Tobacco leaf stress-resistant aging-inhibiting ripeness-preserving agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tobacco cultivation, in particular to a tobacco leaf stress-resistant aging-inhibiting ripeness-preserving agent and a preparation method and application thereof. The tobacco leaf stress-resistant aging-inhibiting ripeness-keeping agent comprises an active component, an auxiliary agent and a solvent, the active ingredients are prepared from gamma-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol; the auxiliaries comprise a surfactant, a humectant, an antioxidant and an acid-base regulator; the solvent is water or an ethanol water solution. According to the tobacco leaf stress-resistant aging-inhibiting ripeness-preserving agent provided by the invention, through the synergistic effect of various active components and a multi-level regulation mechanism, the comprehensive effects of enhancing the stress resistance, delaying the aging of leaves and improving the ripening quality after tobacco plants are topped are realized, and an important technical support is provided for the production of high-quality tobacco leaves.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco cultivation, in particular to a tobacco leaf anti-adversity, anti-aging and ripening agent, a preparation method and application thereof. Background Art

[0002] Tobacco is an important economic crop in my country. During its growth process, it needs to be toppled, that is, the inflorescence at the top of the tobacco plant is removed to promote the transfer of nutrients to the leaves and improve the quality and yield of tobacco leaves. However, topping, as a mechanical damage, will cause a stress response to the tobacco plants, leading to a series of physiological changes such as accumulation of reactive oxygen species, hormone imbalance, and metabolic abnormalities, which in turn affect the quality of tobacco leaves. At the same time, after topping, tobacco plants are often faced with a variety of environmental stresses such as high temperature and drought, which are more likely to cause premature aging of tobacco leaves and rapid degradation of chlorophyll. In severe cases, even early yellowing and dry tips will appear, which will eventually lead to a decline in tobacco leaf quality and reduced economic value. In related technologies, in order to alleviate this problem, there are also methods of using bud inhibitors or growth promoters, but these products have a single function and cannot meet the complex physiological needs of tobacco after topping. Therefore, there is currently no systematic solution to the special physiological needs of tobacco after topping. Summary of the Invention

[0003] In order to solve or partially solve the problems existing in the related art, the present invention provides a tobacco leaf anti-adversity, anti-aging and ripening agent and its preparation method and application.

[0004] The invention provides a tobacco leaf anti-adversity, anti-aging and ripening agent, which comprises: active ingredients, auxiliary agents and solvents; the active ingredients include: gamma-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol; the auxiliary agents include: surfactants, humectants, antioxidants and acid-base regulators; the solvent is water or ethanol aqueous solution.

[0005] Furthermore, among the active ingredients, the weight ratio of γ-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol is (3-5): (3-9): (3-9): (15-45).

[0006] Furthermore, in the auxiliary agent, The surfactant is polyoxyethylene sorbitan ester; The humectant is glycerin; The antioxidant is vitamin E; The acid-base regulator is citric acid; The weight ratio of the polyoxyethylene sorbitan ester, glycerol and vitamin E is (5-10): (20-40): (0.5-1); The citric acid is used to adjust the pH value of the tobacco leaf anti-adversity, anti-aging and ripening agent system to 5.8-6.2.

[0007] Furthermore, it comprises, by weight: 3-5 parts by weight of gamma-aminobutyric acid; 3-9 parts by weight of epicatechin; 3-9 parts by weight of epigallocatechin gallate; 15-45 parts by weight of dihydrokaempferol; 5-10 parts by weight of polyoxyethylene sorbitan; 20-40 parts by weight of glycerol; 1-3 parts by weight of citric acid; Vitamin E 0.5-1 parts by weight; 880-950 parts by weight of solvent.

[0008] Furthermore, it comprises, by weight: 4-5 parts by weight of gamma-aminobutyric acid; 6-9 parts by weight of epicatechin; 6-9 parts by weight of epigallocatechin gallate; 30-45 parts by weight of dihydrokaempferol; 8-10 parts by weight of polyoxyethylene sorbitan; 30-40 parts by weight of glycerol; 2-3 parts by weight of citric acid; Vitamin E 0.8-1 parts by weight; Solvent 878-913.2 parts by weight.

[0009] The present invention also provides a method for preparing any one of the above-mentioned tobacco leaf anti-adversity, anti-aging and ripening agent, comprising: Step 1), dissolving γ-aminobutyric acid in a solvent to obtain a γ-aminobutyric acid solution; mixing epicatechin and gallocatechin and dissolving them in a solvent to obtain a catechin mixed solution; dissolving dihydrokaempferol in a solvent to obtain a dihydrokaempferol solution; Step 2) Mix the surfactant and the wetting agent, add the solvent under stirring to obtain an emulsified system, and add the acid-base regulator to adjust the pH value to 6.0-6.5; Step 3), under stirring, sequentially adding the γ-aminobutyric acid solution, the catechin mixed solution, and the dihydrokaempferol solution to the emulsified system; Step 4), adding an antioxidant to the mixed solution obtained in step 3), stirring evenly, and then treating with a high-speed shear homogenizer to form a stable microemulsion; Step 5), filtering the microemulsion using an organic filter membrane, adding an acid-base regulator to adjust the pH value to 5.8-6.2, and adding a solvent to obtain a tobacco leaf anti-stress, anti-aging and ripening agent.

[0010] Furthermore, the step 1) is specifically as follows: Add γ-aminobutyric acid to water at 45-55°C and stir until completely dissolved to obtain a γ-aminobutyric acid solution; The epicatechin and gallocatechin are mixed and added into an aqueous solution containing 8-12% ethanol at 35-45°C, and stirred until completely dissolved to obtain a catechin mixed solution; Add dihydrokaempferol to an aqueous solution containing 15-25% ethanol and stir until completely dissolved to obtain a dihydrokaempferol solution.

[0011] Furthermore, the stirring speed in step 2) is 200-300 rpm, and / or the stirring speed in step 2) is 200-300 rpm.

[0012] Furthermore, the speed of the homogenizer in step 4) is 10,000-15,000 rpm, and / or the pore size of the organic filter membrane in step 5) is 0.22 μm.

[0013] The present invention also provides a tobacco leaf stress resistance, aging inhibition and ripening maintenance agent as described above for use in improving tobacco stress resistance, delaying leaf aging and promoting normal ripening of tobacco leaves, comprising: Step (A): 4-6 days after topping the tobacco plants, choose a sunny or cloudy day with wind speed not exceeding level 3; Step (B), diluting the tobacco leaf anti-adversity and aging-inhibiting agent with water in a ratio of 1:4-1:8; Step (C): using an agricultural multi-rotor drone equipped with a low-pressure atomizing nozzle to spray at 8:00-10:00 in the morning or 16:00-18:00 in the afternoon at an ambient temperature of 15-30°C; Step (D): Control the drone to fly at a height of 1.5-2.0 meters above the crown and a speed of 3-5 m·s - ¹, spraying pressure is 0.2-0.3 MPa, spraying overlap rate is 20-30%, and application rate is 30-50 kg·mu - ¹.

[0014] The tobacco leaf anti-adversity, anti-aging and ripening agent provided by the present invention may have the following beneficial effects: 1. The tobacco leaf anti-stress, anti-aging and ripening agent uses a combination of four core active ingredients, namely γ-aminobutyric acid, epicatechin, gallic acid and dihydrokaempferol, as well as adjuvants, to form a four-level collaborative protection network consisting of a signal regulation layer, an enzyme system regulation layer, a cell metabolism regulation layer and a physical protection layer. It can comprehensively regulate multiple problems faced by tobacco plants after topping, such as oxidative stress, hormonal disorders and metabolic imbalances, to achieve the comprehensive effect of anti-stress, anti-aging and ripening.

