Application of cinnamyl aldehyde oxime ester in prevention and treatment of bacterial wilt

Abstract

The invention relates to an application of cinnamyl aldehyde oxime ester in prevention and treatment of bacterial wilt, the structure of the cinnamyl aldehyde oxime ester is shown as a formula I, and the cinnamyl aldehyde oxime ester comprises cinnamyl aldehyde phenylpropyl oxime ester, cinnamyl aldehyde pepper oxime ester and cinnamyl aldehyde p-chloro cinnamyl aldehyde oxime ester. The invention provides application of the three compounds in prevention and treatment of mulberry bacterial wilt, the semi-inhibitory concentrations of the three compounds are 0.05 g / L, 0.14 g / L and 0.03 g / L respectively, and the three compounds have a remarkable bacteriostatic effect on ralstonia solanacearum at low concentration and are suitable for being used as a ralstonia solanacearum bacteriostatic agent. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the field of fine chemicals, and in particular to application of cinnamaldehyde oxime ester in preventing and treating bacterial wilt. Background Art

[0002] Bacterial wilt is one of the top ten bacterial diseases that are most harmful to plants in the world. Ralstonia solanacearum is the pathogen of bacterial wilt, which is distributed in tropical, subtropical and temperate regions around the world and can spread through the soil to infect more than 50 families of plants. Mulberry bacterial wilt is commonly known as "mulberry plague". The occurrence of mulberry bacterial wilt has seriously restricted the sustainable development of the sericulture industry.

[0003] The characteristics of Ralstonia solanacearum, such as rich genetics, complex pathogenic pathways and rapid disease spread, make it difficult to control Ralstonia solanacearum. At present, the main control methods of mulberry bacterial wilt are chemical pesticide control, biotechnology control and plant-based control.

[0004] Chemical pesticide control method: Applying 300 times diluted 72% agricultural streptomycin to irrigate the roots of mulberry seedlings and using 1000 times diluted sinoprotinin to disinfect the soil can prevent mulberry bacterial wilt (Northern Sericulture, 2018, 39(01):40-43); the control effect of Longke bacteria on bacterial wilt is 70% (Guangxi Plant Protection, 2012, 25(1):28-29). These chemical pesticides are highly toxic, easily cause environmental pollution, and long-term use will produce drug resistance.

[0005] Biotechnology control method: The "Anti-Qing No. 10" bred by Mao Kengzu et al. (China Sericulture, 1996, 17(4):19-20) has high resistance to mulberry wilt, but the quality of mulberry leaves is poor, and the yield is 10% to 30% lower than that of similar non-resistant varieties. Wang Jicheng et al. (Science of Sericulture, 2020, 46(1):31-36) isolated a Pseudomonas aeruginosa (Pseudomonas aeruginosa YD001) from mulberry root endophytes. Potted plant experiments showed that the control effect of biological control methods was 71.4%. The control effect of biological control methods is affected by environmental factors such as different soils, climates, and hosts, and the control efficiency in the field is poor.

[0006] Plant-derived substances: Patent CN105191940 reported that the inhibition rate of 0.15g / L to 2g / L chitosan derivatives against Ralstonia solanacearum was 47% to 81%, and the antibacterial effect was not high. Patent CN104365598 reported that the inhibition rate of 5g / L to 7g / L caffeic acid alkyl esters against Ralstonia solanacearum was 71% to 85%, but the dosage of the drug was large and the cost was high.

[0007] Cinnamaldehyde is a plant metabolite that has been confirmed as a safe product by the U.S. Food and Drug Administration and the Association of Flavor and Extract Manufacturers. Cinnamaldehyde has a broad-spectrum antibacterial activity and shows strong inhibitory activity against the growth of Escherichia coli, Salmonella, and Staphylococcus aureus. The aldehyde group is an important modification group of cinnamaldehyde. The aldehyde group reacts with ammonia to form an imine structure to generate oxime. This type of compound is easily degraded in nature, is safe, low-toxic, and has little harm to the environment. At present, there is no report on the use of cinnamaldehyde oxime esters for the prevention and treatment of bacterial wilt. Summary of the invention

[0008] The technical problem to be solved by the present invention is the shortcomings of the above-mentioned existing methods for preventing and controlling bacterial wilt, specifically the problems of great environmental harm caused by chemical pesticide methods, unstable effects of biotechnology methods and high drug concentrations used in plant-derived substance methods.

