Naphthoquinone compound with activity of resisting tobacco black shank as well as preparation method and application of naphthoquinone compound

Abstract

The invention belongs to the technical field of agricultural chemistry, and particularly relates to a naphthoquinone compound with tobacco black shank resisting activity as well as a preparation method and application of the naphthoquinone compound. The naphthoquinone compound is prepared by the following steps: by taking honeysuckle branches as raw materials, extracting with high-concentration methanol, high-concentration acetone / water or high-concentration ethanol / water, merging extracting solutions, filtering, and concentrating under reduced pressure to obtain extract; packing the extract by using a silica gel dry method and carrying out silica gel column chromatography; carrying out gradient elution by using a chloroform-acetone solution; and further separating and purifying the 7: 3 part of the eluent by high performance liquid chromatography to obtain the required naphthoquinone compound. The invention further discloses application of the compound, and activity tests show that the compound has a good inhibiting effect on tobacco black shank. The compound disclosed by the invention is novel in structure, has relatively good black shank resisting activity, can be used as a lead compound for resisting tobacco black shank, and is used for research and development of biological pesticides for preventing and treating tobacco black shank.

Description

Technical Field

[0001] The present invention belongs to the technical field of phytochemistry, and particularly relates to a naphthoquinone compound with anti-Phytophthora parasitica var. nicotianae activity, a preparation method thereof, and an application thereof. Background Art

[0002] Phytophthora parasitica var. nicotianae is one of the most devastating diseases in tobacco production, also known as tobacco blight, and tobacco farmers call it "black stem madness", "black root", "aconite disease". At present, the prevention and control of Phytophthora parasitica var. nicotianae are mainly achieved through methods such as crop rotation cultivation, variety gene improvement, and biological pesticides. Among them, the use of biological pesticides for prevention and control is the most commonly used and easiest to implement method. Therefore, searching for more highly efficient biological pesticide lead compounds is of great significance for the prevention and control of Phytophthora parasitica var. nicotianae.

[0003] Lonicera japonica Lonicera japonica Thunb.) is a plant of the genus Lonicera in the family Caprifoliaceae. Its dried flower buds or unopened flowers have extremely high medicinal value and have the effect of clearing heat and detoxifying. It is mainly used to treat exogenous wind-heat or febrile diseases caused by warm pathogens, heat stroke, dysentery with heat-toxicity, carbuncles and sores, sore throat, and various infectious diseases. With the progress of society and the development of science and technology, people's research on the comprehensive development and utilization of Lonicera japonica has become increasingly in-depth. From the original medicinal value, it has developed into the development and production of health care foods, beauty and skin care products, and then to tourism sightseeing and ecological agricultural engineering construction. Research at home and abroad shows that the main chemical components in Lonicera japonica are: organic acids, phenylpropanoids, sterols, quinones, triterpenoid saponins, iridoid glycosides, and terpene volatile oils, etc.

[0004] Naphthoquinone is an important class of natural active ingredients. Naphthoquinone compounds from natural sources are widely distributed in nature, especially in higher plants and also in some microorganisms. Pharmacological experimental studies have shown that they have a wide range of pharmacological activities, such as cytotoxic, antioxidant, anti-inflammatory, and antibacterial activities, etc. The present invention has discovered a new naphthoquinone compound from the branches of Lonicera japonica. It is worth noting that this compound has a rare naphthoquinone structure containing a 3-hydroxymethylfuran ring structure fragment, with a relatively high novelty, and this compound has significant anti-Phytophthora parasitica var. nicotianae activity. Its control effect on Phytophthora parasitica var. nicotianae is (68.9 ± 3.5)%, which is better than the control effect of agricultural chloramphenicol (57.4 ± 3.8)%, and it can be used as a lead compound for anti-Phytophthora parasitica var. nicotianae to develop biological pesticides for the prevention and control of Phytophthora parasitica var. nicotianae. Summary of the Invention

[0005] The first object of the present invention is to provide a naphthoquinone compound with anti-Phytophthora parasitica var. nicotianae activity; the second object is to provide a preparation method of the naphthoquinone compound with anti-Phytophthora parasitica var. nicotianae activity; the third object is to provide an application of the naphthoquinone compound with anti-Phytophthora parasitica var. nicotianae activity.

