Application of a pinosylvin saponin compound in resisting birnavirus

Abstract

This invention relates to the field of plant virus inhibition technology, and particularly to the application of a steroidal saponin compound in the treatment of geminiviruses. The application of the steroidal saponin compound in inhibiting geminiviruses, and / or in the preparation of drugs for inhibiting geminiviruses, specifically tobacco stem rust virus (TbCSV), is as follows: A steroidal saponin aglycone -3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside is weighed, dissolved in dimethyl sulfoxide, vortexed until completely dissolved, and then diluted with deionized water to prepare a stock solution; the stock solution is then applied to the target plants. This invention provides the application of a phenobarbital saponin compound in the fight against bifidobacteria virus, specifically the application of phenobarbital-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside in the inhibition of tobacco stem virus, aiming to fill the gap in the existing technology regarding the anti-TbCSV activity of compounds.

Description

Technical Field

[0001] This invention relates to the field of plant virus inhibition technology, and in particular to the application of a phenobarbital saponin compound in the fight against geminiviruses. Background Technology

[0002] Tobacco curly shoot virus (TbCSV) belongs to the genus *Phragmites australis* in the family Geminidaceae. It is a highly damaging single-stranded circular DNA virus affecting plants. This virus is primarily transmitted persistently by whiteflies and can infect various important economic crops, including tobacco, tomatoes, peppers, and cotton. Infected plants often exhibit leaf curling, yellowing, stunted growth, and reduced fruit set, causing significant economic losses to agricultural production.

[0003] Currently, field control of viral diseases caused by geminiviruses such as TbCSV mainly relies on the following strategies: First, planting crops containing resistance genes (such as...). The methods of control include two main approaches: first, controlling crop varieties (such as [crops]); and second, using chemical pesticides to control the population of the vector insect, the whitefly, thereby interrupting the transmission chain of the virus. However, these existing control methods have significant limitations. Regarding resistant varieties, traditional resistant breeding processes are lengthy and costly, and the rapid mutation rate of geminitrovirus genomes means that long-term selection pressure from a single resistance gene can easily lead to the emergence of resistance-breakthrough strains, causing resistant varieties to lose their resistance. Regarding chemical pesticides, long-term and excessive use has led to whitefly populations developing resistance to multiple pesticides, increasing control costs; simultaneously, pesticide residues pose a certain impact on soil, water bodies, and agricultural product safety. Therefore, there is an urgent need to develop new anti-TbCSV active substances. To this end, we designed a phenobarbital compound for the application of anti-gemini virus, and provided the application of phenobarbital-3β-O-α-L-pyranorhamnosyl-(1→4)-[O-α-L-pyranorhamnosyl-(1→2)]-O-β-D-pyranoside in inhibiting tobacco stem virus, aiming to fill the gap in the existing technology of this compound's anti-TbCSV activity. Summary of the Invention

[0004] Therefore, it is necessary to provide an application of phenobarbital saponins in the treatment of bifidobacteria, in order to solve the technical problems mentioned in the background section.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] The use of steroidal saponins in inhibiting geminiviruses, and / or in the preparation of drugs for inhibiting geminiviruses, wherein the steroidal saponins are at least one of the following:

[0007] (a) Pyrnosapogenin-3β-O-α-L-rhamnosyl-(1→4)-[O-α-L-rhamnosyl-(1→2)]-O-β-D-glucopyranoside;

[0008] (b) Saponin compounds consisting of pennosapogenin and 1-4 glycosyl groups linked by 1→2, 1→3 or 1→4 glycosidic bonds;

[0009] (c) Saponin compounds with pinotore saponin as the aglycone and rhamnose in the sugar chain replaced by glucose, galactose or arabinose;

[0010] (d) Spirostanol or furostanol saponins with a steroidal skeleton similar to that of pinotoresiderin.

[0011] Preferably, the twin virus is a tobacco stem virus.

[0012] Preferably, the application method is as follows:

[0013] Weigh out phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside, dissolve it in dimethyl sulfoxide, vortex until completely dissolved, and then add deionized water to make up to volume to prepare a mother liquor;

[0014] The mother liquor was applied to the target plants.

