Preparation method of flavone or derivative thereof and application of flavone or derivative thereof in preparation of preparation for preventing and treating wheat scab

Abstract

The invention provides a preparation method of flavone or a derivative thereof and an application of the flavone or the derivative thereof in preparing a preparation for preventing and treating wheat scab, which comprises the following steps of: separating an active ingredient 3, 5-dihydroxy-6, 7, 8-trimethyl-1, 3-pentanediol from an artemisia annua extract; the invention relates to a preparation for preventing and treating wheat scab, which is prepared by taking 3, 3 ', 4'-tetramethoxyflavone as an effective component and taking head oil or tail oil of plant essential oil as a solvent. According to the pesticide preparation, active ingredients separated from plants serve as effective ingredients and have excellent environmental compatibility, head oil or tail oil of plant essential oil serves as a solvent in the preparation processing process, toxic aromatic hydrocarbon solvents are avoided, the safety of the pesticide preparation is improved, environmental pollution is reduced, the production cost is reduced, and the pesticide preparation is suitable for industrial production. And the application of a large amount of head oil and tail oil generated in the production process of the plant essential oil is widened.

Description

Technical Field

[0001] This invention belongs to the field of pesticide and plant protection technology, specifically involving the preparation method of flavonoids or their derivatives and their application in the preparation of agents to control wheat scab. Background Technology

[0002] Due to environmental protection requirements, pesticides should now be developed towards highly efficient, low-toxicity, or pollution-free green pesticides. However, the production and processing of pesticides often involve the use of large amounts of organic solvents, such as benzene, toluene, and xylene. This poses a significant threat to the health of producers and users, and also has a huge impact on the ecological environment, such as causing soil compaction and environmental damage due to the volatilization and drift of organic solvents.

[0003] Essential oils are primarily produced using steam distillation. The oil distilled in the initial stages of distillation is called head oil, and the oil distilled at the end is called tail oil. Head and tail oils are byproducts of essential oil production. They are produced in large quantities but are of very poor quality. Compared to the essential oils distilled from the middle fraction, they are not used in industrial production due to their status as byproducts and are often treated as waste. Summary of the Invention

[0004] Purpose of the invention: To solve the above-mentioned technical problems, this invention provides a method for preparing flavonoids or their derivatives and their application in preparing agents for controlling wheat scab. The method utilizes the head or tail oil of plant essential oils as a solvent to prepare the agent for controlling wheat scab. The active ingredient in the agent is 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids, which are active ingredients isolated from plants and have excellent environmental compatibility. This promotes environmental protection and the development of green food, and has significant economic and social benefits.

[0005] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: the application of a flavonoid or its derivative in the preparation of a preparation for preventing and controlling wheat scab.

[0006] Preferably, the flavonoid or its derivative is selected from 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid.

[0007] This invention also provides a method for extracting and separating 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids, comprising the following steps: S1, Artemisia annua powder was extracted with methanol solvent under ultrasonic assistance to obtain Artemisia annua methanol extract; S2, using liquid-liquid extraction, the methanol extract of Artemisia annua was mixed with the extractant and extracted, and the oil phase extract was obtained by standing. S3. The oil phase extract was separated and purified by column chromatography to obtain the yellow needle-like active substance of 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid.

[0008] Preferably, the extractant is petroleum ether, chloroform, ethyl acetate, or n-butanol.

[0009] This invention also provides a formulation for controlling wheat scab, which uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone obtained from Artemisia annua extract as the active ingredient, and mixes it with the head oil and / or tail oil produced during the production of plant essential oils as a solvent to obtain a pesticide formulation for controlling wheat scab.

[0010] Preferably, the head oil and tail oil are selected from eugenol, rose oil, citronella oil, cinnamon oil, spearmint oil, peppermint oil, pepper leaf oil, anise oil, fennel oil, star anise oil, wild thyme oil, wild chrysanthemum oil, chrysanthemum oil, Shennong chrysanthemum oil, wedelia trifoliata oil, marigold oil, Eucalyptus globulus oil, dill oil, calamus oil, garlic oil, litsea cubeba oil, mustard oil, laurel oil, safflower oil, safflower oil, and patchouli. The first and last oils produced during the production of various herbs and plants, including patchouli oil, lavender oil, fragrant grass oil, oregano oil, lantana oil, eucalyptus oil, osmanthus oil, gardenia oil, artichoke oil, motherwort oil, lemongrass oil, lemon oil, catnip oil, wild catnip oil, wormwood oil, artemisia oil, wormwood oil, myristyl ether, carnation oil, birch oil, lily of the valley oil, angelica oil, custard apple oil, and wormwood oil.

