A method for extracting coumarin from sweet clover and its application

Abstract

The invention relates to a method for extracting coumarin from sweet clover and its application. The method for extracting coumarin from sweet clover comprises the following steps: (1) extracting sweet clover powder with ethanol, filtering, and concentrating under reduced pressure to obtain a crude extract 1; (2) adding distilled water to disperse the crude extract 1, extracting with petroleum ether and ethyl acetate in turn, combining the extracts, and concentrating and drying to obtain a crude extract 2; (3) subjecting the crude extract 2 to silica gel column chromatography; and separating through thin layer chromatography to obtain three concentrated solids; (4) dissolving the three concentrated solids with anhydrous ethanol respectively, refluxing, cooling and crystallizing to obtain coumarin, scopoletin, and scopolamine. The coumarin component extracted by the invention has high antibacterial activity. Experiments show that the antibacterial rate of coumarin at a concentration of 1.0 mg / mL is 96.03%, which can well inhibit Phaseolus sphaeroides, indicating that coumarin can be used for the prevention and treatment of diseases caused by Phaseolus sphaeroides, and broadens the development and utilization of sweet clover resources.

Description

Technical Field

[0001] The invention relates to a method for extracting coumarin from sweet clover and application thereof, belonging to the field of botanical pesticides. Background Art

[0002] Sesame diseases are one of the main factors affecting sesame production, causing annual production losses of 15% to 20% of the total output, of which more than 70% are caused by pathogenic fungi. For a long time, chemical pesticides have been the main way to prevent and control fungal diseases. The long-term application of chemical pesticides poses serious risks to human health and the ecological environment. Finding safe and efficient methods to prevent and control fungal diseases is an urgent issue in current agricultural production. Botanical pesticides are natural pesticides derived from nature. They do not cause pesticide residues and are safe for humans and animals. They are one of the preferred biological pesticides to replace chemical pesticides. At present, botanical pesticides have become a new trend in global pesticide research and development.

[0003] Melilotus officinalis belongs to the genus Melilotus in the Leguminosae family. Melilotus officinalis contains many ingredients, such as flavonoids, coumarins, phenolic acids, saponins, terpenes, etc., which have the effects of clearing heat, detoxifying, anti-inflammatory, anti-coagulation, and antibacterial. At present, there are no reports on the application of Melilotus officinalis in agricultural antibacterial research, and the focus is mainly on the treatment of diseases with Melilotus officinalis. For example, the pharmacodynamics study of Melilotus officinalis extract found that it has significant detumescence and anti-inflammatory activity; oral reagents made from Melilotus officinalis are used to treat edema, etc.

[0004] Traditional solvent extraction methods for sweet clover mainly include decoction, maceration, reflux, continuous reflux, percolation, etc. Due to the complex structure and properties of natural products, the above methods generally have problems such as low extraction rate, long cycle, high cost, and complex operation. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for extracting coumarin from sweet clover and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for extracting coumarin from sweet clover, comprising the following steps:

[0008] (1) adding ethanol solution to the powder of sweet clover, extracting under ultrasonic-assisted conditions, filtering and combining the extracts, and concentrating under reduced pressure at 40 to 55° C. into an extract to obtain a crude extract 1;

[0009] (2) adding distilled water to disperse the crude extract 1, extracting with petroleum ether and ethyl acetate in sequence, each for 2 to 3 times, combining the ethyl acetate extracts, concentrating and drying to obtain the crude extract 2;

[0010] (3) The crude extract 2 was adsorbed on silica gel powder to prepare a sample, and then dry-loaded and subjected to silica gel column chromatography; gradient elution was performed with a mixed solvent of petroleum ether and ethyl acetate, and each eluate was collected, separated by thin layer chromatography, and the same components were combined, concentrated and dried to solids to obtain concentrated solids 1, concentrated solids 2, and concentrated solids 3, which were stored at 4° C. for future use;

[0011] (4) Dissolve the concentrated solid 1, concentrated solid 2 and concentrated solid 3 respectively with anhydrous ethanol, reflux at 78° C. for 30 min, then cool to room temperature, wait for crystals to precipitate, filter, and rinse with ice ethanol. The operation method for the three concentrated solids is the same to obtain three crystalline precipitates, namely coumarin, scopoletin, and artemisinin.

