Preparation method of flavonoid compounds and application thereof in anti-tobacco mosaic virus

Abstract

The application discloses a preparation method of a flavone compound and application of the flavone compound in resisting tobacco mosaic virus, and relates to a preparation method of a flavone compound, which comprises the following steps: extracting crushed seed of a fragrant plant by using a solvent 1 to obtain an extract; performing normal phase gradient elution on the extract to obtain a fraction 1; performing reverse phase gradient elution on the fraction 1 to obtain a fraction 2; eluents sequentially include water, a mixture of water and methanol, and methanol; and the fraction 2 is evaporated and recrystallized to obtain a flavone compound shown in formula I. The flavone compound is used for resisting tobacco mosaic virus, and has a direct antiviral effect on the tobacco mosaic virus.

Description

Technical Field

[0001] This invention belongs to the field of phytochemistry technology, specifically relating to a method for preparing flavonoids and their application in combating tobacco mosaic virus. Background Technology

[0002] Plant viral diseases are highly contagious, destructive, and difficult to control, making them the second most prevalent plant disease after fungal diseases. Often referred to as the "cancer" of plant diseases, a fundamental breakthrough has yet to be achieved. While cytosine nucleoside peptide antibiotics, such as ningnanmycin, have been registered for treating Tobacco mosaic virus (TMV), reported treatment efficacy ranges from 30% to 60%, showing instability and failing to eradicate TMV. Therefore, the control of plant viral diseases, particularly TMV, remains a key and challenging area of ​​focus in plant protection both domestically and internationally. Against this backdrop, screening and discovering highly active antiviral lead compounds has become crucial for creating novel plant antiviral agents from the source.

[0003] Flavonoid I, namely epicatechin gallate (EGCG), has the following structural formula:

[0004] Compound I.

[0005] Currently, there are existing literature reports that compound I has significant antiviral activity in the medical field. For example, the literature “Sun Q, Chen X, Zhang J, et al. Diverse Structures of Tea Polyphenols from RouguiWuyi Rock Tea and Their Potential as Inhibitor of 3C-like Protease. Molecules. 2025;30(5):1024” reports that compound I has inhibitory effects on SARS-CoV-2 virus in the medical field; the literature “Ogawa M, Shimojima M, Saijo M, Fukasawa M. Several catechins and flavonols from green tea inhibit severe fever with thrombocytopenia syndrome virus infection in vitro. J Infect Chemother. 2021;27(1):32-39” reports that compound I has antipyretic activity against fever with thrombocytopenia syndrome; the literature “Bamba M, Bordage S, Sahuc ME, et al. Anti-HCV Tannins From Plants Traditionally Used in West Africa and ExtractedWith Green Solvents” reports that compound I has antipyretic activity against fever with thrombocytopenia syndrome; and the literature “Bamba M, Bordage S, Sahuc ME, et al. Anti-HCV Tannins From Plants Traditionally Used in West Africa and ExtractedWith Green Solvents” reports that compound I has antipyretic activity against fever with thrombocytopenia syndrome. The compound was reported in Front Pharmacol. 2022;12:789688. It has anti-HCV effects. Summary of the Invention

[0006] To address the problems of unstable efficacy and difficulty in controlling existing antiviral agents for tobacco mosaic virus (TMV), this invention provides a method for preparing flavonoid compounds and their application in combating TMV. Compound I is used to combat TMV, exhibiting anti-TMV activity and the ability to induce systemic resistance to TMV in the host. It also increases the content of salicylic acid (SA), a stress-related substance in tobacco plants, and promotes the synthesis of resistance proteins (PRs). NPR1 , PR1 , PR2 and PR5 The expression of disease-resistant genes exerts an immune-inducing effect.

[0007] During the experiment, the inventor first obtained the fragrance from the fragrant tree ( Platycarya strobilaceaCompound I was extracted from the seeds of Siebold & Zucc. (the non-medicinal part of the fragrant tree), and the preparation method of the present invention has better environmental compatibility.