[0011] Preferably, an emulsifier is also included. A conventional emulsifier is sufficient.

[0012] Preferably, the active ingredient accounts for 0.01-50% of the total weight.

[0013] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: It can greatly reduce the environmental harm caused by organic solvents, lower production costs, and reduce environmental pollution. This invention not only broadens the uses of the first and last oils of plant essential oils, enriches the market for the development and use of plant essential oils, promotes the production and development of plant essential oils, but also drives the development and application of green, pollution-free, and environmentally friendly pesticides, thus having significant economic and social benefits. Attached Figure Description

[0014] Figure 1 This is a flow chart of the liquid-liquid distribution of the crude methanol extract of Artemisia annua described in the embodiments of the present invention.

[0015] Figure 2 It is 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids 1 H NMR spectrum.

[0016] Figure 3 It is 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids 13 C10 NMR spectrum. Detailed Implementation

[0017] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0018] my country has abundant and widely distributed Artemisia annua resources. Large-scale cultivation bases for Artemisia annua have emerged in some areas, providing raw materials for artemisinin production. Artemisinin accounts for less than 1% of the plant's leaves. After extracting artemisinin with organic solvents, the concentrated mother liquor still contains abundant secondary compounds, such as chlorophyll, artemisinic acid, coumaric acid, flavonoids, stigmasterol, sterol, artemisinone, isoartemisinone, eucalyptol, and volatile oils. Currently, the mother liquor has only undergone limited development and utilization, and the application of its active ingredients in agricultural production has not yet attracted attention. This invention uses a liquid-liquid partition extraction method to preliminarily separate the methanol extract of Artemisia annua obtained by large-scale extraction, yielding petroleum ether, chloroform, ethyl acetate, n-butanol, and aqueous phases. Activity tracking results show that the petroleum ether extract has excellent inhibitory activity against Fusarium graminearum, the causal agent of wheat blight. The petroleum ether extract was subjected to column chromatography for separation, purification, and activity tracking. The molecular structure of the active compound was determined using analytical techniques such as nuclear magnetic resonance (NMR), yielding the active compound 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid. Further indoor and field experiments confirmed the development and application value of this compound in the control of wheat scab. The structural formula of the 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid of this invention is as follows:

[0019] This invention extracts the active ingredient 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids from Artemisia annua. The specific extraction and separation method is as follows: (1) The dried powder of Artemisia annua was extracted with methanol solvent and ultrasonic extraction was used to obtain the methanol extract of Artemisia annua.

[0020] (2) The methanol extract of Artemisia annua extracted above was initially separated by liquid-liquid partition extraction to obtain petroleum ether phase, chloroform phase, ethyl acetate phase, n-butanol phase and aqueous phase, respectively; the activity tracking results showed that the petroleum ether extract had a relatively strong inhibitory activity against Fusarium graminearum.

[0021] (3) The petroleum ether phase extract was subjected to column chromatography for separation, purification, and activity tracking, yielding the active substance as yellow needle-like crystals. The specific liquid-liquid partitioning process of the crude methanol extract of Artemisia annua is shown below: Under ultrasonic assistance, 20 g of dried Artemisia annua powder was placed in a 500 mL stoppered Erlenmeyer flask, 200 mL of methanol solvent was added, the mixture was shaken well, and left in the dark overnight. Ultrasonic extraction was then performed at room temperature for 20 minutes. The mixture was filtered, and the extraction was repeated three times. The extracts were combined and concentrated to dryness under reduced pressure to obtain the methanol extract. Liquid-liquid extraction was used. 10 g of Artemisia annua methanol extract was dissolved in a small amount of methanol, then added to 400 mL of distilled water. The solution was transferred to a 1000 mL separatory funnel and extracted three times with 300 mL of petroleum ether per extraction. This was followed by three extractions with 300 mL of chloroform per extraction, then three extractions with 300 mL of ethyl acetate per extraction, and finally three extractions with 300 mL of n-butanol per extraction. Each extract was concentrated to dryness, weighed, and stored at 4°C for later use. The petroleum ether extract, chloroform extract, ethyl acetate extract, n-butanol extract, and water extract were obtained separately. Activity tracking showed that the active ingredient was present in the petroleum ether extract.

[0022] The petroleum ether extract was separated by silica gel column chromatography. First, the active extract was initially separated using 100-200 mesh silica gel and spotted onto a GF254 silica gel plate. After color development, fractions with similar spotting results were combined. The combined fractions were then subjected to activity determination, and the active fractions were further separated until the active ingredient was obtained. The extraction and separation process is described below. Figure 1 .

