Steroid alkaloid, plant anti-freezing composition and application of plant anti-freezing composition in improvement of plant cold resistance

Abstract

The invention relates to the technical field of plant antifreezing, in particular to steroid alkaloid, a plant antifreezing composition and application of the plant antifreezing composition to improvement of plant cold resistance. The steroidal alkaloid provided by the invention is sprayed on a potato cultivar Desiree, so that the damage of freezing injury to potato leaf cells can be effectively reduced, the freezing injury tolerance and survival rate of plants are improved, the influence of low temperature and freezing injury on crops is reduced, a practical basis is provided for improving the freezing injury tolerance of the plants by utilizing the steroidal alkaloid compound, and the steroidal alkaloid compound has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of plant frost protection, and specifically relates to steroidal alkaloids, plant antifreeze compositions and their applications in improving plant cold tolerance. Background Art

[0002] The ability of plants to resist low-temperature stress depends on the physiological state of the plants, or rather, the different antifreeze abilities of the plants. When the environmental temperature drops below 10°C, plants will be subjected to low-temperature stress to varying degrees. Under low-temperature conditions, the fluidity and stability of cell membranes decrease, the level of malondialdehyde increases, and the enzyme activity and catalytic efficiency in cells decline, resulting in disorders of intracellular metabolic pathways and functions. In addition, the free water in plants will freeze at low temperatures, causing mechanical damage to cells and even leading to the death of plants due to freezing in severe cases.

[0003] As an important food crop, potatoes are relatively sensitive to low temperature and frost damage, which will seriously affect their growth and development and yield. At present, improving the frost resistance of potatoes mainly relies on variety breeding and cultivation management measures. However, the breeding of new crop varieties and low-temperature domestication have a long cycle, and are also affected by regional ecological adaptability and other factors, so the application effect is not obvious. Therefore, it is of great practical application value to develop a class of antifreeze agents that can quickly improve the frost tolerance of potatoes. At present, there is no report on the use of steroidal alkaloids in improving plant cold tolerance. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides steroidal alkaloids and plant antifreeze compositions, and proves that they can effectively reduce the damage caused by low-temperature frost to potatoes, improve the survival rate of potato seedlings, significantly improve the disease resistance, cold resistance and the antifreeze ability against the cold snap in spring of potatoes, effectively prevent the attack of early and late frosts, and avoid or reduce frost damage.

[0005] One object of the present invention is to provide the application of steroidal alkaloids or their derivatives, or their precursor metabolites, or their pharmaceutically acceptable salts in enhancing the frost tolerance of plants.

[0006] The following applications are also within the protection scope of the present invention: The application of steroidal alkaloids or their derivatives in the preparation of drugs for enhancing the frost tolerance of plants.

[0007] The application of steroidal alkaloids or their precursor metabolites in the preparation of drugs for enhancing the frost tolerance of plants.

[0008] The application of steroidal alkaloids or their pharmaceutically acceptable salts in the preparation of drugs for enhancing the frost tolerance of plants.

[0009] Further, the drug uses steroidal alkaloids or their derivatives, or their precursor metabolites, or their pharmaceutically acceptable salts as active ingredients, and is made into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0010] Further, the steroidal alkaloids are selected from solasonine and / or tomatine.

[0011] Unless otherwise indicated, all stereoisomers, geometric isomers, tautomers, N-oxides, hydrates, solvates, metabolites, salts and pharmaceutically acceptable prodrugs of the steroidal alkaloids of the present invention fall within the scope of the present invention.

[0012] A second object of the present invention is to provide a plant antifreeze composition, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is steroidal alkaloids or their derivatives, or their precursor metabolites, or their pharmaceutically acceptable salts.

