Application of chelerythrine and derivative thereof in prevention and treatment of tobacco mosaic disease
By preparing a compound pesticide formulation of chelidonine and dihydrochelidonine, the problem of large-scale infection of tobacco mosaic virus was solved, achieving efficient control of tobacco mosaic disease, and it is safe and harmless to tobacco plants.
Patent Information
- Application Number
- CN202511000080.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-11-28
AI Technical Summary
There is a lack of effective methods for controlling tobacco mosaic virus in the current technology, especially since large-scale infection has a serious impact on the quality of tobacco leaves, and chemical pesticides have limited control effects and pesticide residue problems.
A compound pesticide formulation using chelidonine and its derivative dihydrochelidonine was prepared. The chelidonine extract was prepared by ultrasonic extraction and macroporous resin separation and purification, and the dihydrochelidonine extract was prepared by ethyl acetate extraction and silica gel column chromatography, forming a compound pesticide formulation with a concentration ratio of (0.1-0.20 mg/mL):(0.08-0.16 mg/mL).
It provides highly effective control of tobacco mosaic disease, with a live protective activity of 75.4±3.7, an in vitro passivation activity of 72.6±4.9, and a curative activity of 73.5±5.3. Moreover, it has little impact on the growth of tobacco plants, with no obvious inhibition or poor growth.
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Figure CN121014663A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of plant disease control, and particularly relates to application of chelerythrine and derivatives thereof in prevention and treatment of tobacco mosaic disease. BACKGROUND
[0002] Tobacco mosaic virus (TMV) is a kind of RNA virus, which specifically infects plants, especially tobacco and other solanaceous plants, and can make the infected leaves appear mottled and dirty. Tobacco mosaic disease not only seriously affects the yield of tobacco leaves, reduces the proportion of upper and middle tobacco, but also greatly affects the appearance quality and internal components of tobacco leaves, reduces the availability of tobacco leaves, and causes great losses to tobacco production. At present, chemical control is still the main method for preventing and treating tobacco mosaic virus in agricultural planting, and there are few special preparations against plant viruses. The application of chemical pesticides still has problems such as limited control effect and serious pesticide residues. Plant-derived antiviral agents with natural plant active substances as effective components have the advantages of good environmental compatibility, safety and low development cost, but so far only a small part of substances have been practically applied. In addition, tobacco mosaic virus is mainly prevented, and once the tobacco farmers do not manage well, it will be infected in a large area in the vigorous growth period, and a large area of necrosis will appear. Although it has little effect on yield, it has a very obvious effect on the quality of tobacco leaves, causing serious losses. At present, there is no very effective control measure for the tobacco mosaic virus infected in a large area in the vigorous growth period. Therefore, it is an urgent task for tobacco production to find an economic and effective method for preventing and treating tobacco mosaic disease.
[0003] With the development of natural plant source pesticides, a new way for preventing and treating plant viruses is opened up. Such substances can play a good antiviral and plant resistance enhancing effect under the conditions of green and pollution-free preparation and application. Among them, Chelidonium is a perennial Chelidonium majus plant, which tastes bitter and is cool, also known as Dihuanglian, Tuohuanglian, Duan'gancao and Xionghuangcao. According to records, the whole grass of Chelidonium has the effects of anti-inflammatory, analgesic, relieving cough and detoxification. Studies have shown that the main active ingredients in Chelidonium include alkaloids such as chelerythrine, chelerythrine, sanguinarine and berberine. Among them, chelerythrine is an effective inflammation inhibitor, which has excellent antibacterial, insecticidal and antitumor biological activities. Dihydrochelerythrine belongs to dihydrobenzophenanthridine alkaloids, which is a derivative of chelerythrine. It can affect the biosynthesis process of protein by inhibiting the activity of DNA and RNA polymerase, and has significant antibacterial, antiviral and insecticidal activities. SUMMARY
[0004] The application provides application of chelerythrine and derivatives thereof in prevention and treatment of tobacco mosaic disease, so as to solve the problem that there is no effective control measure for plants infected with tobacco mosaic virus in the prior art.
[0005] One of the purposes of the present application is to provide a complex pesticide preparation comprising chelerythrine extract and dihydrochelerythrine extract, wherein the concentration ratio of chelerythrine extract to dihydrochelerythrine extract in the complex pesticide preparation is (0.1-0.20 mg / mL):(0.08-0.16 mg / mL).
