A naphthoquinone compound with anti-tobacco black shank disease activity and a preparation method and application thereof

Abstract

The application belongs to the technical field of agricultural chemistry, and particularly relates to a naphthoquinone compound with anti-tobacco black shank disease activity and a preparation method and application thereof. The naphthoquinone compound is extracted from honeysuckle branches with high-concentration methanol, high-concentration acetone / water or high-concentration ethanol / water, the extract is combined, filtered, and concentrated into an extract under reduced pressure; the extract is subjected to silica gel column chromatography by dry column packing with silica gel; gradient elution is performed with chloroform-acetone solution; and the 7:3 part of the eluate is further separated and purified by high-performance liquid chromatography to obtain the required naphthoquinone compound. The application also discloses the application of the compound. The active test shows that the compound has a good inhibitory effect on tobacco black shank disease. The compound has a novel structure, good anti-black shank disease activity, and can be used as a lead compound for anti-tobacco black shank disease and for the research and development of anti-tobacco black shank disease biological pesticides.

Description

Technical Field

[0001] This invention belongs to the field of phytochemistry technology, specifically relating to a naphthoquinone compound with activity against tobacco black shank disease, its preparation method, and its application. Background Technology

[0002] Tobacco black shank is one of the most devastating diseases in tobacco production, also known as tobacco blight, and referred to by tobacco farmers as "black stalk madness," "black root," or "aconite disease." Currently, control of black shank mainly relies on methods such as crop rotation, varietal genetic improvement, and biopesticides. Among these, biopesticide control is the most common and easily implemented method. Therefore, finding more highly effective biopesticide lead compounds is of great significance for the control of tobacco black shank.

[0003] honeysuckle( Lonicera japonica Honeysuckle (Lonicera japonica) is a plant belonging to the genus Lonicera in the family Caprifoliaceae. Its dried flower buds or unopened flowers have extremely high medicinal value, possessing heat-clearing and detoxifying effects. It is mainly used to treat exogenous wind-heat or febrile diseases, heatstroke, dysentery due to heat toxins, carbuncles, boils, sore throat, and various infectious diseases. With social progress and the development of science and technology, research on the comprehensive development and utilization of honeysuckle has become increasingly in-depth. From its original medicinal value, it has developed into the development and production of health foods, beauty and skincare products, and further into tourism and ecological agricultural engineering. Domestic and international research shows that the main chemical components of honeysuckle include: organic acids, phenylpropanoids, sterols, quinones, triterpenoid saponins, iridoid glycosides, and terpene volatile oils.

[0004] Naphthoquinones are an important class of natural active ingredients. Naturally derived naphthoquinone compounds are widely distributed in nature, especially in higher plants, and also in some microorganisms. Pharmacological studies have shown that they possess a wide range of pharmacological activities, such as cytotoxicity, antioxidant, anti-inflammatory, and antibacterial activities. This invention discovered a novel naphthoquinone compound from honeysuckle branches. Notably, this compound exhibits a rare naphthoquinone structure containing a 3-hydroxymethylfuran ring fragment, demonstrating high novelty. Furthermore, this compound exhibits significant activity against tobacco black shank, with a control effect of (68.9±3.5)%, superior to the control effect of agricultural chloramphenicol (57.4±3.8)%. It can serve as a lead compound for the development of biopesticides for the control of tobacco black shank. Summary of the Invention

[0005] The first objective of this invention is to provide a naphthoquinone compound with anti-tobacco black shank activity; the second objective is to provide a method for preparing the aforementioned naphthoquinone compound with anti-tobacco black shank activity; and the third objective is to provide applications of the aforementioned naphthoquinone compound with anti-tobacco black shank activity.

[0006] The first objective of this invention is achieved as follows: the naphthoquinone compound with anti-tobacco black shank activity is a light yellow gelatinous substance with the molecular formula: C 16 H 12 O5, named 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione, its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)nap-htho[2,3- b Furan-5,8-dione has the following structure:

[0007] .