[0008] In this invention, the fragrant tree Platycarya strobilacea Siebold & Zucc. is a tall deciduous tree belonging to the genus *Siebold* in the family Juglandaceae. It is native to provinces south of the Qinling Mountains in my country, extending southwest to Yunnan, and is also distributed in Korea and Japan. Its fruit possesses medicinal value, including invigorating qi, dispelling wind, reducing swelling and relieving pain, drying dampness and killing parasites, and clearing heat and detoxifying. The *Siebold* tree used in this invention (…) Platycarya strobilacea The seeds of Siebold & Zucc. are the non-medicinal part of the Siebold tree.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing flavonoids, comprising the following steps: (1) Extract the crushed seeds of the fragrant tree using solvent 1 to obtain an extract; The volume ratio of the crushed seeds of the fragrant tree to the solvent is 1:(3~4), and the solvent 1 is one or more of dichloromethane, methanol and ethanol; (2) The extract was subjected to normal-phase gradient elution to obtain fraction 1; The eluent comprises, in sequence, a mixture of petroleum ether and ethyl acetate, and a mixture of dichloromethane and methanol, wherein the volume ratio of petroleum ether to ethyl acetate in the mixture of petroleum ether and ethyl acetate is (100~1):1, and the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is (20~1):1. (3) The fraction 1 is subjected to reverse-phase gradient elution to obtain fraction 2; the eluent includes water, a mixture of water and methanol, and methanol in sequence; (4) The fraction 2 is evaporated to dryness and recrystallized to obtain the flavonoid compound shown in Formula I:

[0010] Formula I.

[0011] In this invention, in step (1), the solvent 1 is preferably a mixture of dichloromethane and methanol in a volume ratio of 1:1.

[0012] In this invention, in step (1), the crushed material of the incense tree seeds is preferably the material after crushing the incense tree seeds and passing them through a sieve of 30-50 mesh (e.g., 40 mesh).

[0013] In this invention, in step (1), the extraction is preferably a cold soaking extraction at room temperature.

[0014] In this invention, in step (1), the extraction method preferably includes: changing the solvent every 4 to 7 days, and extracting a total of 3 to 4 times.

[0015] In this invention, during step (2) of normal phase gradient elution, preferably, the volume ratio of the petroleum ether and the ethyl acetate is 100:1, 20:1, 10:1, 5:1, 3:1 and 1:1, respectively.

[0016] In this invention, during step (2) of the normal phase gradient elution, preferably, the volume ratio of dichloromethane to methanol is 20:1, 10:1 and 1:1, respectively.

[0017] In a preferred embodiment of the present invention, the elution is first performed sequentially with petroleum ether and ethyl acetate at volume ratios of 100:1, 20:1, 10:1, 5:1, 3:1, and 1:1, followed by sequential elution with dichloromethane and methanol at volume ratios of 20:1, 10:1, and 1:1. Fraction 1 is obtained at a dichloromethane and methanol volume ratio of 10:1; the other volume ratios are used to elute impurities.

[0018] In this invention, in step (2), the positive phase gradient elution preferably uses silica gel as the filler.

[0019] Preferably, the particle size of the silica gel is 100-200 mesh.

[0020] The silicone material can be ordinary silicone or Diol silicone, preferably Diol silicone.

[0021] In this invention, in step (2), the column temperature for the positive phase gradient elution can be room temperature.

[0022] In this invention, in step (2), the flow rate of the mobile phase in the positive phase gradient elution is preferably 1-2 drops / second.

[0023] In this invention, in step (3), the volume of the methanol in the mixture of water and methanol preferably accounts for 20-80%, for example 40% or 60%, of the mixture of water and methanol.

[0024] In a preferred embodiment of the present invention, the eluent sequentially comprises water, a mixture of water and methanol with a methanol content of 20%, a mixture of water and methanol with a methanol content of 40%, a mixture of water and methanol with a methanol content of 60%, a mixture of water and methanol with a methanol content of 80%, and methanol.