[0006] In a preferred embodiment of the present application, the extraction step of chelerythrine is as follows: the artificially cultivated Chelidonium majus plants are washed, dried, and crushed into fine powder using a pulverizer, and then passed through a No. 3 sieve; 95% ethanol is added at a material-liquid ratio of 1:8, and the ultrasonic extraction method is used to set the ultrasonic frequency, and the extraction time is 35 min to obtain an extraction solution; the extraction solution is filtered, concentrated using a rotary evaporator, purified using a macroporous resin, and the conditions are set as follows: an environmental temperature of 30 DEG C, elution treatment using 50% ethanol solution, concentration, dilution using anhydrous ethanol, and chelerythrine extract is obtained.
[0007] In a preferred embodiment of the present application, the ultrasonic frequency is set to 85%, and the flow rate of the elution treatment is 1 mL / min.
[0008] In a preferred embodiment of the present application, the extraction method of dihydrochelerythrine is as follows: the artificially cultivated Chelidonium majus plants are washed, dried, and crushed into fine powder using a pulverizer, and then methanol solution is added at a material-liquid ratio of 1:10, and the mixture is immersed in an environment at 25-40 DEG C for 24 h, stirred, and the mixture after soaking is filtered to separate the solid residue and the extraction solution; the extraction solution is concentrated using a rotary evaporator, liquid-liquid extraction is performed using ethyl acetate, the organic phase and the aqueous phase are separated, the organic phase is separated by silica gel column chromatography, eluted using chloroform, concentrated to dryness, and then redissolved in methanol to obtain dihydrochelerythrine extract.
[0009] The second purpose of the present application is to provide the use of the above-mentioned complex pesticide preparation in the prevention and treatment of tobacco mosaic disease.
[0010] The present application provides a complex pesticide preparation containing chelerythrine extract and dihydrochelerythrine extract, and the use of the complex pesticide preparation in the prevention and treatment of tobacco mosaic disease.
[0011] The protection activity of the chelerythrine provided by the application to living bodies is up to 47.8±3.9, the inactivation activity to ex vivo bodies is up to 49.2±2.4, and the treatment activity is up to 44.5±4.3; the protection activity of the dihydrochelerythrine provided by the application to living bodies is up to 50.8±1.9, the inactivation activity to ex vivo bodies is up to 49.3±3.7, and the treatment activity is up to 58.4±4.8; the protection activity of the compound pesticide preparation provided by the application to living bodies is up to 75.4±3.7, the inactivation activity to ex vivo bodies is up to 72.6±4.9, and the treatment activity is up to 73.5±5.3. It can be seen that the compound pesticide preparation containing chelerythrine extract and dihydrochelerythrine extract provided by the application has the advantages of lower biological toxicity, higher safety, and no phytotoxicity; the treated tobacco plants have less influence on the growth state, and the treated tobacco plants have no obvious inhibition or poor growth phenomenon, and can be applied to agricultural biological control of tobacco mosaic disease. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 Figure 1 is a growth chart of chelidonium majus in an artificial cultivation base in Example 1; Figure 2 Figure 2 is a chelerythrine extraction sample chart in Example 1; Figure 3 Figure 3 is a dihydrochelerythrine extraction sample chart in Example 2; Figure 4 Figure 4 is a chart of plants infected with tobacco mosaic virus treated by applying the compound pesticide preparation in Example 3; A is before treatment; B is after treatment. DETAILED DESCRIPTION
[0013] Those skilled in the art can improve the process parameters as appropriate based on the content herein. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.
[0014] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application will be further described in detail below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.
[0015] Example 1: Extraction of Chelerythrine The artificial cultivation of chelidonium majus plants was used as the material (the growth of chelidonium majus in an artificial cultivation base is as shown in Figure 1).Figure 1 The tobacco leaves were washed and dried in a blast drying oven, and then ground into fine powder using a grinder. 500 g of the dried powdered tobacco leaves were added to 95% ethanol at a solid-liquid ratio of 1:8, and extracted using ultrasonic extraction at an ultrasonic frequency of 85% for 35 min to obtain an extract. The extract was filtered and concentrated using a rotary evaporator, and then purified using a macroporous resin at an ambient temperature of 30°C, eluted with 50% ethanol solution at a flow rate of 1 mL / min, concentrated, and diluted with anhydrous ethanol to obtain a chelerythrine extract (chelerythrine extract sample as shown in FIG. 2). Figure 2 The tobacco leaves were washed and dried in a blast drying oven, and then ground into fine powder using a grinder. 500 g of the dried powdered tobacco leaves were added to 95% ethanol at a solid-liquid ratio of 1:8, and extracted using ultrasonic extraction at an ultrasonic frequency of 85% for 35 min to obtain an extract. The extract was filtered and concentrated using a rotary evaporator, and then purified using a macroporous resin at an ambient temperature of 30°C, eluted with 50% ethanol solution at a flow rate of 1 mL / min, concentrated, and diluted with anhydrous ethanol to obtain a chelerythrine extract (chelerythrine extract sample as shown in FIG. 2).
