Method for extracting scopoletin from artemisia apiacea residues

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a method for extracting scopoletin from artemisia apiacea residues. The method for extracting scopoletin from the artemisia apiacea residues comprises the following steps: S1, carrying out heating reflux extraction on the artemisia apiacea residues, and carrying out suction filtration while the artemisia apiacea residues are hot to obtain filtrate; s2, concentrating the filtrate obtained in the step S1 into extract; s3, dissolving the extract obtained in the step S2 with methanol, diluting with water to obtain an extract suspension, and enriching with macroporous adsorption resin to obtain an artemisia apiacea residue extract; and S4, recrystallizing the artemisia apiacea residue extract obtained in the step S3, and obtaining the high-purity scopoletin by adopting a one-step method. The invention provides a new thought for further mining the utilization value of the artemisia apiacea residues and improving the utilization rate of wastes, and also provides a raw material guarantee for the development of the scopoletin immune resistance inducer.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a method for extracting scopoletin from Artemisia annua residue. Background Art

[0002] Artemisia annua has benefits such as relieving summer heat, reducing fever, and treating malaria. Research has shown that it contains terpenes, flavonoids, coumarins, and volatile oils, making it the primary source plant for artemisinin extraction. However, existing artemisinin extraction processes produce large amounts of artemisinin residues when extracting high-purity artemisinin from the plant. These residues are often used as feed additives or disposed of as waste, resulting in significant resource waste and environmental pollution.

[0003] Scopoletin is a coumarin compound produced in the benzonazole metabolic pathway in plants. It has multiple pharmacological activities such as anti-inflammatory, antioxidant, and anti-tumor. In the field of agricultural research, scopoletin, as a plant growth hormone in plants, can rapidly accumulate in plants when infected by pathogens, inducing plants to produce systemic acquired resistance to resist pathogen infection.

[0004] However, the synthesis process of scopoletin is complex and expensive. Extraction from plant tissues is the only economical way to obtain large quantities of scopoletin. Existing reports indicate that scopoletin can be extracted from various plants, including Angelica dahurica, Angelica sinensis, Solanum belladonna, and Thunbergia paniculata. However, methods for extracting scopoletin from Artemisia annua residues, particularly from discarded residues from which artemisinin has been extracted, have not yet been reported.

[0005] Therefore, in order to fully explore the active ingredients of Artemisia annua residues and improve their utilization rate and utilization value, it is of great practical significance to provide a method for extracting scopoletin from Artemisia annua residues. Summary of the Invention

[0006] In light of this, the present invention aims to provide a method for extracting scopoletin from Artemisia annua residue, aiming to improve the utilization rate of Artemisia annua residue and to explore natural active substances. Furthermore, through formula screening, a microemulsion of Artemisia annua residue extract was developed for field control of tobacco bacterial wilt, achieving a relative efficacy of over 60% during peak disease stages. This provides a theoretical basis for the development of plant-derived immune elicitors.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for extracting scopoletin from Artemisia annua residue, the method comprising the following steps:

[0009] S1. The Artemisia annua residue was heated under reflux for extraction, and the filtrate was filtered while hot;

[0010] S2. The filtrate obtained in step S1 is concentrated to an extract;

[0011] S3. The extract obtained in step S2 was dissolved in methanol, diluted with water to form an extract suspension, and enriched with a macroporous adsorption resin to obtain an extract of Artemisia annua residue;

[0012] S4. Recrystallize the Artemisia annua residue extract obtained in step S3 to obtain high-purity scopoletin using a one-step method, wherein the purity of the scopoletin reaches more than 95%.

[0013] Preferably, the volume ratio of methanol to water in step S3 is 1:4.

[0014] Preferably, the Artemisia annua residue is a discarded residue from which artemisinin has been extracted.

[0015] Preferably, the extraction solvent in the heating reflux extraction is a mixture of ethyl acetate and petroleum ether, and the volume percentage of the ethyl acetate is 82%.

[0016] Preferably, the material-liquid ratio in the heating reflux extraction is 1 g:8 mL, and the extraction time in the heating reflux extraction is 77 min.

[0017] Preferably, the macroporous adsorption resin is macroporous adsorption resin NKA-2, and the eluent is methanol and water in a volume ratio of 4:1.

[0018] Preferably, the solvent for the one-step recrystallization is ethyl acetate, and petroleum ether is slowly added dropwise while stirring.

[0019] In a second aspect, the present invention provides an application of the method described in the present invention in improving the extraction rate of scopoletin.

[0020] It should be noted that extracting scopoletin at high purity is extremely difficult. This is due to multiple factors, including low raw material content, impurity interference, and insufficient process efficiency. Firstly, Artemisia annua residue is a waste product after artemisinin extraction, and the original scopoletin content is typically less than 0.3%. Some processes (such as high-temperature water extraction or extraction of artemisinin using highly polar solvents) may cause partial decomposition of scopoletin or further reduce the residual amount. Secondly, Artemisia annua residue contains a variety of coumarin derivatives (such as hyoscyamine and umbelliferone), whose physical and chemical properties are highly similar to those of scopoletin. Without process integration and innovation, relying solely on traditional silica gel column chromatography (such as a petroleum ether-ethyl acetate system) is difficult to completely separate these analogs. Furthermore, if the ratio of the various components in the silica gel column chromatography eluent is inappropriate (e.g., the proportion of petroleum ether in a petroleum ether-ethyl acetate system is too high or too low), complete separation of these analogs will also be difficult.

[0021] In a third aspect, an Artemisia annua residue extract is provided, which is prepared by the method described in the present invention. The Artemisia annua residue extract includes scopoletin, artemisinic acid, isoflavone, syringetin and deoxyartemisinin, and the purity of scopoletin is above 5%.