[0025] In this invention, in step (3), the column temperature during reverse gradient elution is usually room temperature.

[0026] In this invention, in step (3), the flow rate of the mobile phase in the reverse gradient elution is preferably 1 to 2 drops per second.

[0027] In this invention, in step (3), reverse gradient elution is preferably performed under reduced pressure conditions, with a vacuum degree less than or equal to -0.08 MPa.

[0028] In this invention, in step (3), during the reverse gradient elution, the water is usually pure water.

[0029] In this invention, in step (3), reversed-phase gradient elution uses C18 reversed-phase chromatography as the packing material.

[0030] In this invention, in step (4), the temperature of evaporation is 50~60℃.

[0031] In this invention, in step (4), the solvent 2 used for recrystallization is one or more of dichloromethane, acetone and ethyl acetate; In this invention, in step (4), the amount of solvent 2 added during recrystallization is such that the fraction 2 is in a suspended state at room temperature; In this invention, in step (4), the recrystallization method includes: mixing the fraction 2 with solvent 2, heating to dissolve, filtering, and keeping the solid part at 0~8℃ (e.g. 4℃) for 10~16h; Preferably, the heating temperature is 50~60°C.

[0032] This invention also provides the application of flavonoids as active ingredients in the fight against tobacco mosaic virus, wherein the flavonoids are as shown in Formula I:

[0033] Formula I.

[0034] In this invention, the flavonoids are preferably used as a control agent and immune inducer for tobacco mosaic virus.

[0035] In this invention, the "control agent" is used to directly interact with the virus to produce an antiviral effect, and the "immune inducer" is used to induce plants to develop resistance and thus exert a disease-resistant effect.

[0036] In this invention, the compound can be used directly as a plant virus inhibitor, or it can be used with an agriculturally acceptable vector.

[0037] In this invention, the flavonoids are preferably used in the preparation of expression promoters for disease-resistant protein PRs synthesis-related genes and promoters for SA content in tobacco; the disease-resistant protein PRs synthesis-related genes are, for example,... NPR1 , PR1 , PR2 or PR5 .

[0038] In this invention, the concentration of the flavonoid compound in the pharmaceutical preparation containing the flavonoid compound is preferably 125~500 μg / mL, for example 250 μg / mL.

[0039] In this invention, the flavonoids are preferably flavonoids prepared by the aforementioned method.

[0040] The positive and progressive effects of this invention are as follows: (1) This invention is the first to extract flavonoids of Formula I from the seeds of the Chinese incense tree. The preparation method of this invention has better environmental compatibility.

[0041] (2) The present invention uses the flavonoids shown in Formula I to fight tobacco mosaic virus, which has a direct antiviral effect on tobacco mosaic virus, and can also increase the content of salicylic acid (SA), a stress-related substance in tobacco, and promote the synthesis of disease-resistant proteins PRs. NPR1 , PR1 , PR2 and PR5 The expression of disease-resistant genes exerts an immune-inducing effect. Attached Figure Description

[0042] Figure 1 The figure shows the passivation effect of compound I at different concentrations on TMV in fresh tobacco leaves.

[0043] Figure 2 The figure shows the protective effect of different concentrations of compound I on TMV in fresh tobacco leaves.

[0044] Figure 3 The image shows the therapeutic effects of different concentrations of compound I on TMV in fresh tobacco leaves.

[0045] Figure 4 The diagram shows the effect of compound I on the morphology of TMV virus particles. In the diagram, A shows TMV treated with compound I at 30,000x magnification; B shows TMV treated with compound I at 50,000x magnification; C shows TMV treated with 1% DMSO at 30,000x magnification; and D shows TMV treated with 1% DMSO at 50,000x magnification.

[0046] Figure 5 The figure shows the effect of different concentrations of compound I on SA content in ordinary cigarettes.

[0047] Figure 6 The image shows the effect of different concentrations of compound I on the expression of resistance-related genes in ordinary tobacco. Detailed Implementation

[0048] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0049] The seeds of *Pterocarya stenoptera* used in the following examples were purchased from Shaanxi Xiangju Pharmaceutical Group Co., Ltd.