[0012] Step (1) The particle size of the sweet clover powder is 60-80 meshes; the volume fraction of the ethanol solution is 50%-90%; the solid-liquid ratio of the sweet clover to the ethanol extract is 1kg:15-20L.

[0013] The ultrasonic extraction in step (1) is performed under an ultrasonic time of 50 to 60 minutes, an ultrasonic power of 200 to 400 W, an extraction temperature of 50 to 60° C., and a number of extractions of 2 to 3 times.

[0014] During the extraction in step (2), the volume ratio of petroleum ether to the aqueous solution of the crude extract 1 is 2 to 3:1; the volume ratio of ethyl acetate to the aqueous solution of the crude extract 1 is 2 to 3:1.

[0015] In step (3), the particle size of the silica gel powder is 200-300 mesh, and the mass ratio of the crude extract 2 to the silica gel powder is 1-2:100; during gradient elution, the volume ratios of the petroleum ether and ethyl acetate mixed solvents are 100:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 5:2, 1:1, and 3:5, respectively.

[0016] A method for preparing a coumarin inclusion compound comprises the following steps:

[0017] (1) dissolving the obtained coumarin in anhydrous ethanol to form a saturated coumarin ethanol solution for later use;

[0018] (2) dissolving hydroxypropyl-β-cyclodextrin in an aqueous solution to form a saturated hydroxypropyl-β-cyclodextrin solution, subjecting the solution to ultrasonic treatment for 10 to 30 minutes, stirring the solution at a speed of 1500 r / min at a temperature of 40 to 60° C., adding a saturated ethanol solution of coumarin, continuing to stir the solution for 2 hours, and concentrating the solution until there is no ethanol smell, thereby obtaining a concentrated solution of the inclusion complex; the molar ratio of coumarin to hydroxypropyl-β-cyclodextrin in the concentrated solution of the inclusion complex is 1:1 to 4;

[0019] (3) The concentrated solution of the inclusion complex is frozen for 24 hours, washed with ice ethanol, and freeze-dried to obtain coumarin inclusion complex powder.

[0020] The coumarin extracted by the extraction method is used in inhibiting the activity of pathogenic fungi of crops.

[0021] The crop pathogenic fungus is Ascochyta phaseolus, a pathogen of sesame stem blight.

[0022] The coumarin inclusion compound prepared by the preparation method is used in preparing a medicament for inhibiting sesame stem blight.

[0023] The concentration of the coumarin inclusion compound is 0.8-1.6 mg / L.

[0024] Beneficial effects of the present invention

[0025] The present invention provides a method for extracting coumarin components from sweet clover and application of the extract in inhibiting the activity of agricultural pathogenic fungi. The active ingredients obtained by ultrasonic alcohol extraction mainly include coumarin, scopoletin, artemisinin and other coumarin derivatives, which can effectively inhibit the proliferation of Phaseolus vulgaris. The specific analysis is as follows:

[0026] (1) The present invention makes full use of the cavitation effect of ultrasound to Plant cell wall The rupture of the entire organism is completed in an instant, shortening the crushing time. At the same time, the vibration generated by ultrasound strengthens the release, diffusion and dissolution of intracellular substances, thereby significantly improving the extraction efficiency. The target biologically active components in the cells of the medicinal tissue are rapidly dissociated from the cells and enter the solvent system at the fastest speed, lowest energy consumption and highest yield, achieving a balance in the molecular concentration of effective components inside and outside the cells of the medicinal tissue. Compared with traditional separation methods, it has the advantages of good separation effect, low pollution, high extraction rate (the extraction rate of traditional methods is only 1.0%), short time, convenient operation, easy to repeat, and conducive to industrial production.

