Steroidal alkaloids, plant antifreeze compositions and their use in improving plant cold resistance

Abstract

The present invention relates to the field of plant frost protection technology, specifically to steroidal alkaloids, plant antifreeze compositions, and their use in improving plant cold tolerance. Spraying the potato cultivar Desiree with the steroidal alkaloids provided by the present invention effectively reduces damage to potato leaf cells caused by freezing, improves plant freezing tolerance and survival rate, and mitigates the effects of low temperatures and freezing damage on crops. This provides a practical basis for using steroidal alkaloid compounds to improve plant freezing tolerance and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of plant antifreeze, and in particular to steroidal alkaloids, a plant antifreeze composition and applications thereof in improving the cold resistance of plants. Background Art

[0002] Plants' ability to withstand low-temperature stress varies depending on their physiological state, or in other words, their frost tolerance. When ambient temperatures drop below 10°C, plants experience varying degrees of cold stress. Low temperatures reduce cell membrane fluidity and stability, increase malondialdehyde levels, and decrease enzyme activity and catalytic efficiency, disrupting intracellular metabolic pathways and functions. Furthermore, free water in plants freezes at low temperatures, causing mechanical damage to cells and, in severe cases, freezing to death.

[0003] Potatoes, as an important food crop, are sensitive to low temperatures and freezing damage, which can severely impact their growth, development, and yield. Currently, improving potato frost resistance primarily relies on variety selection and cultivation management practices. However, the long breeding cycle for new crop varieties and their cold acclimation are also affected by regional ecological adaptability and other factors, resulting in limited effectiveness. Therefore, developing antifreeze agents that can rapidly enhance potato frost tolerance has significant practical application value. Currently, there are no reports on the effectiveness of steroidal alkaloids in improving plant cold tolerance. Summary of the Invention

[0004] In order to overcome the shortcomings of the existing technology, the present invention provides a steroidal alkaloid and a plant antifreeze composition, and proves that the steroidal alkaloid and the plant antifreeze composition can effectively reduce the damage caused by low-temperature frost to potatoes, improve the survival rate of potato seedlings, significantly improve the disease resistance, low temperature resistance, and antifreeze ability of potatoes to late spring cold, effectively prevent morning and evening frost attacks, and avoid or reduce freezing damage.

[0005] One of the purposes of the present invention is to provide a method for enhancing plant tolerance to freezing damage by using a steroidal alkaloid or its derivative, or its precursor metabolite, or its pharmaceutically acceptable salt.

[0006] The following applications are also within the scope of protection of the present invention:

[0007] Application of steroidal alkaloids or their derivatives in the preparation of medicines for enhancing the frost damage resistance of plants.

[0008] Application of steroidal alkaloids or their precursor metabolites in the preparation of drugs for enhancing plant tolerance to freezing damage.

[0009] Application of steroidal alkaloids or pharmaceutically acceptable salts thereof in the preparation of drugs for enhancing plant tolerance to freezing damage.

[0010] Furthermore, the drug uses steroidal alkaloids or their derivatives, or their precursor metabolites, or their pharmaceutically acceptable salts as active ingredients, and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0011] Furthermore, the steroidal alkaloid is selected from solanine and / or tomatine.

[0012] 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 are within the scope of the present invention.

[0013] The 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 a steroidal alkaloid or a derivative thereof, or a precursor metabolite thereof, or a pharmaceutically acceptable salt thereof.

[0014] For the purposes of the present invention, "plants" include cereals such as durum wheat and other wheats, rye, barley, triticale, oats, rice or corn (fodder corn and sweet corn / sweet corn and field corn); beets such as sugar beets or fodder beets; fruits such as pome fruits, stone fruits or berries such as apples, pears, plums, peaches, nectarines, almonds, cherries, papayas, strawberries, raspberries, blackberries or gooseberries; leguminous plants such as beans, lentils, peas, alfalfa or soybeans; oilseed plants such as rapeseed (oilseed rape), rapeseed oil, mustard, olives, sunflowers, coconuts, cocoa beans, castor oil plants, oil palm, peanuts or soybeans; cucurbits 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 (e.g. iceberg lettuce), endive, cabbage, asparagus, cabbage, carrots, onions, garlic, leeks, tomatoes, potatoes, gourds or bell peppers; laurel plants, such as avocado, cinnamon or camphor; energy and feedstock plants, such as corn, soybeans, rapeseed, sugarcane 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 (e.g. carnations, petunias, geraniums / scented geraniums, pansies and impatiens), shrubs, broad-leaved trees (e.g. poplars) or evergreen trees, such as conifers; eucalyptus; turf; lawns; grasses, such as grasses for animal feed or ornamental purposes. Preferred plants include potatoes, sugar beets, tobacco, wheat, rye, barley, oats, rice, corn, cotton, soybeans, rapeseed, legumes, sunflowers, coffee, or sugar cane; fruits; grapevines; ornamentals; or vegetables, such as cucumbers, tomatoes, beans, or squash.

