Application of cinnamic acid terpene ester derivative in prevention and treatment of bacterial wilt

Abstract

The invention relates to application of a terpene cinnamate derivative in prevention and treatment of bacterial wilt. The terpene cinnamate derivative comprises geranyl cinnamate, neryl cinnamate, menthyl cinnamate, bornyl cinnamate, carvyl cinnamate, terpinyl cinnamate and linalyl cinnamate. The semi-inhibitory concentration of the seven compounds is below 0.16 g / L, and the seven compounds have a remarkable inhibitory effect on ralstonia solanacearum at low concentration and are suitable for being used as a ralstonia solanacearum bacteriostatic agent.

Description

Technical Field

[0001] The invention relates to the field of plant protection, and in particular to application of cinnamic acid terpene ester derivatives 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. Ralstonia solanacearum is the pathogen of bacterial wilt, which is found in tropical, subtropical and temperate regions around the world. It can spread through the soil and infect more than 50 families of plants, including common economic crops: mulberry and peanuts in the dicotyledonous family, bananas and corn in the monocotyledonous family, and tobacco and potatoes in the Solanaceae family. The occurrence of plant bacterial wilt has seriously restricted the sustainable development of the relevant agricultural economy.

[0003] The fungus invades the host through root wounds or the crown of secondary roots, propagates and spreads through the xylem, destroys the plant vascular system, and causes the plant to wilt rapidly until it dies. The main methods for preventing and controlling bacterial wilt are chemical control and biological control. Chemical control mainly uses chemical pesticides, which have significant control effects but have disadvantages such as high toxicity and environmental pollution. It is reported that the control effect of 50% of solanacearum on solanum bacterial wilt is 73% (Tropical Agricultural Science, 2000, (04): 20-22). And the control effect of 20% of longke bacteria on mulberry bacterial wilt is 70% (Guangxi Plant Protection, 2012, 25 (1): 28-29). Biological control includes biological control bacteria and biological antibacterial control. Wang Jicheng et al. (Science of Sericulture, 2020, 46(1):31-36) isolated a Pseudomonas aeruginosa YD001 from mulberry root endophytes. Potted plant experiments showed that the control effect on Ralstonia serrata was 71.4%. The control effect is easily affected by environmental factors such as different soils, climates, and hosts. Patent CN105191940 reported that the inhibition rate of 0.15g / L to 2g / L chitosan derivatives on Ralstonia serrata was 47% to 81%, which has the disadvantage of a high antibacterial concentration.

[0004] Cinnamic acid is one of the main active ingredients of the traditional Chinese medicinal plant cinnamon, which is both a medicine and food. It is safe, non-toxic, and has good solubility (China Pharmacy, 2018, 29(18): 2581). In addition, cinnamic acid derivatives have multiple biological activities such as antioxidant, anticancer, and antibacterial (Molecules, 2014, 19(12): 19292). Therefore, cinnamic acid has broad application prospects in the pharmaceutical, food, and agricultural industries. It is reported that cinnamic acid itself has inhibitory activity against Ralstonia solanacearum, with a semi-inhibitory concentration of 0.1 mg / mL (International Journal of Biological Macromolecules, 2016, 87: 577). The inhibitory concentration is high and the dosage is large. Studies have shown that cinnamic acid ester modification can improve the antibacterial activity. At present, there are no reports on the use of cinnamic acid terpenoids for the prevention and treatment of bacterial wilt. Summary of the invention

[0005] 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 methods, unstable effects of biological methods or high drug concentrations, and provides an application of cinnamic acid terpene ester derivatives in preventing and controlling bacterial wilt.

[0006] The cinnamic acid terpene ester derivatives of the present invention are synthesized using plant-derived compound cinnamic acid as a raw material, and specifically include 7 compounds, namely, geranyl cinnamate, neryl cinnamate, menthyl cinnamate, bornyl cinnamate, carvyl cinnamate, terpinyl cinnamate and linalyl cinnamate, and their structures are shown in Formula I:

[0007]

[0008] Wherein R is geranyloxy, neryloxy, menthyloxy, bornyloxy, carawayoxy, terpineyloxy or linalyloxy.

[0009] The synthesis conditions of the cinnamic acid terpene ester derivatives of the present invention are as follows: 1-2g of cinnamic acid, 1-2g of oxalyl chloride and 0.05-2mL of N,N-dimethylformamide are dissolved in 5-20mL of dichloromethane, and the reaction is refluxed for 2-6h. After the reaction solution is concentrated to dryness at 30-50°C, 1-2g of terpene alcohol (geraniol, nerol, menthol, borneol, carveol, terpineol or linalool), 0.5-2g of triethylamine and 10-30mL of dichloromethane are added, and the reaction is carried out at 30-50°C for 4-12h.