[0013] The "plants" referred to in the present invention include cereals such as durum wheat and other wheats, rye, barley, triticale, oats, rice or maize (forage maize and sweet maize / sweet corn and field maize); sugar beet, such as sugar beet or fodder beet; fruits, such as pomes, drupes or berries, such as apples, pears, plums, peaches, nectarines, almonds, cherries, papayas, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as kidney beans, lentils, peas, alfalfa or soybeans; oil plants, such as rape (oilseed rape), Brassica rapa, mustard, olive, sunflower, coconut, cocoa bean, castor oil plant, oil palm, peanut or soybean; cucurbitaceous plants, such as squash, pumpkin, cucumber or melon; fiber plants, such as cotton, flax, hemp or jute; citrus fruits, such as oranges, lemons, grapefruits or tangerines; vegetables, such as eggplant, spinach, lettuce (such as iceberg lettuce), chicory, cabbage, asparagus, cabbage, carrots, onions, garlic, leeks, tomatoes, potatoes, gourds or bell peppers; laurel plants, such as avocados, cinnamon or camphor; energy and raw material plants, such as maize, soybeans, rapeseed, sugar cane or oil palm; tobacco; nuts, such as walnuts; pistachios; coffee; tea; bananas; grapevines (table grapes and wine grapes); hops; stevia (also known as Stevia); natural rubber plants or ornamental and forest plants, such as flowers (such as carnations, petunias, geraniums / geraniums, pansies and impatiens), shrubs, broad-leaved trees (such as poplars) or evergreens, such as conifers; eucalyptus; turf; lawn; grasses such as grasses for animal feed or ornamental use. Preferred plants include potatoes, sugar beets, tobacco, wheat, rye, barley, oats, rice, maize, cotton, soybeans, rapeseed, leguminous plants, sunflowers, coffee or sugar cane; fruits; grapevines; ornamental plants; or vegetables, such as cucumbers, tomatoes, kidney beans or squash.

[0014] In some specific embodiments, the plant antifreeze composition further comprises at least one other antifreeze agent that enhances the frost tolerance of plants.

[0015] Those skilled in the art are fully aware that the other antifreeze agents that enhance the frost tolerance of plants referred to in the present invention are substances used to lower the freezing point of the composition liquid and improve the antifreeze ability, and are selected from any one or more of the following: ethylene glycol, propylene glycol, butanol, glycerol, urea.

[0016] In some specific embodiments, the steroidal alkaloids are selected from solasonine and / or tomatine.

[0017] In some specific embodiments, the amount of the active ingredient is 0.001 - 90 wt% relative to the total weight of the composition; for example, 0.02 - 50 wt%; or 0.1 - 20%; or 0.005 - 5%.

[0018] Those skilled in the art are fully aware that the pharmaceutically acceptable excipients are generally recognized for this purpose and serve as inactive ingredients of the medicament.

[0019] The excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants. Among them, the dispersant is used to reduce the aggregation of solid or liquid particles in the dispersion system of the composition. When preparing wettable powders, water-dispersible granules, water-dispersible tablets, suspensions, and oil suspensions, adding a dispersant is easy to form a dispersion and a suspension, and maintain the relative stability of the dispersion system. The dispersants provided by the present invention can be selected from any one or more of the following: phosphate esters, sulfonates, alkylnaphthalene sulfonate condensates, carboxylate polymers, polycarboxylate polymer modified resins, alkyl sulfates, modified alkyl sulfonates, sodium alkylnaphthalene sulfonate condensates, naphthalene sulfonates (condensates), polycarboxylate polymer dispersants, polyphenylethylene phenyl ether phosphate dispersants, naphthalene sulfonate formaldehyde condensates, alkylphenol polyoxyethylene ethers, formaldehyde condensate sulfonates, lignin sulfonates, sodium maleic acid-acrylic acid copolymers, alkyl sulfosuccinates; such as Atlox Metasperse 550S, Dispersol BB4, Dispersol CBZ, Terwet 1004, Tersperse 2700, Morwet D-450, Borresperse CA-SA, sodium tripolyphosphate, 200 solvent oil, Nekal BX, etc.

[0020] An emulsifier can promote the formation of a stable emulsion from two immiscible liquids in a composition and is also a stabilizer for the emulsion. The emulsifier provided in the present invention can be any one or more of the following: calcium dodecylbenzenesulfonate, triphenylethylphenol polyoxyethylene ether, alkylphenol formaldehyde resin polyoxyethylene ether, fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, castor oil polyoxyethylene ether, nonylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene polyoxypropylene ether, modified fatty alcohol polyoxyethylene ether, fatty amine polyoxyethylene ether, alkylaryl polyoxyethylene polyoxypropylene ether, EO-PO block polyether, oleic acid polyoxyethylene ether, phenylethylphenol polyoxyethylene ether, nonylphenol phosphate; such as YUS-110, YUS-EP60P, Ethylan NS-500LQ (nonionic hydroxy polyoxyethylene block copolymer), sodium styrene-maleic anhydride copolymer, etc.