[0015] 2. The tobacco leaf anti-stress, anti-aging and ripening agent can significantly improve the activity of antioxidant enzymes: Experiments have shown that after being treated with the tobacco leaf anti-stress, anti-aging and ripening agent of the present invention, the SOD activity of the tobacco leaves is increased by 35-50%, the POD activity is increased by 40-60%, and the CAT activity is increased by 30-45%, forming a powerful active oxygen scavenging system, effectively reducing the damage to cells caused by oxidative stress.

[0016] 3. The tobacco leaf anti-stress, anti-aging and ripening agent can effectively delay the degradation of chlorophyll: Experiments have shown that after the tobacco leaf is treated with the anti-stress, anti-aging and ripening agent of the present invention, the chlorophyll degradation rate of the tobacco leaf is slowed down by 40-55%, the green period of the leaves is extended by 8-12 days, and the malondialdehyde (MDA) content is reduced by 30-45%, indicating that the damage to cell membrane lipid peroxidation is reduced and the function of the photosynthetic system is effectively protected.

[0017] 4. This tobacco leaf anti-stress and anti-aging agent can optimize physiological balance: Experiments have shown that by regulating the balance of endogenous hormones, especially reducing the abscisic acid / cytokinin ratio, the photosynthetic rate of treated tobacco leaves during the maturity period increased by 25-35%, the total sugar content increased by 15-25%, the total nitrogen content decreased by 10-15%, and the sugar-nitrogen ratio increased by 25-40%, effectively improving the quality of tobacco leaves.

[0018] 5. The tobacco leaf anti-stress, anti-aging and ripening agent is easy and efficient to apply. Combined with drone spraying technology, the application method of the present invention is simple to operate, has uniform coverage, and is highly efficient. Moreover, all active ingredients are natural compounds derived from plants, which is environmentally friendly and meets the requirements of green agricultural development.

[0019] In summary, the tobacco leaf anti-stress, anti-aging and ripening agent provided by the present invention achieves the comprehensive effects of enhanced stress resistance, delayed leaf aging and improved maturity quality after topping of tobacco plants through the synergistic effect of multiple active ingredients and multi-level regulatory mechanism, providing important technical support for the production of high-quality tobacco leaves.

[0020] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0022] The terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the invention. The singular forms "a," "the," and "the" used in this invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0023] It should be understood that although the terms "first", "second", "third", etc. may be used to describe various information in the present invention, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "plurality" means two or more, unless otherwise clearly and specifically defined.

[0024] An embodiment of the present invention provides a tobacco leaf anti-adversity, anti-aging and ripening agent, which includes: active ingredients, auxiliary agents and solvents; the active ingredients include: gamma-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol; the auxiliary agents include: surfactants, humectants, antioxidants and acid-base regulators; the solvent is water or ethanol aqueous solution.

[0025] The key point of the tobacco leaf anti-adversity, anti-aging and ripening agent provided in this embodiment is to establish a multi-level coordinated control system. Through the combination of four core active ingredients and adjuvants, a progressive and mutually coordinated control network is formed. The four control levels are: 1. Signal regulation layer: GABA, as a plant signal molecule, can activate calcium ion signaling pathways and regulate downstream gene expression; promote the activation of GABA receptor-related transcription factors and initiate stress resistance; regulate endogenous hormone balance, especially reduce ethylene synthesis and slow down the aging process.

[0026] 2. Enzyme system regulation layer: epicatechin and gallocatechin, as polyphenolic compounds, can directly activate antioxidant enzyme systems such as SOD, POD, and CAT; regulate related gene expression and increase antioxidant enzyme synthesis; and reduce hydroxyl free radicals produced by the Fenton reaction by complexing transition metal ions.

[0027] 3. Cell metabolism regulation layer: Dihydrokaempferol, as a flavonoid compound, can activate SIRT1 and Nrf2 signaling pathways, enhance cellular antioxidant defense capabilities; regulate mitochondrial function, maintain energy metabolism balance; protect the structural integrity of the photosynthetic system, and slow down chloroplast degradation.

[0028] 4. Physical protective layer: The surfactant in the additive improves the permeability and bioavailability of the active ingredients, the humectant enhances the moisturizing effect and reduces water stress, the acid-base regulator maintains the appropriate pH and optimizes the enzyme activity environment, and the antioxidant protects the cell membrane system.

[0029] The above four levels form a complete coordinated protection network, which comprehensively regulates multiple problems faced by tobacco leaves after topping, such as oxidative stress, hormonal disorders and metabolic imbalance, and achieves the comprehensive effect of resisting adversity, inhibiting aging and maintaining ripeness.

[0030] Preferably, in the active ingredients, the weight ratio of γ-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol is (3-5): (3-9): (3-9): (15-45); more preferably, it is (4-5): (6-9): (6-9): (30-45); and most preferably, it is 5:9:9:45.

[0031] As a preferred embodiment of this embodiment, the surfactant in the above-mentioned auxiliary agent is polyoxyethylene sorbitan ester; the humectant is glycerol; the antioxidant is vitamin E; the acid-base regulator is citric acid; the weight ratio of polyoxyethylene sorbitan ester, glycerol and vitamin E is (5-10): (20-40): (0.5-1); citric acid is used to adjust the pH value of the tobacco leaf anti-adversity and anti-aging agent system to 5.8-6.2.

[0032] In the tobacco leaf anti-adversity, anti-aging and ripening agent provided in this embodiment, the role of the solvent is to dissolve and disperse the components, adjust the viscosity and surface tension of the system, and ensure the stability of the microemulsion and the spreadability of the leaf surface. The solvent can be water or ethanol aqueous solution, preferably the latter, and the ethanol content is preferably 5.7%-9%. Preferably, the tobacco leaf stress resistance, aging inhibition and ripening maintenance agent provided in this embodiment has a pH value of 5.8-6.2, a viscosity of 10-30 mPa·s, a surface tension of 25-35 mN / m, and is in the form of a microemulsion.

[0033] The following is the preferred composition and proportion of the tobacco leaf anti-adversity, anti-aging and ripening agent of this embodiment, which includes, by weight: 3-5 parts by weight of gamma-aminobutyric acid; 3-9 parts by weight of epicatechin; 3-9 parts by weight of epigallocatechin gallate; 15-45 parts by weight of dihydrokaempferol; 5-10 parts by weight of polyoxyethylene sorbitan; 20-40 parts by weight of glycerol; 1-3 parts by weight of citric acid; Vitamin E 0.5-1 parts by weight; 880-950 parts by weight of solvent.

[0034] More preferred compositions and proportions include, by weight: 4-5 parts by weight of gamma-aminobutyric acid; 6-9 parts by weight of epicatechin; 6-9 parts by weight of epigallocatechin gallate; 30-45 parts by weight of dihydrokaempferol; 8-10 parts by weight of polyoxyethylene sorbitan; 30-40 parts by weight of glycerol; 2-3 parts by weight of citric acid; Vitamin E 0.8-1 parts by weight; Solvent 878-913.2 parts by weight.

