A sesquiterpenoid lactone compound and its preparation method and application
By extracting sesquiterpene lactone compounds from the syringae, the problem of ToBRFV prevention and control was solved, efficient and selective viral inhibition effect was achieved, and a basis for the development of new pesticides was provided.
Patent Information
- Application Number
- CN202510730500.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-03
- Publication Date
- 2025-08-19
- Estimated Expiration
- 2045-06-03
AI Technical Summary
The prior art is difficult to effectively prevent and control the tomato brown wrinkle fruit virus (ToBRFV). This virus is prone to mutation and has difficulty in preventing and controlling, affecting crop yield and quality.
Sesquiterpene lactone compounds were extracted from the above-ground part of the squinaceae, and compounds with high efficiency anti-ToBRFV activity were obtained through a multi-step separation and purification method as a novel plant virus inhibitor.
Sesquiterpene lactone compounds have shown superior effects than the broad-spectrum fungicide nynanmycin in the prevention and treatment of ToBRFV, with inhibition rates of 65.11% and 58.6%, respectively, and have efficient and highly selective prevention and treatment effects.
Smart Images
Figure CN120247923B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pesticides, and in particular to a sesquiterpene lactone compound, a preparation method thereof, and application thereof as a pesticide. Background Art
[0002] Currently, the harm caused by plant viral diseases is becoming increasingly serious. The number of disease types and the increase in virus complex infections have a huge impact on the yield and quality of grain, oil, vegetables, flowers, and fruits. How to solve plant viral diseases is an urgent problem in current agricultural production and a guarantee for increasing production and income. Tomato brown rugose fruit virus (ToBRFV) belongs to the Bacillus family ( Virgaviridae Tobacco mosaic virus ( Tobamovirus ToBRFV is a newly discovered RNA virus. Currently, it is being reported in a growing number of tomato-producing areas worldwide, affecting a variety of crops, including tomatoes, peppers, Nicotiana benthamiana, and Nicotiana oleracea. Viruses are known to be prone to mutation, making their control challenging. Under the influence of natural selection, ToBRFV's evolutionary rate and adaptability are increasing. Therefore, finding effective control agents is crucial to mitigating the damage caused by the virus to commercial crops.
[0003] Over the course of long evolution, plants have developed chemical defenses against pests and pathogens. Chemical inducers that stimulate plant immune responses and induce resistance are effective agents for controlling plant viral diseases. These defenses possess high recognition specificity, protecting against infection by foreign pathogens or viruses while maintaining low toxicity to the plant itself and low environmental pollution. Therefore, leveraging plant-derived chemical defenses is an effective approach for developing novel, highly effective, and environmentally friendly botanical pesticides. Summary of the Invention
[0004] The present invention provides a sesquiterpene lactone compound extracted from the aerial parts of Tithonia suffruticosa. This compound serves as a highly effective anti-ToBRFV active lead compound, providing an important basis for subsequently clarifying its target of action and antiviral mechanism, so as to develop new, efficient and highly selective plant virus inhibitors.
[0005] Sesquiterpenoid lactone compound, the structural formula is as follows:
[0006]
[0007] Among them, R1 is α-OH and R2 is OH.
[0008] The preparation method of sesquiterpene lactone compounds is characterized by comprising the following steps:
[0009] S1. Drying and crushing the aerial parts of the Tithonia suffruticosa plant to obtain Tithonia suffruticosa powder; then extracting with methanol under reflux, combining the extracts and concentrating them on a rotary evaporator; extracting the concentrates with ethyl acetate, combining the extracts and concentrating them to obtain an extract;
[0010] S2. The extract was crudely separated by passing it through a 300-400 mesh silica gel column using a mobile phase of dichloromethane:methanol in a volume ratio of 60:1 to 40:1 for gradient elution. When elution was at a volume ratio of 40:1, a petroleum ether:acetone solution system was used for development. After confirming that the target sample had been completely eluted, the eluate was collected and evaporated to dryness;
[0011] S3. The crude sample obtained in step S2 was subjected to 300-400 mesh normal silica gel column chromatography, using petroleum ether:acetone with a volume ratio of 5:1-2:1 as the mobile phase for gradient elution. When the volume ratio was 2:1, the elution was developed with a dichloromethane:methanol solution system. After confirming that the target sample had been completely eluted, the eluate was collected and evaporated to dryness;
[0012] S4. The target sample collected in step S3 is passed through a reverse MCI chromatographic column to remove the pigment in the target sample. A gradient elution is performed using methanol:water with a volume ratio of 30:70-60:40 as the mobile phase. When the methanol ratio of the eluent increases to 60%, a petroleum ether:acetone solution system is used for development. After confirming that the target sample has been completely eluted, the eluent is collected and evaporated to dryness.