[0008] The second objective of this invention is achieved by using honeysuckle branches, a plant that is both medicinal and edible, as raw material, and preparing it through pretreatment, extract extraction, silica gel column chromatography, and purification separation steps, specifically including the following steps:

[0009] A. Pre-treatment: The raw material honeysuckle branches are crushed and passed through a 20-50 mesh sieve to obtain material a;

[0010] B. Extraction of extract: The first solvent is added to material a for reflux extraction. After the extract is concentrated, the second solvent is added for extraction. The extracts of the second solvent are combined and concentrated under reduced pressure to obtain extract b.

[0011] The first solvent is an aqueous ethanol solution with a volume percentage concentration of 90% to 96%.

[0012] The second solvent is ethyl acetate;

[0013] C. Silica gel column chromatography: Dissolve extract b in a third solvent and add 1.5 to 3 times the weight of extract b in 160 to 200 mesh silica gel to pack the column. Elute with a gradient of chloroform-acetone solution with a volume ratio of 20:1 to 1:1. Detect by TLC and combine identical fractions. The third solvent is methanol, ethanol or acetone.

[0014] D. Purification and separation:

[0015] 1) The eluent obtained by elution with a 7:3 chloroform-acetone solution was purified by dextran gel column chromatography to obtain the crude product of the target compound;

[0016] 2) The crude target compound was separated and purified by high performance liquid chromatography to obtain the target compound, a naphthoquinone compound with anti-tobacco black shank activity.

[0017] The specific steps are as follows:

[0018] 1) Dry and pulverize honeysuckle branches, extract with the first solvent, concentrate, extract with the second solvent, combine and concentrate to obtain extract a; the first solvent is a 90% to 96% aqueous ethanol solution; the second solvent is ethyl acetate;

[0019] 2) After the extract a is dissolved in a third solvent, it is separated by silica gel column chromatography and eluted with a gradient of chloroform-acetone solution. The fraction with a volume ratio of 7:3 of chloroform-acetone solution is collected to obtain the eluent component b; the third solvent is methanol, ethanol or acetone.

[0020] 3) The eluent b was purified by dextran gel column chromatography to obtain the crude target compound; the crude target compound was then separated by HPLC to obtain the pure target compound.

[0021] The structures of the naphthoquinone compounds with anti-tobacco black shank activity prepared in the above steps can be identified by the following methods:

[0022] Visual observation revealed that the compound of this invention is a yellow gel-like substance; ultraviolet-visible absorption spectroscopy showed maximum absorption at 215, 268, and 349 nm, proving the presence of an aromatic ring structure in the compound; infrared spectroscopy (potassium bromide tablet) showed the presence of hydroxyl groups (3398 cm⁻¹) in the compound. -1 ), carbonyl (1726 and 1665 cm) -1 ), aromatic rings (1647, 1456, 1345cm) -1 Characteristic functional groups; High-resolution mass spectrometry (HRESIMS) revealed a quasi-molecular ion peak at 307.0581 [M+Na]. + The molecular formula of the compound can be determined to be C. 16 H 12 O5.

[0023] For 1 H NMR, 13Detailed analysis of C10 NMR, DEPT, and HSQC spectral data revealed that the compound contains 16 carbons and 12 hydrogens, including a 1,2,4,5-tetrasubstituted benzene ring (C-1, C-2, C-3, C-4, C-9, C-10, H-1, H-4), two carbonyl groups (C-5 and C-8), two pairs of double bonds (C-6, C-7, C-14, C-15, H-6 and H-15), a 2'-oxopropyl group (-CH2COCH3, C-11~C-13, H2-11 and H3-13), and an oxymethylene group (C-16 and H2-16). Further analysis of its NMR data indicated that one set of double bonds and the two carbonyl groups should be connected to the benzene ring to form 1,4-naphthoquinone, while another set of double bonds should be connected to the benzene ring to form a furan ring, satisfying the 11 degrees of unsaturation in the compound. Furthermore, the inference of the presence of 1,4-naphthoquinone in the compound can be inferred through HMBC correlations between H-6 and C-5 / C-8 / C-10, H-4 and C-5 / C-9 / C-10, and H-1 and C-8 / C-9 / C-10. Figure 3 This was confirmed, and the speculation that another set of double bonds and benzene links form a furan ring can also be confirmed by the HMBC correlation between H-4 and C-3 / C-14, and H-15 and C-2 / C-3 / C-14. Figure 3 This has been confirmed. The compound was identified as naphtho[2,3-] b [Furan-5,8-dione structure]