[0050] The reagents and raw materials used in the following examples are all commercially available.

[0051] Example 1

[0052] The preparation method of flavonoid compound I in this embodiment specifically includes the following steps: Step 1: After the freshly collected seeds of the *Pterocarya stenoptera* tree are air-dried, they are crushed using a plant pulverizer and passed through a 40-mesh sieve. They are then cold-extracted at room temperature using a mixed solvent of dichloromethane and methanol at a volume ratio of 1:1. The volume ratio of the crushed *Pterocarya stenoptera* seeds to the cold-extraction solvent is 1:3. The solvent is changed every 4 days, and the extraction is repeated 3-4 times. Finally, the extracts are combined and concentrated to obtain the crude extract.

[0053] Step 2: The extract from Step 1 was subjected to normal-phase silica gel column chromatography. 100-200 mesh Diol silica gel was used as the packing material. The eluents were, in sequence, a mixture of petroleum ether and ethyl acetate, and a mixture of dichloromethane and methanol. The volume ratios of petroleum ether and ethyl acetate were 100:1, 20:1, 10:1, 5:1, 3:1, and 1:1, respectively, and the volume ratios of dichloromethane and methanol were 20:1, 10:1, and 1:1, respectively, for gradient elution. Fraction 1 was obtained when the volume ratio of dichloromethane to methanol was 10:1. Other volume ratios were used to remove impurities.

[0054] During normal phase gradient elution, the column temperature is room temperature and the mobile phase flow rate is 1-2 drops / second.

[0055] Step 3: Fraction 1 is further loaded onto a reversed-phase silica gel column. The reversed-phase column chromatography uses C18 reversed-phase chromatographic packing material. Gradient elution is performed with the following eluents in sequence: pure water, a mixture of water and methanol with a methanol content of 20%, a mixture of water and methanol with a methanol content of 40%, a mixture of water and methanol with a methanol content of 60%, a mixture of water and methanol with a methanol content of 80%, and finally methanol. Fraction 2 is obtained when the methanol content is 40%.

[0056] During reverse-phase gradient elution, the column temperature is at room temperature, the mobile phase flow rate is 1 drop / second, and reduced pressure is used with a vacuum degree less than or equal to -0.08 MPa.

[0057] Step 4: Evaporate fraction 2 to dryness at 60°C, recrystallize it using dichloromethane as solvent. The amount of dichloromethane added should be such that fraction 2 is in a suspended state at room temperature. Mix fraction 2 and dichloromethane, heat to dissolve at 60°C, filter while hot, and place in a refrigerator at 4°C overnight to obtain substance A.

[0058] The following is a spectroscopic analysis of substance A, with the following morphological and spectroscopic identification data: Substance A, a pale yellow powder, has the following 1H and 1C NMR spectra: 1 H NMR (500 MHz, CD3OD) δ 6.94 (s, 2H, H-2″), 6.92 (d, J = 2.0 Hz, 1H,H-2′), 6.80 (dd, J = 8.2, 2.1 Hz, 1H, H-6′), 6.68 (d, J = 8.2 Hz, 1H, H-5′),5.95 (s, 2H, H-6), 5.58 – 5.43 (m, 1H, H-3), 5.01 (s, 1H, H-2), 2.98 (dd, J =17.4, 4.7 Hz, 1H, H-4a), 2.84 (dd, J = 17.5, 2.5 Hz, 1H, H-4b); 13 C NMR (125MHz, CD3OD) δ 167.6 (C=O), 157.9 (C-9), 157.8 (C-7), 157.3 (C-5), 146.3 (C-3′′, 5′′), 146.0 (C-4′), 145.9 (C-3′), 139.8 (C-4′′), 131.5 (C-1′), 121.5 (C-1′′), 119.4 (C-6′), 116.0 (C-5′), 115.1 (C-2′), 110.2 (C-2′′, 6′′), 99.4 (C-10), 96.6 (C-6), 95.9 (C-8), 78.6 (C-2), 70.0 (C-3), 26.9 (C-4).