[0021] A suspending agent is used to increase the viscosity of the dispersion medium in the composition to reduce the sedimentation rate of the particles or increase the hydrophilicity of the particles. The suspending agent provided in the present invention can be selected from any one or more of the following: phosphate esters, sulfonates, alkylnaphthalenesulfonate condensates, carboxylate polymers, modified polycarboxylate polymer resins, alkyl sulfates, modified alkyl sulfonates, sodium alkylnaphthalenesulfonate condensates, naphthalenesulfonates (condensates), polycarboxylate polymer dispersants, polyphenylethylene phenyl ether phosphate dispersants, naphthalenesulfonate formaldehyde condensates, alkylphenol polyoxyethylene ether formaldehyde condensate sulfonates, lignosulfonates, silica, magnesium aluminum silicate; for example, Tersperse 2500, Tersperse 4894, soybean oil.

[0022] An antifoaming agent is used to remove harmful foams during the production process. The antifoaming agent provided in the present invention can be selected from one or more of GP type antifoaming agent, PE type antifoaming agent, GPES type antifoaming agent, SAG1522 type antifoaming agent, silicone, polyether modified silicone, one or more of polysiloxanes.

[0023] An antifoaming agent is used to remove harmful foams during the production process. The antifoaming agent provided in the present invention can be selected from one or more of GP type antifoaming agent, PE type antifoaming agent, GPES type antifoaming agent, SAG1522 type antifoaming agent, silicone, polyether modified silicone, polysiloxanes.

[0024] A binder is a substance used to bond the surfaces of homogeneous or heterogeneous objects in a composition together. The binder provided in the present invention can be selected from one or more of sodium carboxymethyl cellulose, polyvinylpyrrolidone, starch, polyvinyl alcohol, methyl cellulose, fructose, such as corn starch, cyclohexanone.

[0025] Thickeners are used to increase the viscosity of the dispersion medium in the composition to reduce the sedimentation rate of the fine particles and improve the layering of the liquid composition. The thickeners provided in the present invention are selected from any one or more of the following: xanthan gum, polyethylene glycol 4000, polyethylene glycol 6000, gum arabic, gelatin, epoxy soybean oil.

[0026] Film-forming agents are used to adhere the active ingredients to the surface of the seeds to form a smooth drug film. In the present invention, The film-forming agent can be selected from one or more of polyvinyl alcohol, polyvinyl acetate, carboxymethyl cellulose, arabic gelatin, gelatin, xanthan gum, such as film-forming agent BF308, dodecyl alcohol ester.

[0027] pH regulators are used to adjust the acidity and alkalinity of the composition. The pH regulators provided in the present invention are selected from any one or more of the following: citric acid, sodium bicarbonate, diethylamine, triisopropanolamine, phosphoric acid, glacial acetic acid.

[0028] The suspending agents are selected from any one or more of the following: magnesium aluminum silicate, fumed silica, ammonium sulfate, diatomaceous earth, attapulgite, bentonite, kaolin, calcite, talc, montmorillonite, calcium carbonate, silica, soluble starch, insoluble starch.

[0029] Those skilled in the art are fully aware that the composition can be prepared according to methods known in the art. For this purpose, if necessary, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form a suitable dosage form or administration form for human use.

[0030] In addition, if necessary, colorants, preservatives, fragrances, flavoring agents or other materials can also be added to the composition.

[0031] The dosage form of the above plant antifreeze composition is any one of the following: solution, microemulsion, water-in-oil emulsion, emulsifiable concentrate, wettable powder, suspension, suspoemulsion, water dispersible granule, soluble powder, granule, oil suspension, microcapsule.

[0032] The third object of the present invention is to provide a method for improving the cold tolerance of plants. Before the freeze damage occurs for 3 to 7 days, the above composition is applied to the plants.

[0033] During the application process, the above plant antifreeze composition can be applied to one or more of the stems, leaves, seeds, fruits, roots of plants or the soil, and preferably applied to the leaf surface of plants.

[0034] Among them, the application method of the above plant antifreeze composition can be one or more of dipping, spraying, evaporation, atomization, broadcasting, brushing, etc. Preferably, the spraying method is adopted.