[0024] Table 1. Compounds of the present invention 1 H NMR and 13 C10 NMR data (CDCl3)

[0025]

[0026] Once the parent compound was determined, the positions of the remaining substituents (methylene oxide and 2-oxopropyl) could also be determined through their HMBC correlations. The 2-oxopropyl substitution at C-7 was confirmed by HMBC correlations between H2-11 and C-6 / C-7 / C-8, and between H-6 and C-11. The methyl oxide substitution at C-14 was determined by HMBC correlations between H2-16 and C-3 / C-14 / C-15, and between H-15 and C-16. Thus, the structure of the compound was accurately confirmed. It was named: 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione, its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione.

[0027] Infrared, ultraviolet, and mass spectrometry data of the compound: UV (methanol) λ max (logε)215(4.12), 282(382), 349(3.68);IR(KBr)v max 3398, 3154, 2960, 2838, 1726, 1665, 1647, 1456, 1345, 1240, 1167, 818cm -1 ; 1 H and 13 The C10 NMR data are shown in Table 1; HRESIMS (positive ion mode) m / z 307.0581 [M+Na] + (C 16 H 12 NaO5, calculated value 307.0577).

[0028] The third objective of this invention is achieved by the use of the naphthoquinone compounds in the preparation of drugs for treating tobacco black shank.

[0029] The beneficial effects of this invention are as follows:

[0030] 1) This invention is the first to extract a novel naphthoquinone compound from honeysuckle branches. It is noteworthy that this compound contains a 3-hydroxymethylfuran ring structure fragment, which is rare in natural products. The compound structure has high novelty, and it also has good anti-black shank activity. It can be used as a lead compound for the development of biological pesticides for the prevention and control of tobacco black shank.

[0031] 2) This invention is the first to use honeysuckle branches, a traditional Chinese medicinal herb, as raw material, providing a new research direction for the development of natural products derived from plants that have the effect of preventing and controlling plant diseases.

[0032] 3) The raw materials for preparing this compound are widely available and low in cost, which can provide sufficient and continuous raw material support for the preparation of this compound and make it easy to achieve industrial production. Attached Figure Description

[0033] Figure 1 Carbon NMR spectra of naphthoquinone compounds obtained in Example 1;

[0034] Figure 2 The 1H NMR spectrum of the naphthoquinone compounds prepared in Example 1;

[0035] Figure 3 The diagram shows the main HMBCs of the naphthoquinone compounds prepared in Example 1. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0037] The naphthoquinone compound with anti-tobacco black shank activity described in this invention is a light yellow gelatinous substance with the molecular formula: C 16 H 12 O5, named 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione, its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione has the following structure:

[0038] .

[0039] The method for preparing naphthoquinone compounds with anti-tobacco black shank activity according to the present invention uses honeysuckle branches, a plant that is both medicinal and edible, as raw material, and is prepared through pretreatment, extract extraction, silica gel column chromatography, and purification separation steps, specifically including the following steps:

[0040] A. Pre-treatment: The raw material honeysuckle branches are crushed and passed through a 20-50 mesh sieve to obtain material a;

[0041] B. Extraction of extract: The first solvent is added to material a for reflux extraction. After the extract is concentrated, the second solvent is added for extraction. The extracts of the second solvent are combined and concentrated under reduced pressure to obtain extract b.

[0042] The first solvent is an aqueous ethanol solution with a volume percentage concentration of 90% to 96%.

[0043] The second solvent is ethyl acetate;

[0044] C. Silica gel column chromatography: Dissolve extract b in a third solvent and add 1.5 to 3 times the weight of extract b in 160 to 200 mesh silica gel to pack the column. Elute with a gradient of chloroform-acetone solution with a volume ratio of 20:1 to 1:1. Detect by TLC and combine identical fractions. The third solvent is methanol, ethanol or acetone.

