Application of sanguinarine and simplified derivative thereof in prevention and treatment of plant pathogenic bacteria

Abstract

The invention discloses application of sanguinarine and simplified derivatives thereof in prevention and treatment of plant pathogenic bacteria, and relates to the technical field of medicinal chemistry. The sanguinarine and the simplified derivative thereof have a good inhibition effect on plant pathogenic bacteria such as rice bacterial blight pathogenic bacteria, citrus ulcer pathogenic bacteria and pseudomonas solanacearum pathogenic bacteria, the compound involved in the invention is low in preparation difficulty, the raw materials are cheap and easy to obtain, and the compound is expected to be developed into a novel antibacterial agent.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to the use of sanguinarine and its simplified derivatives in the control of plant pathogenic bacteria. Background Technology

[0002] Microbial infections are a major and complex health problem. Nearly 15 million people die annually from bacterial infections due to the emergence of new infectious diseases and the increasing number of drug-resistant pathogens, many of which are drug-resistant. While commercial fungicides have made significant contributions to agriculture and pharmaceutical treatment over the past few decades, microbial infections continue to limit global agricultural production and increase morbidity in humans and animals. This is due to the low efficiency of existing commercial fungicides against invasive pathogens and the emergence of multidrug-resistant microorganisms caused by various factors, such as the long-term use of traditional fungicides with single modes of action. Therefore, actively discovering innovative fungicidal chemical entities with unique mechanisms of action, simple structures, and high efficiency can provide better alternatives to existing drugs for treating plant bacteria or human pathogens.

[0003] Sanguisorbine ( Sanguinarine Sanguisorbin is a benzophenanthrene alkaloid extracted from poppy plants (Papaveraceae family) and possesses a wide range of biological activities. As early as the 19th century, sanguisorbin was discovered and used in traditional medicine, primarily for its antibacterial, anti-inflammatory, and analgesic effects. In recent years, with the deepening of natural product research, sanguisorbin, due to its unique chemical structure and diverse pharmacological effects, has gradually become a research hotspot in medicinal chemistry, pharmacology, and biomedicine. Despite its broad application prospects, the synthesis and extraction processes of sanguisorbin still face some challenges.

[0004] Therefore, we simplified the structure of sanguinarine, designed and synthesized a series of simplified derivative structures of sanguinarine, and tested their activity against plant pathogenic bacteria and human pathogens. The test results showed that these compounds exhibited certain inhibitory activity against agricultural bacteria such as *Rhizoctonia solani*, as well as human pathogens such as *Staphylococcus aureus*. They showed superior inhibitory effects against *Rhizoctonia solani*, *Citrus canker*, and human pathogens such as *Escherichia coli* and *Staphylococcus aureus*, significantly better than the control drug, and hold promise for development into a novel antibacterial drug. Summary of the Invention

[0005] To address the above shortcomings, this invention provides the use of sanguisorbin and its simplified derivatives in the control of plant pathogenic bacteria. Sanguisorbin and its simplified derivatives are particularly useful for controlling the pathogen of rice bacterial blight (…). Xanthomonas oryzae PXO99A), citrus canker pathogen (PXO99A), Xanthomonas axonopodis jx-6) and Ralstonia solanacearum (JX ... Pseudomonas solanacearum Smith This treatment also shows good inhibitory effects on agricultural diseases caused by BNCC335855, as well as on Escherichia coli ATCC25922, Staphylococcus aureus Newman, and Candida albicans ATCC24433. The specific technical solution is as follows: The use of sanguisorbin and its simplified derivative in the control of plant pathogenic bacteria, wherein the chemical structural formulas of sanguisorbin and its simplified derivative are shown below: .

[0006] Preferably, the plant pathogenic bacteria include *Rhizoctonia solani*, *Rhizoctonia solani*, and *Rhizoctonia solani*. The dosage concentrations of sanguinarine and its simplified derivatives for treating plant pathogenic bacteria are 100, 50, 25, 12.5, 6.25, and 3.12 μg / mL.

[0007] The present invention also provides a fungicide for preventing and controlling plant pathogenic bacteria, the fungicide containing the above-mentioned sanguisorbin and its simplified derivatives.

[0008] Preferably, the fungicide further includes adjuvants and / or agronomically acceptable carriers.

[0009] Preferably, the additive is selected from one or more of dispersants, wetting agents, fillers, solvents, emulsifiers, thickeners, disintegrants, solvents, preservatives, antifreeze agents, defoamers, and water.

[0010] Preferably, the bactericide is formulated as one or more of the following: oil suspension, water dispersible granules, wettable powder, suspension, dry suspension, and emulsifiable concentrate.

[0011] Preferably, the plant pathogenic bacteria include rice bacterial blight pathogen, citrus canker pathogen, and bacterial wilt pathogen.