[0006] The first object of the present invention is achieved as follows. The naphthoquinone compound with anti-Phytophthora nicotianae activity is a light yellow jelly, and its molecular formula is: C 16 H 12 O5, named 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione, and its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)nap-htho[2,3- b furan-5,8-dione, having the following structure: .

[0007] The second object of the present invention is achieved as follows. Using the branches of honeysuckle, a medicinal and edible homologous plant, as the raw material, it is prepared through the steps of pretreatment, extract preparation, silica gel column chromatography, and purification and separation. Specifically, it includes the following steps: A. Pretreatment: The raw material of honeysuckle branches is crushed and sieved through a 20 - 50 mesh sieve to obtain material a; B. Extract preparation: The first solvent is added to material a for reflux extraction. After the extract is concentrated, the second solvent is added for extraction, and the second solvent extraction phase after extraction is combined and concentrated under reduced pressure to obtain extract b; The first solvent is an ethanol aqueous solution with a volume percentage concentration of 90% - 96%; The second solvent is ethyl acetate; C. Silica gel column chromatography: The extract b is dissolved in the third solvent and then added to 160 - 200 mesh silica gel with a weight 1.5 - 3 times that of the extract b to pack the column, and gradient elution is carried out with a chloroform - acetone solution with a volume ratio of 20:1 - 1:1. After TLC detection, the same parts are combined; the third solvent is methanol, ethanol, or acetone; D. Purification and separation: 1) The eluate obtained by eluting with a chloroform - acetone solution with a 7:3 ratio is purified by a Sephadex gel column to obtain the crude target compound; 2) The crude target compound is separated and purified by high - performance liquid chromatography to obtain the target substance, the naphthoquinone compound with anti - Phytophthora nicotianae activity.

[0008] The specific operation is as follows: 1) The honeysuckle branches are dried and crushed, and the first solvent is taken for extraction, concentrated, and the second solvent is taken for extraction, and the concentrated extracts are combined to obtain extract a; the first solvent is an ethanol aqueous solution with a concentration of 90% - 96%; the second solvent is ethyl acetate; 2) After the extract a is dissolved in the third solvent and separated by a silica gel column, gradient elution is carried out with a chloroform - acetone solution, and the fraction with a chloroform - acetone volume ratio of 7:3 is collected to obtain the elution component b; the third solvent is methanol, ethanol or acetone; 3) The elution component b is purified by a Sephadex column to obtain a crude target compound; the crude target compound is further separated by HPLC chromatography to obtain a pure target compound.

[0009] The structure of the naphthoquinone compound with anti - Phytophthora nicotianae activity prepared by the above steps can be identified by the following methods: Appearance observation shows that: the compound of the present invention is a yellow gum; the ultraviolet - visible absorption spectrum shows that it has maximum absorption at 215, 268, 349 nm, proving the existence of an aromatic ring structure in the compound; the infrared spectrum (KBr tablet) shows that there are hydroxyl groups (3398 cm -1 ) and carbonyl groups (1726 and 1665 cm -1 ), and aromatic ring (1647, 1456, 1345 cm -1 ) characteristic functional groups in the compound; high - resolution mass spectrometry (HRESIMS) gives a quasi - molecular ion peak of 307.0581[M + Na] + , and the molecular formula of the compound can be determined as C 16 H 12 O5.

[0010] For its 1 1H NMR, 13 13C NMR, DEPT and HSQC spectral data were analyzed in detail, and it was found that there are 16 carbons and 12 hydrogens in the compound, 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 a methylene oxide group (C - 16 and H2 - 16). Further analysis of its nuclear magnetic resonance data shows that a pair of double bonds and two carbonyl groups should be connected to the benzene ring to form 1,4 - naphthoquinone, and the other pair of double bonds is connected to the benzene ring to form a furan ring to meet the 11 degrees of unsaturation in the compound. In addition, the speculation of the existence of 1,4 - naphthoquinone in the compound can be confirmed by the HMBC correlations of 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) Confirmation, and the speculation that another set of double bonds is connected to benzene to form a furan ring can also be verified by the HMBC correlations of H-4 and C-3 / C-14, H-15 and C-2 / C-3 / C-14 ( Figure 3 ) is confirmed. This compound can be determined to have the structure of naphtho[2,3- b furan-5,8-dione.