[0022] Preferably, the extract of Artemisia annua residue comprises, by mass percentage, 5% scopoletin, 3% artemisinic acid, 3% isofraxidin, 3% syringetin and 2% deoxyartemisinin.

[0023] Also provided is an Artemisia annua residue extract microemulsion, which consists of Artemisia annua residue extract, acetone, ethyl acetate, ethanol, emulsifier EL-20, anionic surfactant 500# and water. The active ingredient of the Artemisia annua residue extract is scopoletin, which is prepared by the method described in the present invention.

[0024] Preferably, the Artemisia annua residue extract microemulsion consists of 4% Artemisia annua residue extract, 15% acetone, 5% ethyl acetate, 5% ethanol, 15% emulsifier EL-20, 5% anionic surfactant 500# and the balance water, calculated by volume percentage.

[0025] Also provided is the use of the Artemisia annua residue extract microemulsion of the present invention in preventing and treating tobacco bacterial wilt.

[0026] Also provided is an Artemisia annua residue extract, wherein the active ingredient of the Artemisia annua residue extract is scopoletin, and the scopoletin is prepared by the method described in the present invention.

[0027] Also provided is the use of the Artemisia annua residue extract microemulsion of the present invention in preventing and treating tobacco bacterial wilt.

[0028] Also provided is the use of the Artemisia annua residue extract microemulsion of the present invention in improving the expression of NtPR1 gene, NtPR2 gene and NtPR5 gene.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) Currently, the main sources of scopoletin include Atropa belladonna L., Angelica decursiva (Miq.) Franch. & Sav., and Angelica sinensis (Oliv.) Diels., etc., which consume a large amount of raw materials during the extraction process, resulting in high costs. The raw materials used in the present invention are waste residues from the extraction of artemisinin, which greatly reduces the cost of raw materials. At the same time, the waste is reused, and the extraction process can be combined with the factory extraction process of artemisinin, which is simple to operate.

[0031] (2) The present invention uses a Box-Behnken response surface design with three factors and three levels to optimize the extraction process of scopoletin. The results show that the optimal process conditions for scopoletin extraction are: 82% ethyl acetate ratio, 1:8 (g·mL-1) solid-liquid ratio, 77 min extraction time, and scopoletin extraction rate (0.091±0.0021)%, which are close to the predicted value, indicating that the process conditions are stable and feasible. At the same time, the purification process of scopoletin was explored, and the macroporous adsorption resin NKA-2 with high efficiency in enriching scopoletin was screened out. The macroporous adsorption resin was enriched to obtain an Artemisia annua residue extract (more than 5% scopoletin); the "one-step method" recrystallization using ethyl acetate dissolution and petroleum ether precipitation can obtain scopoletin crystals with a purity of more than 95%.

[0032] (3) The present invention optimizes and screens a 4% microemulsion formulation of Artemisia annua residue extract. Experimental results confirm that the 4% microemulsion has a 60% or greater efficacy against tobacco bacterial wilt, while having no adverse effects on tobacco growth. This indicates that the 4% microemulsion has the potential to be developed into a plant-derived immune inducer. This invention provides a new approach for further exploring the utilization value of Artemisia annua residue and improving waste utilization, and also provides a theoretical basis for the development of scopoletin immune inducers.

[0033] (4) The results of the field efficacy test of the 4% Artemisia annua residue extract microemulsion of the present invention showed that the prevention and control effect of 1400 times and 800 times of the 4% Artemisia annua residue extract microemulsion on tobacco bacterial wilt reached more than 60%, which was better than the control agent 20% thiophanate-methyl suspension 500 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The control effect of different concentrations of scopoletin on tobacco bacterial wilt. A represents the effect of different concentrations of scopoletin on the tobacco bacterial wilt disease index, B represents the survival rate of tobacco plants inoculated with R. solanacearum at different concentrations of scopoletin, and C represents the disease progression of tobacco plants after scopoletin treatment. Mock: water control; BTH: benzothiadiazole; SC4: scopoletin 4 μM; SC8: scopoletin 8 μM; SC16: scopoletin 16 μM; SC32: scopoletin 32 μM; SC64: scopoletin 64 μM; SC128: scopoletin 128 μM.

[0035] Figure 2 The effect of scopoletin on tobacco defense enzyme activities. A represents catalase, B represents peroxidase, C represents superoxide dismutase, and D represents phenylalanine ammonia lyase. Mock: water control; Scopoletin: scopoletin.

[0036] Figure 3 The effect of scopoletin on resistance gene expression. A represents the relative expression of the NtPR1 gene, B represents the relative expression of the NtPR2 gene, and C represents the relative expression of the NtPR5 gene. Mock: water control; Scopoletin: scopoletin.

[0037] Figure 4 is the standard curve of scopoletin.

[0038] Figure 5 The effect of ethyl acetate ratio on the extraction rate of scopoletin.

[0039] Figure 6 This is the effect of solid-liquid ratio on the extraction rate of scopoletin.

[0040] Figure 7 This is the effect of extraction time on the extraction rate of scopoletin.

[0041] Figure 8 is the response surface plot of the interaction effect. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention.

[0043] An embodiment of the present invention provides a method for extracting scopoletin from Artemisia annua residue, the method comprising the following steps:

[0044] S1. The Artemisia annua residue was heated under reflux for extraction, and the filtrate was filtered while hot;

[0045] S2. The filtrate obtained in step S1 is concentrated to an extract;

[0046] S3. The extract obtained in step S2 was dissolved in methanol, diluted with water to form an extract suspension, and enriched with a macroporous adsorption resin to obtain an extract of Artemisia annua residue;

[0047] S4. Recrystallize the Artemisia annua residue extract obtained in step S3, dissolve it in ethyl acetate, and slowly add petroleum ether dropwise while stirring to obtain high-purity scopoletin.