[0012] Compared with the prior art, the beneficial effects of the present invention are: The sanguisorbin and its simplified derivatives XD1~XD11 of this invention exhibit good inhibitory effects against rice bacterial blight pathogens, citrus canker pathogens, and *Ralstonia solanacearum* pathogens, and also show some inhibitory effects against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*. Furthermore, the sanguisorbin and its simplified derivatives of this invention are easy to prepare, and the raw materials are inexpensive and readily available, making them a promising candidate for development as a novel antibacterial agent. Detailed Implementation

[0013] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0014] Example 1: Preparation of sanguinarine and its simplified derivatives The literature report was adopted. European Journal of Medicinal Chemistry Sanguisorbine and its simplified derivatives were synthesized using method 224 (2021)113723. Pure products were obtained by repeated silica gel column chromatography and other conventional methods. The structures of sanguisorbine and its simplified derivatives XD1~XD11 were determined by mass spectrometry and nuclear magnetic resonance spectroscopy, and their structural formulas are shown below: .

[0015] Example 2: Determination of the anti-agricultural pathogenic bacteria activity of quinine and its simplified derivatives and the results. 1) Test reagents: Sanguisorbine and its simplified derivatives XD1~XD11 prepared in Example 1.

[0016] 2) Test strain: Rice bacterial blight pathogen ( Xanthomonas oryzae PXO99A), citrus canker pathogen (PXO99A), Xanthomonas axonopodis jx-6) and Ralstonia solanacearum (JX ... Pseudomonas solanacearum Smith BNCC335855).

[0017] 3) Antibacterial activity test: The bacterial strains used in this experiment were cryopreserved in the laboratory at -80℃ with 30% glycerol. The cryopreserved strains were removed and streaked onto NA solid medium (beef extract: 3 g, peptone: 5 g, yeast extract: 1 g, sucrose: 10 g, agar: 15 g, distilled water: 1 L, pH 7.0; sterilized at 121℃ for 20 min), and incubated at 28℃ until single colonies appeared. Single colonies from the solid medium were transferred to NB liquid medium (beef extract: 3 g, peptone: 5 g, yeast extract: 1 g, sucrose: 10 g, distilled water: 1 L; sterilized at 121℃ for 20 min), and cultured on a shaker at 28℃ and 180 rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted with NB liquid medium to approximately 10... 6 CFU / mL was prepared for use. The compounds were dissolved separately in DMSO, added to liquid culture medium, and mixed thoroughly to prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. 50 μL of the drug-containing culture medium and the same volume of approximately 10... 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, resulting in a final drug concentration of 100 μg / mL. A control of 100 μL of bacterial culture containing an equal amount of DMSO was used. The 96-well plates were incubated at 28℃ for 24–48 h until bacterial growth was observed in the control group. The OD value (OD) of the bacterial culture in each well was measured using a microplate reader. 600 In addition, the OD values ​​of 100 μL of liquid culture medium and a 100 μg / mL drug concentration were measured to correct for the OD values ​​caused by the culture medium and the drug itself. The formulas for calculating the corrected OD value and inhibition rate are as follows: Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium; Inhibition rate = (OD value of bacterial suspension in control medium after correction - OD value of bacterial suspension in drug-containing medium after correction) / OD value of bacterial suspension in control medium after correction × 100% The drug-containing liquid culture medium was diluted in 96-well plates using a two-fold dilution method to obtain a series of 50 μL drug-containing media. The inhibition rates corresponding to these concentrations were then determined using the same experimental method described above. All experiments were performed in triplicate, and the determined inhibition rates of the compound are shown in Table 1.

[0018] Table 1. Antibacterial activity of sanguinarine and its simplified derivatives XD1~XD11 at 100 μg / mL As shown in Table 1, the sanguinarine and its simplified derivatives XD1~XD11 prepared in this invention exhibited different degrees of inhibitory activity against three plant pathogenic bacteria. Among them, compounds XD10 and XD11 showed excellent antibacterial activity against pathogenic bacteria, with inhibition rates of more than 90% against the test strains at 100 μg / mL.

[0019] Example 3: Determination of the minimum inhibitory concentration (MIC) of sanguinarine and its simplified derivatives The drug-containing liquid culture medium of the active compound was diluted in 96-well plates using a two-fold dilution method to obtain a series of 50 μL drug-containing culture media of various concentrations. Then, the inhibition rate corresponding to the series of concentrations was determined according to the same test method as in Example 2. The lowest concentration with an inhibition rate greater than 90% was defined as the MIC. The measured activity data are shown in Table 2.

