Botanical antiviral preparation as well as preparation method and application thereof

Abstract

The invention discloses a botanical antiviral preparation as well as a preparation method and application thereof, and belongs to the technical field of preparation of botanical antiviral agents. The invention discloses a botanical antiviral preparation, which is prepared from the following components in percentage by weight: 5 to 9 percent of (12R, 13R)-8, 12-epoxy-14-labden-13-ol, 20 to 30 percent of ethanol, 15 to 25 percent of sucrose ester, 6 to 8 percent of lignosulfonate, 0.2 to 0.4 percent of magnesium aluminum silicate, 2 to 4 percent of glycerol and the balance of deionized water. The botanical antiviral preparation disclosed by the invention is high in antiviral activity, the prevention effect of the botanical antiviral preparation on tobacco mosaic virus, potato Y virus and cucumber mosaic virus is obviously superior to that of botanical pesticide amino-oligosaccharin, and the preparation does not contain any toxic organic solvent and is safe to the environment, people and livestock.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of plant-derived antiviral agents, and more particularly to a plant-derived antiviral preparation and a preparation method and application thereof. Background Art

[0002] Plant diterpenes are composed of four isoprene units (C 20 ) are terpenoids widely distributed in higher plants, fungi and marine organisms. The diversity of chemical structure and biological activity makes it widely used in ecology, medicine and agriculture. As the first commercial plant-derived insecticide, azadirachtin is effective against more than 200 pests such as cotton bollworms and aphids, and is safe for bees. In 2024, α-Cembrene from tobacco was approved by the U.S. EPA as a foliar spray (trade name ) to control plant pests and is used in organic vegetable production. Abietic acid, derived from Pinaceae resin, and Tanshinone IIA, derived from Salvia miltiorrhiza, are used for the targeted control of rice blast and wheat fusarium head blight. In addition, Kaurenoic acid can be used as an effective herbicide for barnyardgrass in soybean fields, with a weed control rate of 78% and crop safety reaching Class A. Plant diterpenes have become an important source for the research and development of green pesticides due to their unique chemical structure and ecological compatibility.

[0003] Tobacco (Nicotiana tabacum) is one of the species with the richest diterpenoid components in the Solanaceae family. More than 50 structural types have been identified, mainly including cembranoids, labdanes, kauranes and special modified types (glycosylated diterpenes). Among them, cembranoids are the main components of tobacco glandular secretions, accounting for 60%-80% of the total diterpenoid content, followed by labdanes. Labdanes are the main diterpenoids with a labdane skeleton (C 20 H 32) is based on a tricyclic system (five-membered A ring + six-membered B / C ring, commonly modified with Δ8 or Δ14 double bonds, epoxy, hydroxyl or carboxyl groups, and has diverse stereostructures (such as (12R, 13R)-epoxy or 8R / 8S hydroxylation). Its stereostructure, double bond position, and the type and number of its substituents will result in different biological activities and utilization values. The laurdanoid diterpenoid cis-abieol from tobacco can achieve antibacterial and insecticidal effects by inhibiting the growth of hyphae and inhibiting the feeding of insect larvae; sclareol shows good inhibitory activity against Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans), and can inhibit the metastasis of breast cancer cells by regulating the PPARγ pathway (IC50 = 8.3μM); α-cerene secreted by tobacco glandular hairs has a repellency rate of 65% for tobacco aphids (Myzus persicae) and is safe for bees. Cerebrotriene diol can dissolve cotton bollworms (Helicoverpa armigera epidermal wax layer, leading to dehydration and death (LC50 = 28 μg / cm 2 ), and its efficacy was increased by 40% when combined with the chemical pesticide chlorantraniliprole. Therefore, tobacco labdane-type diterpenoids have important development potential in the green control of plant diseases and insect pests.

