Carnosic acid nano-emulsion as well as preparation method and application thereof

Abstract

The invention discloses application of carnosic acid nano-emulsion as an agricultural bactericide for preventing and treating plant diseases. The plant diseases comprise rhizoctonia solani, colletotrichum rubrum, botrytis cinerea, peronophythora litchii, phytophthora capsici, colletotrichum gloeosporioides, tomato ralstonia solanacearum, citrus canker germs, potato soft rot germs, xanthomonas oryzae pv. Oryzae, mango bacterial angular leaf spot germs and muskmelon bacterial fruit spot germs; the carnosic acid nano-emulsion is prepared from the following raw materials in percentage by mass: 0.5 to 5 percent of carnosic acid, 5 to 20 percent of organic solvent, 4.0 to 10 percent of emulsifier and the balance of water. The carnosic acid nano-emulsion prepared by the invention has the characteristics of simplicity in processing, emulsion stability, good wettability, obvious bactericidal activity and the like, and meanwhile, the carnosic acid is a plant secondary metabolite and is high in use safety in the process of preventing and treating plant diseases.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection, and in particular to a carnosic acid nanoemulsion and a preparation method and application thereof. Background Art

[0002] Plant diseases lead to reduced crop yields and deterioration of quality, resulting in a significant crop yield loss of 10%-30% each year, which seriously restricts the development of agriculture. Although chemical pesticides are currently an effective means of preventing and controlling plant diseases and reducing agricultural production losses, due to the irrational use of chemical pesticides and the emergence of pathogenic bacteria resistance, the dosage and cost of control agents are constantly increasing, while increasing the risk of pesticide residues and environmental pollution. Therefore, agricultural production urgently needs to develop new green pesticides that are safe, effective and environmentally friendly.

[0003] Carnosic acid is a diterpene phenolic compound found in rosemary, sage and other Lamiaceae plants. Its molecular formula is C 20 H 28 O4 (CAS No.: 3650-09-7), easily soluble in oils and difficult to dissolve in water. Carnosic acid has antioxidant, anti-inflammatory, anti-tumor, antibacterial and cardiovascular protection effects. It is safe and low-toxic to humans and animals and is often used in food preservation, cosmetics and health products. Carnosic acid is easily oxidized and decomposed due to its structure and poor water solubility, resulting in low bioaccessibility and bioavailability, which limits its application.

[0004] Nanoemulsion, also known as nanoemulsion, is a stable emulsion liquid preparation in which the water-insoluble active ingredients are coated with emulsifiers and other surfactants and dispersed in water with nanometer-scale particle size. The average particle size of nanoemulsion droplets is 20-500nm. Compared with traditional emulsions, the droplet size is small and the specific surface area is large, which improves the dispersibility and stability of drugs, produces low surface tension and good wettability, and improves the adhesion, deposition and penetration of drugs on the target surface. In recent years, nanoemulsions have been widely studied and applied as excellent drug delivery systems for poorly soluble drugs.

[0005] Therefore, preparing carnosic acid in the form of nanoemulsion to increase the water solubility of carnosic acid and improve its agricultural fungicidal activity is of great significance for broadening its application range in plant diseases. Summary of the invention

[0006] In view of this, the present invention proposes a carnosic acid nanoemulsion and a preparation method and application thereof to solve the above technical problems.

[0007] The technical solution of the present invention is achieved in this way:

[0008] Application of a carnosic acid nanoemulsion as an agricultural fungicide for preventing and controlling plant diseases, wherein the plant diseases include rice sheath blight pathogen Rhizoctonia solani, rubber anthracnose pathogen Colletotrichum gloeosporioides, tomato gray mold pathogen Botrytis cinerea, litchi downy mildew pathogen Peronophthora litchii, pepper phytophthora capsici, grape anthracnose pathogen Colletotrichum viniferum, tomato bacterial wilt pathogen Ralstonia solanacearum, citrus canker pathogen Xanthomonas citri subsp. Citri, potato soft rot pathogen Pectobacterium carotovorum, rice bacterial leaf blight pathogen Xanthomonas oryzae pv.oryzae, mango bacterial angular spot pathogen Xanthomonas compestris pv.mangiferaeindicae, and melon bacterial fruit spot pathogen Acidovorax citrulli;

[0009] The carnosic acid nanoemulsion comprises the following raw materials in percentage by mass: 0.5-5% of carnosic acid, 5-20% of organic solvent, 4.0-10% of emulsifier and the balance of water.