[0010] The bacterial wilt pathogen involved in the present invention is Ralstonia solanacearum MRS-5, and its Taxonomy ID is 1310165.

[0011] When used for preventing and treating bacterial wilt, the cinnamic acid terpene ester derivatives of the invention are mixed with ethanol to form a concentration of 0.02-0.32 g / L for spraying, and the number of times of use per day is 1-5 times.

[0012] The present invention has the following beneficial effects:

[0013] 1) The process for synthesizing cinnamic acid terpene ester derivatives is simple, has low equipment requirements, and is suitable for large-scale operation.

[0014] 2) The semi-inhibitory concentration of cinnamic acid terpene ester derivatives is lower than 0.16 g / L, and the antibacterial effect on Ralstonia solanacearum is significant at low concentrations, making it suitable as a Ralstonia solanacearum antibacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the 1H-NMR diagram of geranyl cinnamate;

[0016] Figure 2 is the 13C-NMR diagram of geranyl cinnamate;

[0017] Figure 3 is the 1H-NMR diagram of neryl cinnamate;

[0018] Figure 4 is the 13C-NMR diagram of neryl cinnamate;

[0019] Figure 5 is the 1H-NMR diagram of menthyl cinnamate;

[0020] Figure 6 is the 13C-NMR diagram of menthyl cinnamate;

[0021] Figure 7 is the 1H-NMR diagram of bornyl cinnamate;

[0022] Figure 8 is the 13C-NMR diagram of bornyl cinnamate;

[0023] Fig. 9 is the 1H-NMR diagram of carvyl cinnamate;

[0024] Fig.10 is the 13C-NMR diagram of carvyl cinnamate;

[0025] Fig.11 is the 1H-NMR diagram of terpinenine cinnamate;

[0026] Fig.12 is the 13C-NMR diagram of terpinenine cinnamate;

[0027] Fig.13 is the 1H-NMR diagram of linalyl cinnamate;

[0028] Fig.14 This is the 13C-NMR spectrum of linalyl cinnamate. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] Synthesis of geranyl cinnamate: 1.48 g of cinnamic acid, 1 mL of N,N-dimethylformamide and 1.38 g of oxalyl chloride were dissolved in 5 mL of dichloromethane, and refluxed for 4 h. After the reaction solution was concentrated to dryness at 50 °C, 1.39 g of geraniol, 0.9 g of triethylamine and 20 mL of dichloromethane were added, and the reaction was carried out at 30 °C for 8 h. The filtrate was concentrated and dried at 50 °C for 8 h to obtain 1.81 g of geranyl cinnamate, with a yield of 64%.

[0032] The structure of the obtained cinnamate geranyl was characterized by hydrogen spectrum, such as Figure 1 Shown: 1H-NMR, δ7.68 (d, J = 16 Hz, 1H), 7.52-7.50 (m, 2H), 7.38-7.36 (m, 3H), 6.44 (d, J = 16 Hz, 1H), 5.43-5.39 (m, 1H), 5.11-5.07 (m, 1H), 4.72 (d, J = 7.6 Hz, 2H), 2.12-2.08 (m, 4H), 1.74 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H).

[0033] The obtained geranyl cinnamate was characterized by carbon spectrum, such as Figure 2 Shown: 13C-NMR, δ 167.12, 144.76, 142.48, 134.57, 131.93, 130.32, 128.98, 128.16, 123.88, 118.45, 118.32, 61.57, 39.66, 26.40, 25.81, 17.81, 16.63.

[0034] Example 2

[0035] Synthesis of neryl cinnamate: 1.24 g cinnamic acid, 0.5 mL N,N-dimethylformamide and 1.45 g oxalyl chloride were dissolved in 10 mL dichloromethane, refluxed for 5 h, the reaction solution was concentrated to dryness at 40 ° C, 1.47 g nerol, 0.4 g triethylamine and 15 mL dichloromethane were added, the reaction was carried out at 40 ° C for 6 h, filtered, and the filtrate was concentrated and dried at 40 ° C for 12 h to obtain 1.90 g geranyl cinnamate, with a yield of 67%.