[0004] (12R,13R)-8,12-epoxy-14-labden-13-ol is a diterpenoid epoxy alcohol compound containing a five-membered ring and two six-membered rings, with an epoxy group (8,12-epoxy), a hydroxyl group (13-ol) and a double bond modification (Δ14). Kingwell.C detected this compound in the resin of Rocky Mountain fir Abies lasiocarpa and proved that this substance can repel pine weevils (Pissodes strobi); this compound from thornless ironwood (Sideroxylon inerme) showed selective inhibitory effects on Staphylococcus aureus (MRSA) and Candida albicans (MIC = 16μg / mL); Alizadeh.N's in vitro test on liver cancer cells (HepG2) showed that (12R,13R)-8,12-epoxy-14-labden-13-ol can induce cell apoptosis through the mitochondrial apoptosis pathway (IC50 = 12.5μM), but has low toxicity to normal liver cells (LO2) (IC50>50μM).

[0005] Identifying compounds from natural products with novel structures, unique mechanisms of action, and environmental friendliness has become a hot topic in modern botanical pesticide research and development. As a natural product, there have been no reports of (12R,13R)-8,12-epoxy-14-labden-13-ol being directly isolated from tobacco, nor have there been any reports of its research and application in treating plant viral diseases.

[0006] Therefore, providing a plant-derived antiviral preparation and its preparation method and application is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a plant-derived antiviral preparation, a preparation method and an application thereof.

[0008] Research in the present invention shows that the compound (12R,13R)-8,12-epoxy-14-labden-13-ol is poorly soluble in water. An appropriate concentration of (12R,13R)-8,12-epoxy-14-labden-13-ol has a good control effect on tobacco virus diseases. However, an application concentration that is too high or too low cannot produce a good control effect. Therefore, the preparation of a pesticide formulation that can be applied to plants is of great significance for the efficient control of (12R,13R)-8,12-epoxy-14-labden-13-ol in plant virus diseases.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides a laudanum type compound derived from the secretion of tobacco glandular hairs

[0011] 12R,13R)-8,12-epoxy-14-labden-13-ol.

[0012] Application of the compound (12R,13R)-8,12-epoxy-14-labden-13-ol in preventing and controlling tobacco mosaic virus (TMV), potato virus Y (PVY) and cucumber mosaic virus (CMV).

[0013] A plant-derived antiviral preparation contains the following components by weight: 5-9% of (12R,13R)-8,12-epoxy-14-labden-13-ol, 20-30% of ethanol as a cosolvent, 15-25% of sucrose ester (SE) as a wetting agent, 6-8% of lignin sulfonate (QL-LS30) as a dispersant, 0.2-0.4% of magnesium aluminum silicate as a thickener, 2-4% of glycerol as an antifreeze agent, and the balance being deionized water.

[0014] Furthermore, the method for preparing the plant-derived antiviral preparation comprises the following steps:

[0015] The compound (12R,13R)-8,12-epoxy-14-labden-13-ol is dissolved in ethanol to obtain an ethanol solution of the compound; sucrose ester, lignin sulfonate and deionized water are stirred with a magnetic rod until completely dissolved, and then mixed with the ethanol solution of the compound; under stirring conditions, the mixture is poured into a sand mill and subjected to cyclic multi-stage grinding; then magnesium aluminum silicate is added and stirred and dissolved with a magnetic rod; finally, propylene glycol is added, stirring is continued, and the mixture is filtered through a 200-mesh sieve to obtain a plant-derived antiviral preparation.

[0016] Furthermore, the plant-derived antiviral preparation is used in preventing and treating tobacco mosaic virus disease, potato virus Y disease and cucumber mosaic virus disease.