[0027] (2) The present invention adopts a column chromatography separation method to effectively separate coumarin components with similar polarity by selecting different eluents, and further obtains crystals of each coumarin component (with a purity of more than 95%) by a recrystallization method. Only when this purity is reached can the structure of the compound be characterized.

[0028] (3) The present invention adopts the method of activity tracking, and performs antibacterial experiments on Phaseolus vulgaris by using the extracted, extracted, column chromatographic and recrystallized coumarin, respectively, to obtain the inhibitory effect of coumarin on Phaseolus vulgaris. The target substance with strong antibacterial activity in the extraction, extraction, column chromatography and recrystallization process is determined.

[0029] (4) The coumarin component extracted by the present invention has high antibacterial activity, wherein coumarin inhibits mycelial growth mainly by causing mycelial distortion, apex swelling, and ablation, and as the concentration of coumarin increases, the germ tubes of the germinating conidia of Phaseolus vulgaris become shorter, and when the coumarin concentration reaches 1.50 mg / mL, the germination of conidia of Phaseolus vulgaris can be completely inhibited. Experiments show that the antibacterial rate of coumarin at a concentration of 1.0 mg / mL is 96.03%, which can effectively inhibit Phaseolus vulgaris, indicating that coumarin can be used for the prevention and treatment of Phaseolus vulgaris and the like, broadening the development and utilization of Melilotus resources.

[0030] (5) The present invention also provides a method for preparing a coumarin inclusion compound, which comprises using ultrasound assistance and heating stirring to fully mix coumarin and hydroxypropyl-β-cyclodextrin; concentrating and freeze-drying the inclusion liquid to prepare a coumarin inclusion compound powder. Experiments have shown that the coumarin inclusion compound has a good preventive effect on sesame stem blight when the concentration is 0.8-1.6 mg / L, and the best preventive effect is achieved when the concentration is 0.8 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The antibacterial effect of different components in Application Example 1 on Phaseolus vulgaris.

[0032] Figure 2 The antibacterial graph of different concentrations of coumarin against Phaseolus vulgaris in Application Example 1.

[0033] Figure 3 Effect of coumarin on the mycelial growth of Phaseolus vulgaris in Application Example 1.

[0034] Figure 4 Effects of different coumarin concentrations on conidia germination of Phaseolus vulgaris in Application Example 1. DETAILED DESCRIPTION

[0035] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0036] Example 1

[0037] A method for extracting coumarin from sweet clover, comprising the following steps:

[0038] (1) adding 1000 L of 50% by volume ethanol solution to 50 kg of sweet clover powder (particle size 60 mesh), and extracting under ultrasonic-assisted conditions, wherein the ultrasonic time is 60 min, the ultrasonic power is 300 W, the extraction temperature is 50° C., and the extraction times are 2 times; filtering and combining the extracts, and concentrating under reduced pressure at 50° C. into an extract to obtain a crude extract 1 (extraction rate is 1.55%);

[0039] (2) adding distilled water to disperse the crude extract 1, extracting it three times with petroleum ether and ethyl acetate in sequence until the extract solution becomes colorless, combining the ethyl acetate extracts, concentrating and drying to obtain the crude extract 2;

[0040] During extraction, the volume ratio of petroleum ether to the aqueous solution of crude extract 1 was 2:1; the volume ratio of ethyl acetate to the aqueous solution of crude extract 1 was 2:1.