[0036] The structure of the obtained cinnamic acid neryl ester was characterized by hydrogen spectrum, such as Figure 3 Shown: 1H-NMR, δ7.68 (d, J = 16 Hz, 1H), 7.52-7.49 (m, 2H), 7.38-7.36 (m, 3H), 6.43 (d, J = 16 Hz, 1H), 5.45-5.41 (m, 1H), 5.12-5.09 (m, 1H), 4.69 (d, J = 7.2 Hz, 2H), 2.15-2.09 (m, 4H), 1.78 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H).

[0037] The obtained cinnamic acid neryl ester was characterized by carbon spectrum, such as Figure 4 Shown: 13C-NMR, δ 167.10, 144.76, 142.86, 134.56, 132.29, 130.33, 128.98, 128.15, 123.70, 119.33, 118.31, 61.28, 32.31, 26.78, 25.83, 23.67, 17.79.

[0038] Example 3

[0039] Synthesis of menthyl cinnamate: 1.57 g of cinnamic acid, 2 mL of N,N-dimethylformamide and 1.64 g of oxalyl chloride were dissolved in 20 mL of dichloromethane and refluxed for 5 h. After the reaction solution was concentrated to dryness at 30 °C, 1.44 g of menthol, 1.2 g of triethylamine and 20 mL of dichloromethane were added, and the reaction was carried out at 40 °C for 10 h. After filtering, the filtrate was concentrated and dried at 40 °C for 6 h to obtain 2.18 g of menthyl cinnamate with a yield of 77%.

[0040] The structure of the obtained menthyl cinnamate was characterized by hydrogen spectrum, such as Figure 5 Shown: 1H-NMR, δ7.66(d,J=16Hz,1H),7.53-7.51(m,2H),7.38-7.36(m,3H),6.42(d,J=16Hz,1H),4.85-4.78(m,1H),2.08-2.04(m,1H),1.94 -1.90(m,1H),1.72-1.68(m,2H),1.54-1.42(m,2H),1.10-1.02(m,2H),0.92(d,J=3.6Hz,3H), 0.90(d,J=4Hz,3H),0.78(d,J=6.8Hz,3H).

[0041] The obtained menthyl cinnamate was characterized by carbon spectrum, such as Figure 6Shown: 13C-NMR, δ 166.53, 144.45, 134.59, 130.23, 128.93, 128.12, 118.77, 74.19, 47.27, 41.12, 34.38, 31.49, 26.43, 23.61, 22.17, 20.89, 16.55.

[0042] Example 4

[0043] Synthesis of bornyl cinnamate: 1.25g of cinnamic acid, 1mL of N,N-dimethylformamide and 1.34g of oxalyl chloride were dissolved in 10mL of dichloromethane, and refluxed for 4h. After the reaction solution was concentrated to dryness at 40℃, 1.32g of borneol, 1.0g of triethylamine and 15mL of dichloromethane were added, and the reaction was carried out at 30℃ for 8h. The filtrate was concentrated and dried at 30℃ for 8h to obtain 2.18g of bornyl cinnamate, with a yield of 77%.

[0044] The structure of the obtained bornyl cinnamate was characterized by hydrogen spectrum, such as Figure 7 Shown: 1H-NMR, δ7.66(d,J=16Hz,1H),7.54-7.52(m,2H),7.38-7.37(m,3H),6.46(d,J=16Hz,1H),5.03-4.99(m,1H),2.45-2.37(m,1H),2 .07-2.00(m,1H),1.81-1.74(m,1H),1.71-1.69(m,1H),1.34-1.24(m,1H),1.07-1.03(m,1H),0.94(s,3H),0.89(s,3H),0.87(s,3H).

[0045] The obtained bornyl cinnamate was characterized by carbon spectrum, such as Figure 8 Shown: 13C-NMR, δ 167.45, 144.33, 134.63, 130.27, 128.96, 128.16, 118.87, 80.09, 49.04, 47.96, 45.04, 36.97, 28.18, 27.33, 19.85, 19.00, 13.69.

[0046] Example 5

[0047] Synthesis of carvone cinnamate: 1.13 g cinnamic acid, 0.5 mL N,N-dimethylformamide and 1.36 g oxalyl chloride were dissolved in 15 mL dichloromethane, refluxed for 5 h, the reaction solution was concentrated to dryness at 50 ° C, 1.48 g carveol, 0.5 g triethylamine and 20 mL dichloromethane were added, the reaction was carried out at 40 ° C for 8 h, filtered, and the filtrate was concentrated and dried at 40 ° C for 10 h to obtain 1.59 g carvone cinnamate, with a yield of 56%.