[0015] In some specific embodiments, the plant antifreeze composition further comprises at least one other antifreeze agent that enhances the plant's tolerance to freezing damage.

[0016] It is well understood by those skilled in the art that the other antifreeze agents for enhancing plant tolerance to freezing damage 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, and urea.

[0017] In some specific embodiments, the steroidal alkaloid is selected from solanine and / or tomatine.

[0018] 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%.

[0019] It is well known to those skilled in the art that the pharmaceutically acceptable excipients are generally recognized for this purpose and serve as inactive ingredients of medicaments.

[0020] The excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, and release retardants.

[0021] Among them, dispersants are 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, dispersants are added to easily form dispersions and suspensions, and maintain the relative stability of the dispersion system. The dispersant provided by the present invention can be selected from any one or more of the following: phosphate esters, sulfonates, alkylnaphthalenesulfonate condensates, carboxylate polymers, polycarboxylate polymer modified resins, alkyl sulfates, modified alkyl sulfonates, alkylnaphthalenesulfonic acid condensates sodium salts, naphthalenesulfonates (condensates), polycarboxylate polymer dispersants, polystyrene phenyl ether phosphate dispersants, naphthalenesulfonate formaldehyde condensates, alkylphenol polyoxyethylene ethers, formaldehyde condensate sulfonates, lignin sulfonates, maleic acid-acrylic acid copolymer sodium salts, 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, and opening powder BX.

[0022] The emulsifier can promote the formation of a stable emulsion between two immiscible liquids in the composition and also serves as a stabilizer for the emulsion. The emulsifier provided by the present invention can be selected from any one or more of the following: calcium dodecylbenzenesulfonate, triphenyl ethylphenol 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, fatty alcohol polyoxyethylene ether modification, 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, EthylanNS-500LQ (nonionic hydroxy polyethylene oxide block copolymer), styrene-maleic anhydride copolymer sodium salt, etc.

[0023] The suspending agent is used to increase the viscosity of the dispersion medium in the composition to reduce the sedimentation rate of the microparticles or increase the hydrophilicity of the microparticles. The suspending agent provided by the present invention can be selected from any one or more of the following: phosphates, sulfonates, alkylnaphthalenesulfonate condensates, carboxylate polymers, polycarboxylate polymer modified resins, alkyl sulfates, modified alkyl sulfonates, alkylnaphthalenesulfonic acid condensates sodium salts, naphthalenesulfonates (condensates), polycarboxylate polymer dispersants, polystyrene phenyl ether phosphate dispersants, naphthalenesulfonate formaldehyde condensates, alkylphenol polyoxyethylene ether formaldehyde condensate sulfonates, lignin sulfonates, white carbon black, magnesium aluminum silicate; for example, Tersperse 2500, Tersperse 4894, and soybean oil.

[0024] Defoaming agent is used to remove harmful foam in the production process. The defoaming agent provided by the present invention can be selected from GP type defoaming agent, PE type defoaming agent, GPES type defoaming agent, SAG1522 type defoaming agent, organic silicon, polyether modified silicon,

[0025] One or more polysiloxanes.

[0026] The defoamer is used to remove harmful foam during the production process. The defoamer provided by the present invention can be selected from one or more of GP type defoamers, PE type defoamers, GPES type defoamers, SAG1522 type defoamers, organic silicon, polyether modified silicon, and polysiloxane.

[0027] The binder is a substance used to bond the surfaces of homogeneous or heterogeneous objects in the composition. The binder provided by the present invention can be selected from one or more of sodium carboxymethyl cellulose, polyvinyl pyrrolidone, starch, polyvinyl alcohol, methyl cellulose, and fructose, such as corn starch and cyclohexanone.

[0028] The thickener is used to increase the viscosity of the dispersion medium in the composition to reduce the sedimentation rate of the particles and improve the stratification of the composition liquid. The thickener provided in the present invention is selected from any one or more of the following: xanthan gum, polyethylene glycol 4000, polyethylene glycol 6000, gum arabic, gelatin, and epoxidized soybean oil.

[0029] The film-forming agent is used to adhere the active ingredients to the surface of the seeds to form a smooth film.

[0030] The film-forming agent can be selected from one or more of polyvinyl alcohol, polyvinyl acetate, carboxymethyl cellulose, gelatin arabic, gelatin, and xanthan gum, such as film-forming agent BF308 and lauryl alcohol ester.

[0031] The pH regulator is used to adjust the pH of the composition. The pH regulator provided in the present invention is selected from any one or more of the following: citric acid, sodium bicarbonate, diethylamine, triisopropanolamine, phosphoric acid, and glacial acetic acid.