[0016] Effect experiment: 1 g of tobacco leaves infected with tobacco mosaic virus were ground into a homogenate in a mortar with quartz sand, filtered and centrifuged, and then 4% polyethylene glycol was added to the supernatant, which was centrifuged again. The precipitate was suspended in 0.2 mol / L phosphate buffer to obtain a crude extract of tobacco mosaic virus. The concentration of the crude extract was determined to be 15.6 mg / L by ultraviolet spectrophotometry, and the crude extract was diluted to 10 mg / L to obtain a solution of tobacco mosaic virus.
[0017] (1) In vivo protection activity test The chelerythrine extract obtained in this example was used to prepare chelerythrine solutions with concentrations of 0.01 mg / mL, 0.1 mg / mL, 0.15 mg / mL and 0.20 mg / mL, respectively. Nicotiana benthamiana leaves with consistent growth and health were sprayed with the chelerythrine solutions, and 48 hours later, a 2000-fold diluted solution of tobacco mosaic virus was inoculated on the leaves. The same amount of water was used as a control. Each treatment was repeated 3 times. The number of dead spots was counted 3 days later, and the preventive effect of chelerythrine on tobacco mosaic virus infection of Nicotiana benthamiana was calculated.
[0018] (2) In vitro inactivation activity test The chelerythrine extract obtained in this example was used to prepare chelerythrine solutions with concentrations of 0.01 mg / mL, 0.1 mg / mL, 0.15 mg / mL and 0.20 mg / mL, respectively. Each solution was mixed with an equal volume of a 1000-fold diluted solution of tobacco mosaic virus at room temperature for 5 hours to allow a full reaction. The same volume of water was used as a control. Each treatment was repeated 3 times. The number of dead spots was counted 3 days later, and the inhibition rate of chelerythrine on the activity of tobacco mosaic virus was calculated.
[0019] (3) In vivo treatment activity test The chelerythrine obtained by extraction in the embodiment is used to prepare chelerythrine solutions with concentrations of 0.01 mg / mL, 0.1 mg / mL, 0.15 mg / mL and 0.20 mg / mL respectively; consistent and healthy N.benthamiana leaves are selected and inoculated with a tobacco mosaic virus solution diluted 2000 times; after 2 days, the chelerythrine solutions with concentrations of 0.01 mg / mL, 0.1 mg / mL, 0.15 mg / mL and 0.20 mg / mL are sprayed respectively, and the same volume of water is sprayed as a control, each treatment is repeated 3 times, and the number of leaf spots is counted after 3 days to calculate the treatment effect of chelerythrine on the diseased tobacco leaves.
[0020]
[0021] The results are shown in Table 1. The chelerythrine prepared in the embodiment has a protection activity of up to 47.8±3.9 in vivo, a passivation activity of up to 49.2±2.4 in vitro, and a treatment activity of up to 44.5±4.3. It can be seen that chelerythrine has strong prevention and control efficacy on tobacco mosaic virus.
[0022] Table 1
[0023] Example 2: Extraction of dihydrochelerythrine The artificially cultivated Chelidonium majus plants are washed and placed in a forced air drying oven for drying. After being ground into fine powder by a grinder, the powder is added with methanol solution at a solid-liquid ratio of 1:10, and then soaked in an environment with a temperature of 25-40°C for 24 hours. The mixture after soaking is stirred and filtered to separate the solid residue and the extraction liquid. The extraction liquid is concentrated by a rotary evaporator, and then subjected to liquid-liquid extraction by using ethyl acetate. The organic phase and the aqueous phase are separated, and the organic phase is separated by silica gel column chromatography, eluted by chloroform, and then concentrated to dryness. After being dissolved in methanol again, dihydrochelerythrine extract (dihydrochelerythrine extraction sample as shown in Figure 3
[0024] Effect experiment In the embodiment, 1 g of tobacco mosaic virus infected leaves is ground into homogenate in a mortar with quartz sand, filtered and centrifuged. Polyethylene glycol is added to a final concentration of 4%, and then centrifuged. The precipitate is suspended in 0.2 mol / L phosphate buffer to obtain a crude tobacco mosaic virus extract. The crude tobacco mosaic virus extract is tested and calculated by ultraviolet spectrophotometry to have a virus mass concentration of 15.6 mg / L, and then diluted to 10 mg / L to obtain a tobacco mosaic virus solution.