[0059] The proton and carbon NMR spectra of substance A are consistent with those in the literature "Hyoung JK, Jun YL, Sung MK, et al. 2009. A new epicatechin gallate and calpain inhibitory activity from..." Orostachys japonicusThe spectral data of epicatechin gallate reported in Fitoterapia, 80(1):73-76” are consistent, and the identified substance A is epicatechin gallate (i.e., compound A). Its structure is shown in the figure below.

[0060] .

[0061] Example 1

[0062] The compound prepared in Example 1 Anti-TMV activity experiments were conducted: The half-leaf necrotic spot method was used, with ningnanmycin as a positive control, to test compound I in new tobacco leaves. Nicotiana glutinosa The effects of L. on the desensitization, protection and treatment of TMV are shown in Table 1.

[0063] Table 1. Compounds at three different concentrations New Leaf Tobacco N. glutinosa Antiviral activity against TMV

[0064] In Table 1, ningnanmycin serves as a positive control. Different letters a, b, and c in the same row indicate values ​​obtained using Duncan's new multiple range test. P The difference was significant at the <0.05 level.

[0065] Figure 1 , Figure 2 Figure 3 shows the passivation, protection, and therapeutic effects of different concentrations of compound I on TMV in fresh tobacco leaves. Figure 1 The left side of each leaf in Figure 2 shows the results after treatment with different concentrations of compound I (500 μg / mL, 250 μg / mL and 125 μg / mL), while the right side of the leaf (i.e. CK) is the 1% DMSO control.

[0066] Combining Table 1 and Figure 1 The results show that: At a concentration of 500 μg / mL, compound I exhibited an 80.27% passivation effect against TMV. Passivation activity is a crucial indicator for evaluating the anti-TMV activity of drugs and is an important method for screening anti-TMV active agents.

[0067] At a concentration of 500 μg / mL, compound I showed a protective effect of 70.28% and a therapeutic effect of 65.17% against TMV, which were superior to those of the commercial drug ningnanmycin (60.32% and 52.58%, respectively).

[0068] At concentrations of 250 and 125 μg / mL, compound I still showed good passivation, protection and therapeutic effects against TMV, with effects superior to or comparable to the positive control ningnanmycin.

[0069] Example 2

[0070] The compound prepared in Example 1 Microscopic observation of TMV virus particles: Equal volumes of TMV (20 μg / mL) and compound Mix (500 μg / mL) and let stand for 1 hour. Then, place the mixture on a carbon-coated grid and negatively stain with 0.01 mL of 2% phosphotungstic acid for 1 minute, rinse once, and dry. An equal volume of 1% DMSO (dimethyl sulfoxide) and TMV (20 μg / mL) was mixed and treated in the same manner as a negative control. The compounds were then identified by observation under a transmission electron microscope. Impact on TMV morphology.

[0071] The concentration of the compound at 500 μg / mL was determined by transmission electron microscopy. The impact on TMV morphology, such as Figure 4 As shown, the compound The compound exhibits a cleavage effect on TMV, causing significant fragmentation of TMV virus particles, breaking them down from a blank size of 400 nm into fragments of 10-200 nm in length, indicating that the compound... It has a significant destructive effect on virus particles.

[0072] Example 3

[0073] The compound prepared in Example 1 Conduct systemic disease resistance experiments: Using ningnanmycin as a positive control, compound I was tested in common tobacco. Nicotiana tabacum Systemic resistance to TMV on cv.K326 (K326).