[0045] D. Purification and separation:

[0046] 1) The eluent obtained by elution with a 7:3 chloroform-acetone solution was purified by dextran gel column chromatography to obtain the crude product of the target compound;

[0047] 2) The crude target compound was separated and purified by high performance liquid chromatography to obtain the target compound, a naphthoquinone compound with anti-tobacco black shank activity.

[0048] Step C includes a mixing step before loading the column with 0.8 to 2.0 times the weight of material b in 80 to 120 mesh silica gel.

[0049] The volume ratios of the chloroform-acetone solution in step C are 20:1, 9:1, 4:1, 7:3, 3:2, and 1:1.

[0050] In step D, step 1), the mobile phase used for dextran gel column purification is methanol.

[0051] In step 2), the HPLC separation was performed using a 21.2 mm × 25 cm chromatographic plate. μ m of C 18 The chromatographic column was used with 48% methanol aqueous solution as the mobile phase at a flow rate of 12 mL / min. The UV detector was used to detect the peak at a wavelength of 349 nm, and the chromatographic peak was collected after 34 min.

[0052] The specific steps are as follows:

[0053] (1) Extraction of extract: Dry and crush honeysuckle branches, extract with the first solvent, concentrate, extract with the second solvent, combine and concentrate to obtain extract;

[0054] (2) The extract was dissolved in a third solvent and separated by silica gel column elution with chloroform / acetone gradient, and the eluent fraction with a volume ratio of chloroform:acetone of 7:3 was collected.

[0055] (3) The eluent obtained in step 2 was purified by dextran gel column to obtain crude target compound, and finally the crude target compound was separated by HPLC to obtain pure target compound.

[0056] The first solvent is a 95% aqueous solution of ethanol.

[0057] In step 1, the sample is refluxed 2-3 times using the first solvent, with each reflux extraction lasting 35-60 minutes.

[0058] The extract obtained by reflux extraction is concentrated and then extracted 2-3 times with a second solvent.

[0059] In step 2, the volume ratios of chloroform and acetone in the gradient elution are 20:1, 9:1, 4:1, 7:3, 3:2, and 1:1, respectively.

[0060] In step 2, a 160-200 mesh silica gel column is used for separation. The extract is dissolved in 1.5 to 3 times the amount of a second solvent and then mixed with 80-120 mesh coarse silica gel.

[0061] During the sample mixing process, the weight of the silica gel used for mixing should be 0.8 to 2.5 times that of the extract.

[0062] In step 3, methanol is used as the mobile phase for dextran gel column purification.

[0063] In step 3, high-performance liquid chromatography (HPLC) separation was performed using a Zorbax PrepHT GF (21.2 mm × 25 cm) reversed-phase column with 48% methanol aqueous solution as the mobile phase, a flow rate of 12 mL / min, and a UV detector with a detection wavelength of 349 nm. The chromatographic peak was collected after 34 min.

[0064] The second solvent is ethyl acetate.

[0065] The third solvent is methanol, ethanol, or acetone.

[0066] This invention also provides the application of the naphthoquinone compounds in the preparation of anti-tobacco black shank formulations. Through anti-tobacco black shank activity tests, this invention demonstrates that the naphthoquinone compounds exhibit a significant inhibitory effect on tobacco black shank.

[0067] The application of this invention is the use of the naphthoquinone compounds with anti-tobacco black shank activity in the preparation of anti-tobacco black shank drugs.

[0068] The present invention will be further described below with reference to specific embodiments:

[0069] Example 1

[0070] In this embodiment, the honeysuckle branch samples were obtained from Kunming, Yunnan. The honeysuckle branches were dried and pulverized to approximately 40 mesh. 3.5 kg of the pulverized sample was weighed and placed in a 20 L glass reactor. 6 L of 95% ethanol aqueous solution was added, and the mixture was refluxed for 50 min. The extract was filtered off. Another 6 L of 95% ethanol aqueous solution was added to the residue, and the mixture was refluxed for 50 min. The extract was then filtered off. The two extracts were combined and concentrated to a small volume, then extracted twice with 6 L of ethyl acetate. The extract was concentrated under reduced pressure to obtain 66.4 g of naphthoquinone extract. The extract was mixed with 80 g (80-120 mesh) coarse silica gel, dried, and eluted using a 200 g silica gel (160-200 mesh) column chromatography with a chloroform:acetone gradient (20:1, 9:1, 4:1, 7:3, 3:2, 1:1), separating the extract into six fractions. The 7:3 elution fraction was selected, concentrated, dissolved in methanol, and purified using a dextran gel column as the mobile phase to obtain the crude compound. The crude compound was further separated by HPLC: using an Agilent Zorbax PrepHTGF (21.2 mm × 25 cm) reversed-phase column, with 48% methanol-water solution as the mobile phase, a flow rate of 12 mL / min, and a UV detector at 349 nm, the chromatographic peak at 34.0 min was collected. After repeated accumulation, the sample was evaporated to dryness to obtain a pale red gel-like substance, which was the pure target compound.