[0013] S5. The target sample collected in step S4 is separated by a methanol gel column and dissolved in pure methanol for later use. After the gel column is rinsed with pure methanol, the sample is loaded; the column flow rate is 5 s / drop, and the petroleum ether: acetone solution system is used for development. The thin layer behavior is consistent with a single compound. After collection, the solvent is recovered using a rotary evaporator to obtain the sesquiterpene lactone compound of the present invention.
[0014] Preferably, the methanol mass concentration in step S1 is 70%.
[0015] The invention relates to an application of the sesquiterpene lactone compounds in a drug for preventing and treating tomato brown wrinkled fruit virus.
[0016] The drug for preventing and treating tomato brown wrinkled fruit virus contains the sesquiterpene lactone compound, and the carrier is a carrier applicable in pesticide science, such as salts.
[0017] The sesquiterpene lactone compounds of the present invention are extracted from the aerial parts of Tithonia suffruticosa. Tithonia suffruticosa, as an alien invasive plant, brings serious harm to crops and the ecological environment. With the continuous deepening of research on Tithonia suffruticosa, it is found that it can be used as a medicinal plant to treat inflammation, tumors, diabetes, fever, trauma, malaria and other diseases. The sesquiterpene lactone compounds in Tithonia suffruticosa have the advantages of stable physical and chemical properties, low toxicity, high activity, and easy metabolism. Using this compound as a highly effective anti-ToBRFV active lead can achieve a win-win goal and provide an important basis for the subsequent clarification of its target and antiviral mechanism to develop new, efficient and highly selective plant virus inhibitors.
[0018] The present invention uses the aerial parts of Tithonia suffruticosa as raw materials and adopts a relatively simple and easy-to-operate extraction and separation method to obtain sesquiterpenoid lactone compounds. Experimental verification shows that the sesquiterpenoid compounds of the present invention have good effects in preventing and controlling ToBRFV. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Structural diagram of the sesquiterpene lactone compound of the present invention.
[0020] Figure 2 Inoculation diagram of the experimental test on the preventive effect of the sesquiterpene lactone compounds of the present invention on ToBRFV;
[0021] Figure a is a diagram showing the effect of the compound of the present invention, Figure b is a diagram showing the effect of Ningnanmycin, and Figure c is a diagram showing the effect of ddH2O, i.e., blank CK.
[0022] Figure 3 Inoculation diagram of the experimental treatment effect of the sesquiterpene lactone compounds of the present invention on ToBRFV;
[0023] Figure a is a diagram showing the effect of the compound of the present invention, Figure b is a diagram showing the effect of Ningnanmycin, and Figure c is a diagram showing the effect of ddH2O, i.e., blank CK. DETAILED DESCRIPTION
[0024] The present invention will be further described below with reference to specific embodiments.