[0074] The assay method was as follows: A compound at a concentration of 500 μg / mL was applied to the lower three leaves of a common tobacco plant as a treatment group. 1% DMSO was applied to the lower three leaves of another common tobacco plant as a negative control. Ningnanmycin treatment served as a positive control. 72 hours later, TMV (10 μg / mL) was inoculated onto the upper three leaves of the common tobacco plant. Five days after inoculation, 0.5 g of the inoculated leaves were ground with 0.01 mol / L, pH 7.4 PBS buffer, and then... (8500×...) gCentrifuge for 3 minutes to obtain the virus extract. After centrifugation, aspirate the supernatant (10 μL) and sequentially couple it with horseradish peroxidase (HRP) and perform a color reaction (30-60 min). Then, incubate it with purified TMV antibody on an ELISA plate at 37°C for 15 minutes. Finally, place the ELISA plate in a microplate reader and read the absorbance of each well at 450 nm. Calculate the TMV content based on a predetermined standard curve showing the relationship between TMV concentration and absorbance. Based on the initial screening results, select compounds with good inducing activity and further determine their inducing activity by setting concentration gradients of 500, 250, and 125 μg / mL. The inhibition rate is calculated using the formula: Inhibition rate (%) = [(Control group - Treatment group) / Control group] × 100.

[0075] compound The effects of different concentrations on inducing resistance to TMV in ordinary tobacco K326 are shown in Table 2.

[0076] Table 2 Compounds Inducing resistance to TMV in common tobacco K326

[0077] In Table 2, ningnanmycin serves as a positive control. Different letters a, b, and c in the same row indicate values ​​obtained using Duncan's new multiple range test. P The difference was significant at the <0.05 level.

[0078] Table 2 shows that compound I, at concentrations of 500 μg / mL, 250 μg / mL, and 125 μg / mL, exhibited resistance to TMV in common tobacco at concentrations of 65.17%, 50.21%, and 32.18%, respectively, which were higher than those of ningnanmycin (49.68%, 35.28%, and 29.12%). Therefore, compound I can be developed as a plant immune inducer.

[0079] Example 4

[0080] The content of salicylic acid (SA), an important stress-resistance secondary metabolite in common tobacco K326, prepared by compound I in Example 1 was tested: Take a 10 g sample of K326 tobacco leaves, grind it thoroughly, add 4 mL of 5% trichloroacetic acid, then add ultrapure water to 20 mL, and then add 30 mL of diethyl ether. After stirring evenly, soak and extract for 12 hours, centrifuge (10000 r / min) for 5 minutes, take the upper ether phase, and repeat the extraction of the lower aqueous phase with diethyl ether twice. Combine the obtained ether phases, evaporate to dryness under reduced pressure using a rotary evaporator, and add 1.0 mL of mobile phase solution to completely dissolve it, which is the free salicylic acid sample. Add an equal volume of 2 mmol / L HCl to the remaining aqueous phase of the extracted free salicylic acid sample, heat in an 80℃ water bath for 1 hour, cool, and extract with diethyl ether 3 times. Combine the ether phases, evaporate to dryness, and add 1 mL of mobile phase solution to dissolve, which is the bound salicylic acid sample. After the sample was filtered through a 0.22 μm microporous filter, the SA content was determined by high performance liquid chromatography (mobile phase: methanol to sodium acetate volume ratio 60:40; flow rate: 0.8 mL / min; chromatographic column: C-18 reversed phase column, 5 μm, 4.6 mm × 150 mm; injection volume: 5 μL).

[0081] Table 3 and Figure 5 The effect of different concentrations of compound I on SA content in ordinary cigarettes was investigated. CK served as the control group, using 1% DMSO by volume.

[0082] Table 3

[0083] In Table 3, the different letters a, b, and c in the same row indicate values ​​tested using Duncan's new multiple range test. P The difference was significant at the <0.05 level.

[0084] Table 3 and Figure 5 This indicates that compound I can increase the content of salicylic acid (SA) in ordinary tobacco products, exhibiting a concentration gradient. The SA content increased over time from 0 to 72 hours, reaching its maximum at 72 hours. At concentrations of 500 g / mL, 250 g / mL, and 125 g / mL, the SA content was 5.70 times, 3.20 times, and 2.88 times that of the control group, respectively. After 72 hours, the activity gradually decreased and returned to normal. This suggests that compound I can increase the content of salicylic acid, a stress-resistance substance in tobacco products, thereby resisting viral invasion.