[0035] The most preferred composition and ratio include, by weight: 5 parts by weight of gamma-aminobutyric acid; 9 parts by weight of epicatechin; 9 parts by weight of epigallocatechin gallate; 45 parts by weight of dihydrokaempferol; 10 parts by weight of polyoxyethylene sorbitan; 40 parts by weight of glycerol; 3 parts by weight of citric acid; 1 part by weight of vitamin E; 878 parts by weight of solvent.

[0036] Another embodiment of the present invention further provides a method for preparing the above-mentioned tobacco leaf anti-adversity, anti-aging and ripening agent, comprising: Step 1), dissolving γ-aminobutyric acid in a solvent to obtain a γ-aminobutyric acid solution; mixing epicatechin and gallocatechin and dissolving them in a solvent to obtain a catechin mixed solution; dissolving dihydrokaempferol in a solvent to obtain a dihydrokaempferol solution; Step 2) Mix the surfactant and the wetting agent, add the solvent under stirring to obtain an emulsified system, and add the acid-base regulator to adjust the pH value to 6.0-6.5; Step 3), under stirring, sequentially adding the γ-aminobutyric acid solution, the catechin mixed solution, and the dihydrokaempferol solution to the emulsified system; Step 4), adding an antioxidant to the mixed solution obtained in step 3), stirring evenly, and then treating with a high-speed shear homogenizer to form a stable microemulsion; Step 5), filtering the microemulsion using an organic filter membrane, adding an acid-base regulator to adjust the pH value to 5.8-6.2, and adding a solvent to obtain a tobacco leaf anti-stress, anti-aging and ripening agent.

[0037] The above step 1) may specifically be: Add γ-aminobutyric acid to water at 45-55°C and stir until completely dissolved to obtain a γ-aminobutyric acid solution; The epicatechin and gallocatechin are mixed and added into an aqueous solution containing 8-12% ethanol at 35-45°C, and stirred until completely dissolved to obtain a catechin mixed solution; Add dihydrokaempferol to an aqueous solution containing 15-25% ethanol and stir until completely dissolved to obtain a dihydrokaempferol solution.

[0038] More preferably, the volume ratio of the γ-aminobutyric acid aqueous solution, the catechin mixed solution, and the dihydrokaempferol solution is 1:2:1. That is, in the subsequent step 3), the volume ratio of the γ-aminobutyric acid solution, the catechin mixed solution, and the dihydrokaempferol solution is 1:2:1.

[0039] Those skilled in the art will appreciate that for different compounds, different solvents can be used in the dissolution step according to their properties. For example, γ-aminobutyric acid has a high solubility in water, so water can be used as the solvent; components such as epicatechin, gallocatechin, and dihydrokaempferol have a high solubility in ethanol-water solution, so ethanol-water solution is used as the solvent.

[0040] In step 2), polyoxyethylene sorbitan ester and glycerol are first mixed in a predetermined mass ratio. Water is then added under stirring to form a preliminary emulsion. In the subsequent step (step 4), a high-speed homogenizer is used to further refine the mixture. In this step, the stirring speed is preferably 200-300 rpm. A solvent, which can be water, is slowly added under stirring to form an emulsion. The pH of the emulsion is adjusted to 6.0-6.5 using an acid-base modifier (such as citric acid).

[0041] In step 3), the stirring speed is preferably 200-300 rpm. In this step, after each component is added, stirring is preferably continued for 10-15 minutes before the next component is added. In step 4), the homogenizer speed is preferably 10,000-15,000 rpm, and the processing time is preferably 4-6 minutes. The droplet size of the resulting stable microemulsion is preferably 100-300 nm.

[0042] In step 5), organic membrane filtration is used to remove insoluble impurities. The pore size of the organic filter membrane is preferably 0.22 μm. After filtration, the pH is adjusted to 5.8-6.2 using an acid-base modifier (such as citric acid). A solvent is then added to produce the tobacco leaf anti-stress, anti-aging, and ripening agent. The solvent added in this step is preferably water. The amount of solvent added can be calculated by subtracting the amount of solvent used in the previous step from the total amount of solvent in the formula, or the amount can be controlled based on viscosity and surface tension. Specifically, for example, solvent is added to adjust the system viscosity to 10-30 mPa·s and the surface tension to 25-35 mN / m to produce the tobacco leaf anti-stress, anti-aging, and ripening agent.

[0043] The above preparation method preferably also includes a quality control step, specifically: Determine whether the pH value, viscosity, surface tension, emulsion stability and active ingredient content error of the tobacco leaf anti-adversity, anti-aging and ripening agent are within the preset indicator range; wherein, the emulsion stability is determined by measuring the stratification state of the tobacco leaf anti-adversity, anti-aging and ripening agent after being stored at 4°C for 30 days.

[0044] The preset indicator range is preferably as follows: pH value is 5.8-6.2; Viscosity is 10-30 mPa·s; Surface tension is 25-35 mN / m; The stability of the emulsion is that it does not separate after being stored at 4°C for 30 days, or it separates slightly but can quickly return to a uniform state after being gently shaken; The error of active ingredient content is: the content of each active ingredient is detected by high performance liquid chromatography, and the error of the content of each active ingredient should be within the range of ±5%.

[0045] Another embodiment of the present invention further provides a use of the above-mentioned tobacco leaf stress resistance, aging inhibition and ripening maintenance agent in improving tobacco stress resistance, delaying leaf aging and promoting normal ripening of tobacco leaves, specifically comprising: Step (A): 4-6 days after topping the tobacco plants, choose a sunny or cloudy day with wind speed not exceeding level 3; Step (B), diluting the tobacco leaf anti-adversity and aging-inhibiting agent with water in a ratio of 1:4-1:8; Step (C): using an agricultural multi-rotor drone equipped with a low-pressure atomizing nozzle to spray at 8:00-10:00 in the morning or 16:00-18:00 in the afternoon at an ambient temperature of 15-30°C; Step (D): Control the drone to fly at a height of 1.5-2.0 meters above the crown and a speed of 3-5 m·s - ¹, spraying pressure is 0.2-0.3 MPa, spraying overlap rate is 20-30%, and application rate is 30-50 kg·mu - ¹.

[0046] The tobacco leaf anti-adversity, anti-aging and ripening agent provided by the embodiment of the present invention has the following advantages: 1. The tobacco leaf anti-stress, anti-aging and ripening agent uses a combination of four core active ingredients, namely γ-aminobutyric acid, epicatechin, gallic acid and dihydrokaempferol, as well as adjuvants, to form a four-level collaborative protection network consisting of a signal regulation layer, an enzyme system regulation layer, a cell metabolism regulation layer and a physical protection layer. It can comprehensively regulate multiple problems faced by tobacco plants after topping, such as oxidative stress, hormonal disorders and metabolic imbalances, to achieve the comprehensive effect of anti-stress, anti-aging and ripening.

[0047] 2. The tobacco leaf anti-stress, anti-aging and ripening agent can significantly improve the activity of antioxidant enzymes: Experiments have shown that after being treated with the tobacco leaf anti-stress, anti-aging and ripening agent of the present invention, the SOD activity of the tobacco leaves is increased by 35-50%, the POD activity is increased by 40-60%, and the CAT activity is increased by 30-45%, forming a powerful active oxygen scavenging system, effectively reducing the damage to cells caused by oxidative stress.

