Sesquiterpenoid lactone compound as well as preparation method and application thereof

Abstract

The invention discloses a sesquiterpene lactone compound as well as a preparation method and application thereof, and relates to the technical field of pesticides. According to the invention, three sesquiterpene lactone compounds are obtained by extracting, separating and purifying overground parts of tithonia diversifolia. According to the present invention, the prepared sesquiterpene lactone compound tithonin A can effectively prevent and control the plant virus ToBRFV, the ToBRFV inhibition effect of the sesquiterpene lactone compound tithonin A is superior to the broad-spectrum bactericide ningnanmycin to a certain extent, the prevention and control effect is good, and the alpha-OH is the main functional group after the sesquiterpene lactone compound tithonin A is compared with the similar compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticides, and particularly relates to sesquiterpene lactone compounds, a preparation method thereof, and an application as pesticides. Background Art

[0002] At present, the harm of plant virus diseases is becoming increasingly serious. The increase in the types of diseases and the phenomenon of virus complex infection have a huge impact on the yield and quality of grains, oils, vegetables, flowers, and fruits. How to solve plant virus diseases is an urgent problem in current agricultural production and also a guarantee for increasing production and income. Tomato brown rugose fruit virus (ToBRFV) belongs to the family Virgaviridae ( Virgaviridae) the genus Tobamovirus ( Tobamovirus ), and is a newly discovered RNA virus. Currently, more and more tomato production areas around the world are infected with this virus, and the virus has been found to infect a variety of crops, such as tomatoes, peppers, Nicotiana benthamiana, and Nicotiana glutinosa. As is well known, viruses are prone to mutation, making their prevention and control very difficult. Under the action of natural selection, the evolution rate and adaptability of ToBRFV show an upward trend. Therefore, finding effective control agents is crucial for reducing the harm of this virus to economic crops.

[0003] During the long-term evolution process, plants have formed chemical defense substances against pests and pathogenic microorganisms. Chemical inducers that stimulate plant immune responses and induce plant resistance are effective agents for controlling plant virus diseases. These defense substances have high recognition specificity and, while resisting the invasion of foreign pathogenic bacteria or viruses, maintain low toxicity to the plants themselves and low pollution to the environment. Therefore, based on the chemical defense substances in plants is an effective way to develop new, efficient, and environmentally friendly plant-derived pesticides. Summary of the Invention

[0004] The present invention provides a sesquiterpene lactone compound extracted from the above-ground part of Tithonia diversifolia. This compound, as a highly effective anti-ToBRFV active lead compound, provides an important basis for subsequently clarifying its action target and antiviral mechanism to develop new, efficient, and highly selective plant virus inhibitors.

[0005] The sesquiterpene lactone compound has the following structural formula:

[0006] Wherein, R1 is α-OH and R2 is OH.

[0007] A preparation method of the sesquiterpene lactone compound, characterized by comprising the following steps: S1. Dry and pulverize the above-ground part of the Tithonia diversifolia plant to obtain Tithonia diversifolia powder; then extract it by refluxing with methanol, combine the extracts and concentrate them using a rotary evaporator; extract the concentrated solution with ethyl acetate, combine and concentrate to obtain an extract; S2. Coarsely separate the extract through a silica gel column with 300 - 400 mesh, use dichloromethane:methanol with a volume ratio of 60:1 - 40:1 as the mobile phase for gradient elution. When eluting with a volume ratio of 40:1, develop with a petroleum ether:acetone solution system. After confirming that the target sample has been completely eluted, collect the eluate and evaporate it to dryness; S3. Subject the crude sample obtained in step S2 to normal-phase silica gel column chromatography with 300 - 400 mesh, use petroleum ether:acetone with a volume ratio of 5:1 - 2:1 as the mobile phase for gradient elution. When eluting with a volume ratio of 2:1, develop with a dichloromethane:methanol solution system. After confirming that the target sample has been completely eluted, collect the eluate and evaporate it to dryness; S4. Remove the pigments in the target sample collected in step S3 through a reverse-phase MCI chromatographic column, use methanol:water with a volume ratio of 30:70 - 60:40 as the mobile phase for gradient elution. When the methanol ratio in the eluate increases to 60%, develop with a petroleum ether:acetone solution system. After confirming that the target sample has been completely eluted, collect the eluate and evaporate it to dryness; S5. Separate the target sample collected in step S4 through a methanol gel column, dissolve it with pure methanol and set it aside. After rinsing the gel column with pure methanol, start loading the sample; the column flow rate is 5 s / drop, develop with a petroleum ether:acetone solution system. When the thin-layer behavior is consistent as a single compound, collect it and use a rotary evaporator to recover the solvent to obtain the sesquiterpene lactone compound of the present invention.