[0048] The structure of the obtained carvum cinnamyl ester was characterized by hydrogen spectrum, such as Fig. 9 Shown: 1H-NMR, δ7.69 (d, J = 16 Hz, 1H), 7.54-7.51 (m, 2H), 7.38-7.37 (m, 3H), 6.47 (d, J = 16 Hz, 1H), 5.78-5.39 (m, 2H), 4.74-4.72 (m, 2H), 2.37-2.21 (m, 2H), 2.10-1.85 (m, 2H), 1.73 (s, 3H), 1.72 (s, 3H), 1.68-1.67 (m, 1H).

[0049] The obtained carvyl cinnamate was characterized by carbon spectrum, such as Fig.10 Shown: 13C-NMR, δ 166.72, 148.62, 144.77, 134.54, 131.19, 130.33, 128.96, 128.17, 125.98, 118.59, 109.46, 70.68, 40.35, 36.00, 33.83, 31.04, 20.82.

[0050] Example 6

[0051] Synthesis of terpineol cinnamate: 1.63 g of cinnamic acid, 1 mL of N,N-dimethylformamide and 1.24 g of oxalyl chloride were dissolved in 20 mL of dichloromethane, and refluxed for 6 h. After the reaction solution was concentrated to dryness at 40 °C, 1.32 g of terpineol, 0.8 g of triethylamine and 30 mL of dichloromethane were added, and the reaction was carried out at 50 °C for 6 h. The filtrate was concentrated and dried at 50 °C for 6 h to obtain 1.53 g of terpineol cinnamate, with a yield of 54%.

[0052] The structure of the obtained terpinenine cinnamate was characterized by hydrogen spectrum, such as Fig.11 Shown: 1H-NMR, δ7.56 (d, J = 16 Hz, 1H), 7.50-7.49 (m, 2H), 7.37-7.35 (m, 3H), 6.36 (d, J = 16 Hz, 1H), 5.38 (s, 1H), 2.17-2.11 (m, 1H), 2.05-1.97 (m, 2H), 1.88-1.83 (m, 2H), 1.64 (s, 3H), 1.52 (s, 3H), 1.49 (s, 3H), 1.35-1.28 (m, 1H).

[0053] Terpinenine cinnamate is characterized by carbon spectrum, such as Fig.12Shown: 13C-NMR, δ 166.38, 143.61, 134.73, 134.12, 130.07, 128.92, 128.07, 120.44, 120.32, 85.31, 42.78, 31.00, 26.53, 24.04, 23.57, 23.42.

[0054] Example 7

[0055] Synthesis of linalyl cinnamate: 1.32 g of cinnamic acid, 2 mL of N,N-dimethylformamide and 1.58 g of oxalyl chloride were dissolved in 10 mL of dichloromethane and refluxed for 8 h. After the reaction solution was concentrated to dryness at 50 °C, 1.15 g of linalool, 0.6 g of triethylamine and 20 mL of dichloromethane were added, and the reaction was carried out at 50 °C for 5 h. The filtrate was concentrated and dried at 50 °C for 8 h to obtain 1.16 g of linalyl cinnamate, with a yield of 41%.

[0056] The structure of the obtained linalyl cinnamate was characterized by hydrogen spectrum, such as Fig.13 Shown: 1H-NMR, δ7.61 (d, J = 16 Hz, 1H), 7.51-7.49 (m, 2H), 7.37-7.36 (m, 3H), 6.39 (d, J = 16 Hz, 1H), 6.07-6.00 (m, 1H), 5.23-5.14 (m, 2H), 5.13-5.09 (m, 1H), 2.04-2.00 (m, 2H), 1.93-1.85 (m, 2H), 1.67 (s, 3H), 1.62 (s, 3H), 1.59 (s, 3H).

[0057] The obtained linalyl cinnamate was characterized by carbon spectrum, such as Fig.14 Shown: 13C-NMR, δ 165.90, 144.11, 141.95, 134.65, 131.99, 130.19, 128.95, 128.11, 123.92, 119.70, 113.31, 83.23, 40.02, 25.80, 23.81, 22.48, 17.72.

[0058] Experiment 1: Determination of the inhibition rate of cinnamic acid terpenoid derivatives on mulberry wilt pathogens:

[0059] 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 cinnamic acid terpene 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. 600The 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.

[0060]

[0061] 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.