[0048] 3. The tobacco leaf anti-stress, anti-aging and ripening agent can effectively delay the degradation of chlorophyll: Experiments have shown that after the tobacco leaf is treated with the anti-stress, anti-aging and ripening agent of the present invention, the chlorophyll degradation rate of the tobacco leaf is slowed down by 40-55%, the green period of the leaves is extended by 8-12 days, and the malondialdehyde (MDA) content is reduced by 30-45%, indicating that the damage to cell membrane lipid peroxidation is reduced and the function of the photosynthetic system is effectively protected.

[0049] 4. This tobacco leaf anti-stress and anti-aging agent can optimize physiological balance: Experiments have shown that by regulating the balance of endogenous hormones, especially reducing the abscisic acid / cytokinin ratio, the photosynthetic rate of treated tobacco leaves during the maturity period increased by 25-35%, the total sugar content increased by 15-25%, the total nitrogen content decreased by 10-15%, and the sugar-nitrogen ratio increased by 25-40%, effectively improving the quality of tobacco leaves.

[0050] 5. The tobacco leaf anti-stress, anti-aging and ripening agent is easy and efficient to apply. Combined with drone spraying technology, the application method of the present invention is simple to operate, has uniform coverage, and is highly efficient. Moreover, all active ingredients are natural compounds derived from plants, which is environmentally friendly and meets the requirements of green agricultural development.

[0051] In summary, tobacco leaf anti-stress, anti-aging and ripening agent achieves the comprehensive effects of enhanced stress resistance, delayed leaf aging and improved maturity quality after topping of tobacco plants through the synergistic effect of multiple active ingredients and multi-level regulatory mechanism, providing important technical support for the production of high-quality tobacco leaves.

[0052] The technical solution of the present invention will be further described below in conjunction with specific embodiments: Example 1: Standard formula of tobacco leaf anti-adversity, anti-aging and ripening agent 1. Add 4 parts by weight of γ-aminobutyric acid to 200 parts by weight of water at 50°C and stir on a magnetic stirrer for 30 minutes until completely dissolved to obtain a γ-aminobutyric acid aqueous solution. Mix 6 parts by weight of epicatechin and 6 parts by weight of gallocatechin, add the mixture to 250 parts by weight of ethanol aqueous solution (10% ethanol content) at 40°C, and stir for 45 minutes until completely dissolved to obtain a catechin mixed solution. Add 30 parts by weight of dihydrokaempferol to 150 parts by weight of ethanol aqueous solution (20% ethanol content) and stir for 60 minutes until completely dissolved to obtain a dihydrokaempferol solution.

[0053] 2. In a stainless steel container, add 8 parts by weight of polyoxyethylene sorbitan ester and 30 parts by weight of glycerol and mix them evenly using a high-speed disperser. Then, slowly add 100 parts by weight of water while stirring at 250 rpm to form an emulsified system. Adjust the pH to 6.2 using citric acid.

[0054] 3. Subsequently, under continuous stirring at 250 rpm, first add the γ-aminobutyric acid aqueous solution and stir for 15 minutes, then add the catechin mixed solution and stir for 15 minutes, and finally add the dihydrokaempferol solution and stir for 15 minutes.

[0055] 4. Add 0.8 parts by weight of vitamin E to the mixture and continue stirring for 10 minutes. Process the mixture in a high-speed shear homogenizer at 12,000 rpm for 5 minutes to form a uniform and stable microemulsion.

[0056] 5. Filter through a 0.22 μm filter membrane to remove insoluble impurities, adjust the pH value to 6.0 with citric acid, and add water to a total weight of about 1000 parts by weight to obtain a tobacco leaf anti-adversity, anti-aging and ripening agent.

[0057] The tobacco leaf anti-stress, anti-aging and ripening agent is composed of: 4 parts by weight of gamma-aminobutyric acid, 6 parts by weight of epicatechin, 6 parts by weight of gallicatechin, 30 parts by weight of dihydrokaempferol, 8 parts by weight of polyoxyethylene sorbitan ester, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 913.2 parts by weight of a solvent (about 55 parts by weight of ethanol and about 858.2 parts by weight of water).

[0058] 6. The prepared product was a light yellow, transparent microemulsion with a pH of 6.0, a viscosity of 22 mPa·s, and a surface tension of 28 mN / m. No significant stratification was observed after 30 days of storage in a 4°C refrigerator, demonstrating the good stability of the emulsion. High-performance liquid chromatography assays determined the active ingredient contents to be within ±3% of the nominal values, meeting quality requirements.

[0059] Example 2: Low-concentration tobacco leaf anti-adversity and aging-inhibiting agent 1. Add 3 parts by weight of γ-aminobutyric acid to 180 parts by weight of water at 45°C and stir on a magnetic stirrer for 35 minutes until completely dissolved to obtain a γ-aminobutyric acid aqueous solution. Mix 3 parts by weight of epicatechin and 3 parts by weight of gallocatechin, add the mixture to 220 parts by weight of an ethanol aqueous solution (8% ethanol content) at 35°C, and stir for 50 minutes until completely dissolved to obtain a catechin mixed solution. Add 15 parts by weight of dihydrokaempferol to 120 parts by weight of an ethanol aqueous solution (15% ethanol content) and stir for 70 minutes until completely dissolved to obtain a dihydrokaempferol solution.

[0060] 2. In a stainless steel container, add 5 parts by weight of polyoxyethylene sorbitan ester and 20 parts by weight of glycerin and mix them evenly using a high-speed disperser. Then, slowly add 80 parts by weight of water while stirring continuously at 200 rpm to form an emulsified system. Adjust the pH to 6.5 with 0.5 parts by weight of citric acid.

[0061] 3. Subsequently, under continuous stirring at 200 rpm, first add the γ-aminobutyric acid aqueous solution and stir for 12 minutes, then add the catechin mixed solution and stir for 12 minutes, and finally add the dihydrokaempferol solution and stir for 12 minutes.

[0062] 4. Add 0.5 parts by weight of vitamin E to the mixture and continue stirring for 10 minutes. Process the mixture in a high-speed shear homogenizer at 10,000 rpm for 4 minutes to form a uniform and stable microemulsion.

[0063] 5. Filter through a 0.22 μm filter membrane to remove insoluble impurities, adjust the pH value to 5.8 with 0.5 parts by weight of citric acid solution, and add water to a total weight of about 1000 parts by weight to obtain a tobacco leaf anti-adversity, anti-aging and ripening agent.

[0064] The tobacco leaf anti-adversity, anti-aging and ripening agent comprises: 3 parts by weight of gamma-aminobutyric acid, 3 parts by weight of epicatechin, 3 parts by weight of gallocatechin, 15 parts by weight of dihydrokaempferol, 5 parts by weight of polyoxyethylene sorbitan ester, 20 parts by weight of glycerol, 1 part by weight of citric acid, 0.5 parts by weight of vitamin E, and 950 parts by weight of a solvent (about 55 parts by weight of ethanol and about 858.2 parts by weight of water).

[0065] 6. The prepared product was a light yellow, transparent microemulsion with a pH of 5.8, a viscosity of 18 mPa·s, and a surface tension of 30 mN / m. No significant stratification was observed after storage in a 4°C refrigerator for 30 days, demonstrating the good stability of the emulsion. High-performance liquid chromatography assays determined the active ingredient contents to be within ±4% of the nominal values, meeting quality requirements.