[0071] Example 2

[0072] The honeysuckle branch samples were obtained from Dali, Yunnan. Honeysuckle branches were dried and pulverized to approximately 40 mesh. 4.0 kg of the pulverized sample was weighed and placed in a 20 L glass reactor. 8 L of 95% ethanol aqueous solution was added, and the mixture was refluxed for 40 min. The extract was filtered off. Another 8 L of 95% ethanol aqueous solution was added to the residue, and the mixture was refluxed for 40 min. The extract was then filtered off. The two extracts were combined and concentrated to a small volume. The mixture was extracted twice with 8 L of ethyl acetate. The combined ethyl acetate phases were concentrated under reduced pressure to obtain 70.22 g of naphthoquinone extract. The extract was mixed with 100 g (80-120 mesh) coarse silica gel, dried, and separated by column chromatography using 220 g (160-200 mesh) silica gel. A chloroform:acetone gradient elution (20:1, 9:1, 4:1, 7:3, 3:2, 1:1) was used to separate the extract into six fractions. The 7:3 elution fraction was selected, concentrated, dissolved in methanol, and purified using a dextran gel column as the mobile phase to obtain the crude compound. The crude compound was further separated by HPLC: using an Agilent Zorbax PrepHT GF (21.2 mm × 25 cm) reversed-phase column, with 48% methanol-water solution as the mobile phase, a flow rate of 12 mL / min, and a UV detector at 349 nm, the chromatographic peak at 34.0 min was collected, accumulated multiple times, and then evaporated to dryness to obtain the pure compound.

[0073] The compound obtained in this embodiment was structurally identified in the same way as in Example 1, confirming that the compound prepared in this embodiment is the naphthoquinone compound described above—3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione.

[0074] Example 3

[0075] The honeysuckle branch samples were obtained from Yuxi, Yunnan. Honeysuckle branches were dried and pulverized to approximately 50 mesh. 4.6 kg of the pulverized sample was weighed and placed in a 20 L glass reactor. 10 L of 95% ethanol aqueous solution was added, and the mixture was refluxed for 30 min. The extract was filtered off. Another 10 L of 95% ethanol aqueous solution was added to the residue, and the mixture was refluxed for 50 min. The extract was then filtered off. The two extracts were combined and concentrated to a small volume, then extracted twice with 10 L of ethyl acetate. The extract was concentrated under reduced pressure to obtain 96.8 g of naphthoquinone extract. The extract was mixed with 130 g (80-120 mesh) coarse silica gel, dried, and separated by column chromatography using 300 g (160-200 mesh) silica gel with a chloroform:acetone gradient elution (20:1, 9:1, 4:1, 7:3, 3:2, 1:1). The extract was separated into six fractions. The 7:3 elution fraction was selected, concentrated, dissolved in methanol, and purified using a dextran gel column as the mobile phase to obtain the crude compound. The crude compound was further separated by HPLC: using an Agilent Zorbax PrepHT GF (21.2 mm × 25 cm) reversed-phase column, with 48% methanol-water solution as the mobile phase, a flow rate of 12 mL / min, and a UV detector at 349 nm, the chromatographic peak at 34.0 min was collected, accumulated multiple times, and then evaporated to dryness to obtain the pure compound.

[0076] The compound obtained in this embodiment was structurally identified in the same way as in Example 1, confirming that the compound prepared in this embodiment is the naphthoquinone compound described above—3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione.