[0062] The results showed that at 0.02-0.32 g / L, the inhibition rates of geranyl cinnamate, neryl cinnamate, menthyl cinnamate, bornyl cinnamate, carvyl cinnamate, terpinyl cinnamate, linalyl cinnamate and lonac were 21.1%-69.0%, 22.5%-66.1%, 21.4%-79.7%, 30.4%-89.8%, 17.5%-81.7%, 14.4%-71.2%, 13.3%-58.8% and 6.7%-86.1%, respectively. Their semi-inhibitory concentrations were 0.13 g / L, 0.12 g / L, 0.08 g / L, 0.04 g / L, 0.07 g / L, 0.15 g / L, 0.16 g / L and 0.09 g / L, respectively. The results showed that the seven cinnamic acid terpenoids had significant antibacterial effects on Ralstonia solanacearum, among which the semi-inhibitory concentrations of menthyl cinnamate, bornyl cinnamate and carvyl cinnamate were lower than that of Ralstonia roqueforti.

[0063] Experiment 2: Determination of the inhibitory effect of cinnamic acid terpenoid ester derivatives on the pathogen of mulberry wilt disease:

[0064] 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 cinnamic acid terpene 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 Longke bacteria. 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.

[0065] The results showed that geranyl cinnamate, neryl cinnamate, menthyl cinnamate, bornyl cinnamate, carvyl cinnamate, terpinyl cinnamate, linalyl cinnamate and Longke bacteria all produced inhibition zones, and the sizes of their inhibition zones were 0.3cm, 0.3cm, 0.6cm, 0.8cm, 0.7cm, 0.2cm, 0.2cm and 0.5cm, respectively. The antibacterial activity of menthyl cinnamate, bornyl cinnamate and carvyl cinnamate was better than that of Longke bacteria.

[0066] Experiment on the control effect of cinnamic acid terpene ester derivatives on mulberry wilt:

[0067] The infected mulberry seedlings inoculated with Ralstonia solanacearum were used as the test objects. 0.02-0.32 g / L cinnamic acid terpene ester ethanol solution was sprayed on the infected mulberry seedlings 3 times a day. The growth of the infected 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 Ralstonia solanacearum. The control rate was the ratio of cured mulberry seedlings to the total number of infected mulberry seedlings.

[0068] The results showed that at 0.02-0.32 g / L, the control rates of geranyl cinnamate, neryl cinnamate, menthyl cinnamate, bornyl cinnamate, carvyl cinnamate, terpinyl cinnamate, linalyl cinnamate and loncosus were 6.3%-56.3%, 6.3%-62.5%, 12.5%-75%, 31.3%-81.3%, 18.8%-75%, 6.3%-50%, 6.3%-56.3% and 6.3%-68.8%, respectively. Among them, the control rate exceeded 69% when the concentration of bornyl cinnamate was higher than 0.1 g / L, and the control effect was significant at lower concentrations.

Claims

1. Application of cinnamic acid terpene ester derivatives in preventing and treating bacterial wilt, wherein the cinnamic acid terpene ester derivatives are any one of the following geranyl cinnamate, neryl cinnamate, menthyl cinnamate, bornyl cinnamate, carvyl cinnamate, terpinyl cinnamate or linalyl cinnamate, and the structure is shown in Formula I: Wherein R is geranyloxy, neryloxy, menthyloxy, bornyloxy, carawayoxy, terpineyloxy or linalyloxy.

2. The use of the cinnamic acid terpene ester derivatives in preventing and treating bacterial wilt according to claim 1, characterized in that The synthesis conditions of cinnamic acid terpene ester derivatives are as follows: 1-2 g cinnamic acid, 1-2 g oxalyl chloride and 0.05-2 mL N,N-dimethylformamide are dissolved in 5-20 mL dichloromethane, and refluxed for 2-6 h. After the reaction solution is concentrated to dryness at 30-50° C., 1-2 g terpene alcohol, 0.5-2 g triethylamine and 10-30 mL dichloromethane are added, and the reaction is carried out at 30-50° C. for 4-12 h.

3. The use of the cinnamic acid terpene ester derivatives in preventing and treating bacterial wilt according to claim 2, characterized in that The terpene alcohol is selected from geraniol, nerol, menthol, borneol, carveol, terpineol or linalool.

4. The use of the cinnamic acid terpene ester derivatives in preventing and treating bacterial wilt according to claim 1, characterized in that The cinnamic acid terpene ester derivative is mixed with ethanol to prepare a solution with a concentration of 0.02-0.32 g / L for spraying when preventing and treating bacterial wilt. The number of times of use per day is 1-5 times.

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