[0085] Example 5

[0086] Compound I prepared in Example 1 was selected in vivo from common tobacco K326 cells for its components related to the synthesis of disease-resistant proteins PRs. NPR1 , PR1 , PR2 , PR5and related to SA synthesis ICS1 , PAL The expression of these six genes was measured.

[0087] When common tobacco plants had grown to about 5 leaves, the undersides of leaves from different plants with similar growth rates were sprayed with Compound I at concentrations of 500 μg / mL, 250 μg / mL, and 125 μg / mL, respectively. Simultaneously, 1% DMSO was sprayed on the undersides of the tobacco leaves as a negative control, and this process was repeated three times. On day 3, samples (0.1 g) were taken, and the expression of the aforementioned genes was determined using RT-qPCR. The primers and internal controls used for gene expression were also analyzed. β-actin 1 See Table 4.

[0088] Table 4 Primer information for resistance-related genes

[0089] Figure 6 Table 5 shows the effects of different concentrations of compound I on the expression of resistance-related genes in ordinary tobacco.

[0090] Table 5

[0091] In Table 5, the different letters a, b, and c in the same row indicate values ​​tested using Duncan's new multiple range test. P The difference was significant at the <0.05 level.

[0092] like Figure 6 As shown in Table 5, compound I has the effect of NPR1 , PR1 , PR2 , PR5 and PAL The expression of these four genes has a promoting effect and exhibits a concentration gradient, while for ICS1 No effect. At concentrations of 500 μg / mL, 250 μg / mL, and 125 μg / mL, NPR1 The expression folds (referring to the comparison values ​​between the direct and internal references) were 3.67, 2.48, and 1.24, respectively; PR1 The expression folds were 3.66, 3.27, and 2.03, respectively; PR2 The expression folds were 5.32, 4.66, and 1.89, respectively; PR5 The expression fold increases were 4.85, 3.24, and 1.39, respectively. PAL The expression fold increases were 13.28, 5.63, and 3.46, respectively. ICS1 The expression fold was not different from that of the control group at any concentration.

[0093] SA is an important secondary metabolite for stress resistance in plants. Its synthesis in plants mainly occurs through two pathways: the phenylalanine ammonia-lyase (PAL) pathway and the heterobranched acid synthase (ICS) pathway. Compound I promotes… PAL Gene expression, on ICS1 The absence of gene involvement indicates that compound 1 exerts its immune-inducing effect through the synthesis of SA via the PAL pathway. Additionally, compound I can promote the synthesis of disease-resistant proteins PRs. NPR1 , PR1 , PR2 and PR5 The expression of these four genes further demonstrates at the molecular level that compound I can enhance tobacco's resistance to diseases.

[0094] Therefore, compound I extracted from the seeds of the Chinese fragrant tree can not only be used to develop plant antiviral agents to directly destroy TMV virus, but also as a plant immune inducer to enhance tobacco resistance and resist TMV virus.

Claims

1. A method for preparing flavonoids, characterized in that, It includes the following steps: (1) Extract the crushed seeds of the fragrant tree using solvent 1 to obtain an extract; The volume ratio of the crushed seeds of the fragrant tree to the solvent is 1:(3~4), and the solvent 1 is one or more of dichloromethane, methanol and ethanol; (2) The extract was subjected to normal-phase gradient elution to obtain fraction 1; The eluent comprises, in sequence, a mixture of petroleum ether and ethyl acetate, and a mixture of dichloromethane and methanol, wherein the volume ratio of petroleum ether to ethyl acetate in the mixture of petroleum ether and ethyl acetate is (100~1):1, and the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is (20~1):

1. (3) The fraction 1 is subjected to reverse-phase gradient elution to obtain fraction 2; the eluent includes water, a mixture of water and methanol, and methanol in sequence; (4) The fraction 2 is evaporated to dryness and recrystallized to obtain the flavonoid compound shown in Formula I: Formula I.