[0010] Preferably, the organic solvent is any one of ethanol, cyclohexanone and methyl oleate.

[0011] Preferably, the emulsifier is at least one of styrylphenol polyoxyethylene ether, tristyrylphenol polyoxyethylene polyoxypropylene ether, castor oil polyoxyethylene ether EL-40, castor oil polyoxyethylene ether EL-60, polyoxyethylene hydrogenated castor oil RH-40, fatty alcohol polyoxyethylene ether AEO-5, peregal O-25, lauryl alcohol polyoxyethylene Brij-35, and isomeric tridecyl alcohol polyoxyethylene ether E-1310.

[0012] Preferably, the emulsifier is a mixture of polyoxyethylene styrylphenol ether and polyoxyethylene hydrogenated castor oil RH-40 in a mass ratio.

[0013] Preferably, the emulsifier is a mixture of polyoxyethylene styrylphenol ether and polyoxyethylene hydrogenated castor oil RH-40 in a mass ratio of 1:1.

[0014] Preferably, the method for preparing the carnosic acid nanoemulsion adopts a low-energy emulsification method, comprising the following steps:

[0015] (1) weighing raw materials according to proportion, completely dissolving the weighed carnosic acid technical in a solvent, then adding an emulsifier, stirring well to obtain an oil phase, and setting aside;

[0016] (2) subjecting the oil phase to magnetic stirring, and under stirring, gradually adding deionized water dropwise into the oil phase;

[0017] (3) After the deionized water is added, the magnetic stirring is continued for 20-40 minutes to form a uniformly dispersed emulsion, thereby obtaining a nanoemulsion of carnosic acid.

[0018] Preferably, the magnetic stirring speed is 500-1000 r / min.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The carnosic acid nanoemulsion provided by the invention has a simple formula composition and uses less materials; adopts a low-energy emulsification method, has simple processing steps and low energy consumption, and obtains a nanoemulsion with fine droplet size, uniform dispersion, not prone to sedimentation, low surface tension, and easy wetting and spreading on the target surface, thereby increasing the water solubility of carnosic acid, improving agricultural bactericidal activity, and broadening its application range in plant diseases. At the same time, the carnosic acid of the invention is a plant secondary metabolite, and is highly safe to use in the process of preventing and controlling plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The following are appearance pictures of the carnosic acid nanoemulsions of Examples 1 and 4.

[0022] Figure 2 This is the particle size distribution diagram of the nanoemulsion described in Example 1.

[0023] Figure 3 This is the ultrastructure of carnosic acid nanoemulsion.

[0024] Figure 4 is the contact angle of carnosic acid nanoemulsion on the leaf surface.

[0025] Figure 5 This is a picture of carnosic acid nanoemulsion inhibiting the growth of pathogenic fungal hyphae. DETAILED DESCRIPTION

[0026] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0027] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0029] Example 1

[0030] A carnosic acid nanoemulsion is composed of the following raw materials in percentage by mass: 2% carnosic acid, 10% ethanol, 3% styrylphenol polyoxyethylene ether, 3% polyoxyethylene hydrogenated castor oil RH-40, and 82% deionized water.

