogram shows the separation of dihydroartemisinic acid standard and artemisinic acid standard by high performance liquid chromatography.

[0032] Figure 2 Artemisinic acid 1 H NMR spectrum.

[0033] Figure 3 Artemisinic acid 13 C NMR spectrum.

[0034] Figure 4 Dihydroartemisinic acid 1 H NMR spectrum.

[0035] Figure 5 Dihydroartemisinic acid 13 C NMR spectrum.

[0036] Figure 6 HPLC chromatogram of methanol.

[0037] Figure 7 This is the HPLC chromatogram of artemisinic acid sample.

[0038] Figure 8 This is the HPLC chromatogram of dihydroartemisinic acid sample.

[0039] Fig. 9 This is a chromatogram of mixed chromatographic peaks of the Artemisia annua extract sample in Comparative Example 1. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and beneficial effects of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0041] Example

[0042] This embodiment provides a method for simultaneously obtaining high-purity artemisinic acid and dihydroartemisinic acid, which specifically comprises 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 to 20 volumes of 80-95% ethanol, each extraction time is 30 to 90 minutes, and the extract is concentrated to dryness to obtain a first crude extract. The raw materials used in this embodiment are plants or extracts containing artemisinic acid and dihydroartemisinic acid, including Artemisia annua, Artemisia annua, and waste residues and waste liquids after extracting artemisinin from Artemisia annua.

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

[0045] (3) preparing an injection solution: dissolving the second crude extract sample obtained in step (2) in acetonitrile, filtering with filter paper, and then filtering through a 0.45 μm filter membrane to obtain an injection solution;

[0046] (4) Liquid phase purification: Reverse phase high performance liquid chromatography was used to separate artemisinic acid and dihydroartemisinic acid in the injection solution.

[0047] The separation conditions are:

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

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

[0050] After separation, the mixture is concentrated in vacuo until the solvent evaporates to obtain an artemisinic acid component and a dihydroartemisinic acid component;

[0051] (5) Filtration: In order to further remove highly polar impurities in artemisinic acid and dihydroartemisinic acid and improve the purity of artemisinic acid and dihydroartemisinic acid, the two components obtained in step (4) are dissolved in petroleum ether and filtered respectively, and the supernatant is vacuum concentrated to evaporate the petroleum ether to obtain high-purity artemisinic acid and dihydroartemisinic acid monomers respectively.

[0052] In the above step (4), the peak elution time of the artemisinic acid and dihydroartemisinic acid fractions is determined by high performance liquid chromatography. When the first separation is performed, the fractions of each chromatographic peak are collected, and 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 dihydroartemisinic acid standards are prepared into solutions, and the peak elution time (retention time) of artemisinic acid and dihydroartemisinic acid standards is determined by high performance liquid chromatography under the same chromatographic conditions to determine the fractions where artemisinic acid and dihydroartemisinic acid are located, and the purity is determined by the area normalization method. In the subsequent separation, only the fractions within the corresponding time are collected.

[0053] The scheme in the embodiment is further described below in conjunction with specific application examples.

[0054] Application Example 1

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

[0056] S1. Weigh 100 g of Artemisia annua, add 800 mL of 95% ethanol, extract twice by ultrasonic, 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, extract 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 in vacuo to obtain 3 g of a second crude extract after preliminary impurity removal;

[0058] S3. The second crude extract obtained in step S2 was dissolved in 4 mL of acetonitrile, passed through a 0.45 μm microporous filter membrane, and subjected to preparative high performance liquid chromatography using the following preparative chromatography conditions: 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] During the first separation, the fractions of each chromatographic peak were collected, and 10 μL of each fraction was injected into an analytical high performance liquid chromatography, and analyzed under the above chromatographic conditions (the chromatographic column was ZORBAX SB-C18 (4.6 mm*250 mm, 5 μm)). At the same time, artemisinic acid and dihydroartemisinic acid standards were prepared into solutions, and the elution time / retention time of artemisinic acid and dihydroartemisinic acid standards were determined by high performance liquid chromatography under the same chromatographic conditions as above ( Figure 1 ) to determine the fractions where artemisinic acid and dihydroartemisinic acid were located, and the area normalization method was used to determine the purity. The subsequent separation only collected the fractions within the corresponding time. At this time, the purities of artemisinic acid and dihydroartemisinic acid were 89% and 70%, respectively.

[0060] In the subsequent separation, only the fractions containing artemisinic acid and dihydroartemisinic acid were collected, and the dihydroartemisinic acid and artemisinic acid fractions were combined. The solvent was recovered in vacuo, dissolved in 5 mL of petroleum ether, filtered, and the supernatant was taken and concentrated and dried to obtain pure artemisinic acid and dihydroartemisinic acid. The product weights were 0.21 g and 0.07 g, respectively, and the purities were 99.6% and 99.8%, respectively.