[0048] Among them, the extract of the Artemisia annua residue mainly includes scopoletin, artemisinic acid, isofraxidin, syringetin and deoxyartemisinin (Table 7).

[0049] The Artemisia annua residue is a waste residue from which artemisinin has been extracted; the extraction solvent in the heating reflux extraction is a mixture of ethyl acetate and petroleum ether, and the volume percentage of the ethyl acetate is 82%; the solid-liquid ratio in the heating reflux extraction is 1 g:8 mL, and the extraction time in the heating reflux extraction is 77 min; the macroporous adsorption resin is macroporous adsorption resin NKA-2, and the eluent is methanol and water in a volume ratio of 4:1.

[0050] Example 1 Materials and Methods

[0051] 1.1 Test materials

[0052] The raw material of Artemisia annua residue is the waste residue from which artemisinin is extracted, which is obtained by evaporating and drying the solvent.

[0053] 1.2 Reagents and Instruments

[0054] Scopoletin standard (98%) was purchased from Nanjing Herbal Source Biotechnology Co., Ltd.; ethyl acetate (99.7%), anhydrous ethanol and acetone (99.7%) were purchased from Shanghai Yien Chemical Technology Co., Ltd.; petroleum ether (99.8%) was purchased from Chongqing Jiyuan Chemical Co., Ltd., and EL-20 and 500# were purchased from Jiangsu Maoxiang Chemical Co., Ltd.

[0055] XD-5205 rotary evaporator was purchased from Shanghai Xiande Experimental Instrument Co., Ltd.; HH-6 digital constant temperature water bath was purchased from Changzhou Tianrui Instrument Co., Ltd.; high performance liquid chromatography was purchased from Agilent (USA); and electric blast drying oven was purchased from Shanghai Boxun Industrial Co., Ltd.

[0056] 1.3 Evaluation of scopoletin-induced antimicrobial activity

[0057] 1.3.1 Indoor potted experiment on the effect of scopoletin on tobacco bacterial wilt

[0058] Tobacco seedlings at the three-leaf, one-heart stage with uniform growth were selected and treated by foliar spraying. Specifically, scopoletin at concentrations of 4, 8, 16, 32, 64, and 128 μM and benzothiadiazole at 50 mg / L (diluted with sterile water) were evenly sprayed onto the leaves and backs of the tobacco seedlings. The treatment was repeated every 3 days for a total of 2 induction treatments. A 0.1% DMSO sterile aqueous solution was used as a blank control. Three days after the second induction treatment, the roots were inoculated with Ralstonia solanacearum at a concentration of 1×10 8 CFU / mL, 10mL was inoculated per plant. The tobacco seedlings were placed in a constant temperature and light greenhouse at 28°C with a photoperiod of 14h / 10h. The incidence of bacterial wilt was investigated daily, and the disease index and relative control efficacy were calculated using the following formula:

[0059]

[0060] 1.3.2 Effects of scopoletin on tobacco defense enzyme activities

[0061] Tobacco leaves were collected 10 min, 1 h, 6 h, 12 h, and 24 h after the spraying and induction treatment. Three tobacco plants were taken from each treatment at each time point, and a total of 0.5 g of tobacco leaves were taken. Tobacco plants that had already been sampled were not sampled again (to avoid errors caused by wounds). The cut tobacco leaf samples were immediately quick-frozen in liquid nitrogen and used immediately or stored in a -80°C refrigerator for later use.

[0062] Tobacco leaves stored at -80°C were placed in a pre-cooled mortar, ground into powder with liquid nitrogen, and evenly distributed into four 10 mL enzyme-free centrifuge tubes, with four tubes for each treatment. SOD, POD, CAT, and PAL extracts were added and fully shaken, centrifuged at 8000g, 4°C for 10 min, and the supernatant was aspirated and distributed into new centrifuge tubes and stored at -80°C for later use. The effects of scopoletin-induced treatment on the activities of tobacco SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), and PAL (phenylalanine ammonia lyase) were determined according to the kit instructions.

[0063] 1.3.3 Effects of scopoletin on the expression of tobacco resistance genes

[0064] Tobacco leaves were collected 10 minutes, 6 hours, 12 hours, and 24 hours after spray treatment. Three tobacco plants were taken from each treatment at each time point, and a total of 0.5 g of tobacco leaves were taken. Tobacco plants that had already been sampled were not sampled again (to avoid errors caused by wounds). The cut tobacco leaf samples were immediately placed in liquid nitrogen for quick freezing and used immediately or stored in a -80°C refrigerator for later use. Total RNA was extracted using the RNA extraction kit, and total RNA was reverse transcribed using the Evo M-MLV reverse transcription premix kit. Real-time fluorescence quantitative PCR was performed using the Pro Taq HS SYBR Green premix qPCR kit. The relative expression of the target gene was calculated using 2 -ΔΔct The calculation formula is as follows:

[0065] Relative expression level = 2^ -[(ct目的基因-ct内参基因)处理组-(ct目的基因-ct内参基因)对照组]

[0066] 1.4 Drawing of the Scopoletin Standard Curve

[0067] Accurately weigh 5.0 mg of scopoletin standard product and dissolve it in 1 mL of methanol to obtain 5000 μg mL -1 Scopoletin standard stock solution: Accurately transfer 160 μL of stock solution with a pipette and dilute to 4 mL with methanol to obtain 200 μg mL -1 , and then serially diluted to obtain 200, 100, 50, 25, 12.5, and 6.25 μg·mL -1, filtered through a 0.22 μm filter, and subjected to liquid phase detection. The detection conditions were: methanol: 0.1% formic acid water = 40%: 60%, flow rate 1 mL / min, detection wavelength 343 nm. A standard curve was drawn with the concentration of the standard solution (C) as the horizontal axis and the peak area (mAU) as the vertical axis. Figure 4 shown.