[0077] Example 4

[0078] The structures of the naphthoquinone compounds prepared by the above method were identified by the following method:

[0079] Visual observation revealed that the compound of this invention is a yellow gel-like substance; ultraviolet-visible absorption spectroscopy showed maximum absorption at 215, 268, and 349 nm, proving the presence of an aromatic ring structure in the compound; infrared spectroscopy (potassium bromide tablet) showed the presence of hydroxyl groups (3398 cm⁻¹) in the compound. -1 ), carbonyl (1726 and 1665 cm) -1 ), aromatic rings (1647, 1456, 1345cm) -1 Characteristic functional groups; High-resolution mass spectrometry (HRESIMS) revealed a quasi-molecular ion peak at 307.0581 [M+Na]. + The molecular formula of the compound can be determined to be C. 16 H 12 O5.

[0080] For 1 H NMR,13 Detailed analysis of C10 NMR, DEPT, and HSQC spectral data revealed that the compound contains 16 carbons and 12 hydrogens, including a 1,2,4,5-tetrasubstituted benzene ring (C-1, C-2, C-3, C-4, C-9, C-10, H-1, H-4), two carbonyl groups (C-5 and C-8), two pairs of double bonds (C-6, C-7, C-14, C-15, H-6 and H-15), a 2'-oxopropyl group (-CH2COCH3, C-11~C-13, H2-11 and H3-13), and an oxymethylene group (C-16 and H2-16). Further analysis of its NMR data indicated that one set of double bonds and the two carbonyl groups should be connected to the benzene ring to form 1,4-naphthoquinone, while another set of double bonds should be connected to the benzene ring to form a furan ring, satisfying the 11 degrees of unsaturation in the compound. Furthermore, the inference of the presence of 1,4-naphthoquinone in the compound can be inferred through HMBC correlations between H-6 and C-8 / C-5 / C-10, H-4 and C-5 / C-9 / C-10, and H-1 and C-8 / C-9 / C-10. Figure 3 This was confirmed, and the speculation that another set of double bonds and benzene links form a furan ring can also be confirmed by the HMBC correlation between H-4 and C-3 / C-14, and H-15 and C-2 / C-3 / C-14. Figure 3 This has been confirmed. The compound was identified as naphtho[2,3-] b [Furan-5,8-dione structure]

[0081] Once the parent compound was determined, the positions of the remaining substituents (methylene oxide and 2-oxopropyl) could also be determined through their HMBC correlations. The 2-oxopropyl substitution at C-7 was confirmed by HMBC correlations between H2-11 and C-6 / C-7 / C-8, and between H-6 and C-11. The methyl oxide substitution at C-14 was determined by HMBC correlations between H2-16 and C-3 / C-14 / C-15, and between H-15 and C-16. Thus, the structure of the compound was accurately confirmed. It was named: 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b Furan-5,8-dione, its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione.

[0082] Example 5

[0083] Anti-tobacco black shank activity test of the compounds of this invention

[0084] I. Inhibition of Tobacco Phytophthora activity test

[0085] Add 1000mL of water to the oatmeal and heat in a boiling water bath for 1 hour. Filter through gauze and add water to make up to 1000mL. Then add sugar and agar, heat until the agar is completely melted, and filter through gauze (with absorbent cotton in between) into an Erlenmeyer flask while hot. Sterilize at 121℃ and 15 psi for 20 minutes. Remove and cool to about 45℃. Add ampicillin (5mg / 100mL) on a sterile work surface, mix well, pour into a petri dish, and incubate at 28℃ for 48 hours. After checking for sterility, it is ready for use.

[0086] Take a 5mm diameter circular filter paper, place it in a petri dish, autoclave at 15 psi for 30 minutes, dry it, and then immerse it in 20 psi. µ The compound prepared in Example 1 of M, 75% ethanol aqueous solution, and sterile distilled water were used. Using a sterile pipette, 0.2 mL of fresh *Phytophthora tobaccoscens* culture was pipetted onto oatmeal agar plates under sterile conditions. The agar was spread evenly with a triangular glass spreader. Filter paper discs were then gently placed onto the corresponding plates using tweezers. The plates were incubated at 28°C to observe the results and determine the size of the inhibition zone. Agricultural chloramphenicol was used as a positive control.

