Steroid saponin, plant anti-freezing composition and application of plant anti-freezing composition to improvement of plant cold resistance

Abstract

The invention relates to the technical field of plant antifreezing, in particular to steroid saponin, a plant antifreezing composition and application of the plant antifreezing composition to improvement of plant cold resistance. The steroidal saponin 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 saponin compound, and the steroidal saponin has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

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

[0002] The difference in 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 all 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 in severe cases, it will lead to the death of the plants due to freezing.

[0003] Potato, as an important food crop, is relatively sensitive to low temperature and freezing injury, which will seriously affect its growth, development and yield. At present, improving the frost resistance of potatoes mainly depends on variety breeding and cultivation management measures. However, the breeding of new crop varieties and low - temperature acclimation have a long cycle, and at the same time, they are affected by regional ecological adaptability and other factors, and the application effect is not obvious. Therefore, it has important practical application value to develop a class of antifreeze agents that can rapidly improve the frost tolerance of potatoes. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides steroidal saponins 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, can significantly improve the disease - resistance, low - temperature resistance and cold - snap resistance of potatoes, effectively prevent the attack of early and late frosts, and avoid or reduce the freezing injury.

[0005] One of the purposes of the present invention is to provide the application of steroidal saponins 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 saponins or their derivatives in the preparation of drugs for enhancing the frost tolerance of plants.

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

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

[0009] Furthermore, the drug uses steroidal saponins 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 saponin is selected from diosgenin and / or smilagenin.

[0011] Unless otherwise indicated, all stereoisomers, geometric isomers, tautomers, N-oxides, hydrates, solvates, metabolites, salts and pharmaceutically acceptable prodrugs of the steroidal saponins of the present invention are 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 a steroidal saponin or its derivative, or its precursor metabolite, or its pharmaceutically acceptable salt.

[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 beets, such as sugar beets or fodder beets; 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 var. oleifera, mustard, olives, sunflowers, coconuts, cocoa beans, castor oil plants, oil palms, peanuts or soybeans; cucurbitaceous plants, such as squashes, pumpkins, cucumbers or melons; fiber plants, such as cotton, flax, hemp or jute; citrus fruits, such as oranges, lemons, grapefruits or tangerines; vegetables, such as eggplants, spinach, lettuce (such as iceberg lettuce), chicory, cabbages, asparagus, cabbages, 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 palms; 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; lawns; 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 cucumber tomatoes, kidney beans or squashes.

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

[0015] Those skilled in the art are fully aware that other active components for enhancing the frost tolerance of plants referred to in the present invention are substances used to lower the freezing point of the liquid of the composition and improve the frost resistance, 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 saponins are selected from diosgenin and / or smilagenin.

[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 conducive to forming a dispersion and a suspension, and maintaining 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] Emulsifiers can promote the formation of a stable emulsion from two immiscible liquids in the composition and also serve as stabilizers for the emulsion. The emulsifiers 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 (non-ionic type hydroxy poly(ethylene oxide) block copolymer), sodium styrene-maleic anhydride copolymer, etc.

[0021] Suspending agents are 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 agents provided in 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, 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] Defoamers are used to remove harmful foams during the production process. The defoamers provided in the present invention can be any one or more of GP type defoamer, PE type defoamer, GPES type defoamer, SAG1522 type defoamer, silicone, polyether-modified silicone, polysiloxane.

[0023] Defoamers are used to remove harmful foams during the production process. The defoamers provided in the present invention can be any one or more of GP type defoamer, PE type defoamer, GPES type defoamer, SAG1522 type defoamer, silicone, polyether-modified silicone, polysiloxane.

[0024] Binders are substances used to bond the surfaces of homogeneous or heterogeneous objects in the composition together. The binders provided in the present invention can be selected from any 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 microparticles 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, epoxidized 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. Provided in the present invention, The film-forming agents 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 administration form or dosage 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 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 frost tolerance of plants. Before the frost damage occurs, the above composition is applied to the plants 3 to 7 days in advance.