[0011] Table 1. 1 H NMR and 13 C NMR data (CDCl3)

[0012] After the parent compound is determined, the positions of the remaining substituents (methylenoxy and 2-oxopropyl) can also be determined by their HMBC correlations. The substitution of 2-oxopropyl at C-7 can be confirmed by the HMBC correlations of H2-11 and C-6 / C-7 / C-8, and H-6 and C-11. The substitution of methylenoxy at C-14 can be determined by the HMBC correlations of H2-16 and C-3 / C-14 / C-15, and H-15 and C-16. Thus far, the structure of the compound has been accurately confirmed. Named: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione, and its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione.

[0013] 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, 818 cm -1 ; 1 H and 13 C 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).

[0014] The third object of the present invention is achieved as follows: the application of the naphthoquinone compound in the preparation of a drug for preventing tobacco black shank.

[0015] The beneficial effects of the present invention are: 1) The present invention is the first to extract a naphthoquinone compound with a novel structure from honeysuckle branches. It is worth noting that: this compound is a rare natural product and contains a 3-hydroxymethylfuran ring structure fragment. The compound structure is highly novel and has good anti-black shank activity. It can be used as a lead compound for tobacco black shank disease resistance in the development of biopesticides for the prevention and control of tobacco black shank disease.

[0016] 2) This invention uses the Chinese medicinal material honeysuckle branches as raw materials for the first time, providing a new research direction for the development of plant-derived natural products with plant disease prevention and treatment effects.

[0017] 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 facilitate industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The carbon NMR spectrum of the naphthoquinone compound prepared in Example 1; Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the naphthoquinone compound prepared in Example 1; Figure 3 This is a main HMBC correlation diagram of the naphthoquinone compounds prepared in Example 1. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0020] The naphthoquinone compound with anti-tobacco black shank activity of the present invention is a light yellow colloid, and its molecular formula is: 16 H 12 O5, named 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b ] Furan-5,8-dione, English name: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b ]furan-5,8-dione, having the following structure: .

[0021] The preparation method of the naphthoquinone compounds with anti-tobacco black shank activity of the present invention is to use the branches of the medicinal and edible plant honeysuckle as raw materials, and to prepare the compounds through pretreatment, extract extraction, silica gel column chromatography and purification and separation steps, and specifically comprises the following steps: A. Pretreatment: The raw honeysuckle branches are crushed and screened through a 20-50 mesh sieve to obtain material a; B. Extract 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 second solvent extraction phase after extraction is combined and concentrated under reduced pressure to obtain extract b; The first solvent is an ethanol aqueous solution with a volume percentage concentration of 90% - 96%; The second solvent is ethyl acetate; C. Silica gel column chromatography: The extract b is dissolved in the third solvent and then loaded onto a column with 160-200 mesh silica gel that is 1.5-3 times the weight of the extract b. A chloroform-acetone solution with a volume ratio of 20:1 - 1:1 is used for gradient elution. After TLC detection, the same parts are combined; the third solvent is methanol, ethanol, or acetone; D. Purification and separation: 1) The eluate obtained by eluting with a chloroform-acetone solution with a 7:3 ratio is purified through a Sephadex gel column to obtain the crude target compound; 2) The crude target compound is separated and purified by high performance liquid chromatography to obtain the target substance, a naphthoquinone compound with anti-Phytophthora nicotianae activity.

[0022] Before loading the column in step C, it also includes a sample mixing step of adding 80-120 mesh silica gel that is 0.8-2.0 times the weight of material b.

[0023] In step C, the volume ratio of the chloroform-acetone solution is 20:1, 9:1, 4:1, 7:3, 3:2, and 1:1.