[0017] It can be seen from the above technical solution that compared with the existing technology, the present invention discloses a plant-derived antiviral preparation, a preparation method and application thereof. The preparation has high antiviral activity and is significantly better than the plant-derived pesticide amino oligosaccharide in preventing tobacco mosaic virus disease, potato Y virus disease and cucumber mosaic virus disease. In addition, the preparation does not contain any toxic organic solvents and is safe for the environment, humans and animals. It solves the problems of poor prevention efficacy of existing plant-derived antiviral preparations, high chemical residues, and severe environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0019] Figure 1 The structural formula of (12R,13R)-8,12-epoxy-14-labden-13-ol of the present invention is:

[0020] Figure 2 The figure shows the effect of the compound (12R,13R)-8,12-epoxy-14-labden-13-ol of the present invention on the morphology of viral particles. A represents the treatment with 150 μg / mL, and the red arrow indicates the swelling of the viral particles. B represents the water control. C represents the treatment with 100 μg / mL, and the red arrow indicates the breakage of the viral particles. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1 Isolation and Identification of (12R, 13R)-8,12-epoxy-14-labden-13-ol

[0023] Tobacco was used as the test material, and was quickly rinsed with 95% ethanol for 1 minute. The residue was filtered to remove the ethanol extraction solution, and the tobacco glandular hair ethanol extract was obtained after nitrogen drying.

[0024] The monomer compounds of tobacco glandular ethanol extract were separated and purified by normal and reverse phase silica gel column chromatography, preparative thin layer chromatography and Sephadex LH-20 gel column chromatography, and their structures were identified by ultraviolet spectroscopy, nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectrometry (ESI).

[0025] Monomer compound: yellow colloid. ESI-MS m / z: 306 [M+H] + , indicating that the molecular formula of the compound is C 20 H 34 O2. 13 C NMR showed a total of 20 carbon signals, including 5 methyl groups, 7 methylene groups, 5 methine groups, and 3 quaternary carbon groups. 1 H NMR (500 MHz, CDCl3) δ H 5.94(dd,J=17.4,11.0Hz,1H),5.29(d,J=16.5Hz,1H),5.11(d,J=10.1Hz,1H),1.94(dt,J=11.9,3.2Hz ,1H),1.73(dq,J=13.8,3.7Hz,1H),1.67(dd,J=9.2,5.5Hz,2H),1.62(td,J=10.1,5.0Hz,1H),1.39(dd q,J=11.9,8.3,4.1Hz,4H),1.34(s,1H),1.27(dd,J=12.8,3.7Hz,1H),1.21(s,3H),1.15(d,J=4.6Hz,1 H),1.13(s,3H),1.00(td,J=12.8,4.1Hz,1H),0.93(dd,J=12.4,3.2Hz,3H),0.85(s,3H),0.80(s,6H); 13CNMR (CDCl3, 125MHz)δ C 144.3(C-14,CH3),112.5(C-15,CH2),85.3(C-12,CH3),81.3(C-8,CH2),73.2(C-1 3,CH3),60.8(C-9,C),57.1(C-5,C),42.5(C-3,C),40.8(C-1,CH2),40.2(C-7,CH2) ,36.4(C-10,CH),33.5(C-18,CH),33.1(C-4,CH),25.4(C-17,CH),24(C-11,CH3), 23.2(C-16,CH), 21.4(C-6,CH2), 21(C-19,CH2), 18.4(C-2,CH2), 15.9(C-20,CH3). After literature search, its NMR data were basically consistent with those reported in the literature, so the monomer compound was identified as (12R,13R)-8,12-epoxy-14-labden-13-ol, with the structural formula shown in Figure 1 .

[0026] Example 2 Evaluation of Antiviral Activity of Monomeric Compounds

[0027] The passivation activity and preventive effect of the compound (12R,13R)-8,12-epoxy-14-labden-13-ol on TMV at different concentrations were compared.

[0028] Take 1 g of K326 tobacco leaves infected with TMV virus, grind them with 100 mL of deionized water and quartz sand on an ice box for 2 minutes, and filter them with gauze to prepare virus juice.