[0025] Example 1: Preparation method of sesquiterpene lactone compounds, the steps are as follows:
[0026] S1. Drying and crushing the aerial parts of the Tithonia suffruticosa plant to obtain Tithonia suffruticosa powder; then extracting the powder three times with methanol under reflux, combining the extracts and concentrating them on a rotary evaporator; extracting the concentrates with ethyl acetate, combining the extracts and concentrating them to obtain an extract;
[0027] S2. The extract was crudely separated by passing it through a 300-400 mesh silica gel column using a mobile phase of dichloromethane:methanol in a volume ratio of 60:1 to 40:1 for gradient elution. When elution was at a volume ratio of 40:1, a petroleum ether:acetone solution system was used for development. After confirming that the target sample had been completely eluted, the eluate was collected and evaporated to dryness;
[0028] S3. The crude sample obtained in step S2 was subjected to 300-400 mesh normal silica gel column chromatography, using petroleum ether:acetone with a volume ratio of 5:1-2:1 as the mobile phase for gradient elution. When the volume ratio was 2:1, the elution was developed with a dichloromethane:methanol solution system. After confirming that the target sample had been completely eluted, the eluate was collected and evaporated to dryness;
[0029] S4. The target sample collected in step S3 is passed through a reverse MCI chromatographic column to remove the pigment in the target sample. A gradient elution is performed using methanol:water with a volume ratio of 30:70-60:40 as the mobile phase. When the methanol ratio of the eluent increases to 60%, a petroleum ether:acetone solution system is used for development. After confirming that the target sample has been completely eluted, the eluent is collected and evaporated to dryness.
[0030] S5. The target sample collected in step S4 is separated by a methanol gel column and dissolved in pure methanol for later use. After the gel column is rinsed with pure methanol, the sample is loaded; the column flow rate is 5 s / drop, and the petroleum ether: acetone solution system is used for development. The thin layer behavior is consistent with a single compound. After collection, the solvent is recovered using a rotary evaporator to obtain the sesquiterpene lactone compound of the present invention.
[0031] The H NMR spectrum of the compound of the present invention ( 1 H-NMR) and carbon spectroscopy ( 13 C-NMR) data ( δ in ppm, J in Hz) as follows:
[0032] 1 H-NMR (400 MHz, DMSO- d 6) δ: 6.15 (1H, d, J = 3.0 Hz, H-13a), 5.47(1H, m, H-8), 5.45 (1H, d, J = 3.0 Hz, H-13b), 4.48 (1H, m, H-6), 4.13 (1H,t, J = 8.3 Hz,H-1), 3.93 (1H, d, J = 3.1 Hz, H-7), 2.36 (1H, m, H-2a), 2.34(1H, m, H-2′), 2.02 (1H, m, H-2b), 1.99 (2H, m, H-5a, 5b), 1.86 (1H, dd, J =14.4, 5.0 Hz, H-4), 1.67 (2H, m, H-9a, 9b), 1.31 (3H, s, H-14), 0.99 (3H, d, J= 5.0 Hz, H-15), 0.96 (3H, d, J = 7.2 Hz, H-3′), 0.94 (3H, d, J = 7.2 Hz, H-4′). 13 C-NMR (CDCl3, 100 MHz) δC (ppm): 176.4 (s, C-1′), 169.7 (s, C-12), 136.8 (s, C-11), 121.7 (t, C-13), 105.3 (s, C-3), 81.8 (s, C-10), 81.3 (d, C-6),78.0 (d, C-1), 69.7 (d, C-8), 47.5 (d, C-7), 46.5 (t, C-2), 44.2 (d, C-4),37.4 (t, C-5), 34.3 (t, C-9), 33.8 (d, C-2′), 24.6 (q, C-14), 18.9 (q, C-15),18.5 (q, C-3′), 18.2 (q, C-4′).
[0033] The sesquiterpene lactone compound prepared in Example 1 of the present invention was used to study and verify the activity of preventing and treating ToBRFV. The specific experiment is as follows:
[0034] a. Preparation of Tomato Brown Rough Fruit Virus
[0035] The common strain of Tomato Brown Rough Fruit Virus was provided by the Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences. It was propagated on common tobacco heart leaves, purified, and its concentration was measured using a UV spectrophotometer before being stored at -80°C for future use.