[0024] In step D1), the mobile phase for the Sephadex gel column purification is methanol.

[0025] In step D2), the HPLC chromatographic separation uses a 21.2 mm×25 cm, 5 μ m C 18 chromatographic column, with a 48% methanol aqueous solution as the mobile phase, a flow rate of 12 mL / min, a detection wavelength of 349 nm for the ultraviolet detector, and collecting the chromatographic peak at 34 min.

[0026] The specific operation is as follows: (1) Extract of extract: The honeysuckle branches are dried and crushed, the first solvent is taken for extraction, concentrated, the second solvent is taken for extraction, and the combined extracts are concentrated to obtain the extract; (2) The extract is dissolved in the third solvent and then separated by a silica gel column, eluted with a chloroform / acetone gradient, and the elution fraction with a chloroform:acetone volume ratio of 7:3 is collected; (3) The elution fraction obtained in step 2 is purified through a Sephadex gel column to obtain the crude target compound, and finally the crude target compound is separated by HPLC chromatography to obtain the pure target compound.

[0027] The first solvent is an aqueous solution of 95% ethanol.

[0028] In the step 1, the sample is subjected to reflux extraction with the first solvent for 2 - 3 times, and the reflux extraction time for each time is 35 - 60 min.

[0029] The extract obtained by reflux extraction is concentrated and then extracted with the second solvent for 2 - 3 times.

[0030] In the step 2, the volume ratios of chloroform to acetone for gradient elution are 20:1, 9:1, 4:1, 7:3, 3:2, and 1:1 in sequence.

[0031] In the step 2, silica gel column with 160 - 200 mesh is used for separation. After the extract is dissolved with 1.5 - 3 times the amount of the second solvent, it is mixed with 80 - 120 mesh coarse silica gel.

[0032] During the mixing process, the weight of the mixing silica gel is 0.8 - 2.5 times that of the extract.

[0033] In the step 3, Sephadex gel column purification is carried out with methanol as the mobile phase.

[0034] In the step 3, high - pressure liquid chromatography separation is carried out using a Zorbax PrepHT GF (21.2 mm × 25 cm) reverse - phase column, with 48% aqueous methanol solution as the mobile phase, the flow rate is 12 mL / min, the detection wavelength of the ultraviolet detector is 349 nm, and the chromatographic peak at 34 min is collected.

[0035] The second solvent is ethyl acetate.

[0036] The third solvent is methanol or ethanol or acetone.

[0037] The present invention also provides the application of the naphthoquinone compound in the preparation of an agent for controlling tobacco black shank. Through the anti - tobacco black shank activity test of the present invention, the naphthoquinone compound has a significant inhibitory effect on tobacco black shank.

[0038] The application of the present invention is the application of the naphthoquinone compound with anti - tobacco black shank activity in the preparation of a drug for controlling tobacco black shank.

[0039] The following further illustrates the present invention with specific examples: Example 1 The honeysuckle branch samples in this example are from Kunming, Yunnan. The honeysuckle branches are dried in the sun and crushed to about 40 mesh. Weigh 3.5 kg of the crushed samples and place them in a 20 L glass reactor. Add 6 L of 95% ethanol aqueous solution, reflux for 50 min, and filter out the extract; add 6 L of 95% ethanol aqueous solution to the residue again, reflux for 50 min, and filter out the extract. Combine the two extracts and concentrate to a small volume, then extract with 6 L of ethyl acetate twice. Concentrate under reduced pressure to obtain 66.4 g of the naphthoquinone part extract. Mix the extract with 80 g of coarse silica gel (80 - 120 mesh), dry it, and perform column chromatography with 200 g of silica gel (160 - 200 mesh), eluting with a gradient of chloroform:acetone (20:1, 9:1, 4:1, 7:3, 3:2, 1:1), dividing it into 6 parts. Select the 7:3 elution part, dissolve it in methanol after concentration, and purify it with a Sephadex column using methanol as the mobile phase to obtain the crude compound. Further separate the crude product by HPLC: use a Zorbax PrepHT GF (21.2 mm × 25 cm) reverse phase column from Agilent, with 48% methanol aqueous solution as the mobile phase, a flow rate of 12 mL / min, a detection wavelength of 349 nm for the UV detector, collect the chromatographic peak at 34.0 min, evaporate to dryness after multiple accumulations to obtain a light red jelly-like substance, which is the pure target compound.