[0020] Table 2. MIC values ​​of sanguinarine and its simplified derivatives As shown in Table 2, the bioassay results indicate that sanguinarine and its simplified derivatives all exhibited certain antibacterial activity against the tested strains. Among them, the effects against plant pathogens such as rice bacterial blight pathogen and citrus canker pathogen were the strongest. Compound XD11 had the lowest MIC value against rice bacterial blight pathogen, reaching 1.56 μg / mL.

[0021] Example 4: 20% Compound XD1 Oil Suspension Compound XD1 20%, tristyrene-phenylphenol polyoxyethylene ether 6% (emulsifier), calcium dodecylbenzenesulfonate 2% (emulsifier), lignin sulfonate 3% (dispersant), silica 0.5% (thickener), and methyl oleate to make up to 100%.

[0022] Preparation method: Weigh all raw materials according to the above formula weight ratio, then add compound XD1 and components such as dispersant, emulsifier, and thickener into a sand mill and grind for a certain time to obtain compound XD1 dispersible oil suspension.

[0023] Example 5: 50% Compound XD2 Water Dispersible Granules Compound XD2 50%, high molecular weight polycarboxylate 5% (dispersant), sodium dodecylbenzenesulfonate 2% (emulsifier), starch 4% (binder), sodium sulfate 3% (disintegrant), silica 1% (disintegrant), kaolin to make up to 100%.

[0024] The preparation method is as follows: the above raw materials are pulverized by air jet milling, granulation, drying and sieving in proportion to prepare water-dispersible granule pesticide.

[0025] Example 6: 35% Compound XD4 Water Dispersible Granules Compound XD4 35%, sodium alkylnaphthalene sulfonate 3% (dispersant), sodium methylene dinaphthalene sulfonate 1.4% (dispersant), sodium dodecylbenzene sulfonate 4% (emulsifier), starch 4% (binder), sodium bicarbonate 2% (disintegrant), silica 1% (disintegrant), bentonite to make up to 100%.

[0026] The preparation method is the same as in Example 5.

[0027] Example 7: 36% Compound XD5 Wettable Powder Compound XD5 36%, alkylnaphthalene sulfonate formaldehyde condensate 3.8% (dispersant), lignin sulfonate 1% (dispersant), sodium dodecylbenzene sulfonate 3.5% (emulsifier), bentonite to make up to 100%.

[0028] The preparation method is as follows: the above raw materials are air-jet pulverized and stirred evenly in proportion to obtain wettable powder pesticide.

[0029] Example 8: 60% Compound XD8 Wettable Powder Compound XD8 60%, sodium alkylnaphthalene sulfonate 3% (dispersant), sodium dodecylbenzene sulfonate 4% (emulsifier), starch 4% (binder), sodium bicarbonate 2% (disintegrant), silica 1% (disintegrant), bentonite to make up to 100%.

[0030] The preparation method is the same as in Example 7.

[0031] Example 9: 30% Compound XD10 Suspension Compound XD10 30%, alkyl alcohol ether phosphate 4.6% (emulsifier), block polyether 2.3% (emulsifier), polycarboxylate 3% (dispersant), ethylene glycol 2%, xanthan gum 0.2% (thickener), magnesium aluminum silicate 0.25% (thickener), organosilicon 0.5% (defoamer), water to 100%.

[0032] The preparation method of the suspension pesticide is as follows: the above raw materials are subjected to high-speed shearing in proportion, and then added to a sand mill for grinding for 2-3 hours to obtain the suspension pesticide formulation.

[0033] Example 10: 40% Compound XD11 Suspension Compound XD11 40%, alkylphenol formaldehyde resin polyoxyethylene ether 5% (emulsifier), alkyl alcohol ether phosphate 2% (emulsifier), polyurethane 3% (dispersant), glycerol 2%, xanthan gum 0.1% (thickener), magnesium aluminum silicate 0.35% (thickener), organosilicon 0.5% (defoamer), water to 100%.

[0034] The preparation method is the same as in Example 9. Example 11 26% Compound XD11 Emulsifiable Concentrate Compound XD11 26%, castor oil polyoxyethylene ether 6% (emulsifier), calcium dodecylbenzenesulfonate 3% (emulsifier), dimethylformamide 10%, xylene to make up to 100%.

[0035] Preparation method: Weigh the above raw materials according to the proportions, and then mix and stir them in a mixing tank to obtain a transparent and uniform liquid, which is the emulsifiable concentrate product of the present invention.

[0036] Field trials for the control of citrus canker Test reagents: Example 4: 20% compound XD1 oil suspension; Example 5: 50% compound XD2 water dispersible granules; Example 6: 35% compound XD4 water dispersible granules; Example 7: 36% compound XD5 wettable powder; Example 8: 60% compound XD8 wettable powder; Example 9: 30% compound XD10 suspension; Example 10: 40% compound XD11 suspension.