[0008] Preferably, the mass concentration of the methanol in step S1 is 70%.

[0009] Application of the sesquiterpene lactone compound of the present invention in the drug for preventing and treating tomato brown rugose fruit virus.

[0010] The drug for preventing and treating tomato brown rugose fruit virus contains the sesquiterpene lactone compound, and the carrier is a carrier applicable in pesticide science, such as salts.

[0011] The sesquiterpene lactone compounds of the present invention are extracted from the aerial parts of Tithonia diversifolia. As an alien invasive plant, Tithonia diversifolia causes serious harm to crops and the ecological environment. With the continuous in-depth study of Tithonia diversifolia, it is found that it can be used as a medicinal plant to treat diseases such as inflammation, tumors, diabetes, as well as fever, trauma, and malaria. The sesquiterpene lactone compounds in Tithonia diversifolia 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 compound can achieve a win-win goal, providing an important basis for subsequent clarification of its action targets and antiviral mechanisms to develop new, highly effective, and highly selective plant virus inhibitors.

[0012] The present invention uses the aerial parts of Tithonia diversifolia as raw materials and adopts a relatively simple and easy-to-operate extraction and separation method to obtain sesquiterpene lactone compounds. Through experimental verification, the sesquiterpene compounds of the present invention have good effects in preventing and controlling ToBRFV. Brief Description of the Drawings

[0013] Figure 1 Structural diagram of the sesquiterpene lactone compound of the present invention.

[0014] Figure 2 Experimental inoculation diagram of the preventive effect of the sesquiterpene lactone compound of the present invention on ToBRFV; Figure a is the effect diagram of the compound of the present invention, Figure b is the effect diagram of ningnanmycin; Figure c is the effect diagram of ddH2O, that is, the blank CK.

[0015] Figure 3 Experimental inoculation diagram of the therapeutic effect of the sesquiterpene lactone compound of the present invention on ToBRFV; Figure a is the effect diagram of the compound of the present invention, Figure b is the effect diagram of ningnanmycin; Figure c is the effect diagram of ddH2O, that is, the blank CK. Detailed Description of the Invention

[0016] The present invention will be further described below in conjunction with specific embodiments.

[0017] Example 1: Preparation method of sesquiterpene lactone compounds, the steps are as follows: S1. Dry and crush the aerial parts of the Tithonia diversifolia plant to obtain Tithonia diversifolia powder; then reflux and extract 3 times with methanol, combine the extracts and concentrate them with a rotary evaporator; extract the concentrated solution with ethyl acetate, combine and concentrate to obtain an extract; S2. Coarsely separate the extract through a silica gel column of 300 - 400 mesh, use dichloromethane:methanol with a volume ratio of 60:1 - 40:1 as the mobile phase for gradient elution, when eluting with a volume ratio of 40:1, develop with a petroleum ether:acetone solution system, confirm that the target sample has been completely eluted, then collect the eluate and evaporate it to dryness; S3. Subject the crude sample obtained in step S2 to silica gel column chromatography with a mesh size of 300 - 400 in the positive direction. Use petroleum ether:acetone with a volume ratio of 5:1 - 2:1 as the mobile phase for gradient elution. When eluting with a volume ratio of 2:1, develop using a dichloromethane:methanol solution system. Confirm that the target sample has been completely eluted, then collect the eluate and evaporate it to dryness. S4. Subject the target sample collected in step S3 to a reverse MCI chromatographic column to remove pigments in the target sample. Use methanol:water with a volume ratio of 30:70 - 60:40 as the mobile phase for gradient elution. When the methanol ratio in the eluate increases to 60%, develop using a petroleum ether:acetone solution system. Confirm that the target sample has been completely eluted, then collect the eluate and evaporate it to dryness. S5. Separate the target sample collected in step S4 through a methanol gel column, dissolve it with pure methanol for standby, and start loading the sample after rinsing the gel column with pure methanol; the column flow rate is 5 s / drop, develop using a petroleum ether:acetone solution system. The thin layer behavior is consistent as a single compound. After collection, use a rotary evaporator to recover the solvent to obtain the sesquiterpene lactone compound of the present invention.