[0031] The preparation steps of the carnosic acid nanoemulsion are as follows:

[0032] S1. Weigh the original drug according to the mass ratio, dissolve carnosic acid completely in ethanol solvent, add emulsifier (styrylphenol polyoxyethylene ether and polyoxyethylene hydrogenated castor oil RH-40), stir and disperse evenly to form a uniform oil phase;

[0033] S2, subjecting the oil phase obtained in step S1 to magnetic stirring at a stirring speed of 1000 r / min, and during the stirring process, adding deionized water drop by drop into the oil phase;

[0034] S3. After the deionized water is added dropwise, stirring is continued at a speed of 800 r / min for 30 min to obtain a 2% carnosic acid nanoemulsion.

[0035] The nanoemulsion prepared in this embodiment has a light yellow transparent liquid appearance (such as Figure 1 The average particle size of the emulsion diluted 100 times is 19.42nm, and the dispersion coefficient is 0.09. The particle size distribution diagram is shown in Figure 2 shown.

[0036] Example 2

[0037] A carnosic acid nanoemulsion is composed of the following raw materials in percentage by mass: 2% carnosic acid, 10% methyl oleate, 3% styrylphenol polyoxyethylene ether, 3% castor oil polyoxyethylene ether EL-60, and 82% deionized water.

[0038] The preparation steps of the carnosic acid nanoemulsion are as follows:

[0039] S1. Weigh the original drug according to the mass ratio, dissolve carnosic acid completely in methyl oleate solvent, add emulsifier (styrylphenol polyoxyethylene ether and castor oil polyoxyethylene ether EL-60), stir and disperse evenly to form a uniform oil phase;

[0040] S2, subjecting the oil phase obtained in step S1 to magnetic stirring at a stirring speed of 800 r / min, and during the stirring process, adding deionized water drop by drop into the oil phase;

[0041] S3. After the deionized water is added dropwise, stirring is continued at a speed of 800 r / min for 30 min to obtain a 2% carnosic acid nanoemulsion.

[0042] The nanoemulsion prepared in this example has a light yellow emulsion appearance. The average particle size of the emulsion diluted 100 times is 152.3 nm, and the dispersion coefficient is 0.620.

[0043] Example 3

[0044] A carnosic acid nanoemulsion is composed of the following raw materials in percentage by mass: 2% carnosic acid, 10% cyclohexanone, 4% styrylphenol polyoxyethylene ether, 2% castor oil polyoxyethylene ether EL-40, and 82% deionized water.

[0045] The preparation steps of the carnosic acid nanoemulsion are as follows:

[0046] S1. Weigh the original drug according to the mass ratio, dissolve carnosic acid completely in cyclohexanone solvent, add emulsifier (styrylphenol polyoxyethylene ether and castor oil polyoxyethylene ether EL-40), stir and disperse evenly to form a uniform oil phase;

[0047] S2, subjecting the oil phase obtained in step S1 to magnetic stirring at a stirring speed of 500 r / min, and during the stirring process, adding deionized water drop by drop into the oil phase;

[0048] S3. After the deionized water is added dropwise, stirring is continued at a speed of 1000 r / min for 30 min to obtain a 2% carnosic acid nanoemulsion.

[0049] The nanoemulsion prepared in this example has a light yellow emulsion appearance. The average particle size of the emulsion diluted 100 times is 192.57 nm, and the dispersion coefficient is 0.681.

[0050] Example 4

[0051] A carnosic acid nanoemulsion is prepared from the following raw materials in percentage by mass:

[0052] Carnosic acid 4%, ethanol 10%, polyoxyethylene styrylphenol ether 4%, polyoxyethylene hydrogenated castor oil RH-40 4%, deionized water input 78%.

[0053] The preparation steps of the carnosic acid nanoemulsion are as follows:

[0054] S1. Weigh the original drug according to the mass ratio, dissolve carnosic acid completely in ethanol solvent, add emulsifier (styrylphenol polyoxyethylene ether and polyoxyethylene hydrogenated castor oil RH-40), stir and disperse evenly to form a uniform oil phase;

[0055] S2, subjecting the oil phase obtained in step S1 to magnetic stirring at a stirring speed of 1000 r / min, and during the stirring process, adding deionized water drop by drop into the oil phase;

[0056] S3. After the deionized water is added dropwise, stirring is continued at a speed of 800 r / min for 30 min to obtain a 2% carnosic acid nanoemulsion.