[0025] (1) In vivo protection activity test The dihydrosanguinarine obtained by the extraction of the present embodiment is prepared into dihydrosanguinarine solutions with concentrations of 0.01 mg / mL, 0.08 mg / mL, 0.12 mg / mL and 0.16 mg / mL respectively; consistent and healthy N. benthamiana leaves are selected and sprayed with the dihydrosanguinarine solutions with different concentrations, and 48 hours later, a 2000-fold diluted tobacco mosaic virus solution is inoculated; the same amount of water is used as a control, each treatment is repeated 3 times, and the number of dry spots is counted after 3 days to calculate the preventive effect of dihydrosanguinarine on tobacco mosaic virus infection of N. benthamiana.
[0026] (2) In vitro passivation activity test The dihydrosanguinarine obtained by the extraction of the present embodiment is prepared into dihydrosanguinarine solutions with concentrations of 0.01 mg / mL, 0.08 mg / mL, 0.12 mg / mL and 0.16 mg / mL respectively; consistent and healthy N. benthamiana leaves are selected and sprayed with the dihydrosanguinarine solutions with different concentrations, and 48 hours later, a 2000-fold diluted tobacco mosaic virus solution is inoculated; the same amount of water is used as a control, each treatment is repeated 3 times, and the number of dry spots is counted after 3 days to calculate the preventive effect of dihydrosanguinarine on tobacco mosaic virus infection of N. benthamiana.
[0027] (3) In vivo treatment activity test The dihydrosanguinarine obtained by the extraction of the present embodiment is prepared into dihydrosanguinarine solutions with concentrations of 0.01 mg / mL, 0.08 mg / mL, 0.12 mg / mL and 0.16 mg / mL respectively; consistent and healthy N. benthamiana leaves are selected and sprayed with the dihydrosanguinarine solutions with different concentrations, and 48 hours later, a 2000-fold diluted tobacco mosaic virus solution is inoculated; the same amount of water is used as a control, each treatment is repeated 3 times, and the number of dry spots is counted after 3 days to calculate the preventive effect of dihydrosanguinarine on tobacco mosaic virus infection of N. benthamiana.
[0028]
[0029] The results are shown in Table 2. When the concentration of dihydrosanguinarine prepared by the present embodiment is 0.12 mg / mL, the in vivo protective activity is as high as 50.8±1.9, the in vitro passivation activity is as high as 49.3±3.7, and the treatment activity is as high as 58.4±4.8. It can be seen that dihydrosanguinarine has strong prevention and treatment effect on tobacco mosaic virus.
[0030] Table 2
[0031] Example 3: Preparation of the complex pesticide preparation and its application in the prevention and treatment of tobacco mosaic disease Using the chelerythrine obtained by extraction in Example 1, chelerythrine solutions with concentrations of 0.01 mg / mL, 0.1 mg / mL, 0.15 mg / mL and 0.20 mg / mL were prepared, respectively; using the dihydrochelerythrine obtained by extraction in Example 3, dihydrochelerythrine solutions with concentrations of 0.01 mg / mL, 0.08 mg / mL, 0.12 mg / mL and 0.16 mg / mL were prepared, respectively; the complex pesticide preparation was prepared according to the concentration ratio of the chelerythrine extract and the dihydrochelerythrine extract described in Table 3.
[0032] Effect experiment: In this example, 1 g of tobacco mosaic disease susceptible leaf was ground into homogenate in a mortar with quartz sand, filtered and centrifuged, polyethylene glycol was added to a final concentration of 4%, centrifuged, and 0.2 mol / L phosphate buffer was added to the precipitate to obtain a tobacco mosaic virus crude extract; the virus mass concentration in the virus crude extract was tested and calculated by ultraviolet spectrophotometry to be 15.6 mg / L, which was then diluted to 10 mg / L to obtain a tobacco mosaic virus solution.