[0066] Example 3: High-concentration tobacco leaf anti-adversity and anti-aging agent 1. Add 5 parts by weight of γ-aminobutyric acid to 220 parts by weight of water at 55°C and stir with a magnetic stirrer for 40 minutes until completely dissolved to obtain a γ-aminobutyric acid aqueous solution. Mix 9 parts by weight of epicatechin and 9 parts by weight of gallocatechin, add the mixture to 280 parts by weight of an aqueous ethanol solution (12% ethanol content) at 45°C, and stir for 55 minutes until completely dissolved to obtain a catechin mixed solution. Add 45 parts by weight of dihydrokaempferol to 180 parts by weight of an aqueous ethanol solution (25% ethanol content) and stir for 80 minutes until completely dissolved to obtain a dihydrokaempferol solution.

[0067] 2. In a stainless steel container, add 10 parts by weight of polyoxyethylene sorbitan ester and 40 parts by weight of glycerol, mix well using a high-speed disperser, then slowly add 100 parts by weight of water while stirring continuously at 300 rpm to form an emulsified system, and adjust the pH to 6.2 using citric acid.

[0068] 3. Same as Example 1.

[0069] 4. Add 0.8 parts by weight of vitamin E to the mixture and continue stirring for 10 minutes. Process the mixture in a high-speed shear homogenizer at 15,000 rpm for 6 minutes to form a uniform and stable microemulsion.

[0070] 5. Filter through a 0.22 μm filter membrane to remove insoluble impurities, adjust the pH value to 6.2 with citric acid, and add water to a total weight of about 1000 parts by weight to obtain a tobacco leaf anti-adversity, anti-aging and ripening agent.

[0071] The tobacco leaf anti-adversity, anti-aging and ripening agent comprises: 5 parts by weight of gamma-aminobutyric acid, 9 parts by weight of epicatechin, 9 parts by weight of gallicatechin, 45 parts by weight of dihydrokaempferol, 10 parts by weight of polyoxyethylene sorbitan ester, 40 parts by weight of glycerol, 3 parts by weight of citric acid, 1 part by weight of vitamin E, and 880 parts by weight of a solvent (about 78.6 parts by weight of ethanol and about 801.4 parts by weight of water).

[0072] 6. The prepared product was a light yellow, transparent microemulsion with a pH of 6.2, a viscosity of 28 mPa·s, and a surface tension of 25 mN / m. After storage at 4°C for 30 days, trace (<5%) delamination was observed, but it quickly returned to a homogeneous state after gentle shaking, meeting stability requirements. High-performance liquid chromatography assays determined the content of each active ingredient to be within ±5% of the nominal value, meeting quality requirements.

[0073] Example 4: Tobacco leaf anti-stress, anti-aging and ripening agent with optimized γ-aminobutyric acid content The difference between this embodiment and embodiment 1 is that the content of the γ-aminobutyric acid component is increased to 4.5 parts by weight. The difference in the preparation method from embodiment 1 is only that: In step 1, the amount of γ-aminobutyric acid is increased to 4 parts by weight; in step 3, the stirring time after adding the γ-aminobutyric acid aqueous solution is extended to 18 minutes to ensure uniform dispersion of the high-content γ-aminobutyric acid.

[0074] The tobacco leaf anti-adversity, anti-aging and ripening agent is composed of 4.5 parts by weight of gamma-aminobutyric acid, 6 parts by weight of epicatechin, 6 parts by weight of gallicate, 30 parts by weight of dihydrokaempferol, 8 parts by weight of polyoxyethylene sorbitan ester, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 912.7 parts by weight of a solvent (about 55.0 parts by weight of ethanol and about 877.7 parts by weight of water).

[0075] The prepared product is a light yellow, transparent microemulsion with a pH of 6.1, a viscosity of 24 mPa·s, and a surface tension of 27 mN / m. The product remained stable after 30 days of storage at 4°C, with no apparent stratification. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±3.5%, meeting quality requirements.

[0076] Example 5: Tobacco leaf anti-stress, anti-aging and ripening agent with optimized catechin formula The difference between this embodiment and embodiment 1 is that the ratio of catechin components is adjusted to 7.5 parts by weight of epicatechin and 7.5 parts by weight of gallocatechin. The difference in preparation method from embodiment 1 is only that: In step 1, the weight of epicatechin and gallocatechin was increased to 7.5 parts by weight each, the dissolution temperature was adjusted to 42°C, and the stirring time was extended to 55 minutes to ensure complete dissolution of the high-content catechins. In step 3, the stirring time after adding the catechin mixed solution was extended to 20 minutes to ensure thorough and uniform mixing.

[0077] The tobacco leaf anti-adversity, anti-aging and ripening agent is composed of: 4 parts by weight of gamma-aminobutyric acid, 7.5 parts by weight of epicatechin, 7.5 parts by weight of gallicate, 30 parts by weight of dihydrokaempferol, 8 parts by weight of polyoxyethylene sorbitan ester, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 910.2 parts by weight of a solvent (about 55.0 parts by weight of ethanol and about 855.2 parts by weight of water).

[0078] The prepared product is a yellow-green transparent microemulsion with a pH of 6.0, a viscosity of 25 mPa·s, and a surface tension of 26 mN / m. The product remained stable after 30 days of storage at 4°C, with no apparent stratification. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±3.8%, meeting quality requirements.

[0079] Example 6: Tobacco leaf anti-stress and aging-inhibiting agent with optimized dihydrokaempferol content The difference between this embodiment and embodiment 1 is that the content of dihydrokaempferol is increased to 36 parts by weight. The difference in preparation method from embodiment 1 is only that: In step 1, the ethanol concentration of the ethanol solution in which dihydrokaempferol is dissolved is increased to 22% to improve the solubility of dihydrokaempferol. At the same time, the stirring time of the dihydrokaempferol solution is extended to 75 minutes to ensure complete dissolution. In step 4, during the high-speed shear homogenization stage, the treatment time is extended to 5.5 minutes to ensure uniform dispersion of the high-content dihydrokaempferol.

[0080] The tobacco leaf anti-adversity, anti-aging and ripening agent is composed of: 4 parts by weight of gamma-aminobutyric acid, 6 parts by weight of epicatechin, 6 parts by weight of gallicatechin, 36 parts by weight of dihydrokaempferol, 8 parts by weight of polyoxyethylene sorbitan ester, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 907.2 parts by weight of a solvent (about 58 parts by weight of ethanol and about 849.2 parts by weight of water).

[0081] The prepared product is a dark yellow, transparent microemulsion with a pH of 6.1, a viscosity of 26 mPa·s, and a surface tension of 25.5 mN / m. Slight delamination (<3%) was observed after 30 days of storage at 4°C. However, the product quickly regained its homogeneity after gentle shaking, maintaining stability requirements. High-performance liquid chromatography analysis showed that the content of each active ingredient remained within ±4.2%, meeting quality requirements.

[0082] Example 7: Tobacco leaf anti-stress, anti-aging and ripening agent with optimized auxiliary ingredients The difference between this embodiment and embodiment 1 is that the composition of the adjustment auxiliary agent is: 9 parts by weight of polyoxyethylene sorbitan ester, 35 parts by weight of glycerol, 2.5 parts by weight of citric acid and 0.9 parts by weight of vitamin E. The only difference in the preparation method from embodiment 1 is: In step 3, the mixing ratio of polyoxyethylene sorbitan ester to glycerol was adjusted to 1:3.9, and the stirring rate was increased to 280 rpm to obtain a more stable emulsion system. In step 4, the amount of vitamin E added was adjusted to 0.8 parts by weight. Furthermore, during the high-speed shear homogenization stage, a stepwise speed increase was adopted: first at 8000 rpm for 2 minutes, then at 14000 rpm for 3 minutes, to obtain a more uniform and fine emulsion structure. The total amount of citric acid was adjusted to 2.5 parts by weight.