[0057] The nanoemulsion prepared in this embodiment has a milky white appearance (such as Figure 1 As shown), the average particle size of the emulsion diluted 100 times is 171.87nm and the dispersion coefficient is 0.428.

[0058] Example 5 Observation of the morphology of carnosic acid nanoemulsion

[0059] The carnosic acid nanoemulsion prepared in Example 1 of the present invention was diluted 100 times, and a drop of the diluted emulsion was deposited on a 200-mesh copper grid of the carbon coating, dried at room temperature, and observed by transmission electron microscopy (HT700). The ultrastructure of the carnosic acid nanoemulsion was as shown in the attached figure. Figure 3 As shown, the nanoparticles are wrapped by the hydrophilic and lipophilic amphiphilic emulsifier, forming a stable hydrophilic layer in the water phase and are evenly dispersed.

[0060] Example 6 Determination of contact angle and surface tension

[0061] The contact angle and surface tension of the carnosic acid nanoemulsions and 1% carnosic acid ethanol solutions obtained in the above four examples were measured by diluting the test solutions containing 200 mg / L carnosic acid with deionized water.

[0062] The contact angle was measured using the sessile drop method of KRUSSDSA100 (Krues Scientific Instruments). Fresh, clean rubber leaves were cut into strips and fixed on a glass slide with the front side facing up. The strips were placed on the sample stage of the measuring instrument. A syringe SY-3601 with a diameter of 0.517 mm was used to aspirate 2-3 μL of the test solution each time. The static contact angle was taken when the droplet contacted the leaf for 30 seconds, and the average value of the parallel tests was taken. The surface tension was measured using the hanging drop method of KRUSSDSA100. A syringe SY-3601 with a diameter of 1.83 mm was used. The shape parameter range of the hanging drop was 0.56-0.65. The test was repeated 3 times in parallel and the average value was taken.

[0063] The results are shown in Table 1:

[0064] Table 1 Contact angle and surface tension test results of carnosic acid nanoemulsion dilution (treatment concentration 200 mg / L)

[0065]

[0066] Figure 4 is the contact angle of the leaf surface of carnosic acid ethanol solution and carnosic acid nanoemulsion, Figure 4 As can be seen from the test results in Table 1, the surface tension of the carnosic acid nanoemulsions obtained in Examples 1-4 of the present invention is relatively low, and they are easier to wet and spread on the leaf surface.

[0067] Example 7 Inhibitory activity of carnosic acid nanoemulsion against plant pathogens

[0068] 1. Test strains

[0069] Rice sheath blight pathogen Rhizoctonia solani, citrus fruit rot Lasiodiplodia pseudotheobromae, tomato gray mold pathogen Botrytis cinerea, litchi downy mildew pathogen Peronophthoralitchii, pepper phytophthora capsici, grape anthracnose pathogen Colletotrichum viniferum, tomato bacterial wilt pathogen Ralstonia solanacearum, citrus canker pathogen Xanthomonas citrisubsp.Citri, potato soft rot pathogen Pectobacterium carotovorum, rice bacterial leaf blight pathogen Xanthomonas oryzae pv.oryzae, mango bacterial angular spot pathogen Xanthomonas compestris pv.mangiferaeindicae, melon bacterial fruit spot pathogen Acidovorax citrulli. The above strains were provided by the pesticide research group of the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences.