[0029] Passivation test: using the juice friction method to measure different concentrations (50-150μg / mL)

[0030] The inactivation activity of (12R,13R)-8,12-epoxy-14-labden-13-ol against TMV was determined by first selecting healthy, uniformly growing, 5-6-leaf Nicotiana tabacum seedlings and evenly spreading quartz sand (600 mesh) over the inoculated leaves. (12R,13R)-8,12-epoxy-14-labden-13-ol was then diluted to 100, 200, and 300 μg / mL, and a 150-fold dilution of 0.5% amino oligosaccharide solution was prepared. The monomer compound at various concentrations and the 150-fold dilution of amino oligosaccharide were then mixed with an equal volume of virus sap. After 30 minutes, a sterile cotton swab dipped in the same concentration of virus sap (mixed with deionized water and an equal volume of virus sap) was gently rubbed onto half a leaf (CK). The corresponding half leaf was then inoculated with the mixture of the monomer compound and virus. A botanical pesticide, amino oligosaccharide solution, served as a positive control (CK1). 20 minutes after inoculation, rinse the leaves with distilled water to remove any remaining sap. Inoculate the top three expanded leaves of each tobacco plant. Repeat for 5 plants per treatment, repeat 3 times, and for a total of 15 Sansheng tobacco plants per treatment. 2-3 days after inoculation, when necrosis symptoms become apparent, collect the test results and calculate the control efficacy using the following formula:

[0031] Necrosis spot inhibition rate (%) = (number of control necrosis spots - number of treated necrosis spots) × 100 / number of control necrosis spots Prevention test: Select healthy and uniformly growing Sansheng tobacco seedlings at the 4-5 leaf stage, and use the same treatment concentration (50-150 μg / mL) as the passivation test. Spray 1 ml per plant, and spray again after 7 days. Inoculate TMV 24 hours later. The inoculation method and calculation of control effect are the same as those in the passivation test.

[0032] The results of the passivation activity and preventive activity are shown in Table 1.

[0033] Table 1 Anti-TMV activity of (12R,13R)-8,12-epoxy-14-labden-13-ol

[0034]

[0035] The results of the passivation and preventive activity tests showed that the compound's preventive and therapeutic efficacy increased with increasing concentration within the 50-150 μg / mL range, with the highest efficacy at 150 μg / mL. The passivation-induced necrosis inhibition rate was 72.34%, significantly higher than the positive control (35.64%) of amino oligosaccharides; the preventive-induced necrosis inhibition rate was 85.27%, significantly higher than the positive control (37.16%) of amino oligosaccharides. The compound's preventive activity against TMV was higher than its passivation activity (Table 1).

[0036] Observation of TMV granule morphology

[0037] The morphology of viral particles after inactivation by L (100 μg / mL) and H (150 μg / mL) (12R,13R)-8,12-epoxy-14-labden-13-ol was observed using transmission electron microscopy. Figure 2 As shown, TMV in normal physiological state presents regular long rod shape ( Figure 2 B). After H treatment, longitudinal expansion occurs, and the expansion at the end is more obvious ( Figure 2 A); L treatment resulted in transverse point-like fractures, which were short rod-like ( Figure 2 C), therefore, the treatment with better protective effect of the compound is more harmful to the virus.

[0038] Example 3 Preparation of (12R, 13R)-8,12-epoxy-14-labden-13-ol suspension

[0039] First, the (12R,13R)-8,12-epoxy-14-labden-13-ol compound from tobacco was dissolved in ethanol to obtain an ethanol solution of the compound. Then, sucrose ester, lignin sulfonate (QL-LS30), and deionized water were stirred with a magnetic rod until completely dissolved according to the proportions in Table 2, and mixed with the ethanol solution of the compound. Under stirring conditions, the mixture was poured into a sand mill and subjected to cyclic multi-stage grinding. Subsequently, magnesium aluminum silicate was added and dissolved by stirring with a magnetic rod. Finally, glycerol was added and stirring was continued for 30 minutes. The mixture was filtered through a 200-mesh sieve to remove unground particles and grinding medium residues, thereby obtaining a tobacco (12R,13R)-8,12-epoxy-14-labden-13-ol suspension (antiviral preparation).