[0037] Control agent: 20% thiabendazole copper suspension, registration certificate PD20086024, Zhejiang Longwan Chemical Co., Ltd., purchased from the market.

[0038] Blank control: Water Experimental site conditions and citrus varieties: The experimental site is located in Ningming County, Chongzuo City, Guangxi Province, with poor soil fertility and low terrain. The tested variety is Wogan mandarin orange, with a tree age of 4 years and average tree vigor.

[0039] Experimental Design and Methods: Application of pesticides was carried out during the spring shoot stage, focusing on the leaves and ensuring thorough spraying on both the upper and lower surfaces. The experiment consisted of 10 treatment plots, each randomly assigned to a block design. Each plot contained 15 Wogan mandarin orange trees, with three replicates. A conventional electric sprayer (16 kg capacity) was used. The dosage of each pesticide is shown in Table 3. Two applications were made on March 2nd and March 22nd, 2024, during mostly cloudy or overcast weather. The control group (water) received no pesticide treatment for canker.

[0040] Investigation Methods: The occurrence of leaf canker was observed and recorded on days 5 and 10 after each spraying. The control efficacy was investigated 10 days after the last spraying, and relevant data were recorded. A diagonal sampling method was used, with five fixed-point surveys. One citrus tree was surveyed at each point, and samples were taken from each tree at five points (east, south, west, north, and center). One branch was surveyed at each point, and five leaves were examined on each branch. The number of diseased leaves at each level was recorded.

[0041] The severity of citrus canker is classified according to the number of lesions on a single leaf, and the grading standards are as follows: Level 0: No disease; Grade 1: 1-5 lesions per leaf; Grade 3: 6-10 lesions per leaf; Grade 5: 11-15 lesions per leaf; Grade 7: 16-20 lesions per leaf; Grade 9: More than 21 lesions per leaf.

[0042] Calculate the disease index and relative efficacy using the following formulas: Disease index (%) = [∑(number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value)] × 100.

[0043] Relative efficacy (%) = (Disease index after treatment in the control area - Disease index after treatment in the treatment area) / Disease index after treatment in the control area × 100. Results are shown in Table 3. Table 3 Results of field efficacy trials for controlling citrus canker As shown in Table 3 above, the agents prepared in Examples 4 to 10 showed 12.95%-40% higher efficacy in controlling citrus canker than the 20% thiabendazole copper suspension. Among them, the 40% compound XD11 suspension (2000 times dilution) and the 30% compound XD10 suspension (1500 times dilution) performed best, with relative efficacy of over 90% in controlling citrus canker. This indicates that when the dosage of the simplified sanguinarine derivative of this invention is less than that of the control agent, the antibacterial activity of most compounds is higher than that of the control drug thiabendazole copper. However, due to long-term use, thiabendazole copper has developed certain resistance. To achieve a certain control effect in controlling citrus canker, the dosage must be increased.

[0044] In summary, the simplified sanguinarine derivative of this invention exhibits good inhibitory effects against three plant pathogenic bacteria: rice bacterial blight pathogen, citrus canker pathogen, and Ralstonia solanacearum pathogen. Furthermore, this compound is easy to prepare, and the raw materials are inexpensive and readily available, making it a promising candidate for development as a novel antibacterial agent.

[0045] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The use of sanguinarine and its simplified derivative in the control of plant pathogenic bacteria, characterized in that, The chemical structural formulas of sanguinarine and its simplified derivatives are shown below: 。 2. The use of sanguinarine and its simplified derivative in the control of plant pathogenic bacteria according to claim 1, characterized in that, The plant pathogenic bacteria include rice bacterial blight pathogen, citrus canker pathogen, and bacterial wilt pathogen.

3. A fungicide for controlling plant pathogenic bacteria, characterized in that, The bactericide contains sanguinarine and its simplified derivatives as described in claim 1.

4. The fungicide for controlling plant pathogenic bacteria according to claim 3, characterized in that, The fungicide also includes adjuvants and / or agronomically acceptable carriers.

5. The fungicide for controlling plant pathogenic bacteria according to claim 4, characterized in that, The additives are selected from one or more of the following: dispersants, wetting agents, fillers, solvents, emulsifiers, thickeners, disintegrants, solvents, preservatives, antifreeze agents, defoamers, and water.

6. The fungicide for controlling plant pathogenic bacteria according to claim 3, characterized in that, The bactericide is formulated as one or more of the following: oil suspension, water dispersible granules, wettable powder, suspension, dry suspension, and emulsifiable concentrate.

7. The fungicide for controlling plant pathogenic bacteria according to claim 3, characterized in that, The plant pathogenic bacteria include rice bacterial blight pathogen, citrus canker pathogen, and bacterial wilt pathogen.

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