[0070] 2. Test methods

[0071] 2.1 In vitro antibacterial activity against plant pathogenic fungi

[0072] The in vitro antibacterial activity of carnosic acid against plant pathogenic fungi was determined by the growth rate method with reference to the agricultural industry standard of the People's Republic of China "Guidelines for Indoor Bioassay Tests of Pesticides". The treatment agent is a 2% carnosic acid nanoemulsion and a 1% carnosic acid ethanol solution prepared in Example 2. The treatment agent is diluted with sterile water to form carnosic acid solutions of various concentrations, which are mixed with sterile molten PDA and poured into a culture dish. After cooling and solidification, a plant pathogenic fungus plate (5 mm in diameter) is inoculated in the center of the culture dish. The control group is treated with a drug-free nanoemulsion dilution. After the culture dish of the control group is completely covered with fungal hyphae, the hyphae growth diameter is measured. According to the colony growth diameter, the hyphae growth inhibition rate is calculated according to formula (1), the toxicity curve is constructed, and the inhibition concentration EC is calculated. 50 value.

[0073] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial plate diameter) × 100% (1)

[0074] The results are shown in Table 2:

[0075] Table 2 Indoor toxicity of 2% carnosic acid nanoemulsion to seven plant pathogenic fungi

[0076]

[0077] The test results show that carnosic acid has a high indoor toxicity to the seven tested plant pathogenic fungi, and its toxicity to pepper phytophthora, grape anthracnose and citrus fruit rot is higher than that of other pathogenic fungi. In contrast, the nanoemulsion increased the indoor toxicity of carnosic acid to plant pathogenic fungi. The antibacterial effect of carnosic acid nano solution on the growth of litchi downy mildew is shown in Figure 2. Figure 5 As shown in the figure, it can be seen that with the increase of the concentration of carnosic acid, its inhibitory effect on plant pathogens also increases to varying degrees.

[0078] 2.2 In vitro inhibitory activity against plant pathogenic bacteria

[0079] The present invention adopts the turbidity method (96-well culture plate) to determine the inhibitory effect of carnosic acid nanoemulsion on plant pathogenic bacteria. The prepared plant pathogenic bacteria suspension (bacterial suspension OD 600 Value is about 1.0) was added to a 96-well culture plate, and 10 μL was added to each well. The control group added a culture medium without medicine, and the amount added to each well was 190 μL; the experimental group diluted the carnosic acid nanoemulsion prepared in Examples 1 to 4 with the culture medium to the required drug-containing culture medium, and 190 μL was added to each well. The drug-containing culture medium and the bacterial suspension were blown and mixed, and each treatment was repeated 3 times. At the same time, a background group without inoculation of bacterial solution was set up for both the control group and the experimental group. The culture plate was placed in an incubator at 28 to 30°C and vibrated (450 r / min) for 10 to 18 hours. The OD of the culture solution in each well was measured with an enzyme marker. 600 The inhibitory effect of the compound on pathogenic bacteria was calculated using formula (2).

[0080] Inhibition rate (%) = {(OD 600 -Control background group OD 600 )-(OD of experimental group 600 -Pharmacy background group OD 600 )} / (control group OD 600 -Control background group OD 600 )×100%(2)

[0081] The results are shown in Table 3:

[0082] Table 3 In vitro antibacterial activity of carnosic acid nanoemulsion against 6 plant pathogenic bacteria (treatment concentration 100 mg / L)

[0083]

[0084] The test results show that the carnosic acid nanoemulsion has different degrees of inhibitory effect on the six tested plant pathogenic bacteria, among which the inhibitory activity against tomato bacterial wilt and citrus canker is higher. The antibacterial activity of the carnosic acid nanoemulsion prepared in Example 1 is generally better than the antibacterial activity of the carnosic acid nanoemulsions in other examples.

[0085] Example 8 Potted plant test of carnosic acid nanoemulsion against tomato bacterial wilt

[0086] Treatment group: the nanoemulsions of Examples 1 to 4 were diluted into solutions containing carnosic acid at concentrations of 1000 and 2000 mg / mL;

[0087] Control group: 3% Zhongshengmycin wettable powder diluted into 1000 and 2000 mg / L solution;

[0088] Blank control group: water.