[0018] The proton nuclear magnetic resonance spectrum ( 1 H-NMR) and carbon nuclear magnetic resonance spectrum ( 13 C-NMR) data ( δ in ppm, J in Hz) of the compound of the present invention are as follows: 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′). 1313C-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′).

[0019] The activity study and verification of controlling ToBRFV were carried out using the sesquiterpene lactone compounds prepared in Example 1 of the present invention. The specific experiments are as follows: a. Preparation of Tomato brown rugose fruit virus The common strain of Tomato brown rugose fruit virus was provided by the Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences. It was propagated on the common tobacco Nicotiana glutinosa, and after purification, its concentration was measured by an ultraviolet spectrophotometer and then stored in a -80 °C refrigerator for later use. b. Inoculating virus and applying drugs Inhibitory effect of the compound on the primary infection of ToBRFV (preventive effect): Select healthy tobacco seedlings of Nicotiana glutinosa at the 5-6 true leaf stage with consistent growth. Uniformly apply 2 mL of the compound of this example with a concentration of 200 μg / mL on the 4th and 5th true leaves until the compound preparation is used up; after 10 min, rinse the leaf surface with clean water, and after 6 h, inoculate by rubbing with ToBRFV with a concentration of 100 μg / mL, and rinse with clean water after 10 min. Set four controls. Control group 1 is to apply the same amount of ddH2O as the compound of this example, control group 2 is to apply the same amount of ningnanmycin as the compound of this example, and control groups 3 and 4 are to apply the same amount of sesquiterpene lactone compound 1 and sesquiterpene lactone compound 2 as the compound of this example respectively. The remaining treatment steps are the same.

[0020] The above three groups of experiments were repeated three times for each sample, and after culturing in an insect-free greenhouse for 3-4 days, the number of necrotic spots was counted, and the inhibition rate was calculated. The calculation method of the inhibition rate is as follows: Inhibition rate = (number of control necrotic spots - number of treated necrotic spots) / number of control necrotic spots × 100%.

[0021] Inhibitory effect of the compound on the replication and proliferation of ToBRFV (therapeutic effect): Select healthy Nicotiana glutinosa seedlings at the 5-6 true leaf stage with consistent growth, rub and inoculate ToBRFV with a concentration of 100 μg / mL on the 4th and 5th true leaves. After 10 minutes, rinse the leaf surface with clean water, and evenly apply the compound of this example with a concentration of 200 μg / mL after 24 hours; Set up four controls. Control group 1 is to apply the same amount of ddH2O as the compound of this example, control group 2 is to apply the same amount of ningnanmycin as the compound of this example, and control groups 3 and 4 are to apply the same amount of sesquiterpene lactone compound 1 and sesquiterpene lactone compound 2 as the compound of this example respectively, and the remaining treatment steps are the same.

[0022] The above three groups of experiments are repeated three times for each sample, and after culturing in an insect-free greenhouse for 3-4 days, count the number of necrotic spots and calculate the inhibition rate. The calculation method of the inhibition rate is the same as above.