[0068] 1.5 Scopoletin Extraction Process

[0069] 1.5.1 Basic extraction process

[0070] Accurately weigh 10.0 g of Artemisia annua residue and place it in a 250 mL volumetric flask. Add the corresponding volume of a mixture of ethyl acetate and petroleum ether, weigh the total weight, and perform heating reflux extraction under different conditions. The temperature is controlled at 60°C. Filter the filtrate while it is hot, make up the weight loss, and accurately transfer 5 mL of the filtrate to a penicillin bottle. After the solvent evaporates, dilute the volume to 5 mL with methanol. Pass the filter through a 0.22 μm filter membrane and perform liquid chromatography detection according to the method described in 1.4. Substitute the obtained peak area into the regression equation to obtain the sample solution concentration. After conversion, the extraction rate of scopoletin in the sample can be calculated.

[0071] 1.5.2 Single-factor experiment

[0072] Taking the extraction rate of scopoletin as an indicator, the effects of different ethyl acetate ratios, different solid-liquid ratios, and different extraction times on the extraction rate of scopoletin were explored. The single factor level settings are shown in Table 1. The three variable factors initially set for exploration were 50% ethyl acetate, a solid-liquid ratio of 1:10, and an extraction time of 60 min. The sample weight of each sample was 10.0 g.

[0073] Table 1 Single factor test

[0074]

[0075] 1.5.2.1 Effect of different solvent ratios on the extraction rate of scopoletin

[0076] Referring to the method described in 1.5.1, using the ratio of ethyl acetate to petroleum ether as a variable, compare the effects of 10% ethyl acetate, 20% ethyl acetate, 50% ethyl acetate, 80% ethyl acetate, and 100% ethyl acetate on the extraction rate of scopoletin when the solid-liquid ratio is 1:10 and the extraction time is fixed at 60 min.

[0077] 1.5.2.2 Effect of different material-liquid ratios on the extraction rate of scopoletin

[0078] Referring to the method described in 1.5.1, with the solid-liquid ratio as the variable, compare the effects of solid-liquid ratios of 1:6, 1:8, 1:10, 1:12, and 1:14 on the extraction rate of scopoletin when the ethyl acetate ratio is 50% and the extraction time is fixed at 60 min.

[0079] 1.5.2.3 Effect of different extraction times on the extraction rate of scopoletin

[0080] Referring to the method described in 1.5.1, with extraction temperature as the variable, compare the effects of extraction times of 40 min, 50 min, 60 min, 70 min, and 80 min on the extraction rate of scopoletin when the ethyl acetate ratio is 50% and the solid-liquid ratio is fixed at 1:10.

[0081] 1.5.3 Response surface methodology optimization

[0082] Based on the optimal values of each factor screened out by the single-factor experiment, a three-factor three-level Box-Behnken experiment was designed for the ethyl acetate ratio (A), solid-liquid ratio (B) and extraction time (C) using Design Expert 13 software (as shown in Table 2). The extraction rate of scopoletin (Y) was used as the response index to determine the optimal process conditions for scopoletin extraction.

[0083] Table 2 Response surface test factors and levels

[0084]

[0085] 1.6 Scopoletin Purification Process

[0086] 1.6.1 Screening of macroporous adsorption resins

[0087] Twelve macroporous adsorption resins were selected. Their physical and chemical properties and applicable ranges are shown in Table 3 below:

[0088] Table 3 Types, physical and chemical properties and uses of 12 macroporous adsorption resins

[0089]

[0090]

[0091] 1.6.1.1 Pretreatment of macroporous adsorption resin

[0092] Macroporous resins often contain unpolymerized monomers, cross-linking agents, porogens and other chemical residues during the synthesis process. Therefore, the resin needs to be soaked in methanol / ethanol for 24 hours before use. Weigh 5g of macroporous resins of different types, add 50mL of methanol and soak for 24 hours, load them onto the column, elute with methanol until the outflowing solution is no longer turbid after diluting with water (proving that the resin activation is complete), and finally rinse with pure water until there is no obvious alcohol smell before loading.

[0093] 1.6.1.2 Screening of macroporous adsorption resins

[0094] Using the static adsorption method, the pretreated macroporous adsorption resin was placed in a 250mL triangular flask, 13g of extract was weighed, 260mL of methanol was added to dissolve, and pure water was added to 1300mL, which was a 20% methanol sample solution. The initial concentration was detected by liquid phase. 100mL of the prepared sample solution was added to each resin, 120rpm, 25℃, shaken overnight, the supernatant was aspirated, liquid phase detection was performed, and the adsorption rate was calculated. The supernatant was discarded, 50mL of methanol was added, 120rpm, 25℃, shaken for 4-5h, the supernatant was aspirated, liquid phase detection was performed, and the desorption rate was calculated according to the following formula:

[0095]

[0096]

[0097] Qe: adsorption capacity at equilibrium (mg / g dry weight); A: adsorption rate at equilibrium (%); D: desorption rate at desorption equilibrium. Co and Ce are the initial concentration and adsorption equilibrium concentration (mg / mL), respectively; Vi and Vd are the initial sample volume and desorption volume (mL), respectively; and W represents the dry weight of the resin (g).

[0098] 1.6.2 Sample enrichment with macroporous adsorption resin

[0099] 1.6.2.1 Pretreatment of macroporous adsorption resin

[0100] Weigh 40 g of macroporous adsorption resin, add 200 mL of methanol and soak for 24 h, then load onto the column and elute with pure water until there is no alcohol smell.