2. The method for preparing flavonoids according to claim 1, characterized in that, In step (1), solvent 1 is a mixture of dichloromethane and methanol in a volume ratio of 1:1; And / or, in step (1), the crushed material of the incense tree seeds is the material after crushing the incense tree seeds and passing them through a 30-50 mesh sieve.

3. The method for preparing flavonoids according to claim 1, characterized in that, In step (1), the extraction is a cold soaking extraction at room temperature; And / or, in step (1), the extraction method includes: changing the solvent every 4 to 7 days, for a total of 3 to 4 extractions.

4. The method for preparing flavonoids according to claim 1, characterized in that, It meets one or more of the following conditions: ① In step (2), during normal-phase gradient elution, the volume ratio of petroleum ether to ethyl acetate is 100:1, 20:1, 10:1, 5:1, 3:1 and 1:1, respectively; ② In step (2), during normal phase gradient elution, the volume ratio of dichloromethane to methanol is 20:1, 10:1 and 1:1 respectively; ③ In step (2), when performing normal phase gradient elution, the mobile phase flow rate is 1-2 drops / second; In step (2) and ④, the positive phase gradient elution uses silica gel as the filler; preferably, the particle size of the silica gel is 100~200 mesh; preferably, the silica gel is Diol silica gel.

5. The method for preparing flavonoids according to claim 1, characterized in that, It meets one or more of the following conditions: ① In step (3), the volume of methanol in the mixture of water and methanol accounts for 20-80% of the volume of the mixture, for example, 40% or 60%; Preferably, the eluent comprises, in sequence, water, a mixture of water and methanol with a methanol content of 20%, a mixture of water and methanol with a methanol content of 40%, a mixture of water and methanol with a methanol content of 60%, a mixture of water and methanol with a methanol content of 80%, and methanol. ② In step (3), the flow rate of the mobile phase in the reverse gradient elution is 1~2 drops / second; ③ In step (3), the reverse gradient elution is performed under reduced pressure conditions, with a vacuum degree less than or equal to -0.08 MPa; In step (3) and step (4), reversed-phase gradient elution uses C18 reversed-phase chromatography as the packing material.

6. The method for preparing flavonoids according to claim 1, characterized in that, In step (4), the temperature for evaporation is 50~60℃.

7. The method for preparing flavonoids according to claim 1, characterized in that, It meets one or more of the following conditions: ① In step (4), the solvent 2 used for recrystallization is one or more of dichloromethane, acetone and ethyl acetate; ② In step (4), the amount of solvent 2 added in the recrystallization is such that the fraction 2 is in a suspended state at room temperature; In step (4) of section ③, the recrystallization method includes: mixing the fraction 2 with solvent 2, heating to dissolve, filtering, and keeping the solid part at 0~8℃ for 10~16h; Preferably, the heating temperature is 50~60°C.

8. The application of a flavonoid compound as an active ingredient in the treatment of tobacco mosaic virus, wherein the flavonoid compound is as shown in Formula I: Formula I.

9. The application of the flavonoids as described in claim 8 as an active ingredient in the treatment of tobacco mosaic virus, characterized in that, The flavonoids are used as a control agent and immune inducer for tobacco mosaic virus.

10. The application of the flavonoids as described in claim 8 as an active ingredient in the treatment of tobacco mosaic virus, characterized in that, It meets one or more of the following conditions: ① The application of the flavonoids in the preparation of expression promoters for disease-resistant protein PRs synthesis-related genes and promoters for SA content in tobacco; the disease-resistant protein PRs synthesis-related genes are, for example, NPR1 , PR1 , PR2 or PR5 ; ② In the pharmaceutical preparation containing the flavonoid compound, the concentration of the flavonoid compound is 125~500 μg / mL, for example 250 μg / mL; The flavonoids mentioned in ③ are flavonoids prepared by the method described in any one of claims 1 to 7.

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