[0009] The present invention provides a new use of cinnamaldehyde oxime ester in the prevention and treatment of bacterial wilt. Cinnamaldehyde ester uses plant-derived compound cinnamaldehyde as a raw material to synthesize a novel cinnamaldehyde ester, which can effectively inhibit bacterial wilt at a low concentration. The structure of the cinnamaldehyde ester is shown in Formula I:

[0010]

[0011] The synthesis conditions of the cinnamaldehyde oxime ester are as follows: 1-2g of cinnamaldehyde, 0.5-1.5g of hydroxylamine hydrochloride and 1-2g of sodium carbonate are added to 10-100mL of ethanol, reacted at 40-60°C for 1-6h, poured into 10-50mL of ice water, filtered, and the filter cake, 1-2g of potassium carbonate and 1-2g of acyl chloride compound (phenylpropionyl chloride, piperonyl chloride or p-chlorocinnamoyl chloride) are added to 10-50mL of dichloromethane, and reacted at 20-60°C for 1-6h.

[0012] The bacterial wilt pathogen is Ralstonia solanacearum MRS-5, and its Taxonomy ID is 1310165.

[0013] When the cinnamaldehyde oxime ester of the present invention is used for preventing and controlling bacterial wilt, the ethanol solution of the compound of formula I with a concentration of 0.02-0.32 g / L is sprayed, and the number of times of use per day is 1-5 times.

[0014] The present invention has the following beneficial effects:

[0015] 1) Cinnamaldehyde oxime was used for the first time in the prevention and treatment of bacterial wilt. The synthesis process is simple, the equipment requirements are low, and it is suitable for large-scale operation.

[0016] 2) The semi-inhibitory concentration of cinnamaldehyde oxime ester is 0.03-0.14 g / L. The inhibition rate of Ralstonia solanacearum is high at low concentrations, and the antibacterial effect is significant. It is suitable as a Ralstonia solanacearum antibacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The following are the synthetic routes of cinnamaldehyde oxime esters described in the three embodiments;

[0018] Figure 2 1H-NMR diagram of cinnamaldehyde phenylpropionoxime ester;

[0019] Figure 3 is the 13C-NMR diagram of cinnamaldehyde phenylpropionoxime ester;

[0020] Figure 4 1H-NMR diagram of cinnamaldehyde piperonyl oxime ester;

[0021] Figure 5 is the 13C-NMR diagram of cinnamaldehyde piperonyl oxime ester;

[0022] Figure 6 is the 1H-NMR diagram of cinnamaldehyde p-chlorocinnamoxime ester;

[0023] Figure 7 This is the 13C-NMR diagram of cinnamaldehyde p-chlorocinnamaldehyde oxime ester. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0025] Example 1

[0026] Synthesis of cinnamaldehyde phenylpropionyl oxime ester: According to Figure 1 The synthetic route is as follows: 1.35 g of cinnamaldehyde is put into a three-necked flask, 50 mL of ethanol is added and stirred to dissolve, 1.21 g of sodium carbonate and 0.78 g of hydroxylamine hydrochloride are added, and the reaction is carried out at 60°C for 2 h. The reactant is poured into 15 mL of ice water and filtered. The filter cake and 1.87 g of potassium carbonate are put into a three-necked flask, 20 mL of dichloromethane is added, and the mixture is stirred in an ice bath for 20 min. Then 1.51 g of phenylpropionyl chloride is slowly added, and the reaction is carried out at room temperature for 4 h. The mixture is filtered and the filtrate is concentrated and dried at 50°C for 8 h to obtain 1.48 g of cinnamaldehyde phenylpropionyl oxime ester with a yield of 52%.

[0027] The structure of the obtained cinnamaldehyde phenylpropionyl oxime ester was characterized by hydrogen spectrum, such as Figure 2 Shown: 1H-NMR, δ8.11-8.08 (m, 1H), 7.48-7.45 (m, 2H), 7.38-7.35 (m, 3H), 7.31-7.28 (m, 2H), 7.24-7.19 (m, 3H), 6.99-6.97 (m, 2H), 3.04 (t, J = 8 Hz, 2H), 6.36 (t, J = 8 Hz, 2H).

[0028] Cinnamaldehyde phenylpropionyl oxime ester is characterized by carbon spectrum, such as Figure 3Shown: 13C-NMR, δ 170.54, 157.39, 143.82, 135.21, 131.35, 129.99, 129.08, 128.71, 128.47, 127.53, 126.53, 120.36, 34.65, 30.90.