[0032] The suspending agent is 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, and insoluble starch.

[0033] It is well known to those skilled in the art that the compositions can be prepared according to methods known in the art. For this purpose, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants, if necessary, to prepare a suitable administration form or dosage form for human use.

[0034] Furthermore, if necessary, coloring agents, preservatives, perfumes, flavoring agents or other materials may be added to the composition.

[0035] The dosage form of the plant antifreeze composition is any one of the following: solution, microemulsion, aqueous emulsion, emulsifiable concentrate, wettable powder, suspension, suspoemulsion, water-dispersible granules, soluble powder, granules, oil suspension, and microcapsule.

[0036] The third object of the present invention is to provide a method for improving the cold resistance of plants, wherein the composition is applied to the plants 3 to 7 days before the onset of frost damage.

[0037] During application, the plant antifreeze composition can be applied to the stems, leaves, seeds, fruits, roots,

[0038] or one or more in the soil, preferably applied to the leaves of plants.

[0039] The plant antifreeze composition may be applied by one or more of dipping, spraying, evaporation, atomization, broadcasting, brushing, etc. Preferably, spraying is used.

[0040] 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.

[0041] Beneficial effects: Spraying the potato cultivar Desiree with the steroidal alkaloids provided by the present invention can effectively reduce the damage to 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. This provides a practical basis for using steroidal alkaloids to improve plant frost damage tolerance and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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;

[0043] 1A is a diagram of plant phenotypic differences, and 1B is a diagram of plant survival;

[0044] Figure 2 The graph of the changes of electrical conductivity of potato plants after steroidal alkaloids treatment; *** indicates that compared with the blank group, P <0.001. DETAILED DESCRIPTION

[0045] 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 embodiments of the present invention.

[0046] The present invention is described in detail. The experimental methods in the following examples, for which specific conditions are not specified, are generally 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, were 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 meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0047] The endpoints of ranges and any values disclosed herein are not limited to the precise range or value.

[0048] Ranges or values should be understood to include values approaching these ranges or values.

[0049] The ranges can be divided into two groups, between the endpoints of the ranges and the individual point values, and between the individual point values.

[0050] To obtain one or more new numerical ranges by combining with each other, these numerical ranges should be regarded as having

[0051] The body is open.

[0052] The technical solution of the present invention is further described in detail below with reference to specific embodiments and drawings.

[0053] It should be understood that the following examples are only used to explain the present invention and are not intended to limit the present invention.

[0054] Example 1 Plant antifreeze composition

[0055] Weigh 10 mg of solanine, add 11.3 mL of dimethyl sulfoxide solution, and mix well.

[0056] Example 2

[0057] Weigh 5 mg of tomatidine, add 12 mL of dimethyl sulfoxide solution, and mix well.

[0058] Example 3 Plant antifreeze composition

[0059] Weigh 5 mg of solanine, add 1 mL of dimethyl sulfoxide solution, and mix well.

[0060] Example 4 Plant antifreeze composition

[0061] Weigh 150 mg of solanine, add 1 mL of dimethyl sulfoxide solution, and mix well.

[0062] Example 5 Plant antifreeze composition

[0063] Weigh 1 mg of tomatidine, add 1 mL of dimethyl sulfoxide solution, and mix well.

[0064] Example 6 Plant antifreeze composition

[0065] Weigh 0.2 mg of tomatidine, add 1 mL of dimethyl sulfoxide solution, and mix well.

[0066] Example 6 Plant antifreeze composition

[0067] Weigh 1 g of tomatidine, add 1 mL of dimethyl sulfoxide solution, and mix well.

[0068] Example 7: Investigating the effect of reducing the degree of freeze damage to potatoes

[0069] Experimental materials: The plant material used in this study was the cultivated potato material Desiree, which was grown on MS medium supplemented with 2% sucrose and 0.31% agar at 20±1°C for 2 weeks. After that, the 2-week-old tissue culture seedlings were transferred to plastic pots (10×10 cm) containing a substrate (nutrient soil and perlite volume ratio of approximately 3:1) and planted in a plant growth climate chamber (22±2°C, 16 h light / 8 h dark photoperiod).

[0070] Preparation of antifreeze agent: Add the plant antifreeze composition to pure water, mix well and prepare a 10 μM antifreeze agent.

[0071] Experimental method: Four-week-old plants grown under normal conditions were moved to a 4°C cold room and cold-acclimated for 7 days under a 16-h light / 8-h dark photoperiod. During the cold acclimation period, the plants were sprayed with the antifreeze prepared in Example 1, the antifreeze prepared in Example 2, or the control reagent (pure water containing the same proportion of DMSO) once a day using a portable sprayer, with approximately 10 mL sprayed per plant. Then, the cold-acclimated plants were treated at −2°C for 12 hours, and the growth status of the plants and (e.g. Figure 1 shown).