[0033] (1) In vivo protection activity test The complex pesticide preparations with different concentration ratios prepared in this example according to Table 3 were sprayed on the healthy tobacco leaves of Nicotiana benthamiana with consistent growth, and 48 hours later, the leaves were inoculated with a tobacco mosaic virus solution diluted 2000 times, with the same volume of water being used as a control, each treatment was repeated 3 times, and the number of dead spots was counted after 3 days to calculate the prevention effect of the complex pesticide preparation on the infection of Nicotiana benthamiana with tobacco mosaic virus.
[0034] (2) In vitro inactivation activity test The complex pesticide preparations with different concentration ratios prepared in this example according to Table 3 were mixed with the same volume of a tobacco mosaic virus solution diluted 1000 times at room temperature for 5 hours to allow them to react fully; then, the same volume of water was used for the same treatment on the healthy tobacco leaves of Nicotiana benthamiana with consistent growth, each treatment was repeated 3 times, and the number of dead spots was counted after 3 days to calculate the prevention effect of the complex pesticide preparation on the infection of Nicotiana benthamiana with tobacco mosaic virus.
[0035] (3) In vivo treatment activity test Healthy tobacco leaves of Nicotiana benthamiana with consistent growth were selected and inoculated with a tobacco mosaic virus solution diluted 2000 times; 2 days after inoculation, the complex pesticide preparations with different concentration ratios prepared in this example according to Table 3 were sprayed on the inoculated tobacco leaves of Nicotiana benthamiana, with the same volume of water being used as a control, each treatment was repeated 3 times, and the number of dead spots was counted after 3 days to calculate the prevention effect of the complex pesticide preparation on the infection of Nicotiana benthamiana with tobacco mosaic virus.
[0036]
[0037] The results are shown in Table 3, the protection activity of the complex pesticide preparation prepared according to the concentration ratio of 0.15 mg / mL chelerythrine + 0.12 mg / mL dihydrochelerythrine is as high as 75.4±3.7 for living body, the passivation activity is as high as 72.6±4.9 for in vitro, and the treatment activity is as high as 73.5±5.3; the tobacco plant after applying the complex pesticide preparation is shown in Figure 4 It can be seen that the complex pesticide preparation has strong prevention and treatment effect on tobacco mosaic virus.
[0038] Table 3
[0039] The content not described in detail in the specification of the present application is the technology known to the person skilled in the art. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.
Claims
1. A compound pesticide formulation, characterized in that, The compound pesticide formulation contains chelidonine extract and dihydrochelidonine extract, and the concentration ratio of chelidonine extract and dihydrochelidonine extract in the compound pesticide formulation is (0.1-0.20 mg / mL): (0.08-0.16 mg / mL).
2. The compound pesticide formulation according to claim 1, characterized in that, The extraction steps of celandine are as follows: using artificially cultivated celandine plants as material, wash, dry, and pulverize into fine powder using a pulverizer and pass through a No. 3 sieve; add 95% ethanol at a material-to-liquid ratio of 1:8, and use ultrasonic extraction method with the ultrasonic frequency set and the extraction time set to 35 min to obtain the extract; filter the above extract, concentrate it using a rotary evaporator, separate and purify it using macroporous resin, set the conditions to an ambient temperature of 30℃, use 50% ethanol solution for elution treatment, concentrate, and dilute with anhydrous ethanol to obtain celandine extract.
3. The compound pesticide formulation according to claim 2, characterized in that, The ultrasonic frequency is set to 85%, and the elution flow rate is 1 mL / min.
4. The compound pesticide formulation according to claim 1, characterized in that, The extraction method of dihydrocelein is as follows: using artificially cultivated celebrine plants as material, wash, dry, and pulverize into fine powder using a pulverizer, add methanol solution at a material-to-liquid ratio of 1:10, place in an environment of 25-40℃ for 24 hours for extraction, stir, filter the mixture after soaking, and separate the solid residue and extract; concentrate the above extract using a rotary evaporator, perform liquid-liquid extraction using ethyl acetate, separate the organic phase and aqueous phase, separate the organic phase by silica gel column chromatography, elute with chloroform, concentrate to dryness, and then add methanol to redissolve to obtain dihydrocelein extract.
5. The application of the compound pesticide formulation according to any one of claims 1 to 4 in the prevention and control of tobacco mosaic disease.