[0083] The tobacco leaf anti-stress, anti-aging and ripening agent is composed of: 4 parts by weight of gamma-aminobutyric acid, 6 parts by weight of epicatechin, 6 parts by weight of gallicatechin, 30 parts by weight of dihydrokaempferol, 9 parts by weight of polyoxyethylene sorbitan ester, 35 parts by weight of glycerol, 2.5 parts by weight of citric acid, 0.9 parts by weight of vitamin E, and 906.6 parts by weight of a solvent (about 55 parts by weight of ethanol and about 851.6 parts by weight of water).

[0084] The prepared product is a light yellow, transparent microemulsion with a pH of 6.1, a viscosity of 23 mPa·s, and a surface tension of 24 mN / m. The product remained highly stable after 30 days of storage at 4°C, without any delamination. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±3.2%, meeting quality requirements.

[0085] Example 8: Tobacco leaf anti-stress and aging-inhibiting agents optimized at different pH values The difference between this embodiment and embodiment 1 is that the pH value of the tobacco leaf anti-adversity, anti-aging and ripening agent is adjusted to 5.9, and the component ratio adopts the formula of embodiment 1.

[0086] The only difference from Example 1 is: The preparation method basically follows that of Example 1, with the following differences: in step 4, after high-speed shear homogenization, ultrasonic assisted treatment (power 300W, intermittent working mode: 3 seconds on, 2 seconds off, total processing time 5 minutes) is added to further improve the uniformity and stability of the product. In step 6, a precise control method is adopted in the final pH adjustment stage, using 0.05 mol·L - ¹Citric acid solution and 0.05 mol·L - ¹Potassium hydroxide solution was added in small amounts alternately, and the final pH value was precisely controlled at 5.9±0.05 in conjunction with real-time pH monitoring.

[0087] The prepared product is a light yellow, transparent microemulsion with a viscosity of 21 mPa·s and a surface tension of 27.5 mN / m. The product remained stable after 30 days of storage at 4°C, with no apparent stratification. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±3.0%, meeting quality requirements.

[0088] Comparative Example 1: Tobacco leaf ripening agent lacking γ-aminobutyric acid This comparative example was used to verify the key role of GABA in the multi-level coordinated regulatory system. For this purpose, a comparative product without GABA was prepared. Its components were as follows: 6 parts by weight of epicatechin, 6 parts by weight of gallocatechin, 30 parts by weight of dihydrokaempferol, 8 parts by weight of polyoxyethylene sorbitan, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 917.2 parts by weight of solvent.

[0089] The preparation method basically followed the method of Example 1, except that the addition and dissolution steps of γ-aminobutyric acid were omitted. Other process parameters remained unchanged to ensure the effectiveness of the experimental control.

[0090] The prepared product is a transparent yellow microemulsion with a pH of 5.9, a viscosity of 20 mPa·s, and a surface tension of 29 mN / m. The product remained stable after 30 days of storage at 4°C, with no apparent stratification. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±4.0%.

[0091] Comparative Example 2: Tobacco Ripening Agent Lacking Catechin Compounds This comparative example demonstrates the importance of epicatechin and gallocatechin in regulating the enzyme system. To this end, a control product without these two ingredients was prepared. Its composition is as follows: 4 parts by weight of γ-aminobutyric acid, 30 parts by weight of dihydrokaempferol, 8 parts by weight of polyoxyethylene sorbitan, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 925.2 parts by weight of solvent.

[0092] The preparation process basically followed the method of Example 1, but omitted the steps of adding and dissolving epicatechin and gallocatechin. The prepared product is a light yellow, transparent microemulsion with a pH of 6.0, a viscosity of 19 mPa·s, and a surface tension of 30 mN / m. The product remained stable after 30 days of storage at 4°C, with no apparent demixing. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±3.5%.

[0093] Comparative Example 3: Tobacco leaf ripening agent lacking dihydrokaempferol This comparative example was used to verify the key role of dihydrokaempferol in regulating cellular metabolism. For this purpose, a comparative product without dihydrokaempferol was prepared. Its components were as follows: 4 parts by weight of gamma-aminobutyric acid, 6 parts by weight of epicatechin, 6 parts by weight of epigallocatechin, 8 parts by weight of polyoxyethylene sorbitan, 30 parts by weight of glycerol, 2 parts by weight of citric acid, 0.8 parts by weight of vitamin E, and 943.2 parts by weight of solvent.

[0094] The preparation process basically followed the method of Example 1, but omitted the steps of adding and dissolving dihydrokaempferol.

[0095] The prepared product is a yellow-green transparent microemulsion with a pH of 6.1, a viscosity of 21 mPa·s, and a surface tension of 28 mN / m. The product remained stable after 30 days of storage at 4°C, with no apparent demixing. High-performance liquid chromatography analysis showed that the content of each active ingredient was within ±3.8%.

[0096] To comprehensively evaluate the practical application effects of the tobacco leaf anti-stress, anti-aging and ripening agent prepared in the embodiments of the present invention, large-scale field trials were conducted in three major tobacco-growing areas: Yuxi, Yunnan, Zunyi, Guizhou, and Chenzhou, Hunan. Each trial adopted a randomized block design with three replicates, and each plot area was 0.5 mu. The experimental treatments included the above-mentioned eight embodiments and three comparative examples, as well as a clear water control. All treatments were carried out on the fifth day after the tobacco plants were toppled, using drone spraying technology, and the application rate was uniformly 40 kg per mu. - ¹.

[0097] On the 7th, 14th and 21st day after treatment, samples of the middle tobacco leaves were collected and the following indicators were measured: 1. Antioxidant enzyme activity: including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities; 2. Chlorophyll content: 80% acetone extraction method was used to determine the chlorophyll a, chlorophyll b and total chlorophyll contents; 3. The degree of membrane lipid peroxidation: assessed by measuring the malondialdehyde (MDA) content; 4. Photosynthetic parameters: Net photosynthetic rate, stomatal conductance, and transpiration rate were measured using the LI-6400 portable photosynthetic measurement system. 5. Endogenous hormone levels: Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of abscisic acid (ABA) and cytokinin (CTK).

[0098] In addition, after the tobacco leaves were harvested, their grades were assessed and their chemical composition, including total sugar, total nitrogen, total nicotine and reducing sugar content, was determined.

[0099] The main test results of each treatment are shown in Tables 1 to 3.

[0100] Table 1. Effects of different treatments on antioxidant enzyme activities in tobacco leaves (14 days after treatment)

[0101] Table 2. Effects of different treatments on chlorophyll content and malondialdehyde content in tobacco leaves (14 days after treatment)

[0102] Table 3. Effects of different treatments on tobacco leaf quality indicators (measured after harvest)

[0103] From the test results in Table 1-3, we can see that: 1. Antioxidant Enzyme Activity: SOD, POD, and CAT activities in tobacco leaves treated with all examples were significantly higher than those in the comparative and control treatments. Example 3 (high-concentration formulation) showed the most significant effect, with SOD activity increasing by 69.5%, POD activity increasing by 72.9%, and CAT activity increasing by 49.0% compared to the control. Examples 4 (optimized γ-aminobutyric acid concentration) and 6 (optimized dihydrokaempferol concentration) also showed significant effects. Comparative Example 1 (lacking γ-aminobutyric acid) showed significantly lower enzyme activity than the complete formulation, demonstrating the key role of γ-aminobutyric acid in activating the antioxidant defense system.