[0072] from Figure 1 A It can be seen that after frost damage treatment, the leaves, petioles and young stems of potato plants sprayed with the control reagent were severely wilted and damaged; in comparison, the potato plants sprayed with solanine or tomatine grew well and no obvious tissue damage was observed.

[0073] from Figure 1 As shown in Figure B, after the frost-damaged potato plants were transferred to a plant growth climate chamber (22±2°C, 16h light / 8h dark photoperiod) for recovery and cultivation for one 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 solanine and tomatine was above 60%, which was significantly higher than that of the control group materials.

[0074] Example 8 Investigation of the effect on enhancing the freezing tolerance of potatoes

[0075] Experimental Materials: The plant material used in this study was the cultivated potato material Desiree. The plants were grown on MS medium supplemented with 2% sucrose and 0.31% agar at 20 ± 1°C for 2 weeks. Two-week-old tissue culture seedlings were then transplanted into plastic pots (10 × 10 cm) containing a substrate (nutrient soil and perlite in a volume ratio of approximately 3:1).

[0076] The plants were grown in a plant growth climate chamber (22 ± 2°C, 16 h light / 8 h dark photoperiod).

[0077] Preparation of antifreeze agent: 1 mL of the plant antifreeze composition was added to 100 mL of pure water, and the mixture was mixed to prepare a 10 μM antifreeze agent.

[0078] Experimental Methods: Four-week-old plants grown under normal conditions were transferred to a 4°C cold room and cold-acclimated for 7 days under a 16-h light / 8-h dark photoperiod. During the cold acclimation period, the plants were sprayed once daily with the antifreeze agent of Example 1, the antifreeze agent of Example 2, or a control agent (pure water containing the same ratio of DMSO) using a portable sprayer, with approximately 10 mL applied to each plant. The cold-acclimated plants were then kept at −2°C for 12 h, and their leaves were then sampled for conductivity testing. The specific testing process was as follows: the top leaf was placed in a centrifuge tube, 10 mL of deionized water was added, and the conductivity was measured as ECi. The leaves were shaken at approximately 100 rpm for 4 h at room temperature, and the conductivity was measured as ECf. After boiling for 20 min, the leaves were shaken at approximately 100 rpm for 1 h at room temperature, and the conductivity was measured as ECt. Electrolyte leakage was calculated using the following formula: (ECf - ECi) / (ECt - ECi) × 100. Experimental data were calculated using the Student's method. t The results of the test statistical software analysis are as follows Figure 2 shown.

[0079] from Figure 2 It can be seen that before frost damage treatment, the electrolyte leakage rate of all potato plant leaves was similar and maintained at a low level; after frost damage treatment, the electrolyte leakage rate of potato plant leaves sprayed with control reagent increased by more than 3 times, indicating serious cell damage; while the electrolyte leakage rate of potato leaves sprayed with solanine and tomatine only increased slightly, which was significantly lower than that of the control group material.

[0080] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make various similar expressions without violating the purpose and claims of the present invention. Such changes fall within the scope of protection of the present invention.

Claims

1. Application of steroidal alkaloids in enhancing plant tolerance to freezing damage, including: The steroidal alkaloid is selected from solanine and tomatine; and the plant is potato.

2. The use according to claim 1, characterized in that Application in the preparation of drugs for enhancing plant tolerance to freezing damage.

3. The use according to claim 2, characterized in that: The medicine uses steroidal alkaloids as active ingredients and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

4. A method for improving the cold resistance of plants, characterized in that: The plant antifreeze composition is applied to the plant 3 to 7 days before the onset of frost damage; the plant antifreeze composition comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is a steroidal alkaloid; the steroidal alkaloid is selected from one or more of solanine and tomatine; and the plant is potato.

5. The method according to claim 4, characterized in that The plant antifreeze composition further comprises an antifreeze agent, which is one or more of ethylene glycol, propylene glycol, butanol, glycerol and urea.

6. The method according to claim 4, characterized in that The amount of the active ingredient is 0.001-90 wt % relative to the total weight of the plant antifreeze composition.

7. The method according to claim 4, characterized in that The amount of the active ingredient is 0.02-50 wt % relative to the total weight of the plant antifreeze composition.

8. The method according to claim 4, characterized in that The amount of the active ingredient is 0.1-20% relative to the total weight of the plant antifreeze composition.

9. The method according to claim 4, characterized in that The amount of the active ingredient is 0.005-5% relative to the total weight of the plant antifreeze composition.

10. The method according to claim 4, characterized in that Before foliar application, dissolve the plant antifreeze composition in water to prepare an antifreeze concentration of 5-15 μM; the application amount per plant is 10 mL / day.

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