[0061] S4. Structural identification of artemisinic acid and dihydroartemisinic acid obtained in step S3:

[0062] Artemisinic acid dehydrogenation 15 H 22 O2-H]- The theoretical accurate mass number of is 233.1542, and the accurate mass number of the artemisinic acid dehydrogenation ion obtained in step S3 of this embodiment measured by high-resolution mass spectrometry is 233.1546, and the two data are basically consistent; dihydroartemisinic acid dehydrogenation [C 15 H 24 O2-H] - The theoretical accurate mass number is 235.1698, and the accurate mass number of the dihydroartemisinic acid reduced hydrogen ion obtained in step S3 of this embodiment measured by high-resolution mass spectrometry is 235.1700, and the two data are basically consistent.

[0063] Take 20 mg of artemisinic acid and dihydroartemisinic acid obtained in step S3 respectively in a nuclear magnetic resonance tube, add 0.6 mL of deuterated chloroform to dissolve, and use 600 MHz nuclear magnetic resonance to detect 1 H. 13 C NMR scan of artemisinic acid 1 H. 13 C NMR spectrum Figure 2 , Figure 3 As shown, dihydroartemisinic acid 1 H. 13 C NMR spectrum Figure 4 , Figure 5 shown.

[0064] Artemisinic acid 1 H 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. 6Hz,1H,H-7),2.61(s,1H,H-6),1.93(m,1H,H-2),1.87(dd,J=12.0,4.2Hz,1H,H-3),1.77(dd, J=16.8,5.4Hz,1H,H-3),1.71(dd,J=12.6,3.0Hz,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=6.0Hz,3H,H-14); 13CNMRδ(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 H NMR(CDCl3): δ(ppm)5.12(s,1H,H-5),2.50(m,2H,H-11and H-6),1.95(m,2H,H-9and H-8),1.81(m,1H,H-8),1.64(s,3H,H-15),1.61(m,2H,H-2and H-1),1.43(m,1H,H-10),1.27(m,1H,H-7),1.19(d,J=6.6Hz,3H,H-13),1.11(m,1H,H-3),0.97(m,1H,H-2),0.87(d,J=6.6Hz,3H,H-14); 13 C NMRδ(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 show 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 performs purity analysis on the artemisinic acid and dihydroartemisinic acid obtained in Example 1.

[0069] Preparation method of test samples: 2.0 mg of pure artemisinic acid and dihydroartemisinic acid obtained in Example 1 were accurately weighed and placed in a 5 mL volumetric flask, methanol was added to dissolve and the volume was made up to 5 mL, and filtered through a 0.45 μm filter membrane as the test sample solution for purity detection of the prepared artemisinic acid and dihydroartemisinic acid.

[0070] Chromatographic conditions: Agilent 1260 high performance liquid chromatograph, DAD diode array detector, chromatographic column: ZORBAXSB-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, determine the chromatographic peak of the solvent (methanol) used, then determine the chromatographic peaks of artemisinic acid and dihydroartemisinic acid sample solutions, and perform area normalization method to quantify the sample chromatographic peaks after deducting the solvent chromatographic peak. 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 should be at least 3 times the retention time of the chromatographic peak.

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

[0074] Table 1: Peak list of artemisinic acid sample HPLC detection

[0075]

[0076] Table 2: Peak list of dihydroartemisinic acid sample HPLC detection

[0077]

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

[0079] Application Example 3

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

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

[0082] S2. Add 30 mL of water to the first crude extract obtained in step S2, extract 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 in vacuo to obtain 4.3 g of the second crude extract after preliminary impurity removal;

[0083] S4. The second crude extract obtained in step S3 was dissolved in 8 mL of acetonitrile, passed through a 0.45 μm microporous filter membrane, and subjected to preparative high performance liquid chromatography using the following preparative chromatography conditions: 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 1500 μL.

[0084] The determination method of the fractions and purities of artemisinic acid and dihydroartemisinic acid is consistent with that in Example 2. At this time, the purities of artemisinic acid and dihydroartemisinic acid are 91% and 67%, respectively. The dihydroartemisinic acid and artemisinic acid fractions are combined, the solvent is recovered in vacuo, 5 mL of petroleum ether is dissolved, filtered, the supernatant is taken and concentrated and dried to obtain pure 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 consistent with 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 comprises the following steps:

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

[0088] S2. Add 70 mL of water to the first crude extract obtained in step S2, extract 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 in vacuo to obtain 11.5 g of the second crude extract after preliminary impurity removal;

[0089] S4. The second crude extract obtained in step S3 was dissolved in 20 mL of acetonitrile, passed through a 0.45 μm microporous filter membrane, and subjected to preparative high performance liquid chromatography using the following preparative chromatography conditions: 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 2000 μL.

[0090] The determination method of the fractions and purities of artemisinic acid and dihydroartemisinic acid is consistent with that in Example 2. At this time, the purities of artemisinic acid and dihydroartemisinic acid are 87% and 63%, respectively. The dihydroartemisinic acid and artemisinic acid fractions are combined, the solvent is recovered in vacuo, 10 mL of petroleum ether is dissolved, filtered, the supernatant is taken and concentrated and dried to obtain pure artemisinic acid and dihydroartemisinic acid. The product weights are 0.54 g and 0.12 g, respectively, and the purities are 98.6% and 99.1%, respectively. The structural identification of the prepared compound is consistent with that in Example 1.