[0101] 1.6.2.2 Sample solution preparation and sample adsorption

[0102] Weigh 20 g of the extract, first add 200 mL of methanol to dissolve it, then add pure water to 1000 mL, determine the initial concentration of the sample solution, pour the sample solution into the resin column, and detect the scopoletin content of the effluent while adsorbing until the macroporous adsorption resin is saturated with adsorption, and stop loading.

[0103] 1.6.2.3 Sample desorption

[0104] Elution was performed with industrial methanol, and the scopoletin content was detected while the eluate was collected until the eluted solution no longer contained scopoletin and the desorption was completed. The extract was concentrated under reduced pressure to obtain the extract.

[0105] 1.6.3 Recrystallization

[0106] The extract enriched by the macroporous adsorption resin is dissolved in ethyl acetate, petroleum ether is slowly added, and the mixture is continuously stirred to obtain scopoletin crystals with a purity of more than 95%.

[0107] 1.7 Preparation of microemulsion of Artemisia annua residue extract

[0108] Add 4.0g of Artemisia annua residue extract to each stoppered test tube, then add 25mL of a candidate solvent to each. Observe the dissolution behavior. The solvent with the best solubility is selected as the primary solvent, and other solvents with better solubility are selected as cosolvents. Cross-design multiple formulations and measure the solubility of the original drug in each solvent combination. If the solubility is greater than 30% and there is no precipitation or crystallization after storage in a refrigerator (0°C) for 4 hours, the original drug will proceed to the next round of screening.

[0109] Add 15% (mass fraction) of nonionic surfactant (single or combined) and 5% of anionic surfactant (500#) to the solution, mix well, and let it stand at room temperature for 24 hours. Then, select an emulsifier based on the appearance and emulsification properties of the preparation. Emulsifiers that form a transparent, uniform, single-phase liquid, exhibit no crystallization during cold storage, no significant precipitation during hot storage, and a relatively stable dilution are used as the standby mix. Then, design several different content mixes for trial mixing, evaluate them according to the four indicators mentioned above, and determine the final emulsifier formula.

[0110] 1.8 Safety test of microemulsion of Artemisia annua residue extract on tobacco

[0111] Experimental design and investigation: Each tobacco variety safety test was conducted with four treatments, including 2000-fold, 1400-fold, and 800-fold dilutions of Artemisia annua residue extract (active ingredient scopoletin) microemulsion and a water control. Each plot was approximately 18 m2. 2 Application Method: The experiment involved two applications, one at the three-leaf, one-heart stage after transplanting, and another application every 7-10 days. Each spray volume per plot was 1.36L. Survey Method: The height of each treated tobacco plant was surveyed before the first application and 21 days after the last application. Starting from the first application, daily visual inspections were conducted to assess whether the tobacco had any signs of damage, such as discoloration, necrosis, wilting, or deformity.

[0112] Result statistics: The safety of 4% (mass percentage) Artemisia annua residue extract microemulsion to tobacco is described according to the following formula.

[0113]

[0114] 1.9 Field efficacy test of Artemisia annua residue microemulsion in controlling tobacco bacterial wilt

[0115] Test locations: Zunyi, Guizhou; Weng'an, Guizhou; Qianjiang and Pengshui, Chongqing; and Yibin, Sichuan.

[0116] Experimental treatment: There are 4 treatments in total, namely 2000 times, 1400 times, 800 times of microemulsion of Artemisia annua residue, and 500 times of 20% (mass percentage) thiophanate-methyl suspension concentrate. Each treatment is repeated 3 times. The area of each plot is about 30m 2 .

[0117] Application method: The experiment applied pesticides twice, once during the clustering stage and once during the vigorous growth stage after tobacco transplanting, and the spraying liquid volume per plot was 2.25L each time.

[0118] Survey method: Surveys were conducted in the early stages of tobacco bacterial wilt according to GB / 23222-2008 "Grading and Survey Methods for Tobacco Pests and Diseases". The grading standards are as follows:

[0119] Level 0: The whole plant is disease-free;

[0120] Level 1: There are occasional chlorotic spots on the stem, or less than 1 / 2 of the leaves on the diseased side wilt;

[0121] Level 3: There are black streaks on the stem, but not exceeding 1 / 2 of the stem height, or 1 / 2-2 / 3 of the leaves on the diseased side are withered;

[0122] Level 5: Black streaks on the stem exceed 1 / 2 of the stem height but do not reach the top of the stem, or more than 2 / 3 of the leaves on the diseased side wilt;

[0123] Level 7: Black streaks on the stem reach the top of the stem, or all leaves of the diseased plant wilt;

[0124] Level 9: The diseased plants are basically dead.

[0125] The disease index and prevention efficacy were calculated according to the following formula.

[0126]

[0127] Example 2 Result Analysis

[0128] 2.1 Evaluation of scopoletin-induced antimicrobial activity

[0129] 2.1.1 Effectiveness of Scopoletin against Tobacco Bacterial Wilt in Indoor Pot Plants

[0130] Depend on Figure 1 It can be seen that by screening different concentrations of scopoletin, it was found that when the concentration of scopoletin was 8μM (1.54mg / L), it had the best prevention and control effect on tobacco bacterial wilt, with a relative prevention efficiency of more than 60%, which was significantly higher than the control agent benzothiadiazole (BTH). Subsequently, 1.54mg / L will be used to evaluate the induced resistance activity.