[0040] Example 2 The honeysuckle branch samples are from Dali, Yunnan. The honeysuckle branches are dried in the sun and crushed to about 40 mesh. Weigh 4.0 kg of the crushed samples and place them in a 20 L glass reactor. Add 8 L of 95% ethanol aqueous solution, reflux for 40 min, and filter out the extract; add 8 L of 95% ethanol aqueous solution to the residue again, reflux for 40 min, and filter out the extract. Combine the two extracts and concentrate to a small volume, extract with 8 L of ethyl acetate twice, combine the ethyl acetate phases of the extraction, and concentrate under reduced pressure to obtain 70.22 g of the naphthoquinone part extract. Mix the extract with 100 g of coarse silica gel (80 - 120 mesh), dry it, and perform column chromatography with 220 g of silica gel (160 - 200 mesh), eluting with a gradient of chloroform:acetone (20:1, 9:1, 4:1, 7:3, 3:2, 1:1), dividing it into 6 parts. Select the 7:3 elution part, dissolve it in methanol after concentration, and purify it with a Sephadex column using methanol as the mobile phase to obtain the crude compound. Further separate the crude product by HPLC: use a Zorbax PrepHT GF (21.2 mm × 25 cm) reverse phase column from Agilent, with 48% methanol aqueous solution as the mobile phase, a flow rate of 12 mL / min, a detection wavelength of 349 nm for the UV detector, collect the chromatographic peak at 34.0 min, evaporate to dryness after multiple accumulations to obtain the pure compound.

[0041] The structural identification of the compound obtained in this example is the same as that in Example 1. It is confirmed that the compound prepared in this example is the naphthoquinone compound - 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione.

[0042] Example 3 The honeysuckle branch samples are from Yuxi, Yunnan. The honeysuckle branches are dried in the sun and crushed to about 50 mesh. Weigh 4.6 kg of the crushed samples and place them in a 20 L glass reactor. Add 10 L of 95% ethanol aqueous solution, reflux for 30 min, and filter out the extract; add 10 L of 95% ethanol aqueous solution to the residue again, reflux for 50 min, and filter out the extract. Combine the two extracts and concentrate to a small volume, then extract with 10 L of ethyl acetate twice. Concentrate under reduced pressure to obtain 96.8 g of naphthoquinone extract. The extract is mixed with 130 g of coarse silica gel (80 - 120 mesh), dried, and separated by column chromatography with 300 g of silica gel (160 - 200 mesh), eluting with chloroform:acetone (20:1, 9:1, 4:1, 7:3, 3:2, 1:1) gradient, divided into 6 parts. Select the 7:3 elution part, dissolve it in methanol after concentration, and purify it with a Sephadex column using methanol as the mobile phase to obtain the crude compound. The crude product is further separated by HPLC: use an Agilent Zorbax PrepHT GF(21.2 mm×25 cm) reverse phase column, with 48% methanol aqueous solution as the mobile phase, a flow rate of 12 mL / min, the detection wavelength of the ultraviolet detector is 349 nm, collect the chromatographic peak at 34.0 min, evaporate to dryness after multiple accumulations to obtain the pure compound.

[0043] The structural identification of the compound obtained in this example is the same as that in Example 1. It is confirmed that the compound prepared in this example is the naphthoquinone compound - 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione.