[0041] (3) adsorbing the crude extract 2 on silica gel powder (the particle size of the silica gel powder is 200 mesh) to prepare a sample, then dry-loading the sample and performing silica gel column chromatography; the mass ratio of the crude extract 2 to the silica gel powder is 1 to 2:100;

[0042] Gradient elution was performed with a mixed solvent of petroleum ether and ethyl acetate in volume ratios of 100:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 5:2, 1:1, and 3:5, respectively, and the eluates were collected and separated by thin layer chromatography. The same components were combined and concentrated and dried to solids to obtain concentrated solid 1, concentrated solid 2, and concentrated solid 3, which were stored at 4° C. for future use;

[0043] (4) Dissolving the concentrated solid 1, concentrated solid 2, and concentrated solid 3 obtained in step (3) in anhydrous ethanol, respectively, and refluxing at 78° C. for 30 min, then cooling to room temperature, filtering and washing with ice ethanol after crystals are precipitated. The operation methods for the three concentrated solids are the same to obtain three kinds of crystal precipitates, respectively. 1 H spectrum, 13 The components were determined by C spectrum, namely coumarin (45 mg, yield 9×10 -5 %), scopoletin (50 mg, yield 9×10 -4 %), artemisinin (58 mg, yield 1.16 × 10 -4 %).

[0044] 1 H spectrum, 13 The C spectrum information is as follows:

[0045] Coumarin: 1H-NMR (600MHz, DMSO), δH: 8.06 (1H, d, J=9.6Hz, H-4), 7.72 (2H, q, J=7.6Hz, H-6, H-8), 7.43 (2H, q, J=12Hz, H-7, H-5), 6.48 (1H, d, J=9.6Hz, H-3);

[0046] 13 In the C-NMR spectrum, δC159.85 (s, C-2), 116.14 (d, C-3), 144.12 (d, C-4), 28.36 (d, C-5), 124 .39(d, C-6), 131.86(d, C-7), 116.19(d, C-8), 153.44(s, C-9), 118.67(s, C-10).

[0047] Scopolamine: 1 H-NMR (600MHz, DMSO) δH: 7.92 (d, J=12Hz, 1H), 7.22 (s, 1H), 6.79 (s, 1H), 6.23 (d, J=9.5Hz) 3.82 (s, 3H, -OCH3);

[0048] 13 C-NMR (600MHz,) δ161.13, 151.59, 149.95, 145.70, 144.93, 112.14, 110.99, 110.01, 103.22, 56.44.

[0049] Artemisinin: 1 H-NMR (600MHz, DMSO), δH: 3.87 (3H, s, -OCH3), 3.81 (3H, s, -OCH3), 6.30 (1H, d, J=6.0Hz), 7.08 (s, 1H), 7.26 (s, 1H), 7.96 (d, J=12Hz, 1H);

[0050] 13 C-NMR (600MHz) δ: 161 (C-2), 153.03 (C-7), 149.90 (C-6), 146.35 (C-9), 144.75 (C-4), 113.11(C-10), 111.65(C-3), 109.65(C-5), 100.5(C-8), 56.62(-OMe), 56.37(-OMe).

[0051] Example 2

[0052] The extraction method of coumarin in this embodiment is the same as that in embodiment 1, except that the volume concentration of ethanol during extraction is changed from 50% to 70%, and the other parameters are the same.

[0053] Three crystalline precipitates were obtained, coumarin (41 mg, yield 8.2 × 10 -5 %), scopoletin (52 mg, yield 1.04 × 10 -4 %), artemisinin (55 mg, yield 1.1 × 10 -4 %).

[0054] Example 3

[0055] The extraction method of coumarin in this embodiment is the same as that in Example 1, except that the raw material of sweet clover powder is changed from 50 kg to 55 kg, and the ultrasonic extraction temperature is changed from 50° C. to 60° C., and the other parameters are the same.

[0056] Three crystalline precipitates were obtained, coumarin (46 mg, yield 9.2 × 10 -5 %), scopoletin (52 mg, yield 1.04 × 10 -4 %), artemisinin (60 mg, yield 1.2 × 10 -4 %).