[0023] The active compound, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone, was isolated by nuclear magnetic resonance analysis and identified as a yellow needle-like crystalline substance, indicating that the compound is 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone.

[0024] Structural parameters of 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids: Yellow crystals. mp 210-212. IR (KBr) 3440-3100, 2960, 2940, 1680, 1620, 1560, 1540, 1465, 1420, 1385, 1235, 1210, 1200, 1180, 1010, 810 cm⁻¹ -1 ; 1H NMR(400Mz, CDCl3, TMS): (d3.85(3H,s,OMe),3.91(3H,s,OMe),3.98(3H,sOMe),4.01(3H,sOMe),6.01(1H,s,OH ), 6.52(1H,s,H-8) ,7.02(1H, d, J=8.5 Hz,H-5'), 7.65 (1H, d,J=8.5 Hz,H- 2'), 7.73(1H,sa, H-6'), 12.65 (1H,s OH). 13 CNMR(75Mz,CDCl3,TMS): (d56.04 (OMe at C-3'), 56.23(OMe at C-4'),60.07(OMe at C-7), 60.79(OMe at C-6), 90.23(C-8), 104.80(C-10), 110.84(C-2'),114.49(C-5'),122.37(C-1'), 122.51(C-6'), 138.61(C-3), 146.26(C-6), 148.25(C-2, C-3'), 152.21(C-4'), 152.69(C-5), 155.86(C-7), 158.68(C-9), 178.80 (C-4).MS (m / z) 374 ([M + ], 100,C 19 H 18 O8), 373(27), 360(16), 359(62), 355(15), 331(13),316(5), 181(6), 164(7), 151(12). Activity test 1: The activity of the active compound 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone against Fusarium graminearum ( Fusarium gramineae bactericidal activity Test strain: Fusarium graminearum, the pathogen of wheat scab (Fusarium graminearum). Fusarium gramineae (This is a routinely tested pathogen, purchased from the Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University.)

[0025] Test compounds: 97.5% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone, 98% prothioconazole.

[0026] Experimental method: The activity of the tested compounds against fungicides by the mycelial growth rate method was determined, and the median inhibitory concentration (EC50) of the compound against wheat scab pathogen was calculated. 50The Fusarium head blight strain was inoculated onto potato dextrose agar and activated at 28°C. After colony growth stabilized, 5 mm diameter mycelial cakes were collected using a sterile punch and stored at 4°C for later use. Five concentrations of each of the above compounds were prepared. 1 mL of the prepared test solution was added to 100 mL of sterile melted potato dextrose agar, thoroughly mixed, and poured into 9 cm diameter sterile Petri dishes to prepare drug-containing plates. After solidification, 5 mm diameter mycelial cakes were inoculated and incubated at 28°C. Each treatment was replicated in triplicate, with a control treatment using potato dextrose agar inoculated with 1 mL of sterile water. After 3 days of incubation at 28°C, the colony diameter was measured using the cross-cross method, and the mycelial growth inhibition rate was calculated using the following formula.

[0027] Mycelial growth inhibition rate = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100% Using the logarithmic values ​​of the mass concentrations of each compound as the x-axis and the inhibition rate as the y-axis, the toxicity regression equation, correlation coefficient (r), and median inhibitory concentration (EC50) were obtained using DPS software. 50 value).

[0028] Table 1. Effects of 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids on Fusarium graminearum (… Fusarium grasses ) activity

[0029] Table 1 shows that the median inhibitory concentration (EC5) of 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids on Fusarium graminearum is... 50 The value was 1.66 mg / L; while the median inhibitory concentration (EC50) of prothioconazole against Fusarium graminearum was 1.66 mg / L. 50 The concentration of the active ingredient was 1.46 mg / L, and there was no significant difference in activity between the two. However, the active ingredient selected in this invention, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid, is a plant-derived active ingredient derived from Artemisia annua, which has a greater market advantage in terms of environmental safety. The following describes the extraction of the active ingredient in combination with different head oils and / or tail oils: I. Preparation of Examples Example 1: 0.01% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and Litsea cubeba oil and cinnamon oil as solvents. The oil is prepared by dissolving, stirring and dispensing emulsifiable concentrates, with 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounting for 0.01%, Litsea cubeba oil for 0.10%, and cinnamon oil for 94.89%. The emulsifier is added to make 100%.