[0087] Test results showed that the diameter of the inhibition zone of the naphthoquinone compound of the present invention was 16.6±2.6 mm, while the diameter of the inhibition zone of the positive control, agricultural chloramphenicol, was 13.5±2.4 mm. This indicates that the compound of the present invention has a significantly better inhibitory effect on Phytophthora tobaccois than the positive control, agricultural chloramphenicol, and exhibits outstanding inhibitory activity against black shank disease.

[0088] II. Testing the effectiveness of tobacco black shank control

[0089] Tobacco seedlings were transplanted into pots 10cm in diameter and 10cm in height. The cultivation substrate consisted of sterilized soil, peat moss, and perlite (2:2:1), with one seedling per pot. After transplanting and allowing the seedlings to recover, 10g of mycelium was added to the roots per seedling. The seedlings were then placed in an artificial climate chamber with a daytime temperature of 30℃ and a nighttime temperature of 28℃, a light-to-dark ratio of 12h:12h, and a relative humidity of 95% to induce disease. Before the onset of black shank disease, the seedlings were treated with a 20µM solution of the compound of this invention, 10mL per seedling, for a total of two applications. Each treatment consisted of 10 seedlings, repeated three times. After 14 days, the disease incidence was assessed, and the disease index was calculated.

[0090] Results: The compound of this invention showed a control effect of (68.9±3.5)% on tobacco black shank, while the control effect of agricultural chloramphenicol was (57.4±3.8)%. The control effect of the compound of this invention exceeded that of agricultural chloramphenicol, indicating that the naphthoquinone compound of this invention has a significant control effect on tobacco black shank.

Claims

1. A naphthoquinone compound with activity against tobacco black shank, characterized in that, The naphthoquinone compound with anti-tobacco black shank activity is a light yellow gelatinous substance with the molecular formula: C 16 H 12 O5, named 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3-b]furan-5,8-dione, has the English name: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3-b]furan-5,8-dione has the following structure: The method for preparing the naphthoquinone compounds with anti-tobacco black shank activity involves using honeysuckle branches (a plant used in both medicine and food) as raw material, followed by pretreatment, extract extraction, silica gel column chromatography, and purification separation steps. Specifically, the method includes the following steps: A. Pre-treatment: The raw material honeysuckle branches are crushed and passed through a 20-50 mesh sieve to obtain material a; B. Extraction of extract: The first solvent is added to material a for reflux extraction. After the extract is concentrated, the second solvent is added for extraction. The extracts of the second solvent are combined and concentrated under reduced pressure to obtain extract b. The first solvent is an aqueous ethanol solution with a volume percentage concentration of 90% to 96%. The second solvent is ethyl acetate; C. Silica gel column chromatography: After dissolving extract b in a third solvent, add 1.5 to 3 times its weight of extract b in 160 to 200 mesh silica gel and pack the column. Before loading the column, add 0.8 to 2.0 times its weight of extract b in 80 to 120 mesh silica gel for mixing. Perform gradient elution with chloroform-acetone solutions at volume ratios of 20:1, 9:1, 4:1, 7:3, 3:2, and 1:

1. Detect by TLC and combine identical fractions. The third solvent is methanol, ethanol, or acetone. D. Purification and separation: 1) The eluent obtained by elution with a 7:3 chloroform-acetone solution was purified by dextran gel column chromatography, wherein methanol was used as the mobile phase for dextran gel column chromatography to obtain the crude product of the target compound. 2) The crude target compound was separated and purified by high performance liquid chromatography (HPLC) to obtain the target compound, a naphthoquinone compound with anti-tobacco black shank activity. The HPLC separation was performed using a 21.2 mm × 25 cm, 5 μm C18 column, with 48% methanol aqueous solution as the mobile phase, a flow rate of 12 mL / min, and a UV detector with a detection wavelength of 349 nm. The chromatographic peak was collected after 34 min.

2. The application of a naphthoquinone compound with anti-tobacco black shank activity as described in claim 1, characterized in that, The application of the naphthoquinone compounds with anti-tobacco black shank activity in the preparation of anti-tobacco black shank drugs.

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