[0091] Comparative Example 1

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

[0093] S2. Add 1.5 mL of acetonitrile to the crude extract obtained in step S1 and resuspend it, vacuum filter, and pass the filtrate through a 0.45 μm microporous filter membrane, and perform preparative high performance liquid chromatography using the following preparative chromatography conditions: Shimadzu PRC-ODS, reverse phase column, inner diameter 20 mm, length 250 mm; mobile phase acetonitrile / water (volume ratio 60:40); flow rate 10 mL / min; detection wavelength 192 nm; column temperature 40 ° C; injection volume 400 μL, the chromatographic peaks of the comparative sample were mixed (such as Fig. 9 Artemisinic acid and dihydroartemisinic acid were not separated.

[0094] Comparative Example 2

[0095] S1. Weigh 20 g of dried Artemisia annua sample, add 40 mL of 95% ethanol, extract under reflux for 3 times, each time for 1 h, combine the extracts, filter, concentrate and recover the solvent, and dissolve in petroleum ether.

[0096] S2. Add an equal volume of 5% NaOH solution to the petroleum ether solution in step S1, extract three times, let stand to separate the layers, 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 to extract three times, let stand to separate the layers, separate, and combine the organic phases.

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

[0099] S5. The refined product of Artemisia annua was added to a pretreated silica gel column and eluted with an eluent of petroleum ether: ethyl acetate in a volume ratio of 10:1. TLC (thin layer chromatography) detection was performed, and the eluents of artemisinic acid and dihydroartemisinic acid were collected and dried. The extracts obtained in each extraction step in the preparation process were tested for the content and purity of artemisinic acid and dihydroartemisinic acid. The testing method was the same as that in Example 2, and the test results are shown in Table 3.

[0100] Table 3: Determination results of artemisinic acid and dihydroartemisinic acid 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 dihydroartemisinic acid, and silica gel column chromatography separation could not obtain high-purity dihydroartemisinic acid.

[0103] The extraction scheme provided by the present disclosure extracts the first crude extract obtained by ultrasonic extraction, preliminarily removes the highly polar and water-soluble impurities in the extract, and then re-dissolves and filters the artemisinic acid component and the dihydroartemisinic acid component separated by reversed-phase high-performance liquid chromatography, further removing the highly polar impurities in artemisinic acid and dihydroartemisinic acid, and finally obtains high-purity artemisinic acid and dihydroartemisinic acid, and the product purity is above 98%. In addition, since a mobile phase containing acetic acid is added in the reversed-phase high-performance liquid chromatography, the dissociation of the sample components can be suppressed, the retention of the components on the stationary phase is increased, and the stability of artemisinic acid and dihydroartemisinic acid is ensured while accelerating the separation effect.

[0104] The embodiments and application examples described above are merely illustrative descriptions of the present disclosure and are not intended to limit the scope of the present disclosure. Without departing from the design spirit of the present disclosure, various modifications and improvements made to the technical solutions of the present disclosure by ordinary technicians in this field should all 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: The following steps are involved: S1. performing ultrasonic extraction on the 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 an injection solution of the second crude extract, and separating 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 an acetonitrile-acetic acid aqueous 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, the raw material is soaked in 8 to 20 times the volume of 70 to 100% ethanol and then ultrasonically extracted. The ultrasonic extraction is performed twice, and the extraction time for each time is 30 to 90 minutes.

3. The method according to claim 2, characterized in that The raw material is soaked in 8 to 15 times the volume of 95% ethanol and then ultrasonically extracted. The extraction time for each extraction is 60 to 90 minutes.

4. The method according to claim 1, characterized in that: In step S2, the solvent is petroleum ether.

5. The method according to claim 4, characterized in that Step S2 includes: S21. placing the first crude extract in water to form a suspension; S22. The suspension is extracted with the petroleum ether; S23. After combining the organic phases, washing the organic phases with water; S24. Drying and filtering the organic phase to remove the petroleum ether to obtain the second crude extract.

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

7. The method according to claim 6, characterized in that The acetic acid aqueous solution contains 0.1% volume fraction of acetic acid, the flow rate of the mobile phase is 10 mL / min, the volume ratio of the acetonitrile and the acetic acid aqueous solution is 70:30, and the injection volume of the injection liquid is 400-2000 μL.

8. The method according to claim 1, characterized in that The inner diameter of the reverse phase column was 20 mm, the length was 250 mm, the column temperature was 40°C, and the detection wavelength was 220 nm.

9. The method according to claim 1, characterized in that: In step S3, the second crude extract is dissolved in acetonitrile, and the acetonitrile solution of the second crude extract is filtered through filter paper and then filtered through a 0.45 μm filter membrane to obtain the injection solution.

10. The method according to claim 1, characterized in that The raw materials include plants or extracts containing artemisinic acid and dihydroartemisinic acid, including Artemisia annua, Artemisia annua, and waste liquid and waste residue after artemisinin is extracted from Artemisia annua.

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