[0015] Preferably, applying the mother liquor to the target plant includes at least one of the following:

[0016] Injection, foliar spraying, root irrigation, seed soaking, and wound application.

[0017] Preferably, the target plant is:

[0018] Solanaceae crops or all other host plants that TbCSV can infect.

[0019] Preferably, applying the mother liquor to the target plant includes:

[0020] Used in combination with antiviral compounds, and / or, plant immune inducers, and / or, chemical pesticides, and / or, biological control agents.

[0021] Preferably, the twin viruses also include any one of the following: Tomato Yellow Leaf Curl Virus (TYLCV), Chinese Tomato Yellow Leaf Curl Virus (TYLCCNV), and Tobacco Stem Curl Virus.

[0022] The application of phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside in reducing the localization of V1 protein in the cell nucleus, and / or the application in the preparation of drugs that reduce the localization of V1 protein in the cell nucleus.

[0023] 8. It is obvious without a doubt that the technical problems to be solved by the present invention can be solved by the above-described technical solutions of the present invention.

[0024] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects:

[0025] 1. The present invention provides the application of a phenobarbital saponin compound in the fight against geminiviruses, specifically the application of phenobarbital-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside in the inhibition of tobacco stem virus, aiming to fill the gap in the prior art of this compound in the field of the fight against geminiviruses.

[0026] 2. This invention is the first to discover that phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside has the activity of inhibiting TbCSV, which opens up new application directions for phenobarbitone compounds and provides new candidate molecules for screening antiviral active substances from natural products. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the chemical structural formula of the compound of the present invention;

[0029] Figure 2 A schematic diagram showing the symptoms of *Nicotiana benthamiana* treated with the foliar spraying method of the present invention 7 days after inoculation with virus;

[0030] Figure 3 This is a confocal microscope schematic diagram illustrating the effect of the compound of the present invention on the subcellular localization of V1-GFP.

[0031] Figure 4 This is a schematic diagram of the qPCR results of virus accumulation after foliar spraying according to the present invention;

[0032] Figure 5 This is a schematic diagram of the qPCR results of viral accumulation after injection in this invention;

[0033] Figure 6 This is a schematic diagram illustrating the effect of the compounds of the present invention on the fluorescence signal intensity of V1-GFP;

[0034] Figure 7 The three-dimensional structure of bait protein V1;

[0035] Figure 8 This is a schematic diagram of the molecular docking between the compound and the V1 protein. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0038] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.

[0039] The compound of this invention is phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside ( Figure 1 ), belonging to the steroidal saponin class of phenobarbital saponins, this compound has the CAS number 55916-52-4 and the Chinese name: phenobarbital-3β-O-α-L-pyranorhamnosyl-(1→4)-[O-α-L-pyranorhamnosyl-(1→2)]-O-β-D-pyranoside.

[0040] The English name is: Pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside. Its chemical structural formula is as follows: Figure 1 .

[0041] This compound is a natural steroidal saponin compound, which can be obtained by extraction and isolation from plants or by chemical synthesis.

[0042] To facilitate the representation of the compound "partanosapogenin-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside", in Figures 3-8 This compound is labeled as Compound B in China.

[0043] Example 1: This example mainly discloses the application of phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside in inhibiting tobacco stalk virus (TbCSV).

[0044] The application provided by this invention includes the following steps in its specific implementation:

[0045] (1) Preparation of the medicine solution

[0046] Weigh 5 mg of phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside and dissolve it in 100 μL of dimethyl sulfoxide (DMSO). Vortex until completely dissolved, then add deionized water to make up to 10 mL to prepare a stock solution.

[0047] Before use, dilute the stock solution with deionized water to the required concentration. If it is to be used for spraying, add 0.05% by volume of Tween-20 as a surfactant.

[0048] (2) Application method

[0049] This invention provides the following two application methods:

[0050] Method 1: Injection method

[0051] The stock solution of the compound of this application was mixed with Agrobacterium tumefaciens culture carrying a TbCSV infectious clone to achieve a final concentration of 20 μg / mL (or within the range of 10-100 μg / mL). The mixture was injected into the leaf tissue of *Tobacco Benzovia* using a sterile syringe. Three days after application, the accumulation of TbCSV in the leaves was detected by qPCR.