[0029] Example 2

[0030] Synthesis of cinnamaldehyde piperonyl oxime ester: According to Figure 1 The synthetic route is as follows: 1.24 g of cinnamaldehyde is put into a three-necked flask, 30 mL of ethanol is added and stirred to dissolve, 1.13 g of sodium carbonate and 0.78 g of hydroxylamine hydrochloride are added, and the reaction is carried out at 40°C for 3 h. The reactant is poured into 30 mL of ice water and filtered. The filter cake and 1.65 g of potassium carbonate are put into a three-necked flask, 40 mL of dichloromethane is added, and the mixture is stirred in an ice bath for 40 min. Then 1.66 g of piperic acid chloride is slowly added, and the mixture is reacted at room temperature for 6 h. The mixture is filtered, and the filtrate is concentrated and dried at 40°C for 12 h to obtain 1.39 g of cinnamaldehyde piperonyl oxime ester with a yield of 47%.

[0031] The structure of the obtained cinnamaldehyde piperonyl oxime ester was characterized by hydrogen spectrum, such as Figure 4 Shown: 1H-NMR, δ8.31-8.29 (m, 1H), 7.74-7.71 (m, 1H), 7.53-7.48 (m, 3H), 7.41-7.37 (m, 3H), 7.08-7.05 (m, 2H), 6.87 (d, J = 8.4 Hz, 1H), 6.06 (s, 2H).

[0032] The obtained cinnamaldehyde piperonyl oxime ester was characterized by carbon spectrum, such as Figure 5 Shown: 13C-NMR, δ 163.51, 157.68, 152.20, 147.99, 143.74, 135.27, 129.98, 129.09, 127.56, 125.85, 122.32, 120.52, 109.67, 108.30, 102.06.

[0033] Example 3

[0034] Synthesis of cinnamaldehyde p-chlorocinnamoyl oxime ester: According to Figure 1The synthetic route is as follows: 1.58 g of cinnamaldehyde is put into a three-necked flask, 70 mL of ethanol is added and stirred to dissolve, 1.48 g of sodium carbonate and 0.86 g of hydroxylamine hydrochloride are added, and the reaction is carried out at 50°C for 3 h. The reactant is poured into 40 mL of ice water and filtered. The filter cake and 1.95 g of potassium carbonate are put into a three-necked flask, 60 mL of dichloromethane is added, and the mixture is stirred in an ice bath for 40 min. Then 1.81 g of p-chlorocinnamic acid chloride is slowly added, and the reaction is carried out at room temperature for 2 h. The mixture is filtered and the filtrate is concentrated and dried at 50°C for 6 h to obtain 1.51 g of p-chlorocinnamic acid oxime ester of cinnamaldehyde in a yield of 48%.

[0035] The structure of the obtained cinnamaldehyde p-chlorocinnamoyl oxime ester was characterized by hydrogen spectrum, such as Figure 6 1H-NMR shown, δ 8.22-8.20 (m, 1H), 7.79 (d, J = 16 Hz, 1H), 7.48-7.46 (m, 4H), 7.38-7.35 (m, 5H), 7.04-7.02 (m, 2H), 6.48 (d, J = 16 Hz, 1H).

[0036] The obtained cinnamaldehyde p-chlorocinnamoyl oxime ester was characterized by carbon spectrum, such as Figure 7 Shown: 13C-NMR, δ 164.59, 157.60, 145.20, 143.94, 136.77, 135.22, 132.75, 130.02, 129.54, 129.38, 129.10, 127.57, 120.40, 115.90.

[0037] Experiment 1 for determining the inhibition rate of cinnamaldehyde oxime ester on mulberry wilt pathogens:

[0038] Using the spectrophotometer method, 0.04 mL of Ralstonia solanacearum suspension, 0.14 mL of CPG liquid culture medium, and 0.02 mL of 0.2-3.2 g / L cinnamaldehyde oxime ester ethanol solution were added to a 96-well plate, respectively, and cultured at 30°C and 200 rpm. The blank control was an equal amount of ethanol, and the positive control was an equal amount of Longke bacteria. The OD of the culture solution at 0 h and 24 h was measured respectively. 600 The inhibition rate was calculated according to Formula II, and then the Dose Response model was used to fit the inhibition rate data to calculate the half inhibition concentration.

[0039]

[0040] Where T0 Sample and TF Sample Respectively represent the OD before and after adding compound solution bacterial suspension culture 600 Value reading, T0 Blank and TF Blank Respectively represent the OD before and after adding solvent bacterial suspension culture 600 Value reading.