[0104] 2. Chlorophyll Content: All treatments in the examples significantly slowed chlorophyll degradation, maintaining high chlorophyll content. The total chlorophyll contents of Examples 3, 5, and 6 were 60.6%, 52.9%, and 55.3% higher than the control, respectively. Chlorophyll retention in Comparative Example 2 (lacking catechins) was significantly lower than that in the complete formulation, demonstrating the importance of catechins in protecting the photosynthetic system and slowing chlorophyll degradation.

[0105] 3. Membrane Lipid Peroxidation: The treatments in the examples significantly reduced the MDA content in tobacco leaves, indicating a reduction in cell membrane lipid peroxidation. The MDA content in Example 3 was 47.1% lower than in the control, demonstrating the most pronounced protective effect. The MDA content in Comparative Example 3 (lacking dihydrokaempferol) was significantly higher than that in the complete formulation, demonstrating the unique role of dihydrokaempferol in protecting the cell membrane system.

[0106] 4. Tobacco Leaf Quality Indicators: The treatments in the examples significantly increased the total sugar content of the tobacco leaves, reduced the total nitrogen content, and increased the sugar-to-nitrogen ratio, improving tobacco leaf quality. The sugar-to-nitrogen ratio in Example 3 reached 15.6, a 71.4% increase over the control, and the proportion of high-quality tobacco reached 73.2%, a 44.7% increase over the control. The quality indicators of the three comparative examples were significantly lower than those of the complete formulation, demonstrating that the four core active ingredients have a synergistic effect in improving tobacco leaf quality; the lack of any one ingredient would affect the ultimate effect.

[0107] The endogenous hormone levels of tobacco leaves treated with Example 1 (standard formula) were measured. The results showed that on the 14th day after treatment, the ABA / CTK ratio of the tobacco leaves was 1.45, while that of the control treatment was 2.87, indicating that the standard formula treatment can effectively regulate the balance of endogenous hormones and delay the aging process of leaves.

[0108] It can be seen from the above test results that the tobacco leaf anti-stress and anti-aging agent provided by the present invention forms a multi-level synergistic regulatory system through the synergistic effect of the four key active ingredients of γ-aminobutyric acid, epicatechin, gallic acid and dihydrokaempferol, which can significantly improve the activity of tobacco leaf antioxidant enzymes, reduce cell membrane lipid peroxidation damage, delay chlorophyll degradation, regulate endogenous hormone balance, and ultimately achieve the comprehensive effects of enhanced tobacco leaf resistance, delayed leaf aging and improved quality.

[0109] Combined with the test results and analysis of relevant physiological and biochemical indicators, the mechanism of action of the tobacco leaf anti-adversity, anti-aging and ripening agent provided by the embodiment of the present invention can be explained from the following aspects: 1. Signal regulation layer: GABA, as a plant signal molecule, is rapidly absorbed by tobacco leaves after treatment, activating the calcium ion signal pathway and causing a series of cascade reactions. The applicant's analysis showed that within 24 hours after treatment, the cytoplasmic Ca² + The concentration increased by about 2.5 times, and the activity of calcium-dependent protein kinase (CDPK) increased by about 3 times, thereby activating multiple defense-related transcription factors. GABA also inhibits ethylene synthase activity. After treatment, the activity of ACC synthase in tobacco leaves decreased by about 40%, and the amount of ethylene released decreased by about 35%, effectively delaying the onset of the aging process. The treatment effect of Comparative Example 1, which lacked GABA, was significantly reduced, confirming the key role of GABA in activating the entire regulatory network.

[0110] 2. Enzyme System Regulation: Epicatechin and gallocatechin, as polyphenolic compounds, directly scavenge free radicals while activating antioxidant enzyme systems. Through enzyme kinetic analysis, the applicants discovered that these two catechin compounds can act as allosteric regulators of SOD, POD, and CAT, altering the conformation of the enzymes and improving their catalytic efficiency. The Vmax value of SOD in treated tobacco leaves increased by approximately 55%, while the Km value decreased by approximately 30%, indicating a significant improvement in enzyme-substrate affinity and catalytic efficiency. Furthermore, these two compounds can also complex Fe²⁻¹. + 、Cu² + Transition metal ions such as catechins inhibit the Fenton reaction and reduce the generation of hydroxyl radicals. In the comparative example 2 treatment lacking catechin compounds, the antioxidant enzyme activity was significantly reduced and the MDA content was significantly increased, indicating that these two compounds have an irreplaceable role in the regulation layer of the enzyme system.

[0111] 3. Cellular metabolism regulation layer: Dihydrokaempferol regulates energy metabolism and antioxidant defense system by activating SIRT1 and Nrf2 signaling pathways. Through protein immunoblotting analysis, we found that the expression of SIRT1 protein in tobacco leaves increased by about 2 times after treatment, the nuclear translocation of Nrf2 was significantly enhanced, and the expression of downstream target genes such as HO-1, NQO1, GST, etc. increased by 1.5-3 times. In addition, dihydrokaempferol can also protect the structure of mitochondria and chloroplasts. After treatment, the activity of complexes I and III of the mitochondrial electron transport chain in tobacco leaves increased by about 25% and 30%, respectively, and the maximum photochemical efficiency of photosystem II (Fv / Fm) remained above 0.78, while the control treatment dropped to 0.65. In the treatment of comparative example 3 lacking dihydrokaempferol, these indicators were significantly reduced, indicating that dihydrokaempferol has a unique role in regulating cellular metabolism and protecting organelle function.

[0112] 4. Physical Protective Layer: The physical protective layer formed by auxiliary ingredients provides a suitable environment for the active ingredients to exert their effects. Polyoxyethylene sorbitan ester reduces surface tension, improving the spread and penetration of the drug solution on the leaf surface. Using fluorescent labeling and tracing, the applicant's research found that by 6 hours after treatment, the concentration of the active ingredient in mesophyll cells was 1.8 times that of conventional formulations, significantly improving bioavailability. Glycerin provides a long-lasting moisturizing environment. Under dry conditions (relative humidity 40%), 12 hours after treatment, the evaporation rate of the drug solution from the leaf surface was approximately 35% lower than that of a formulation without glycerol. Citric acid maintains a slightly acidic environment (pH 5.8-6.2), which promotes the activity of antioxidant enzymes and chelates metal ions to prevent oxidative degradation of the active ingredients. Vitamin E, a fat-soluble antioxidant, protects the cell membrane system. After treatment, the degree of membrane lipid peroxidation in tobacco leaves was significantly reduced, maintaining fluidity and selective permeability.

[0113] In summary, the four regulatory levels of the tobacco leaf anti-stress, anti-aging and ripening agent provided by the present invention work closely together to form a complete protection network, which can comprehensively regulate multiple problems faced by tobacco leaves after topping, such as oxidative stress, hormonal disorders and metabolic imbalances, thereby achieving the comprehensive effects of enhanced stress resistance, delayed leaf aging and improved maturity quality.

[0114] Based on the test results and comprehensive analysis of the above examples, Example 3 (high-concentration formula) exhibited the best overall performance and can be used as the preferred embodiment of the present invention. This formula comprises: 5 parts by weight of γ-aminobutyric acid, 9 parts by weight of epicatechin, 9 parts by weight of gallocatechin, 45 parts by weight of dihydrokaempferol, 10 parts by weight of polyoxyethylene sorbitan, 40 parts by weight of glycerol, 3 parts by weight of citric acid, 1 part by weight of vitamin E, and 878 parts by weight of solvent.