[0052] In one specific embodiment of the present invention, a leaf instantaneous co-inoculation system was used, with a compound treatment group and a solvent control group set on the left and right sides of the same leaf, respectively. The results showed that the relative accumulation of TbCSV in the compound treatment group was significantly reduced, approximately 50% of that in the control group.

[0053] Note: The "Agrobacterium tumefaciens culture carrying TbCSV infectious clones" in this method is only used for subsequent qPCR detection of virus accumulation and has no function related to compound adjuvants.

[0054] Method 2: Foliar spraying

[0055] The stock solution of the compound of this application was diluted with deionized water to 200 μg / mL (or in the range of 100-500 μg / mL), and 0.05% (volume fraction) of Tween-20 was added as a surfactant. The prepared solution was then evenly sprayed onto the surface of plant leaves by foliar spraying.

[0056] In one specific embodiment of the present invention, an application strategy of "pre-inoculation prevention + post-inoculation inhibition" is adopted: the first application is carried out 24 hours before TbCSV inoculation, and the second application is carried out 48 hours after TbCSV inoculation. The amount of pesticide solution sprayed per plant each time is 0.8 mL, ensuring that the spraying amount is the same for the treatment group and the control group. Seven days after inoculation, symptoms are observed and leaves are collected for DNA extraction, and the virus accumulation is detected by qPCR. The results show that the TbCSV accumulation in plants treated with the compound of this application is significantly reduced by about 30% compared with the control group, and the leaf curling symptoms are significantly alleviated (see...). Figure 2 ).

[0057] (3) Subcellular localization observation

[0058] In one embodiment of the present invention, confocal microscopy was used to observe the effect of the compound of this application on the subcellular localization of the V1-GFP fusion protein. The V1 protein is a TbCSV-encoded capillary protein (CP), which has been reported to participate in viral nucleoplasmic transport.

[0059] Figure 7 The three-dimensional structure of bait protein V1 is shown. The pink area represents the critical NLS region 1-77 aa, and the right image shows the surface form of this structure. Figure 8 A schematic diagram of the molecular docking between the compound and the V1 protein is shown.

[0060] The compound of this application was mixed with Agrobacterium tumefaciens bacterial suspension carrying the V1-GFP expression vector and TbCSV infectious clone (final compound concentration 20 μg / mL), and injected into tobacco leaves. A control was prepared by adding an equal volume of the same bacterial suspension (0.04% DMSO). The fluorescence signal distribution of V1-GFP was observed under a confocal microscope 72 hours after injection.

[0061] The observation results showed that, under the same imaging parameters, the fluorescence signal of V1-GFP in the cell nucleus of leaf cells treated with the compound of this application was reduced compared with the control group. Figure 3The fluorescence expression levels of cell nuclei were analyzed using ImageJ software under the same field of view. The results showed that the fluorescence expression levels in the compound-treated group were significantly lower than those in the control group. This indicates that this compound can reduce the localization of V1 in the cell nucleus.

[0062] (4) Detection of inhibitory effect

[0063] After application, the inhibitory effect can be evaluated by observing changes in plant symptoms, detecting the accumulation of TbCSV in the plant, and observing changes in the subcellular localization of viral proteins. The accumulation of virus can be detected using real-time quantitative PCR (qPCR), with a plant internal reference gene (such as 25S rRNA) as a control, to calculate the relative content of TbCSV. Figure 4 and Figure 5 ).

[0064] 3. Scope of application of the technical solution

[0065] The technical solution of this invention is applicable to host plants infected by tobacco stem rot virus (TbCSV), including but not limited to Nicotiana benthamiana, Nicotiana tabacum, tomato (Solanumlycopersicum), and pepper (Capsicum annuum) and other Solanaceae crops.

[0066] Of course, the technical solution of the present invention can be used alone or in combination with other antiviral substances or agricultural control measures.

[0067] Example 2, which discloses an analogue substitution based on the phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside based on Example 1.