[0030] Example 2: 2.5% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient and rose oil as the solvent. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounts for 2.5%, rose oil accounts for 90.5%, and emulsifier is added to make 100%. It is processed by dissolving, stirring, and dispensing emulsifiable oil.

[0031] Example 3: 10% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and peppermint oil head oil and citrus peel oil tail oil as solvents. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounts for 10%, peppermint oil head oil 40%, and citrus peel oil tail oil 43%. Emulsifier is added to 100%, and the mixture is processed using the emulsifiable concentrate processing method of dissolving, stirring, and dispensing.

[0032] Example 4: 50% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and the solvents are jasmine oil (top oil), spearmint oil (tail oil), and lemongrass oil (top oil). 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 50%, jasmine oil (top oil) 17%, spearmint oil (tail oil) 15%, and lemongrass oil (top oil) 10%. Other adjuvants are added to make 100%. The mixture is processed using the emulsifiable concentrate processing method of dissolving, stirring, and dispensing.

[0033] Example 5: 5% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and cinnamon oil head oil and laurel oil tail oil as solvents. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounts for 5%, cinnamon oil head oil accounts for 37%, and laurel oil tail oil accounts for 43%. The emulsifier is added to 100%, and the product is processed by dissolving, stirring, and dispensing emulsifiable concentrate processing methods.

[0034] Example 6: 0.3% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and rose oil, eugenol tail oil, and artemisia annua oil as solvents. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounts for 0.3%, rose oil 68.4%, eugenol tail oil 16.0%, and artemisia annua oil 7.0%. Emulsifier is added to bring the total to 100%. The mixture is processed using a dissolving, stirring, and packaging emulsion processing method.

[0035] Example 7: 10% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and rose oil head oil and laurel oil tail oil as solvents. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounts for 10%, rose oil head oil 62%, and laurel oil tail oil 11%. Emulsifier is added to 100%, and the mixture is processed using the emulsifiable concentrate processing method of dissolving, stirring, and dispensing.

[0036] Example 8: 50% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsifiable concentrate This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and garlic oil tail oil, Litsea cubeba oil head oil, and mustard oil tail oil as solvents. Among them, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone accounts for 50%, garlic oil tail oil 16%, Litsea cubeba oil head oil 15%, and mustard oil tail oil 9.2%. The emulsifier is added to 100%, and it is processed by dissolving, stirring, and dispensing emulsifiable concentrate processing method.

[0037] Example 9: 40% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsifiable concentrate The active ingredient used in this embodiment is 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids, and the solvents are Artemisia annua oil (head oil), Artemisia capillaris oil (head oil), Artemisia argyi oil (tail oil), and Artemisia argyi oil (tail oil). Among them, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 40.0%, Artemisia annua oil (head oil) 8.0%, Artemisia capillaris oil (head oil) 19.2%, Artemisia argyi oil (tail oil) 19.0%, and Artemisia argyi oil (tail oil) 6.2%. The emulsifier is added to 100%, and it is processed according to the emulsifiable concentrate processing method of dissolving, stirring, and dispensing.

[0038] Example 10: 12% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and the solvents are sclerotium truncatum oil (head oil), sclerotium truncatum oil (tail oil), patchouli oil (tail oil), patchouli oil (head oil), and lavender oil (head oil). 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 120%. Sclerotium truncatum oil (head oil) accounts for 19.0%, sclerotium truncatum oil (tail oil) 18.1%, patchouli oil (tail oil) 8.6%, patchouli oil (head oil) 22.4%, and lavender oil (head oil) 12.8%. Emulsifier is added to bring the total to 100%. The mixture is processed using a dissolving, stirring, and dispensing emulsifiable concentrate method.

[0039] Example 11: 3% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and Angelica sinensis oil, custard apple oil, and Artemisia capillaris oil as solvents. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 3.0%, Angelica sinensis oil 41.0%, custard apple oil 10%, and Artemisia capillaris oil 30.0%. Emulsifier is added to bring the total to 100%. The mixture is processed using a dissolving, stirring, and packaging emulsifiable concentrate method.

[0040] Example 12: 20% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and the solvents are lantana oil (top oil), oregano oil (top oil), lantana oil (tail oil), eucalyptus oil (tail oil), and osmanthus oil (tail oil). Among them, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 20.0%, lantana oil (top oil) 5.0%, oregano oil (top oil) 10.2%, lantana oil (tail oil) 21.0%, eucalyptus oil (tail oil) 20.0%, and osmanthus oil (tail oil) 13.0%. The emulsifier is added to 100%, and it is processed according to the emulsifiable concentrate processing method of dissolving, stirring, and dispensing.