[0035] In some specific embodiments, before foliar application, the composition is dissolved in water to prepare an antifreeze agent with a concentration of 5-15 μM; the application amount per plant is 10 mL / day.

[0036] Beneficial effects: Spraying the potato cultivar Desiree with the steroidal alkaloids provided by the present invention can effectively reduce the damage of potato leaf cells caused by frost damage, improve the frost damage tolerance and survival rate of the plants, and reduce the impact of low temperature and frost damage on crops, providing a practical basis for using steroidal alkaloids to improve plant frost damage tolerance, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the effect of steroidal alkaloids on the degree of freeze damage of potato; * indicates that compared with the blank group, P <0.05; 1A is a diagram of plant phenotypic differences, and 1B is a diagram of plant survival; Figure 2 The graph of the effect of steroidal alkaloids on the conductivity of potato plants; *** indicates that compared with the blank group, P <0.001. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following is a detailed description of the present invention. The present invention is described in detail. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar to or equivalent to the recorded contents can be applied to the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0039] The endpoints of ranges and any values ​​disclosed herein are not limited to the precise range or value. Ranges or values ​​should be understood to include values ​​close to these ranges or values. The ranges can be between the endpoints of the ranges, between the endpoints of the ranges and the individual point values, and between the individual point values. The numerical ranges can be combined with each other to obtain one or more new numerical ranges, which should be regarded as having The body is open.

[0040] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. Solution: The following examples are only used to explain the present invention and are not used to limit the present invention.

[0041] Example 1 Plant antifreeze composition Weigh 10 mg of solasonine and add it to 11.3 mL of dimethyl sulfoxide solution, and mix evenly.

[0042] Example 2 Weigh 5 mg of tomatine and add it to 12 mL of dimethyl sulfoxide solution, and mix evenly.

[0043] Example 3 Plant antifreeze composition Weigh 5 mg of solasonine and add it to 1 mL of dimethyl sulfoxide solution, and mix evenly.

[0044] Example 4 Plant antifreeze composition Weigh 150 mg of solasonine and add it to 1 mL of dimethyl sulfoxide solution, and mix evenly.

[0045] Example 5 Plant antifreeze composition Weigh 1 mg of tomatine and add it to 1 mL of dimethyl sulfoxide solution, and mix evenly.

[0046] Example 6 Plant antifreeze composition Weigh 0.2 mg of tomatine and add it to 1 mL of dimethyl sulfoxide solution, and mix evenly.

[0047] Example 6 Plant antifreeze composition Weigh 1 g of tomatine and add it to 1 mL of dimethyl sulfoxide solution, and mix evenly.

[0048] Example 7 Investigation of the effect on reducing the degree of potato freezing injury Experimental materials: The plant materials used in this study were the cultivated potato variety Desiree. They were grown on MS medium supplemented with 2% sucrose and 0.31% agar at 20 ± 1 °C for 2 weeks. Then, the 2-week-old tissue culture seedlings were transplanted into plastic pots (10 × 10 cm) containing a substrate (the volume ratio of nutrient soil to perlite was approximately 3:1) and planted in a plant growth climate chamber (22 ± 2 °C, 16 h light / 8 h dark photoperiod).

[0049] Prepare the antifreeze agent: Take the plant antifreeze composition and add it to pure water, and mix evenly to prepare a 10 μM antifreeze agent.

[0050] Experimental method: Four-week-old plants grown under normal conditions were transferred to a cold room at 4°C and cold acclimated for 7 days under a 16 h light / 8 h dark light cycle. During the low-temperature acclimation period, the plants were sprayed once a day with the antifreeze agent prepared in Example 1, the antifreeze agent prepared in Example 2, or a control reagent (pure water containing the same proportion of DMSO) using a portable sprayer, and about 10 mL was sprayed on each plant material. Then, after the cold-acclimated plants were treated at -2°C for 12 h, the growth status of the plants was observed and (as Figure 1 shown).

[0051] As Figure 1 can be seen from A, after the freeze injury treatment, the leaves, petioles, and young stems of the potato plants treated with the control reagent were severely wilted and severely damaged; in contrast, the potato plants sprayed with solasonine or tomatine grew well and no obvious tissue damage was observed.