[0057] Example 4 Preparation of coumarin inclusion compound

[0058] The preparation method of the coumarin inclusion compound comprises the following steps:

[0059] (1) dissolving the coumarin obtained in Example 1 in anhydrous ethanol to form a saturated coumarin ethanol solution for later use;

[0060] (2) dissolving hydroxypropyl-β-cyclodextrin in an aqueous solution to form a saturated hydroxypropyl-β-cyclodextrin solution, subjecting the solution to ultrasonic treatment for 30 minutes, stirring the solution at a speed of 1500 r / min at a temperature of 50° C., adding a saturated ethanol solution of coumarin, continuing to stir the solution for 2 hours, and concentrating the solution until there is no ethanol smell, thereby obtaining a concentrated solution of the inclusion complex;

[0061] The molar ratio of coumarin to hydroxypropyl-β-cyclodextrin in the concentrated solution of the inclusion complex is 1:1;

[0062] (3) The concentrated solution of the inclusion complex is frozen for 24 hours, washed with ice ethanol, and freeze-dried to obtain coumarin inclusion complex powder.

[0063] Application Example 1 Inhibitory Effect of Coumarin on Phaseolus vulgaris

[0064] (1) The inhibitory activity of the crude extract of sweet clover coumarin 1 (the crude extract of sweet clover obtained in Example 1) and the crystals obtained after recrystallization (coumarins, scopolamine, and scopoletin obtained in Example 1) against Coccidioides phaseoli was tested using the mycelium growth rate method. The specific process is as follows:

[0065] The crude extract 1, coumarin, scopoletin and artemisinin were respectively dissolved in DMSO (dimethyl sulfoxide) solution to obtain corresponding sample solutions (10 mg / mL); the sample solutions were respectively added to PDA culture medium cooled to 50-60° C., so that the final concentration of the sweet clover crude extract 1 sample in the PDA culture medium was 7.5 mg / mL, and the final concentration of the coumarin, scopoletin and artemisinin samples in the PDA culture medium was 1 mg / mL.

[0066] After the culture medium was cooled and solidified, one Coccidioides phaseoli was inoculated into the center of each culture medium, and the PDA culture medium without adding the sample solution was used as a blank control, and each treatment was repeated 3 times. The culture was cultured at 28°C, and when the mycelium on the blank control PDA culture medium grew to about 9 cm (after 3 days), the colony diameter of each culture medium was measured, and the antibacterial rate was calculated to screen out the best antibacterial component in Melilotus officinalis.

[0067] Inhibition rate = (colony diameter of control group - colony diameter of treatment group) / (colony diameter of control group - diameter of bacterial cake) × 100%.

[0068] The experimental results are as follows Figure 1 As shown in the figure, after 3 days on the blank PDA medium, the mycelium of Phaseolus phaseolus turned gray and black and produced sclerotia; the antibacterial rate of the crude extract 1 of Melilotus officinalis was 70%, the antibacterial rate of coumarin was 98%, the antibacterial rate of artemisinin was 80%, and the antibacterial rate of scopoletin was 50%. That is, the antibacterial ability of each component in Melilotus officinalis was coumarin>artemisinin>Crude extract 1 of Melilotus officinalis>scopoletin.

[0069] (2) The toxic medium method was used to detect the effect of coumarin on the mycelial growth of Phaseolus vulgaris.

[0070] The coumarin obtained in Example 1 was prepared into coumarin solutions of different concentrations, so that the final concentrations of coumarin in PDA medium were 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL, respectively. After the culture medium was cooled and solidified, a cake of Phaseolus vulgaris was inoculated into the center of each culture medium, and the PDA culture medium without coumarin solution was used as a blank control. Each treatment was repeated 3 times. Cultured at 28°C, when the mycelium on the blank control PDA grew to about 7.5-8 cm, the colony diameter of each culture medium was measured, the inhibition rate was calculated, and the minimum inhibitory concentration was obtained.