[0041] Example 13: 1.8% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and anise oil, fennel oil, and star anise oil as solvents. 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 1.8%, anise oil 25.0%, fennel oil 32.0%, and star anise oil 31.0%. Emulsifier is added to bring the total to 100%. The mixture is processed using a dissolving, stirring, and packaging emulsion processing method.

[0042] Example 14: 20% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsifiable concentrate This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and the solvents are the head oil, tail oil, and patchouli oil of *Cinnamomum camphora*. The composition is as follows: 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids 20.0%, head oil of *Cinnamomum camphora* 12.0%, tail oil of *Cinnamomum camphora* 25.0%, and head oil of patchouli 31.8%. Emulsifier is added to bring the total to 100%. The mixture is processed using a dissolving, stirring, and packaging emulsifiable concentrate method.

[0043] Example 15: 20% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and lemongrass oil head oil, lemon oil head oil, catnip oil tail oil, wild catnip oil tail oil, and artemisia oil tail oil as the solvents. Among them, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids account for 20%, lemongrass oil head oil 4.1%, lemon oil head oil 14.0%, catnip oil tail oil 9.4%, wild catnip oil tail oil 6.4%, and artemisia oil tail oil 35%. The emulsifier is added to 100%, and it is processed by dissolving, stirring, and dispensing emulsifiable oil processing methods.

[0044] Example 16: 10% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and myristole head oil and pepper leaf tail oil as solvents. The active ingredient is 10% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone, 44.0% myristole head oil, and 40.0% pepper leaf tail oil. The emulsifier is added to 100%, and the product is processed by dissolving, stirring, and dispensing emulsifiable concentrate.

[0045] Example 17: 5.0% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and wild thyme oil (head oil), wild chrysanthemum oil (head oil), chrysanthemum oil (tail oil), *Chrysanthemum indicum* oil (head oil), *Wedelia trifoliata* oil (head oil), marigold oil (head oil), and nutmeg oil (tail oil) as solvents. Specifically, 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids comprise 5.0%, wild thyme oil (head oil) 12.0%, wild chrysanthemum oil (head oil) 7.2%, chrysanthemum oil (tail oil) 11.8%, *Chrysanthemum indicum* oil (head oil) 12.0%, *Wedelia trifoliata* oil (head oil) 18.0%, marigold oil (head oil) 21.0%, and nutmeg oil (tail oil) 12.0%. Emulsifier is added to bring the total to 100%. The mixture is processed using a dissolving, stirring, and packaging emulsifiable concentrate method.

[0046] Example 18: 40% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsifiable concentrate This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and Eucalyptus globulus oil (top oil), dill oil (top oil), and Acorus calamus oil (tail oil) as solvents. The composition of 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone is 40%, Eucalyptus globulus oil (top oil) is 20%, dill oil (top oil) is 15%, and Acorus calamus oil (tail oil) is 23%. The emulsifier is added to bring the total to 100%. The emulsifiable concentrate is processed using a dissolving, stirring, and packaging method.

[0047] Example 19: 4.5% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone as the active ingredient, and carnation oil (top oil), birch oil (tail oil), and lily of the valley oil (tail oil) as solvents. The proportions are as follows: 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone 4.5%, carnation oil (top oil) 21.0%, birch oil (tail oil) 18.0%, and lily of the valley oil (tail oil) 50.0%. The emulsifier is added to 100%, and the mixture is processed using a dissolving, stirring, and dispensing emulsifiable concentrate processing method.

[0048] Example 20: 25% 3,5-Dihydroxy-6,7,3',4'-Tetramethoxyflavonoid emulsion This embodiment uses 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids as the active ingredient, and gardenia oil (head oil), ageratum oil (head oil), motherwort oil (tail oil), lemongrass oil (head oil), and lemon oil (tail oil) as solvents. The composition is as follows: 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids 25.0%, gardenia oil (head oil) 19.8%, ageratum oil (head oil) 18.0%, motherwort oil (tail oil) 6.1%, lemongrass oil (head oil) 9.5%, lemon oil (tail oil) 20.0%. The emulsifier is added to 100%, and the mixture is processed using the dissolving, stirring, and packaging emulsifiable concentrate processing method.

[0049] II. Activity and Efficacy Testing Activity test 1: 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate against Fusarium graminearum (Fusarium graminearum) Fusarium gramineae bactericidal activity assay Test strain: Fusarium graminearum, the pathogen of wheat scab (Fusarium graminearum). Fusarium gramineae (This is a routinely tested pathogen, purchased from the Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University.)