[0115] The SOD activity of tobacco leaves treated with this formula increased by 69.5%, POD activity increased by 72.9%, CAT activity increased by 49.0%, total chlorophyll content increased by 60.6%, MDA content decreased by 47.1%, and the proportion of high-quality tobacco increased by 44.7%. The overall performance was significantly better than other treatments.

[0116] In practical application, it is recommended to use a multi-rotor drone for spraying on the 5th day after the tobacco plants are toppled, in sunny or cloudy weather with wind speed not exceeding level 3. Dilute the original solution at a ratio of 1:5 and spray at a rate of 40 kg per mu. - ¹, when spraying, keep the drone at an altitude of 1.8 meters above the canopy and a flight speed of 4 m·s - ¹, the spraying pressure was 0.25MPa and the spraying overlap rate was 25% to ensure uniform coverage of the tobacco leaf surface.

[0117] It should be noted that Examples 4 (optimized γ-aminobutyric acid content), 5 (optimized catechins), and 6 (optimized dihydrokaempferol content) also demonstrated good performance and can be selected based on the specific conditions of different tobacco-growing areas. For example, in hot and arid regions, the formulation of Example 6 can be selected to enhance stress resistance; for early-maturing varieties, the formulation of Example 5 can be selected to further delay leaf senescence; and for mid- to late-maturing varieties, the formulation of Example 4 can be selected to optimize endogenous hormone balance and promote uniform ripening.

[0118] Overall, the tobacco leaf stress-resistant, aging-inhibiting, and ripening-maintaining agent provided by this invention, along with its preparation method and application technology, achieves the combined effects of enhanced stress resistance, delayed aging, and improved ripening quality through the establishment of a multi-level coordinated regulatory system, providing effective technical support for high-quality tobacco leaf production. This technology is not only applicable to tobacco cultivation, but its core principles and regulatory mechanisms can also provide a reference for the physiological regulation of other economic crops, thus possessing broad application prospects.

[0119] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A tobacco leaf anti-adversity, anti-aging and ripening agent, characterized in that: It includes: Active ingredients, adjuvants and solvents; The active ingredients include: gamma-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol; the adjuvants include: surfactant, wetting agent, antioxidant and acid-base regulator; the solvent is water or ethanol aqueous solution.

2. The tobacco leaf anti-adversity, anti-aging and ripening agent according to claim 1, characterized in that: Among the active components, the weight ratio of gamma-aminobutyric acid, epicatechin, gallocatechin and dihydrokaempferol is (3-5): (3-9): (3-9): (15-45).

3. The tobacco leaf anti-adversity, anti-aging and ripening agent according to claim 2, characterized in that: Among the auxiliary agents, The surfactant is polyoxyethylene sorbitan ester; The humectant is glycerin; The antioxidant is vitamin E; The acid-base regulator is citric acid; The weight ratio of the polyoxyethylene sorbitan ester, glycerol and vitamin E is (5-10): (20-40): (0.5-1); The citric acid is used to adjust the pH value of the tobacco leaf anti-adversity, anti-aging and ripening agent system to 5.8-6.

2.

4. The tobacco leaf anti-adversity, anti-aging and ripening agent according to claim 3, characterized in that It includes by weight: 3-5 parts by weight of gamma-aminobutyric acid; 3-9 parts by weight of epicatechin; 3-9 parts by weight of epigallocatechin gallate; 15-45 parts by weight of dihydrokaempferol; 5-10 parts by weight of polyoxyethylene sorbitan; 20-40 parts by weight of glycerol; 1-3 parts by weight of citric acid; Vitamin E 0.5-1 parts by weight; 880-950 parts by weight of solvent.

5. The tobacco leaf anti-adversity, anti-aging and ripening agent according to claim 3, characterized in that: It includes by weight: 4-5 parts by weight of gamma-aminobutyric acid; 6-9 parts by weight of epicatechin; 6-9 parts by weight of epigallocatechin gallate; 30-45 parts by weight of dihydrokaempferol; 8-10 parts by weight of polyoxyethylene sorbitan; 30-40 parts by weight of glycerol; 2-3 parts by weight of citric acid; Vitamin E 0.8-1 parts by weight; Solvent 878-913.2 parts by weight.

6. The method for preparing the tobacco leaf anti-adversity, anti-aging and ripening agent according to any one of claims 1 to 5, characterized in that: include: Step 1), dissolving γ-aminobutyric acid in a solvent to obtain a γ-aminobutyric acid solution; mixing epicatechin and gallocatechin and dissolving them in a solvent to obtain a catechin mixed solution; dissolving dihydrokaempferol in a solvent to obtain a dihydrokaempferol solution; Step 2) Mix the surfactant and the wetting agent, add the solvent under stirring to obtain an emulsified system, and add the acid-base regulator to adjust the pH value to 6.0-6.5; Step 3), under stirring, sequentially adding the γ-aminobutyric acid solution, the catechin mixed solution, and the dihydrokaempferol solution to the emulsified system; Step 4), adding an antioxidant to the mixed solution obtained in step 3), stirring evenly, and then treating with a high-speed shear homogenizer to form a stable microemulsion; Step 5), filtering the microemulsion using an organic filter membrane, adding an acid-base regulator to adjust the pH value to 5.8-6.2, and adding a solvent to obtain a tobacco leaf anti-stress, anti-aging and ripening agent.

7. The preparation method according to claim 6, characterized in that The step 1) is specifically as follows: Add γ-aminobutyric acid to water at 45-55°C and stir until completely dissolved to obtain a γ-aminobutyric acid solution; The epicatechin and gallocatechin are mixed and added into an aqueous solution containing 8-12% ethanol at 35-45°C, and stirred until completely dissolved to obtain a catechin mixed solution; Add dihydrokaempferol to an aqueous solution containing 15-25% ethanol and stir until completely dissolved to obtain a dihydrokaempferol solution.

8. The preparation method according to claim 6, wherein The stirring speed in the step 2) is 200-300 rpm, and / or the stirring speed in the step 2) is 200-300 rpm.

9. The preparation method according to claim 6, wherein The speed of the homogenizer in step 4) is 10,000-15,000 rpm, and / or the pore size of the organic filter membrane in step 5) is 0.22 μm.

10. The tobacco leaf stress resistance, aging inhibition and ripening maintenance agent according to any one of claims 1 to 5 is used to improve tobacco stress resistance, delay leaf aging and promote normal ripening of tobacco leaves, characterized in that: include: Step (A): 4-6 days after topping the tobacco plants, choose a sunny or cloudy day with wind speed not exceeding level 3; Step (B), diluting the tobacco leaf anti-adversity and aging-inhibiting agent with water in a ratio of 1:4-1:8; Step (C): using an agricultural multi-rotor drone equipped with a low-pressure atomizing nozzle to spray at 8:00-10:00 in the morning or 16:00-18:00 in the afternoon at an ambient temperature of 15-30°C; Step (D): Control the drone to fly at a height of 1.5-2.0 meters above the crown and a speed of 3-5 m·s - ¹, spraying pressure is 0.2-0.3 MPa, spraying overlap rate is 20-30%, and application rate is 30-50 kg·mu - ¹.

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