[0068] Clearly, the compound in this application is pennosapogenin-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside, belonging to the pennosapogenin class of steroidal saponins. Based on the structure-activity relationship of this class of compounds, we infer that the following structural analogs have similar anti-TbCSV activity:

[0069] Saponin compounds composed of phenobarbital and different numbers (1-4) and different linkages (1→2, 1→3, 1→4, etc.);

[0070] Saponin compounds with pinotoresiderin as the aglycone and rhamnose in the sugar chain replaced by other monosaccharides such as glucose, galactose, and arabinose.

[0071] Other spirostanol or furostanol saponins with similar steroidal skeletons.

[0072] Example 3, this example is used to disclose the plant extract substitution based on the phenobarbitone-3β-O-α-L-pyranorhamnosyl-(1→4)-[O-α-L-pyranorhamnosyl-(1→2)]-O-β-D-pyranoside based on Example 1.

[0073] The compounds described in this application are naturally found in plants of the genera *Paris* and *Dioscorea*. Therefore, it is reasonable to expect that:

[0074] Plant extracts containing the compounds of this application (such as Paris rhizome extract and Dioscorea rhizome extract) also exhibit TbCSV inhibitory activity;

[0075] The above-mentioned plant extracts can be obtained through simple extraction processes (such as alcohol extraction, water extraction, ultrasonic extraction, etc.) without separation and purification to a single compound, and can be used as effective components against TbCSV.

[0076] Example 4, this example is mainly used to disclose alternative solutions related to the application method based on Example 1.

[0077] Example 1 has verified the effectiveness of injection and foliar spraying. Based on this, the following alternative application methods can be reasonably expected to be equally effective:

[0078] Root irrigation treatment: Dilute the compound of this application and irrigate it to the roots of plants. The compound is absorbed by the roots and transported to the above-ground parts to inhibit viral infection.

[0079] Seed soaking treatment: Before sowing, soak the seeds in the solution of the compound of this application to make the seeds drugged and prevent viral infection during the seedling stage;

[0080] Wound application: When plants are wounded due to agricultural operations (such as topping or pruning), the compound of this application is applied to the wound to block the invasion of viruses through the wound.

[0081] Example 5, which mainly discloses alternatives related to the application timing based on Example 1.

[0082] Example 1 verified the effectiveness of "spraying 24 hours before and 48 hours after infection". Based on this, it is reasonable to expect that the following application timing adjustments will also be effective:

[0083] Apply only before infection: for preventative protection, suitable for early prevention and control in areas with high viral prevalence;

[0084] Apply only after exposure: for therapeutic intervention after the onset of illness;

[0085] Multiple consecutive sprayings: For example, spraying once every 3-5 days for long-term control;

[0086] Adjustments should be made based on the virus infection patterns: for example, concentrated application should be made before the peak migration period of whiteflies.

[0087] Example 6, this example is mainly used to disclose alternative solutions related to the concentration range based on Example 1.

[0088] Example 1 validated the effectiveness of both injection (20 μg / mL) and foliar spraying (200 μg / mL). Based on this, the following concentration ranges are reasonably expected to be equally effective:

[0089] Injection method: 1-500 μg / mL, preferably 10-100 μg / mL, more preferably 20-50 μg / mL;

[0090] Foliar spraying: 50-1000 μg / mL, preferably 100-500 μg / mL, more preferably 150-300 μg / mL.

[0091] The specific optimal concentration can be determined by the applicant through multiple creative experiments, taking into account factors such as crop type, growth stage, and viral infection pressure.

[0092] Example 7, this example is mainly used to disclose alternative solutions related to the formulation and preparation based on Example 1.

[0093] The formulation used in Example 1 consisted of DMSO dissolution + water dilution + Tween-20 adjuvant. Based on this, it is reasonable to expect the following formulations to be equally effective:

[0094] Different solvents: Organic solvents such as ethanol, methanol, and acetone can replace DMSO;

[0095] Different surfactants: Tween-80, Span, sodium dodecyl sulfate (SDS), organosilicon additives, etc. can replace Tween-20;

[0096] Different formulations: It can be prepared into commonly used agricultural formulations such as soluble concentrates, emulsifiable concentrates, microemulsions, water-in-oil emulsions, suspensions, wettable powders, and water-dispersible granules;

[0097] Sustained-release formulations: The compounds of this application are encapsulated in a sustained-release carrier (such as cyclodextrin, chitosan, nanomaterials, etc.) to prolong the duration of effect.