[0044] Example 4 The structure of the naphthoquinone compound prepared by the above method was identified by the following method: Observation of appearance found that: the compound of the present invention is a yellow gum; the ultraviolet-visible absorption spectrum shows that it has maximum absorption at 215, 268, and 349 nm, proving the existence of an aromatic ring structure in the compound; the infrared spectrum (KBr tablet) shows that the compound has hydroxyl groups (3398 cm -1 ), carbonyl groups (1726 and 1665 cm -1 ), and aromatic ring (1647, 1456, 1345 cm -1 ) characteristic functional groups; high-resolution mass spectrometry (HRESIMS) gives the quasi-molecular ion peak 307.0581[M+Na]+ , the molecular formula of the compound can be determined as C 16 H 12 O5.

[0045] For its 1 H NMR, 13 C NMR, DEPT and HSQC spectral data were analyzed in detail, and it was found that there were 16 carbons and 12 hydrogens in the compound, 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 a methylene oxide group (C-16 and H2-16). Further analysis of its nuclear magnetic resonance data showed that a group of double bonds and two carbonyl groups should be connected to the benzene ring to form 1,4-naphthoquinone, while the other group of double bonds was connected to the benzene ring to form a furan ring to meet the 11 degrees of unsaturation in the compound. In addition, the speculation of the presence of 1,4-naphthoquinone in the compound could be confirmed by the HMBC correlations of 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 ), and the speculation that the other group of double bonds was connected to the benzene to form a furan ring could also be confirmed by the HMBC correlations of H-4 and C-3 / C-14, H-15 and C-2 / C-3 / C-14 ( Figure 3 ). The compound could be determined to have a naphtho[2,3- b furan-5,8-dione structure.

[0046] After the parent body of the compound was determined, the positions of the remaining substituents (methylene oxide and 2-oxopropyl) could also be determined by their HMBC correlations. The substitution of 2-oxopropyl at the C-7 position could be confirmed by the HMBC correlations of H2-11 and C-6 / C-7 / C-8, and H-6 and C-11. By the HMBC correlations of H2-16 and C-3 / C-14 / C-15, and H-15 and C-16, it could be determined that the methylene oxide was substituted at C-14. 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, and its English name was: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione.

[0047] Example 5 Anti-Phytophthora nicotianae Activity Test of the Compounds of the Present Invention I. Activity Test for Inhibiting Phytophthora nicotianae Take oatmeal and add 1000 mL of water. Heat it in a boiling water bath for 1 hour, filter it through a gauze, make up the volume to 1000 mL with water, then add sugar and agar. Heat until the agar is completely melted, and then filter it through a gauze (with absorbent cotton in the middle) while it is still hot into an Erlenmeyer flask. Sterilize it at 121 °C and 15 pounds for 20 min (minutes). Take it out and cool it to about 45 °C. Add ampicillin (5 mg / 100 mL) on a sterile operating table, mix well and pour it into petri dishes. Incubate at 28 °C for 48 hours. After checking for sterility, it is ready for use.

[0048] Take round filter papers with a diameter of 5 mm, put them into petri dishes, sterilize them at 15 pounds of high pressure for 30 min, dry them, and then immerse them in the compound prepared in Example 1 with a concentration of 20 µ M, 75% aqueous ethanol solution and sterilized distilled water respectively. On a sterile operating table, use a sterile pipette to suck 0.2 mL of fresh bacterial solution of Phytophthora nicotianae onto the oatmeal agar medium plate respectively. Spread it evenly with a triangular glass spreading rod, gently stick the filter papers on the corresponding plates with forceps respectively, place them at 28 °C for culturing and observing the experimental results, and measure the size of the inhibition zone. At the same time, use agricultural chloramphenicol as a positive control.

[0049] The test results show that: the diameter of the inhibition zone of the naphthoquinone compounds of the present invention is 16.6 ± 2.6 mm, and the diameter of the inhibition zone of the positive control agricultural chloramphenicol is 13.5 ± 2.4 mm. It shows that the compounds of the present invention have a significantly better effect on inhibiting Phytophthora nicotianae than the positive control agricultural chloramphenicol, and have outstanding activity against black shank.