[0036] b. Virus inoculation and pesticide application
[0037] Inhibitory effect (preventive effect) of the compound on primary infection of ToBRFV:
[0038] Healthy, uniformly growing heartleaf tobacco seedlings with 5-6 true leaves were selected. 2 mL of the compound of this example at a concentration of 200 μg / mL was evenly applied to the 4th and 5th true leaves until the compound preparation was used up. After 10 minutes, the leaf surface was rinsed with clean water. After 6 hours, the leaf surface was inoculated with 100 μg / mL ToBRFV by friction and then rinsed with clean water after 10 minutes.
[0039] Four controls were set up, control group 1 was smeared with an equal amount of ddH2O as the compound of this example, control group 2 was smeared with an equal amount of ningnanmycin as the compound of this example, control group 3 and control group 4 were smeared with an equal amount of sesquiterpene lactone compound 1 and sesquiterpene lactone compound 2 as the compound of this example, respectively, and the rest of the treatment steps were the same.
[0040] The above three experiments were repeated three times for each sample. After culturing in an insect-free greenhouse for 3-4 days, the number of dead spots was counted and the inhibition rate was calculated. The inhibition rate calculation method is as follows:
[0041] Inhibition rate = (number of control necrotic spots - number of treated necrotic spots) / number of control necrotic spots × 100%.
[0042] The inhibitory effect of the compound on ToBRFV replication and proliferation (therapeutic effect):
[0043] Healthy, uniformly growing heartleaf tobacco seedlings with 5-6 true leaves were selected, and the 4th and 5th true leaves were inoculated with 100 μg / mL of ToBRFV by friction. After 10 minutes, the leaf surface was rinsed with clean water. After 24 hours, the compound of this example was evenly applied at a concentration of 200 μg / mL.
[0044] Four controls were set up, control group 1 was smeared with an equal amount of ddH2O as the compound of this example, control group 2 was smeared with an equal amount of ningnanmycin as the compound of this example, control group 3 and control group 4 were smeared with an equal amount of sesquiterpene lactone compound 1 and sesquiterpene lactone compound 2 as the compound of this example, respectively, and the rest of the treatment steps were the same.
[0045] The above three experiments were repeated three times for each sample, and the number of dead spots was counted after culturing in an insect-free greenhouse for 3-4 days, and the inhibition rate was calculated. The calculation method of the inhibition rate was the same as above.
[0046] The structural formula of the sesquiterpene lactone compound 1 is as follows:
[0047]
[0048] Among them, R1 is α-OCH3 and R2 is OH.
[0049] The structural formula of the sesquiterpene lactone compound 2 is as follows:
[0050]
[0051] Among them, R1 is H and R2 is OH.
[0052] Table 1 shows the results of the verification of the anti-ToBRFV activity of sesquiterpene lactone compounds.
[0053] Table 1
[0054]
[0055] The experimental results are the mean of three experiments, and letters represent significant differences.
[0056] The in vivo anti-ToBRFV experiment showed that the sesquiterpene lactone compounds of the embodiments of the present invention showed good preventive and therapeutic effects on ToBRFV, with the preventive inhibition rate of 65.11%, which was higher than the 55.94% of the positive control Ningnanmycin, and the therapeutic inhibition rate of 58.6%, which was higher than the 46.18% of the positive control Ningnanmycin.
[0057] In addition, compared with sesquiterpene lactone compound 1 and sesquiterpene lactone compound 2, although only the substituents are different, the preventive and therapeutic effects on ToBRFV are not as good as the compound of the present invention. The two substituents of the sesquiterpene lactone compound of the present invention are both hydroxyl groups, which enhances the hydrophilicity and increases the binding force of small molecules to virus-related proteins compared to compound 1 and compound 2. Therefore, the effect of the compound of the present invention is better than the other two compounds.
[0058] It should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and may have other variations. All variations directly or indirectly derived from the present disclosure by those skilled in the art should be considered to be within the scope of protection of the present invention.
Claims
1. Use of a sesquiterpenoid lactone compound as a drug for preventing and treating tomato brown wrinkle fruit virus, characterized in that: The structural formula of the sesquiterpenoid lactone compound is as follows: Among them, R1 is α-OH and R2 is OH.