[0098] Example 8, this example is mainly used to disclose related alternatives based on the combination of Example 1.

[0099] The compounds in this application can be used alone or in combination with other antiviral substances or agricultural control measures to enhance control efficacy. Reasonably foreseeable combination regimens include:

[0100] It can be used in combination with other antiviral compounds (such as ningnanmycin, lentinan, ribavirin, etc.);

[0101] It can be used in combination with plant immune inducers (such as salicylic acid, jasmonic acid, chitosan, etc.);

[0102] Used in combination with biological control agents (such as Beauveria bassiana, Metarhizium anisopliae, etc.).

[0103] Example 9, which is mainly used to disclose the expanded alternatives to the target virus and host plant range based on Example 1.

[0104] Example 1 has verified the inhibitory effect of this compound on TbCSV in Nicotiana benthamiana. Based on the conservation of geminiviruses and the known activities of similar compounds, it is reasonable to expect:

[0105] Expansion of target viruses: This compound may also have inhibitory effects on other isolates such as Tomato Yellow Leaf Curl Virus (TYLCV), Tomato Yellow Leaf Curl Virus (TYLCCNV), and Tobacco Stem Curl Virus.

[0106] Host plant expansion: This compound can be used on all host plants that TbCSV can infect, including but not limited to tobacco, tomato, pepper, cotton, cassava, soybean and other economic crops.

[0107] Example 10: This example mainly discloses alternative solutions related to the mechanism of action of Example 1.

[0108] Based on the subcellular localization observation results of Example 1 (reduced intranuclear fluorescence signal of V1-GFP), it is reasonable to expect:

[0109] This compound may exert its antiviral effect by interfering with the nuclear input process of the V1 protein;

[0110] Based on this mechanism, the compound may also be effective against other viruses that rely on similar nuclear input mechanisms, especially other geminiviruses.

[0111] Therefore, any related applications that use "interference with viral capsid protein nuclear input" as a mechanism should be considered as equivalent alternatives to this patent.

[0112] Based on the above, this application is not limited to Embodiment 1, and all embodiments should be within the protection scope of this application.

[0113] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The use of steroidal saponins in inhibiting geminiviruses, and / or in the preparation of drugs for inhibiting geminiviruses, characterized in that, The steroidal saponin compound is at least one of the following: (a) Pyrnosapogenin-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside; (b) Saponin compounds consisting of pennosapogenin and 1-4 glycosyl groups linked by 1→2, 1→3 or 1→4 glycosidic bonds; (c) Saponin compounds with pinotore saponin as the aglycone and rhamnose in the sugar chain replaced by glucose, galactose or arabinose; (d) Spirostanol or furostanol saponins with a steroidal skeleton similar to that of pinotoresiderin.

2. The application according to claim 1, characterized in that, The aforementioned twin virus is tobacco stem virus.

3. The application according to claim 1, characterized in that, The application method is as follows: Weigh out 3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside, dissolve it in dimethyl sulfoxide, vortex until completely dissolved, and then add deionized water to make up to volume to prepare a stock solution. The mother liquor was applied to the target plants.

4. The application according to claim 3, characterized in that, The application of the stock solution to the target plant includes at least one of the following: Injection, foliar spraying, root irrigation, seed soaking, and wound application.

5. The application according to claim 3, characterized in that, The target plant is: Solanaceae crops or all other host plants that TbCSV can infect.

6. The application according to claim 3, characterized in that, The application of the mother liquor to the target plant includes: Used in combination with antiviral compounds, and / or, plant immune inducers, and / or, chemical pesticides, and / or, biological control agents.

7. The application according to claim 2, characterized in that, The twin viruses also include any one of the following: Tomato Yellow Leaf Curl Virus (TYLCV), Chinese Tomato Yellow Leaf Curl Virus (TYLCCNV), and Tobacco Stem Curl Virus.

8. The application of phenobarbitone-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside in reducing the localization of V1 protein in the cell nucleus, and / or the application in the preparation of drugs that reduce the localization of V1 protein in the cell nucleus.

Images