[0040] Table 2 (12R,13R)-8,12-epoxy-14-labden-13-ol suspension concentrate formulation

[0041]

[0042] The obtained antiviral preparations were tested for active ingredient content using HPLC (GB / T14825-2023), with the active ingredient contents reaching 5%, 7%, and 9%, respectively. Three different concentrations of (12R,13R)-8,12-epoxy-14-labden-13-ol preparations were also quality-tested. The test results are shown in Table 3.

[0043] Table 3 Quality Specifications of (12R,13R)-8,12-epoxy-14-labden-13-ol Suspension Concentrate

[0044]

[0045] Example 4: Prevention and Control of Viral Diseases in Fields Using Antiviral Preparations

[0046] 1) Experimental design

[0047] The experiment was conducted at the Qingdao Jimo Experimental Farm of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences from 2023 to 2024. The (12R,13R)-8,12-epoxy-14-labden-13-ol antiviral preparation (active ingredient 7%) was diluted into 200, 300 and 500 times of solution, respectively, and PVY, CMV and TMV viral disease prevention and control tests were carried out.

[0048] 2) Experimental materials and methods

[0049] The test materials were the flue-cured tobacco variety K326, a 200 / 300 / 500-fold dilution of the antiviral preparation (12R,13R)-8,12-epoxy-14-labden-13-ol, and a 150-fold dilution of a 0.5% amino oligosaccharide solution. Applications were made once during the clustering stage of the tobacco plants and again 10 days later for a total of two sprayings. Disease was allowed to develop naturally in the field, and disease activity was monitored during the vigorous growth phase.

[0050] 3) Statistical methods

[0051] The disease index is implemented in accordance with GB / T 23222-2008 Grading and investigation methods for tobacco pests and diseases.

[0052] Disease index (DI)

[0053]

[0054] Where: DI - disease index

[0055] Ni - number of diseased leaves (plants) at each level

[0056] i - the corresponding severity level of the disease

[0057] N-Total number of leaves (plants) surveyed

[0058] Control effect = (disease index of drug treatment - disease index of blank control) / disease index of drug treatment * 100%.

[0059] 4) The test results are shown in Table 4.

[0060] Table 4 Antiviral efficacy of (12R,13R)-8,12-epoxy-14-labden-13-ol antiviral suspension concentrate in the field

[0061]

[0062] As can be seen from Table 4, different concentrations of antiviral suspension concentrates have good control effects on tobacco TMV, CMV and PVY, and are significantly better than the plant-based antiviral agent amino oligosaccharides. Among them, the 300-fold solution has the best control effect, and the 500-fold solution has the least control effect.

[0063] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A plant-derived antiviral preparation, characterized in that: The invention contains the following components by weight: 5-9% of (12R, 13R)-8,12-epoxy-14-labden-13-ol, 20-30% of ethanol, 15-25% of sucrose ester, 6-8% of lignin sulfonate, 0.2-0.4% of magnesium aluminum silicate, 2-4% of glycerol, and the balance is deionized water.

2. The method for preparing a plant-derived antiviral preparation according to claim 1, characterized in that: The following steps are involved: The compound (12R,13R)-8,12-epoxy-14-labden-13-ol was dissolved in ethanol to obtain an ethanol solution of the compound; sucrose ester, lignin sulfonate and deionized water were stirred with a magnetic bar until completely dissolved, and then mixed with the ethanol solution of the compound; Under stirring conditions, the mixed liquid is poured into a sand mill for cyclic multi-stage grinding; then magnesium aluminum silicate is added and stirred and dissolved with a magnetic rod; finally, propylene glycol is added, stirring is continued, and the mixture is filtered through a 200-mesh sieve to obtain a plant-derived antiviral preparation.

3. Use of the plant-derived antiviral preparation according to claim 1 in preventing and treating tobacco mosaic virus disease, potato virus Y disease and cucumber mosaic virus disease.

4. Application of the compound (12R,13R)-8,12-epoxy-14-labden-13-ol in the prevention and treatment of tobacco mosaic virus disease, potato virus Y disease and cucumber mosaic virus disease.

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