[0033] During the application process, the above plant antifreeze composition can be applied to one or more of the stems, leaves, seeds, fruits, roots or soil of the plants, and preferably applied to the leaf surface of the 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 steroidal saponin provided by the present invention on the potato cultivar Desiree can effectively reduce the damage of freezing injury to potato leaf cells, improve the freezing tolerance and survival rate of plants, reduce the impact of low temperature and freezing injury on crops, provide a practical basis for using steroidal saponin compounds to improve plant freezing tolerance, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shows the effect of steroidal saponin on the degree of potato freezing injury; * indicates compared with the blank group, P <0.05; Figure 1A is a diagram of plant phenotype differences, and Figure 1B is a diagram of plant survival; Figure 2 Shows the change in electrical conductivity of potato plants treated with steroidal saponin; *** indicates compared with the blank group, P <0.001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the following detailed description of the present invention is provided in conjunction with specific embodiments. The experimental methods without specific conditions indicated in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples are, unless otherwise specified, purchased from regular biochemical reagent stores. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0039] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0040] The technical solutions of the present invention will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0041] Example 1 Plant antifreeze composition Weigh 5 mg of diosgenin, add 12 mL of dimethyl sulfoxide solution, and mix evenly.

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

[0043] Example 3 Plant antifreeze composition Weigh 10 mg of diosgenin, add 1 mL of dimethyl sulfoxide solution, and mix evenly.

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

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

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

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

[0048] Example 7 Investigation of the effect on reducing the degree of frost damage to potatoes Experimental materials: The plant material used in this study was the cultivated potato variety Desiree. It was 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. After mixing evenly, it is prepared into an antifreeze agent with a concentration of 10 μM.

[0050] Experimental method: Transfer 4-week-old plants grown under normal conditions into a 4 °C cold room, with a 16 h light / 8 h Cold acclimation was carried out for 7 days under a 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, the cold-acclimated plants were treated at -2°C for 12 h, and the growth status of the plants was observed (as shown). Figure 1

[0051] As can be seen from Figure 1 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 diosgenin or sarsasapogenin were in good growth condition, and no obvious tissue damage was observed.

[0052] As can be seen from Figure 1 B, after transferring the freeze-injured potato plants to a plant growth climate chamber (22 ± 2°C, 16 h light / 8 h dark light cycle) for 1 day of recovery culture, 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 diosgenin and sarsasapogenin was above 60%, which was significantly higher than that of the control group materials.

[0053] Example 8 Investigation of the effect on enhancing the frost tolerance of potatoes Experimental materials: The plant materials used in this study were 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. 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 well to prepare a 10 μM antifreeze agent.

[0055] Experimental method: The 4-week-old plants grown under normal conditions were transferred to a 4°C cold room, with a 16 h light / 8 h ​Cold acclimation was carried out for 7 days under a 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 electrical conductivity. The specific detection process was as follows: The top leaves were put into a centrifuge tube, and 10 mL of deionized water was added. The measured value of the electrical conductivity was ECi; it was shaken at about 100 rpm at room temperature for 4 hours, and the measured value of the electrical conductivity was ECf; after boiling for 20 min, it was shaken at about 100 rpm at room temperature for 1 hour, and the measured value of the electrical conductivity 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 , 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 diosgenin and smilagenin only increased slightly, which 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, under the inspiration of the present invention and without departing from the purpose and claims of the present invention, can make various similar representations, and such transformations all fall within the protection scope of the present invention.

Claims

1. Use of a steroidal saponin or its derivative, or its precursor metabolite, or its pharmaceutically acceptable salt in enhancing the frost tolerance of plants; the steroidal saponin is selected from diosgenin and smilagenin, and the plant is potato.

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

3. The application according to claim 1 or 2, characterized in that: The drug uses a steroidal saponin 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 saponin 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 at least one other active component for enhancing the frost tolerance of plants.

6. The plant antifreeze composition according to claim 4 or 5, characterized in that, The steroidal saponin is selected from diosgenin and / or smilagenin.

7. The plant antifreeze composition obtained 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 composition; or 0.02 - 50 wt%; or 0.1 - 20%; or 0.005 - 5%.

8. A method for improving the freezing tolerance of plants, characterized in that, Before the frost disaster comes for 3 to 7 days, apply the composition according to claims 4 - 7 to the plants.

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

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