[0131] 2.1.2 Effects of scopoletin on tobacco defense enzyme activities

[0132] Changes in the activity of defense enzymes in plants reflect the disease resistance response of plants. The applicant measured the changes in the activities of four defense enzymes, SOD, POD, CAT, and PAL, in tobacco after treatment with scopoletin by ultraviolet spectrophotometry. The results showed that the activity of CAT enzyme showed a trend of first increasing and then decreasing within 24 hours after treatment. CAT activity reached its highest level 6 hours after treatment, which was 4.80 times that of the control group ( Figure 2 Middle A). One hour after scopoletin treatment, the SOD enzyme activity in tobacco reached a peak, which was 1.37 times that of the control group, and then began to decline ( Figure 2 POD enzyme activity gradually increased after treatment, reaching a maximum value 6 hours after treatment, which was 5.80 times that of the control group, and then began to decline, reaching the level of the control group at 24 hours after treatment ( Figure 2 Middle B). The PAL enzyme activity in tobacco leaves increased slowly within 6 hours, reaching 2.55 times that of the control group at 6 hours, and then began to slowly decrease ( Figure 2 Middle D).

[0133] 2.1.3 Effects of scopoletin on the expression of tobacco resistance genes

[0134] Depend on Figure 3 It can be seen that resistance-related PR genes were selected for qRT-PCR analysis. The results showed that scopoletin treatment, especially in the early stage after treatment, can cause significant upregulation of PR genes. Among them, 10 minutes after treatment, the expression levels of NtPR1, NtPR2, and NtPR5 genes increased by 4.29 times, 3.57 times, and 2.63 times respectively compared with the control group. 6 hours after treatment, the expression levels of NtPR1, NtPR2, and NtPR5 genes increased by 1.98 times, 2.28 times, and 1.50 times respectively compared with the control group, indicating that scopoletin treatment can cause the expression of tobacco resistance genes and enhance the plant resistance to infection by bacterial wilt.

[0135] 2.2 Drawing of the Scopoletin Standard Curve

[0136] The standard curve was drawn with the concentration X and peak area Y of the scopoletin standard solution, and the linear regression equation Y = 41.483X-11.061 (R 2 =0.9999). Figure 4 shown.

[0137] 2.3 Single-factor test results

[0138] 2.3.1 Effect of different ethyl acetate ratios on the extraction rate of scopoletin

[0139] Depend on Figure 5It can be seen that as the proportion of ethyl acetate increases, the extraction rate of scopoletin increases continuously, reaching a peak at 80% ethyl acetate. This is because as the ethyl acetate concentration increases, the solubility of scopoletin in the sample is better. However, when the proportion continues to increase to 100% ethyl acetate, the extraction rate of scopoletin shows a downward trend. This may be because as the ethyl acetate concentration increases, more fat-soluble substances are extracted, affecting the extraction of scopoletin and reducing the extraction rate of scopoletin. Therefore, 80% ethyl acetate was selected as the optimal extraction solvent, and the extraction rate at this time was 0.096%. Therefore, 50% ethyl acetate, 80% ethyl acetate, and 100% ethyl acetate were selected as factors for the design of the response surface experiment.

[0140] 2.3.2 Effect of different material-liquid ratios on the extraction rate of scopoletin

[0141] Depend on Figure 6 It can be seen that when the solid-liquid ratio increases from 1:6 to 1:8, the most scopoletin is extracted and the extraction effect is the best. Then, as the solid-liquid ratio continues to increase, the extraction rate shows a downward trend. When the solvent dosage reaches a certain value, scopoletin has been completely extracted. Continuing to increase the solvent dosage will not only cause the concentration of scopoletin per unit extract to decrease, but also cause waste of resources. Therefore, solid-liquid ratios of 1:6, 1:8, and 1:10 are selected as factors for designing the response surface experiment.

[0142] 2.3.3 Effect of different extraction times on the extraction rate of scopoletin

[0143] Depend on Figure 7 It can be seen that the extraction rate of scopoletin increases with the increase of extraction time, reaching a maximum of 0.092% at 70 minutes. However, when the extraction time continues to increase, the extraction rate of scopoletin decreases. Therefore, 70 minutes was selected as the optimal extraction time, and 60 minutes, 70 minutes, and 80 minutes were selected as factors for the response surface design experiment.

[0144] 2.4 Response surface optimization results

[0145] 2.4.1 Response surface optimization design test results

[0146] The response surface design results are shown in Table 4. Design Expert 13 software was used to fit the effects of the independent variables ethyl acetate ratio (A), solid-liquid ratio (B), and extraction time (C) on the scopoletin extraction rate (Y), and the resulting multiple regression equation was as follows:

[0147] Y=0.0923-0.0001A-0.0003B+0.0016C-0.0077AB+0.0070AC+0.0072BC-0.0079A 2-0.0127B 2 -0.0029C 2 .

[0148] Table 4 Response surface experimental design and results

[0149]

[0150]

[0151] As shown in Table 5, the P value of the regression model is 0.0123 (P < 0.05), so the model is significant. The P value of the lack of fit term is 0.9471 (P > 0.05), and the lack of fit term is not significant. It can be seen that the model has high credibility and good fit, and can better predict the optimal extraction process. 2 、B 2 The interaction terms AB, AC, and BC had significant effects on the extraction of scopoletin (P < 0.05), and the first-order terms A, B, C and the second-order term C 2 The extraction effect on scopoletin was not significant (P>0.05).The influence of each factor was in the following order: C (extraction time)>B (material-liquid ratio)>A (ethyl acetate ratio).