[0041] The results showed that at 0.02-0.32 g / L, the inhibition rates of cinnamaldehyde phenylpropionyl oxime ester, cinnamaldehyde piperonyl oxime ester, and cinnamaldehyde against chlorocinnamoxime ester and Roncosporin were 33.5%-91.1%, 14.2%-70.4%, 42.6%-97.3%, and 6.7%-86.1%, respectively. Their semi-inhibitory concentrations were 0.05 g / L, 0.14 g / L, 0.03 g / L, and 0.09 g / L, respectively. This indicated that the three cinnamaldehyde phenylpropionyl oxime esters all had significant antibacterial effects against Ralstonia solanacearum, and the semi-inhibitory concentrations of cinnamaldehyde phenylpropionyl oxime ester and cinnamaldehyde against chlorocinnamoxime ester were lower than those of Roncosporin.

[0042] Experiment 2 on the inhibitory effect of cinnamaldehyde oxime on the pathogen of mulberry wilt:

[0043] Using the plate confrontation method, 0.2 mL of a 10-fold diluted Ralstonia solanacearum suspension was spread on a CPG solid culture medium (hydrolyzed casein: glucose: tryptone: agar powder: water mass ratio of 0.2:1:2:6.8:200), and three small holes were punched in the culture medium with a hole puncher. 0.03 mL of 10 g / L cinnamaldehyde oxime ester ethanol solution was added to the holes. After culturing at 30 ° C for 24 hours, the bacterial growth in the plate was observed. The blank control was an equal amount of ethanol, and the positive control was an equal amount of Ralstonia longica. If there is an antibacterial zone near the small hole, it means that it has an antibacterial effect, and if there is no antibacterial zone, it means that it has no antibacterial effect.

[0044] The results showed that cinnamaldehyde phenylpropionate oxime ester, cinnamaldehyde piperonyl oxime ester and cinnamaldehyde all produced inhibition zones against chlorocinnamaldehyde oxime ester and Longke bacteria, and the sizes of their inhibition zones were 0.8cm, 0.3cm, 1.0cm and 0.5cm, respectively. The antibacterial activity of cinnamaldehyde phenylpropionate oxime ester and cinnamaldehyde against chlorocinnamaldehyde oxime ester was better than that of Longke bacteria.

[0045] Experiment on the control effect of cinnamaldehyde oxime on mulberry wilt:

[0046] The infected mulberry seedlings inoculated with Ralstonia solanacearum were used as the test objects. 0.02-0.32 g / L cinnamaldehyde oxime ester ethanol solution was sprayed on the diseased mulberry seedlings 3 times a day. The growth of the diseased mulberry seedlings was observed after 7 days. The blank control was an equal amount of ethanol, and the positive control was an equal amount of Longke bacteria. The control rate was the ratio of cured mulberry seedlings to the total diseased mulberry seedlings.

[0047] The results showed that at 0.02-0.32 g / L, the control rates of cinnamaldehyde phenylpropionyl oxime ester, cinnamaldehyde piperonyl oxime ester and cinnamaldehyde against chlorocinnamoxime ester and Loncodium were 12.5%-81.3%, 6.3%-56.3%, 37.5%-87.5% and 6.3%-68.8%, respectively. Among them, when the concentration of cinnamaldehyde against chlorocinnamoxime ester was higher than 0.08 g / L, the control rate exceeded 75%, and the control effect was significant at lower concentrations.

Claims

1. Application of cinnamaldehyde oxime ester in the prevention and treatment of bacterial wilt, wherein the chemical structure of the cinnamaldehyde oxime ester is shown in Formula I:

2. The use of cinnamaldehyde oxime ester in the prevention and treatment of bacterial wilt according to claim 1, characterized in that The synthesis conditions of cinnamaldehyde oxime ester are as follows: add 1-2g cinnamaldehyde, 0.5-1.5g hydroxylamine hydrochloride and 1-2g sodium carbonate to 10-100mL ethanol, react at 40-60°C for 1-6h, pour into 10-50mL ice water, filter, add the filter cake, 1-2g potassium carbonate and 1-2g acyl chloride compound to 10-50mL dichloromethane, and react at 20-60°C for 1-6h.

3. The use of cinnamaldehyde oxime ester in the prevention and treatment of bacterial wilt according to claim 2, characterized in that The acyl chloride compound is selected from phenylpropionyl chloride, piperonyl chloride or p-chlorocinnamoyl chloride.

4. The use of cinnamaldehyde oxime ester according to claim 1, characterized in that When the cinnamaldehyde oxime ester is used to prevent and treat bacterial wilt, the cinnamaldehyde oxime ester is mixed with ethanol to prepare a solution with a concentration of 0.02-0.32 g / L for spraying, and the number of times of use per day is 1-5 times.

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