[0050] Test reagents: 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared in this invention, with Eucalyptus globulus oil, Dill oil, and Acorus calamus oil as solvents), 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared with xylene as solvent), and 40% prothioconazole·tebuconazole suspension (commercially available formulation).

[0051] Experimental methods: The mycelial growth rate method was used to determine the activity of the three prepared formulations against the fungicide *Fusarium graminearum*, the pathogen of wheat scab, and to calculate their median inhibitory concentration (EC50). 50 The Fusarium head blight strain was inoculated onto potato dextrose agar and activated at 28°C. After colony growth stabilized, 5 mm diameter mycelial cakes were collected using a sterile punch and stored at 4°C for later use. Five concentrations of each agent were prepared for the experiment. 1 mL of the prepared test solution was added to 100 mL of sterile melted potato dextrose agar, thoroughly mixed, and poured into 9 cm diameter sterile Petri dishes to prepare drug-containing plates. After solidification, 5 mm diameter mycelial cakes were inoculated and incubated at 28°C. Each treatment was replicated in triplicate, with a control treatment using potato dextrose agar inoculated with 1 mL of sterile water. After 3 days of incubation at 28°C, the colony diameter was measured using the cross-hatching method, and the mycelial growth inhibition rate was calculated using the following formula.

[0052] Mycelial growth inhibition rate = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100% Using the logarithmic mass concentration of each drug as the abscissa (x) and the inhibition rate as the ordinate (y), the toxicity regression equation, correlation coefficient (r), and median inhibitory concentration (EC50 value) were obtained using DPS software.

[0053] Table 2. Effects of 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate on Fusarium graminearum (… Fusarium grasses ) activity

[0054] Table 2 shows the median concentration (EC50) of conventional 40% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate prepared with toluene as a solvent and newly prepared 40% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate prepared with head oil or tail oil as solvents against wheat scab. 50The concentrations were 3.41 mg / L and 1.82 mg / L, respectively. Furthermore, the newly formulated 40% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate using head or tail oil as solvents showed no significant difference in activity against wheat scab compared to the commercially available chemical pesticide 40% prothioconazole·tebuconazole suspension. The formulations using Eucalyptus oil head oil, dill oil head oil, and Acorus calamus oil tail oil as solvents exhibited significantly higher antifungal activity against Fusarium graminearum than the conventional emulsifiable concentrate formulation prepared with toluene, with an activity 1.87 times higher. Additionally, the active ingredient 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid selected in this invention is a plant-derived active ingredient derived from Artemisia annua, offering a greater market advantage in terms of environmental safety.

[0055] Activity test 2: 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate against Fusarium graminearum (… Fusarium gramineae bactericidal activity assay Test strain: Fusarium graminearum, the pathogen of wheat scab (Fusarium graminearum). Fusarium gramineae (This is a routinely tested pathogen, purchased from the Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University.)

[0056] Test reagents: 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared in this invention, with gardenia oil, artichoke oil, motherwort oil, lemongrass oil, and lemon oil as solvents), 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared with xylene as solvent), and 25% cyazofamid suspension concentrate (commercially available formulation).

[0057] Experimental methods: The mycelial growth rate method was used to determine the activity of the three prepared formulations against the fungicide *Fusarium graminearum*, the pathogen of wheat scab, and to calculate their effective inhibitory concentration (EC50). 50 The Fusarium head blight strain was inoculated onto potato dextrose agar and incubated at 28°C for activation. After colony growth stabilized, 5 mm diameter mycelial cakes were collected using a sterile punch and stored at 4°C for later use. Five concentrations of each agent were prepared for the experiment. 1 mL of the prepared test solution was added to 100 mL of sterile melted potato dextrose agar, thoroughly mixed, and poured into 9 cm diameter sterile Petri dishes to prepare drug-containing plates. After solidification, 5 mm diameter mycelial cakes were inoculated and incubated at 28°C. Each treatment was replicated in triplicate; a control group was prepared by inoculating mycelial cakes onto potato dextrose agar with 1 mL of sterile water. After 3 days of incubation at 28°C, the colony diameter was measured using the cross-hatching method, and the mycelial growth inhibition rate was calculated using the following formula.

[0058] Mycelial growth inhibition rate = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100% Using the logarithmic values ​​of each drug's mass concentration as the abscissa (x) and the inhibition rate as the ordinate (y), the toxicity regression equation, correlation coefficient (r), and effective inhibitory median concentration (EC) were obtained using DPS software. 50 value).