[0152] Table 5 Analysis of variance

[0153] Sources of variance sum of squares degrees of freedom mean square F-number P-value Model 0.0014 9 0.0002 9.25 0.0123 A (ethyl acetate ratio) <![CDATA[1.250×10 -7 ]]> 1 <![CDATA[1.250×10 -7 ]]> 0.0072 0.9357 B (material-liquid ratio) <![CDATA[5.000×10 -7 ]]> 1 <![CDATA[5.000×10 -7 ]]> 0.0288 0.8720 C (extraction time) 0.0000 1 0.0000 1.22 0.3205 AB 0.0002 1 0.0002 13.82 0.0137 AC 0.0002 1 0.0002 11.28 0.0202 BC 0.0002 1 0.0002 12.09 0.0177 <![CDATA[A 2 ]]> 0.0002 1 0.0002 13.31 0.0148 <![CDATA[B 2 ]]> 0.0006 1 0.0006 34.08 0.0021 <![CDATA[C 2 ]]> 0.0000 1 0.0000 1.81 0.2366 residual 0.0001 5 0.0000 / / Loss of Fit 0.0000 3 <![CDATA[4.083×10 -6 ]]> 0.1094 0.9471 error 0.0001 2 0.0000 / / Total value 0.0015 14 / / /

[0154] 2.4.2 Response surface analysis

[0155] Depend on Figure 8 As can be seen, the surface plots for each interaction response all open downward and have their highest points. The center of the minimum ellipse in the contour plot lies within the range of the experimental factor conditions, indicating that the scopoletin extraction rate reaches its maximum value within the range set by each factor. The response surfaces for each factor interaction show that the AB response surface (ethyl acetate ratio and solid-liquid ratio) has the steepest slope, indicating that the interaction between ethyl acetate ratio and solid-liquid ratio has the most significant effect on the scopoletin extraction rate. The scopoletin extraction rate peaks around 80% ethyl acetate and around a solid-liquid ratio of 1:8. The BC response surface (solid-liquid ratio and extraction time) has a lower steepness than the AB response surface, indicating that the interaction between solid-liquid ratio and extraction time has the second greatest impact on the scopoletin extraction rate. The scopoletin extraction rate peaks between a solid-liquid ratio of 1:8 and an extraction time of 70-80 minutes. Through response surface analysis, it can be concluded that the significance of the interaction effects of various factors on the extraction rate of scopoletin is AB>BC>AC, and the results of surface analysis are consistent with the results of significance analysis of the regression model.

[0156] 2.5 Optimal extraction process verification

[0157] The regression equation model was fitted and analyzed using Design Expert 13 software, and the optimal process conditions for scopoletin extraction were obtained as follows: 81.525% ethyl acetate ratio, 1:8.247 solid-liquid ratio, and 77.464 min extraction time. Considering the feasibility of actual operation, the ethyl acetate ratio was adjusted to 82% and the solid-liquid ratio was adjusted to 1:8 (g·mL -1 ), the extraction time was adjusted to 77 min, and the test was repeated three times for verification. The test results showed that the extraction rate of scopoletin was (0.091±0.0021)%, which was consistent with the expectation.

[0158] 2.6 Macroporous adsorption resin screening results

[0159] As shown in Table 6, by evaluating the static adsorption capacity of 12 different types of macroporous adsorption resins, the NKA-2 macroporous adsorption resin was screened out to have the strongest adsorption capacity for scopoletin, which was 92.3%. Subsequently, the NKA-2 macroporous resin was used to study the purification process of scopoletin. The purity of scopoletin was increased from 1% to 5% by enrichment with the macroporous adsorption resin, and an Artemisia annua residue extract containing 5% scopoletin was obtained.

[0160] Table 6 Static adsorption capacity of scopoletin by different types of macroporous adsorption resins

[0161]

[0162]

[0163] Table 7 Main components of Artemisia annua residue extract

[0164] Element content(%) Scopoletin 5 Artemisinic acid 3 Isofraxin 3 Lilac Pavilion 3 Deoxyartemisinin 2 Tagetesin 1 Scopoletin 1 Zedoaria flavonoids 1 Kaempferol 0.5 Apigenin 0.5

[0165] 2.7 Preparation results of Artemisia annua residue microemulsion

[0166] 2.7.1 Results of Solvent System Screening for Artemisia annua Residue Extract

[0167] The solubility of Artemisia annua residue extract in different solvents was determined. It can be seen that the solubility of Artemisia annua residue extract in acetone is the best, and the solubility in ethyl acetate, N,N-dimethylformamide and ethanol is relatively large. The solubility and cold storage stability in other solvents are poor.

[0168] Table 8 Solubility results of Artemisia annua residue extract in different solvents

[0169] solvent Solubility Cold storage stability 4h Quality compliance (yes / no) acetone Completely dissolved Completely dissolved yes Ethyl acetate Completely dissolved Microprecipitation yes N,N-Dimethylformamide Completely dissolved A small amount of precipitation yes Cyclohexanone Almost insoluble Large amounts of precipitation no Xylene A small amount of dissolution Large amounts of precipitation no ethanol Completely dissolved Microprecipitation yes Methanol Completely dissolved A small amount of precipitation no dimethyl sulfoxide A small amount of dissolution Large amounts of precipitation no

[0170] As shown in Table 8, the solubility and stability of the Artemisia annua residue extract in acetone, ethyl acetate, benzene, N,N-dimethylformamide, and ethanol were good, while the solubility and stability in other solvents were average. Considering cost factors and to reduce the use of restrictive organic solvents, a combination of acetone, ethyl acetate, and ethanol was selected, and 42 solvent combinations were screened.

[0171] According to the results in Table 9, taking into account factors such as solubility, cost, and toxicity, the solvent combination of acetone:ethyl acetate:ethanol was finally selected at 3:1:1 (volume ratio) (total solvent amount was 25%) for the next step of emulsifier screening.

[0172] Table 9 Solvent combinations with good solubility for Artemisia annua residue extracts

[0173]

[0174]

[0175]

[0176]

[0177] 2.7.2 Results of screening of emulsifiers for Artemisia annua residue extract

[0178] Non-ionic surfactants were screened under the above-mentioned solvent ratio. From the appearance of the preparation, cold storage stability, hot storage stability, and dilution stability, it can be seen from Table 10 that EL-20 meets the relevant requirements. Therefore, EL-20 was selected as the emulsifier for the microemulsion of Artemisia annua residue extract.