[0089] When the potted tomato seedlings grew to 3-4 pieces, 10 mL of each test solution was taken for root irrigation, once every 2 days, for a total of 3 times. One day after the third root irrigation, 5 mL of bacterial wilt solution (2×10 8 cfu / mL), 20 plants were treated for each treatment, and cultured for 15 days at 27-30℃ under a 13h / 11h photoperiod. The number of diseased plants was observed and recorded, and the statistics were recorded according to Part 32 "Fungicides for Control of Tomato Bacterial Wilt" (NY / T 1464.32-2010) of the "Guidelines for Efficacy", and the control efficacy was calculated according to Formula 3 and Formula 4.

[0090] Incidence rate (%) = number of diseased plants / number of investigated plants × 100% (3)

[0091] Prevention effect % = (the incidence rate of the control group after application of the drug - the incidence rate of the treatment group after application of the drug) / the incidence rate of the control group after application of the drug × 100% (4) The results are shown in Table 4:

[0092] Table 4 Potted plant protection effect of carnosic acid nanoemulsion on tomato bacterial wilt

[0093]

[0094]

[0095] The test results show that the higher the treatment concentration of the carnosic acid nanoemulsion, the higher the control effect on tomato wilt potted plants; at a treatment concentration of 2000 mg / L, the control effect of the nanoemulsion of Example 1 is higher than that of Examples 3 and 4, and also higher than the control effect of the sinoproline in the control agent; at a treatment concentration of 1000 mg / L, the control effect of the carnosic acid nanoemulsions prepared in Examples 1-4 is better than the control effect of the sinoproline in the control agent.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of a carnosic acid nanoemulsion as an agricultural fungicide for preventing and controlling plant diseases, characterized in that: The plant diseases include rice sheath blight, rubber anthracnose, tomato gray mold, litchi downy mildew, pepper phytophthora, grape anthracnose, tomato bacterial wilt, citrus canker, potato soft rot, rice bacterial leaf blight, mango bacterial angular leaf spot, melon bacterial fruit spot; The carnosic acid nanoemulsion comprises the following raw materials in percentage by mass: 0.5-5% of carnosic acid, 5-20% of organic solvent, 4.0-10% of emulsifier and the balance of water.

2. The use according to claim 1, characterized in that: The organic solvent is any one of ethanol, cyclohexanone and methyl oleate.

3. The use according to claim 1, characterized in that: The emulsifier is at least one of styrylphenol polyoxyethylene ether, tristyrylphenol polyoxyethylene polyoxypropylene ether, castor oil polyoxyethylene ether EL-40, castor oil polyoxyethylene ether EL-60, polyoxyethylene hydrogenated castor oil RH-40, fatty alcohol polyoxyethylene ether AEO-5, peregal O-25, lauryl alcohol polyoxyethylene Brij-35, and isomeric tridecyl alcohol polyoxyethylene ether E-1310.

4. The use according to claim 3, characterized in that: The emulsifier is prepared by mixing styrylphenol polyoxyethylene ether and polyoxyethylene hydrogenated castor oil RH-40 according to a mass ratio.

5. The use according to claim 4, characterized in that: The emulsifier is prepared by mixing styrylphenol polyoxyethylene ether and polyoxyethylene hydrogenated castor oil RH-40 in a mass ratio of 1:

1.

6. The use according to claim 1, characterized in that: The preparation method of the carnosic acid nanoemulsion adopts a low-energy emulsification method and comprises the following steps: (1) weighing raw materials according to proportion, completely dissolving the weighed carnosic acid technical in a solvent, then adding an emulsifier, stirring well to obtain an oil phase, and setting aside; (2) subjecting the oil phase to magnetic stirring, and under stirring, gradually adding deionized water dropwise into the oil phase; (3) After the deionized water is added, the magnetic stirring is continued for 20-40 minutes to form a uniformly dispersed emulsion, thereby obtaining a nanoemulsion of carnosic acid.

7. The use according to claim 1, characterized in that: The magnetic stirring speed is 500-1000 r / min.

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