[0071] The experimental results are as follows Figure 2As shown. From the figure, we can intuitively see the growth status of Phaseolus vulgaris on the third day. The blank group is completely full, and when the coumarin concentration is 0.5mg / mL and 1.0mg / mL, the mycelium is light brown and white, respectively, and no sclerotia are produced. The antibacterial rates of coumarin concentrations of 0.125mg / mL, 0.25mg / mL, and 0.5mg / mL are 4.7%, 16.24%, and 59.33%, respectively; the antibacterial rate of coumarin concentration of 1.0mg / mL is 96.03%, which can effectively inhibit Phaseolus vulgaris.

[0072] (3) The effect of coumarin on the germination of spores of Phaseolus vulgaris was observed under a microscope.

[0073] Select a mature conidiophore of Phaseolus vulgaris, gently press the glass slide to release the conidia, and then use a 200μL pipette to draw up the spore liquid. The liquid is filtered through 4 layers of gauze, and the spore concentration is determined by hemocytometer to obtain a spore suspension of Phaseolus vulgaris, which is placed in a 1.5mL centrifuge tube for later use. Pipette the spore liquid into coumarin solutions of different concentrations, so that the final concentrations of the coumarin solutions are 0.75mg / mL, 1.0mg / mL, 1.25mg / mL, and 1.5mg / mL, respectively. The spore liquid without coumarin solution is used as a blank control, and the process is repeated 3 times. After culturing at 28°C for 16h, draw up 10μL of the spore suspension, observe 200 spores under a 10X objective, and record the spore germination ( Figure 3 ).

[0074] Spore germination rate = number of spores germinated / (number of spores germinated + number of spores not germinated) × 100%.

[0075] Table 1 shows that when the coumarin concentrations were 0.75, 1.0, 1.25, and 1.50 mg / mL, the spore germination rates were 96.33%, 6.17%, 5.33%, and 0, respectively, and the germination rate of the blank control group was 99.17%. Coumarin inhibits hyphae growth mainly by causing hyphae to twist, apex to swell, and ablate ( Figure 3 As the concentration of coumarin increases, the germ tubes of the germinating conidia of Phaseolus vulgaris become shorter. When the coumarin concentration reaches 1.50 mg / mL, the germination of the conidia of Phaseolus vulgaris is completely inhibited ( Figure 4 ).

[0076] Table 1 Effect of different concentrations of coumarin on spore germination rate of Phaseolus vulgaris

[0077] Coumarin concentration (mg / mL) Spore germination rate (%) 0.75 96.33±0.60bB 1.00 6.17±0.44cC 1.25 5.33±0.73cC 1.50 0.00±0.00dD Blank control 99.17±0.33aA

[0078] Note: The values ​​in the table are mean ± standard error. Different lowercase letters indicate significant differences (P < 0.05) tested by Duncan's new multiple range method.

[0079] Application Example 2 Test on the efficacy of coumarin inclusion compound in preventing sesame stem blight

[0080] Preparation of mycelial liquid of Phaseolus vulgaris: Inoculate the Phaseolus vulgaris cake into PD medium and culture for 5-7 days. After filtering, break the culture with a homogenizer and dilute it with sterile water to 1×10 4 cfu·mL -1 .

[0081] The sterilized sesame seeds were sown in a flower pot filled with sterile soil and cultured to the 4-6 leaf stage, with 4 seedlings per pot. Five treatments were set up in the test group: the coumarin inclusion compound (obtained in Example 4) was diluted with sterile water to 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, and 1.6 mg / L, respectively, and root irrigation was performed; the control group used: 100 billion spores / gram of Bacillus subtilis wettable powder sterile water dilution (diluted 1000 times) for root irrigation; only the bean shell spore mycelium liquid was inoculated as a positive control; and sterile water root irrigation was used as a blank control. Each treatment was repeated 3 times, with 12 plants per repeat, and 5 mL was irrigated to the root of each plant.