[0179] Table 10 Emulsifier screening results

[0180]

[0181]

[0182] 2.7.3 Screening results of emulsifier content in Artemisia annua residue extract

[0183] The selected emulsifier, EL-20, was set at six concentration gradients. After adding the emulsifier to the aforementioned solvent formulation, the mixture was topped up to 100% with deionized water. As shown in Table 11, increasing emulsifier content gradually improved the microemulsion's appearance, low-temperature stability, thermal storage stability, and dilution stability. At 15% EL-20, the formulation essentially met the relevant standards. Considering factors such as cost and environmental impact, the final EL-20 content in the Artemisia annua residue extract microemulsion was determined to be 15%.

[0184] Table 11 Emulsifier content determination results

[0185] Test number Content setting Appearance of preparation Low temperature stability Hot storage stability Dilution stability 1 5％ turbid turbid Large amounts of precipitation turbid 2 10％ Uniform and transparent Uniform, translucent Uniform and transparent Uniform, translucent 3 15％ Uniform and transparent Uniform and transparent Uniform and transparent Uniform and transparent 4 20％ Uniform and transparent Uniform and transparent Uniform and transparent Uniform and transparent 5 25％ Uniform and transparent Uniform and transparent Uniform and transparent Uniform and transparent 6 30％ Uniform and transparent Uniform and transparent Uniform and transparent Uniform and transparent

[0186] 2.7.4 Determination of the formula of microemulsion of Artemisia annua residue extract

[0187] Based on the above series of test results, the formula composition of the Artemisia annua residue extract microemulsion was finally determined, as shown in Table 12 below:

[0188] Table 12 Microemulsion formulation composition

[0189]

[0190]

[0191] 2.8 Effects of Artemisia annua residue extract microemulsion on tobacco safety

[0192] The results of the investigation on the direct effects of the test microemulsion of Artemisia annua residue extract on the growth of three tobacco varieties are shown in Table 13. Within 21 days after treatment with 800-2000 times dilution of the microemulsion of Artemisia annua residue extract, the leaves of the three tobacco varieties did not show symptoms of discoloration, necrosis, deformity, and wilting.

[0193] The effects of the test agent, Artemisia annua residue extract microemulsion, on tobacco growth and development are shown in Table 14. As shown in Table 14, after treatment with 800-2000 times the Artemisia annua residue extract microemulsion, there was no significant difference in plant height compared to the water control for the three tobacco varieties, and the growth rate inhibition rate was less than 10%, indicating that the test agent is safe for tobacco growth.

[0194] In conclusion, treatment with 800-2000 times dilution of microemulsion of Artemisia annua residue extract had no adverse effects on the growth of the three tobacco varieties, with a safety factor of 4.

[0195] Table 13 Effect of Artemisia annua residue extract microemulsion on tobacco safety

[0196]

[0197]

[0198] Table 14 Effects of Artemisia annua residue extract microemulsion on tobacco growth

[0199]

[0200] 2.9 Field control effect of Artemisia annua residue extract microemulsion on tobacco bacterial wilt

[0201] Microemulsions of Artemisia annua residue extract were prepared and field efficacy tests were carried out. The results showed that the control effect of 1400-fold and 800-fold dilutions of microemulsions of Artemisia annua residue extract on tobacco bacterial wilt reached more than 60%, which was better than the control agent 500-fold dilution of 20% thiophanate-methyl suspension (Table 15).

[0202] Table 15 Control effect of Artemisia annua residue extract microemulsion on tobacco bacterial wilt

[0203]

[0204]

[0205] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

Claims

1. A method for extracting scopoletin from Artemisia annua residue, characterized in that: The method comprises the following steps: S1. The Artemisia annua residue was heated under reflux for extraction, and the filtrate was filtered while hot; S2. The filtrate obtained in step S1 is concentrated to an extract; S3. The extract obtained in step S2 was dissolved in methanol, diluted with water to form an extract suspension, and enriched with a macroporous adsorption resin to obtain an extract of Artemisia annua residue; S4. Recrystallize the Artemisia annua residue extract obtained in step S3 to obtain high-purity scopoletin using a one-step method.

2. The method according to claim 1, characterized in that The artemisia annua residue is the discarded residue from which artemisinin has been extracted.

3. The method according to claim 1, characterized in that The extraction solvent in the heating reflux extraction is a mixture of ethyl acetate and petroleum ether, and the volume percentage of the ethyl acetate is 82%.

4. The method according to claim 1, wherein The solid-liquid ratio in the heating reflux extraction is 1 g:8 mL, and the extraction time in the heating reflux extraction is 77 min.

5. The method according to claim 1, characterized in that The macroporous adsorption resin is macroporous adsorption resin NKA-2, and the eluent is methanol and water in a volume ratio of 4:

1.

6. The method according to claim 1, characterized in that The solvent for the one-step recrystallization is ethyl acetate, and petroleum ether is slowly added dropwise while stirring.

7. Use of the method according to any one of claims 1 to 6 in improving the extraction rate of scopoletin.

8. An extract of Artemisia annua residue, characterized in that The Artemisia annua residue extract is prepared by the method according to any one of claims 1 to 6, and the Artemisia annua residue extract comprises scopoletin, artemisinic acid, isofraxidin, syringetin and deoxyartemisinin.

9. A microemulsion of an Artemisia annua residue extract, characterized in that: The Artemisia annua residue extract microemulsion consists of Artemisia annua residue extract, acetone, ethyl acetate, ethanol, emulsifier EL-20, anionic surfactant 500# and water. The active ingredient of the Artemisia annua residue extract is scopoletin, which is prepared by the method described in any one of claims 1 to 6.

10. Use of the microemulsion of Artemisia annua residue extract according to claim 9 in the prevention and treatment of tobacco bacterial wilt.

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