[0059] Table 3. Effects of 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate on Fusarium graminearum (… Fusarium grasses ) activity

[0060] Table 3 shows the median inhibitory concentration (EC5) of newly formulated 20% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (EC5) against wheat scab using either the head or tail oil as solvent, and the conventional 20% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (EC5) formulated with toluene as solvent. 50 The values ​​were 1.75 mg / L and 3.05 mg / L, respectively; and the formulations prepared with gardenia oil, artichoke oil, motherwort oil, lemongrass oil, and lemon oil as solvents showed significantly higher antibacterial activity against Fusarium graminearum than conventional emulsifiable concentrates prepared with xylene as solvent, with an activity 1.74 times higher.

[0061] Efficacy Trial Implementation 3: Wheat Fusarium Head Blight Control Trial 1 Test reagents: 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared in this invention, with Eucalyptus globulus oil, Dill oil, and Acorus calamus oil as solvents), 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared with xylene as solvent), and 40% prothioconazole·tebuconazole suspension (commercially available formulation).

[0062] Experiment location: Wheat planting base in Gongdao Town, Hanjiang District, Yangzhou City, Jiangsu Province.

[0063] Experimental Design: The experiment consisted of 4 treatments, with a control treatment sprayed with an equal volume of water. Each treatment was replicated 3 times, for a total of 12 plots, each plot being 120 m². 2 The cells were randomly arranged. Treatment 1: 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (solvent is the head oil or tail oil of this invention) 40 mL / 667m 2 Treatment 2: 40.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (solvent-flavored xylene) 40 mL / 667m2 Treatment 3: 40% prothioconazole·tebuconazole suspension 40 mL / 667m 2 The pesticide was applied twice, once at the early flowering stage (April 27, 2025) and once at the heading stage (May 3, 2025), using an electric sprayer with a water volume of 15 L / 667m². 2 Spray the control area with an equal amount of water.

[0064] Investigation content and calculation method: The incidence of disease in each treatment was investigated and recorded 20 days after the last application of medication. A 5-point investigation method was used, with each point measuring 0.25 m. 2 Record the occurrence level and the corresponding number of diseased ears, and calculate the diseased ear rate, disease index, and disease control efficacy.

[0065] Grading standards: Grade 1: The area of ​​dead ears accounts for less than 1 / 4 of the total ear area; Grade 2: The area of ​​dead ears accounts for 1 / 4 to 1 / 2 of the total ear area; Grade 3: The area of ​​dead ears accounts for 1 / 2 to 3 / 4 of the total ear area; Level 4: The area of ​​dead ears accounts for 3 / 4 or more of the total ear area.

[0066] Calculation formula: Diseased ear rate (%) = (Number of diseased ears / Total number of ears surveyed) × 100; Disease control efficacy (%) = ((Disease rate in control area - Disease rate in treatment area) / Disease rate in control area) × 100; Disease index = (∑(number of diseased leaves at each level × disease severity value) / (total number of leaves surveyed × 4)) × 100; Disease control efficacy (%) = ((Disease index in control area - Disease index in treatment area) / Disease index in control area) × 100; Data processing and analysis: Statistical analysis was performed using WPS 2024 and SPSS 22.0. The final data were analyzed using one-way ANOVA, and Duncans' new multiple range method was used for multiple comparisons to determine the significance of differences between treatments (P < 0.05).

[0067] Table 4. Control efficacy of each treatment against wheat scab

[0068] Efficacy Trial Implementation 4: Wheat Fusarium Head Blight Control Trial II Test reagents: 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared in this invention, with gardenia oil, artichoke oil, motherwort oil, lemongrass oil, and lemon oil as solvents), 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (prepared with xylene as solvent), and 25% cyazofamid suspension concentrate (commercially available formulation).

[0069] Experiment location: Wheat planting base in Touzao Town, Dongtai City, Yancheng City, Jiangsu Province.

[0070] Experimental Design: The experiment included four treatments, with the control treatment sprayed with an equal volume of water. Each treatment was replicated three times, for a total of 12 plots, each plot being 120 m². 2 The cells were randomly arranged. Treatment 1: 200 mL / 667m³ of 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsion (solvent being the head or tail oil of this invention) 2 Treatment 2: 25.0% 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid emulsifiable concentrate (xylene solvent) 200 mL / 667m 2 25% Cyazofamid suspension concentrate (commercially available formulation) 200 mL / 667m 2 The pesticide was applied twice, once at the early flowering stage (April 28, 2025) and once at the heading stage (May 4, 2025), using an electric sprayer with a water volume of 15 L / 667m². 2 Spray the control area with an equal amount of water.