[0052] As Figure 1 can be seen from B, after the potato plants treated with freeze injury were transferred to a plant growth climate chamber (22 ± 2°C, 16 h light / 8 h dark light cycle) for recovery culture for 1 day, the survival rate of the potato plants sprayed with the control reagent was less than 20%, while the survival rate of the potato seedlings sprayed with solasonine and tomatine was above 60%, which was significantly higher than that of the control group materials.

[0053] Example 8 Investigation of the effect on enhancing the freeze tolerance of potatoes Experimental materials: The plant materials used in this study were the cultivated potato variety Desiree. They were grown on MS medium supplemented with 2% sucrose and 0.31% agar at 20 ± 1°C for 2 weeks, and then the 2-week-old tissue culture seedlings were transplanted into plastic pots (10 × 10 cm) containing a substrate (the volume ratio of nutrient soil to perlite was about 3:1) and planted in a plant growth climate chamber (22 ± 2°C, 16 h light / 8 h dark light cycle).

[0054] Preparation of antifreeze agent: 1 mL of the plant antifreeze composition was added to 100 mL of pure water and mixed to prepare a 10 μM antifreeze agent.

[0055] Experimental method: Four-week-old plants grown under normal conditions were transferred to a cold chamber at 4°C and cold acclimated for 7 days under a 16 h light / 8 h dark light cycle. During the low-temperature acclimation period, the plants were sprayed once a day with the antifreeze agent of Example 1, the antifreeze agent of Example 2, or a control reagent (pure water containing the same proportion of DMSO) using a portable sprayer, and about 10 mL was sprayed on each plant material. Then, after the cold-acclimated plants were treated at -2°C for 12 h, the leaves were taken to detect the conductivity. The specific detection process was as follows: The top leaves were put into a centrifuge tube, 10 mL of deionized water was added, and the measured conductivity value was ECi; shaken at about 100 rpm for 4 hours at room temperature, and the measured conductivity value was ECf; after boiling for 20 min, shaken at about 100 rpm for 1 hour at room temperature, and the measured conductivity value was ECt. The electrolyte leakage rate was calculated according to the following formula: (ECf - ECi) / (ECt - ECi) × 100. The experimental data were analyzed using Student’s t test statistical software, and the results are as Figure 2 shown.

[0056] As can be seen from Figure 2 it, before the freeze injury treatment, the electrolyte leakage rates of the leaves of all potato plants were similar and maintained at a low level; after the freeze injury treatment, the electrolyte leakage rate of the leaves of the potato plants treated with the control reagent increased by more than 3 times, indicating serious cell damage; while the electrolyte leakage rate of the potato leaves sprayed with solasodine and tomatine only increased slightly and was significantly lower than that of the control group materials.

[0057] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without departing from the purpose and claims of the present invention. Such transformations all fall within the protection scope of the present invention.

Claims

1. Use of a steroid alkaloid or its derivative, or its precursor metabolite, or its pharmaceutically acceptable salt in enhancing the frost tolerance of plants, wherein, The steroidal alkaloids are selected from solasonine and tomatine; the plant is potato.

2. The application according to claim 1, wherein Use in the preparation of a medicament for enhancing the frost tolerance of plants.

3. The application according to claim 2, characterized in that: The medicament uses a steroidal alkaloid or its derivative, or its precursor metabolite, or its pharmaceutically acceptable salt as the active ingredient, and is made into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

4. A plant antifreeze composition, characterized in that, It includes an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is a steroidal alkaloid or its derivative, or its precursor metabolite, or its pharmaceutically acceptable salt.

5. The plant antifreeze composition according to claim 4, characterized in that, It further includes other antifreezes, and the antifreeze is one or more of ethylene glycol, propylene glycol, butanol, glycerol, and urea.

6. The plant antifreeze composition according to claim 4 or 5, characterized in that The steroidal alkaloids are selected from solasonine and / or tomatine.

7. The plant antifreeze composition according to claim 4, wherein The amount of the active ingredient is 0.001 - 90 wt% relative to the total weight of the composition; or 0.02 - 50 wt%; or 0.1 - 20%; or 0.005 - 5%.

8. A method for improving the cold tolerance of plants, characterized in that, 3 to 7 days before the onset of frost damage, apply the plant antifreeze composition according to claims 4 - 7 to the plants.

9. The method according to claim 8, wherein Before use for foliar application, dissolve the plant antifreeze composition in water to prepare an antifreeze with a concentration of 5 - 15 μM; the application amount per plant is 10 mL / day.

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