[0050] II. Detection of the Effect of Controlling Tobacco Black Shank Transplant tobacco seedlings into flower pots with a diameter of 10 cm and a height of 10 cm. The cultivation substrate is: sterilized soil, peat, and perlite (2:2:1), with 1 plant per pot. After transplanting and slow seedling establishment, add 10 g / plant of bacterial grains to the roots, place the tobacco seedlings in an artificial climate chamber for cultivation, at 30 °C during the day, 28 °C at night, light:dark (12 h:12 h), and relative humidity 95% to make the tobacco seedlings diseased. Before the onset of black shank, use the compound of the present invention with a concentration of 20 µM to irrigate the roots of the tobacco seedlings, pour 10 mL per plant, and pour 2 times in total. There are 10 plants in each treatment, repeated 3 times. After 14 d, investigate the disease incidence and calculate the disease index.

[0051] Result: The control effect of the compound of the present invention on tobacco black shank is (68.9 ± 3.5)%, while the control effect of the control agricultural chloramphenicol is (57.4 ± 3.8)%. Its control effect exceeds that of the control agricultural chloramphenicol, indicating that the naphthoquinone compounds described in the present invention have a significant control effect on tobacco black shank.

Claims

1. A naphthoquinone compound with anti-Phytophthora parasitica var. nicotianae activity, characterized in that, The naphthoquinone compound with anti-Phytophthora nicotianae activity is a light yellow gum, and its molecular formula is: C 16 H 12 O5, named 3-(hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione, and its English name is: 3-(Hydroxymethyl)-7-(2'-oxopropyl)naphtho[2,3- b furan-5,8-dione, having the following structure: 。 2. A method for preparing a naphthoquinone compound having anti-Phytophthora nicotianae activity as described in claim 1, characterized in that, It is prepared from the branches of honeysuckle, a plant with both medicinal and edible uses, through the steps of pretreatment, extract preparation, silica gel column chromatography and purification and separation, specifically including the following steps: A. Pretreatment: The raw material of honeysuckle branches is crushed and screened through a 20-50 mesh sieve to obtain material a; B. Extract preparation: The first solvent is added to material a for reflux extraction, the extract is concentrated, and then the second solvent is added for extraction. The second solvent extraction phase after extraction is combined and concentrated under reduced pressure to obtain extract b; The first solvent is an ethanol aqueous solution with a volume percentage concentration of 90% - 96%; The second solvent is ethyl acetate; C. Silica gel column chromatography: The extract b is dissolved with the third solvent and then loaded onto a column with 160-200 mesh silica gel that is 1.5-3 times the weight of the extract b. Gradient elution is carried out with a chloroform-acetone solution with a volume ratio of 20:1 - 1:

1. After TLC detection, the same parts are combined; the third solvent is methanol, ethanol or acetone; D. Purification and separation: 1) The eluate obtained by eluting with a chloroform-acetone solution with a ratio of 7:3 is purified by a Sephadex gel column to obtain a crude target compound; 2) The crude target compound is separated and purified by high performance liquid chromatography to obtain the target substance, a naphthoquinone compound with anti-Phytophthora nicotianae activity.

3. The preparation method according to claim 2, characterized in that, Before loading onto the column in step C, a sample mixing step is also included, in which silica gel with a mesh size of 80-120 and a weight 0.8-2.0 times that of material b is added.

4. The preparation method according to claim 2, characterized in that, In step C, the volume ratio of the chloroform-acetone solution is 20:1, 9:1, 4:1, 7:3, 3:2 and 1:

1.

5. The preparation method according to claim 2, characterized in that, In step D1), the mobile phase for purification by the Sephadex gel column is methanol.

6. The preparation method according to claim 2, characterized in that, In step D 2), the HPLC chromatographic separation was carried out using a 21.2 mm×25 cm, 5 μ μm C 18 chromatographic column, with an aqueous solution of 48% methanol as the mobile phase, a flow rate of 12 mL / min, a detection wavelength of 349 nm for the UV detector, and collecting the chromatographic peaks at 34 min.

7. Use of a naphthoquinone compound having anti-Phytophthora nicotianae activity as described in claim 1, characterized in that, Application of the naphthoquinone compound with anti-Phytophthora nicotianae activity in the preparation of anti-Phytophthora nicotianae drugs.

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