[0071] Investigation content and calculation method: The incidence of disease in each treatment was investigated and recorded 20 days after the last application of medication. A 5-point investigation method was used, with each point measuring 0.25 m. 2 Record the occurrence level and the corresponding number of diseased ears, and calculate the diseased ear rate, disease index, and disease control efficacy.

[0072] Grading standards: Grade 1: The area of ​​dead ears accounts for less than 1 / 4 of the total ear area; Grade 2: The area of ​​dead ears accounts for 1 / 4 to 1 / 2 of the total ear area; Grade 3: The area of ​​dead ears accounts for 1 / 2 to 3 / 4 of the total ear area; Level 4: The area of ​​dead ears accounts for 3 / 4 or more of the total ear area.

[0073] Calculation formula: Diseased ear rate (%) = (Number of diseased ears / Total number of ears surveyed) × 100; Disease control efficacy (%) = ((Disease rate in control area - Disease rate in treatment area) / Disease rate in control area) × 100; Disease index = (∑(number of diseased leaves at each level × disease severity value) / (total number of leaves surveyed × 4)) × 100; Disease control efficacy (%) = ((Disease index in control area - Disease index in treatment area) / Disease index in control area) × 100; Data processing and analysis: Statistical analysis was performed using WPS 2024 and SPSS 22.0. The final data were analyzed using one-way ANOVA, and Duncans' new multiple range method was used for multiple comparisons to determine the significance of differences between treatments (P < 0.05).

[0074] Table 5. Control efficacy of each treatment against wheat scab.

[0075] The above-mentioned indoor activity tests and field efficacy tests show that the agent for controlling wheat scab, prepared by using 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavone, an active ingredient isolated from Artemisia annua extract, as the effective component and using the head or tail oil of plant essential oil as a solvent, has significantly improved biological activity and enhanced the efficacy of the agent. Furthermore, it effectively utilizes industrial by-products. Therefore, this invention has strong operability and applicability.

[0076] The above description is only a partial embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of flavonoids or their derivatives in the preparation of agents for the prevention and control of wheat scab.

2. The application according to claim 1, wherein the flavonoid or its derivative is selected from 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids.

3. A method for extracting and separating 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids, characterized in that... Includes the following steps: S1, Artemisia annua powder was extracted with methanol solvent under ultrasonic assistance to obtain Artemisia annua methanol extract; S2, using liquid-liquid extraction, the methanol extract of Artemisia annua was mixed with the extractant and extracted, and the oil phase extract was obtained by standing. S3. The oil phase extract was separated and purified by column chromatography to obtain the yellow needle-like active substance of 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoid.

4. The extraction and separation method for 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids according to claim 3, characterized in that: The extractant used is petroleum ether, chloroform, ethyl acetate, or n-butanol.

5. An agent for controlling wheat scab, characterized in that: Using 3,5-dihydroxy-6,7,3',4'-tetramethoxyflavonoids isolated from Artemisia annua extract as the active ingredient, and mixing them with the head oil and / or tail oil produced during the production of plant essential oils as a solvent, a pesticide formulation for controlling wheat scab was obtained.

6. The preparation for controlling wheat scab according to claim 5, characterized in that: The head and tail oils are selected from eugenol, rose oil, lemongrass oil, cinnamon oil, spearmint oil, peppermint oil, pepper leaf oil, anise oil, fennel oil, star anise oil, wild thyme oil, wild chrysanthemum oil, chrysanthemum oil, Shennong chrysanthemum oil, wedelia trifoliata oil, marigold oil, Eucalyptus globulus oil, dill oil, calamus oil, garlic oil, litsea cubeba oil, mustard oil, laurel oil, safflower oil, wampee oil, patchouli oil, and more. The first and last oils produced during the production of various herbs and herbal oils, including patchouli oil, lavender oil, fragrant grass oil, oregano oil, lantana oil, eucalyptus oil, osmanthus oil, gardenia oil, artichoke oil, motherwort oil, lemongrass oil, lemon oil, catnip oil, wild catnip oil, wormwood oil, artemisia oil, mugwort oil, myristyl ether, carnation oil, birch oil, lily of the valley oil, angelica oil, custard apple oil, and wormwood oil.

7. The preparation for controlling wheat scab according to claim 5, characterized in that: It also includes emulsifiers.

8. The preparation for controlling wheat scab according to claim 7, characterized in that: The active ingredient accounts for 0.01-50% of the total weight.

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