[0082] After the sesame seedlings were treated as above, they were immediately inoculated with the mycelial liquid of Phaseolus vulgaris, and 5 mL was irrigated into the roots of each plant. The disease status of the sesame seedlings was investigated 7-14 days after the inoculation of the pathogen.

[0083] Incidence rate = number of diseased plants / total number of plants × 100%;

[0084] Disease index = Σ(number of diseased plants × representative values ​​of each level) / (total number of plants × representative value of the most severe disease level) × 100;

[0085] Control effect = (disease index of positive control area - disease index of treated area) / disease index of positive control area × 100%.

[0086] The experimental results are shown in Table 2. After root irrigation with coumarin inclusion compounds at concentrations of 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, and 1.6 mg / L, the control effects of each treatment on sesame stem blight were 20.84%, 31.25%, 32.29%, 48.96%, and 43.75%, respectively. The control effect of 1000-fold dilution of Bacillus subtilis wettable powder on sesame stem blight was 21.88%. There was no significant difference in the control effect of 0.8 mg / L and 1.6 mg / L coumarin inclusion compounds on sesame stem blight, but it was significant with other treatments. The results showed that coumarin inclusion compounds had a good control effect on sesame stem blight when the concentration was 0.8-1.6 mg / L, and the control effect was the best when the coumarin inclusion compound concentration was 0.8 mg / L.

[0087] Table 2 Test on the efficacy of coumarin inclusion compound against sesame stem blight

[0088]

[0089] Note: The values ​​in the table are mean ± standard error. Different lowercase letters indicate significant differences (P < 0.05) tested by Duncan's new multiple range method.

Claims

1. A method for extracting coumarin from sweet clover, characterized in that: The following steps are involved: (1) adding ethanol solution to the powder of sweet clover, extracting under ultrasonic-assisted conditions, filtering and combining the extracts, and concentrating under reduced pressure at 40-55° C. into an extract to obtain a crude extract 1; The ultrasonic extraction time is 50-60 min, the ultrasonic power is 200-400 W, the extraction temperature is 50-60 ° C, and the extraction times are 2-3 times; (2) adding distilled water to disperse the crude extract 1, extracting with petroleum ether and ethyl acetate in sequence, each for 2 to 3 times, combining the ethyl acetate extracts, concentrating and drying to obtain the crude extract 2; During extraction, the volume ratio of petroleum ether to the aqueous solution of crude extract 1 is 2-3:1; the volume ratio of ethyl acetate to the aqueous solution of crude extract 1 is 2-3:1; (3) The crude extract 2 was adsorbed on silica gel powder to prepare a sample, and then dry-loaded and subjected to silica gel column chromatography; gradient elution was performed with a mixed solvent of petroleum ether and ethyl acetate, and each eluate was collected, separated by thin layer chromatography, and the same components were combined, concentrated and dried to solids to obtain concentrated solids 1, concentrated solids 2, and concentrated solids 3, which were stored at 4°C for future use; (4) Dissolving the concentrated solid 1, concentrated solid 2 and concentrated solid 3 respectively with anhydrous ethanol, reflux at 78° C. for 30 min, and then cooling to room temperature. After crystals are precipitated, filtering and washing with ice ethanol are performed. The operation method for the three concentrated solids is the same to obtain three crystalline precipitates, namely coumarin, scopoletin and artemisinin.

2. The extraction method according to claim 1, characterized in that Step (1) The particle size of the sweet clover powder is 60-80 mesh; the volume fraction of the ethanol solution is 50%-90%; and the solid-liquid ratio of the sweet clover to the ethanol extract is 1 kg:15-20 L.

3. The extraction method according to claim 1, characterized in that In step (3), the particle size of the silica gel powder is 200-300 mesh, and the mass ratio of the crude extract 2 to the silica gel powder is 1-2:100; during gradient elution, the volume ratios of the petroleum ether and ethyl acetate mixed solvents are 100:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 5:2, 1:1, and 3:5, respectively.

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