[0023] The structural formula of the sesquiterpene lactone compound 1 is as follows:

[0024] Among them, R1 is α-OCH3 and R2 is OH.

[0025] The structural formula of the sesquiterpene lactone compound 2 is as follows:

[0026] Among them, R1 is H and R2 is OH.

[0027] Table 1 shows the verification results of the anti-ToBRFV activity of sesquiterpene lactone compounds.

[0028] Table 1

[0029] The experimental results are all three-time averages, and the letters represent significant differences.

[0030] The in vivo anti-ToBRFV experiment shows that the sesquiterpene lactone compounds of the examples of the present invention exhibit good preventive and therapeutic effects on ToBRFV. The inhibition rate of its preventive effect is 65.11%, which is higher than that of the positive control ningnanmycin (55.94%), and the inhibition rate of its therapeutic effect is 58.6%, which is higher than that of the positive control ningnanmycin (46.18%).

[0031] In addition, compared with sesquiterpene lactone compound 1 and sesquiterpene lactone compound 2, although they only differ in substituents, their preventive and therapeutic effects against ToBRFV are inferior to those of the compounds of the present invention. Both substituents of the sesquiterpene lactone compounds of the present invention are hydroxyl groups, which enhances the hydrophilicity and increases the binding force between small molecules and virus-related proteins compared with compound 1 and compound 2. Therefore, the effects of the compounds of the present invention are better than those of the other two compounds.

[0032] It should be noted that the above are only several specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and there can be other variations. All variations directly derived or indirectly extended by those skilled in the art from the disclosed content of the present invention should be considered as within the protection scope of the present invention.

Claims

1. A sesquiterpene lactone compound, with the structural formula as follows: Among them, R1 is α-OH and R2 is OH.

2. Preparation method of sesquiterpene lactone compounds, characterized in that It includes the following steps: S1. Dry and crush the above-ground part of the Tithonia diversifolia plant to obtain Tithonia diversifolia powder; then extract it by refluxing with methanol, combine the extracts and concentrate them using a rotary evaporator; extract the concentrated solution with ethyl acetate, combine and concentrate to obtain an extract. S2. Coarsely separate the extract through a silica gel column with 300 - 400 mesh, use dichloromethane:methanol with a volume ratio of 60:1 - 40:1 as the mobile phase for gradient elution. When eluting with a volume ratio of 40:1, develop with a petroleum ether:acetone solution system. After confirming that the target sample has been completely eluted, collect the eluate and evaporate it to dryness. S3. Subject the crude sample obtained in step S2 to normal-phase silica gel column chromatography with 300 - 400 mesh, use petroleum ether:acetone with a volume ratio of 5:1 - 2:1 as the mobile phase for gradient elution. When eluting with a volume ratio of 2:1, develop with a dichloromethane:methanol solution system. After confirming that the target sample has been completely eluted, collect the eluate and evaporate it to dryness. S4. Remove the pigments in the target sample collected in step S3 through a reverse MCI chromatographic column, use methanol:water with a volume ratio of 30:70 - 60:40 as the mobile phase for gradient elution. When the methanol ratio in the eluate increases to 60%, develop with a petroleum ether:acetone solution system. After confirming that the target sample has been completely eluted, collect the eluate and evaporate it to dryness. S5. Separate the target sample collected in step S4 through a methanol gel column, dissolve it with pure methanol and set it aside. After rinsing the gel column with pure methanol, start loading the sample; the column flow rate is 5 s / drop, develop with a petroleum ether:acetone solution system. When the thin-layer behavior is consistent as a single compound, collect it and use a rotary evaporator to recover the solvent to obtain the sesquiterpene lactone compound of the present invention.

3. The preparation method according to claim 2, wherein, In step S1, the mass concentration of the methanol is 70%.

4. Use of the sesquiterpene lactone compound according to claim 1 as a drug for preventing and treating tomato brown rugose fruit virus.

5. The application according to claim 4, wherein The drug for preventing and treating tomato brown rugose fruit virus contains the sesquiterpene lactone compound.

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