A nanoemulsion formulation containing d-limonene and its application

Abstract

This invention provides a nanoemulsion formulation containing d-limonene, comprising the following components in weight percentage: 1-15% d-limonene, 2-30% emulsifier, 1-15% co-emulsifier, and distilled water to make up to 100%. This application improves the dispersibility of d-limonene in an aqueous environment, enhances the stability of d-limonene, and increases the delivery efficiency of d-limonene by developing a nanoemulsion formulation of d-limonene, thereby enhancing its antibacterial activity.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to a nanoemulsion formulation containing d-limonene and its application. Background Technology

[0002] The application of plant-derived pesticides and other natural compounds has become one of the alternatives to chemically synthesized pesticides, and their application in agricultural management is receiving increasing attention. Limonene, also known as limonene, is a monoterpene compound and a common component of plant essential oils. It is mainly found in citrus fruits such as oranges, lemons, tangerines, grapefruits, and kumquats, and is also present in other plants such as peppermint, camphor, fir oil, pine needles, and spearmint. d-Limonene is the dextrorotatory isomer of limonene. d-Limonene possesses various agricultural biological activities, including bactericidal, insecticidal, and acaricidal effects. However, a search on pesticide information websites shows that currently registered products only contain d-limonene soluble concentrates, indicating relatively limited formulation development.

[0003] Nanoemulsion formulations are thermodynamically stable, isotropic, transparent or translucent diffusion distribution systems with particle sizes ranging from 1 to 100 nm, composed of an oil phase (O), an aqueous phase (W), a surfactant (S), and a co-surfactant (A) in a specific ratio. In terms of particle size, nanoemulsions are a transitional product between micelles and emulsions, possessing characteristics shared by both.

[0004] d-Limonene is a plant-derived pesticide characterized by low toxicity and low residue, which helps maintain the high quality of agricultural products. However, its plant-derived bioactivity is also relatively low. d-Limonene has natural hydrophobic properties, making it difficult to integrate with other substances, and it is prone to volatility and oxidation during use, leading to a decrease in its activity. To protect the functional properties of d-limonene and improve its efficacy, this application will develop a nanoemulsion formulation of d-limonene to enhance its stability and bioactivity. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides the following technical solution:

[0006] A nanoemulsion formulation containing d-limonene comprises the following components in weight percentage: d-limonene 1-15%, emulsifier 2-30%, co-emulsifier 1-15%, and distilled water to make up 100%.

[0007] A nanoemulsion formulation containing d-limonene comprises the following components in weight percentage: 1-10% d-limonene, 2-20% emulsifier, 1-10% co-emulsifier, and distilled water to make up 100%.

[0008] A nanoemulsion formulation containing d-limonene comprises the following components in weight percentage: 2-10% d-limonene, 2-15% emulsifier, 2-10% co-emulsifier, and distilled water to make up 100%.

[0009] The emulsifier in the nanoemulsion formulation is Ethylan NS-500LQ or TERMUL2507; the co-emulsifier is one or more of n-butanol, ethylene glycol, or glycerol; the mass ratio of emulsifier to co-emulsifier is 1:1 to 6:1.

[0010] The emulsifier in the nanoemulsion formulation is Ethylan NS-500LQ, and the co-emulsifier is n-butanol; the mass ratio of the emulsifier and the co-emulsifier is 1:1-5:1, preferably 1:1-4:1 or 1:1-3:1.

[0011] The mass ratio of d-limonene to the total amount of emulsifier and co-emulsifier in the nanoemulsion formulation is 1:2-1:9. Preferably, it is 1:2-1:7 or 1:2-1:5.

[0012] The nanoemulsion formulation described above can be used for the prevention and control of crop diseases and pests; especially for the prevention and control of crop diseases; preferably for the prevention and control of tomato gray mold.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects:

[0014] Nanoemulsification can improve the water solubility of d-limonene, allowing lipophilic components to quickly integrate into systems with water as the main solvent. The nanoemulsion has a smaller particle size, better dispersibility, and a larger specific surface area, making it easier for active ingredients to come into contact with the target organism, thereby enhancing antibacterial activity.

[0015] Compared with known 5% d-limonene soluble concentrates, the d-limonene nanoemulsion formulation prepared in this invention can significantly improve the control effect of the agent, and field observations have shown that it is safe for crops. Attached Figure Description

[0016] Figure 1 Pseudo-ternary phase diagram of Ethylan NS-500LQ and ethylene glycol combination

[0017] Figure 2 Ternary phase diagram of Ethylan NS-500LQ and n-butanol combination

[0018] Figure 3 Pseudo-ternary phase diagram of Ethylan NS-500LQ and glycerol combination Detailed Implementation

[0019] The present invention will be further explained below with reference to specific embodiments.

[0020] Example 1: Screening of emulsifiers and co-emulsifiers in d-limonene nanoemulsion formulations

[0021] Materials and Methods

[0022] Active ingredient: d-limonene;

[0023] Emulsifiers: TERMUL2507, Ethylan NS-500LQ, Tween 80, Agricultural Emulsion 500#;

[0024] Co-emulsifiers: n-Butanol, ethylene glycol, glycerin;

[0025] Solvent: Distilled water

[0026] Test method:

[0027] Criteria for judging nanoemulsions: The prepared liquid is clear and transparent or translucent; it has a blue opalescence; it is stable and does not separate into layers after high-speed centrifugation at 13,000 rpm for 45 min; it exhibits the Tyndall effect after parallel light incident; and its particle size is between 1 and 100 nm as detected by transmission electron microscopy.

[0028] Selection of combinations of emulsifiers and co-emulsifiers

[0029] Emulsifiers TERMUL2507, EthylanNS-500LQ, Tween 80, and Agricultural Emulsifier 500# were mixed with co-emulsifiers n-butanol, ethylene glycol, and glycerin at a mass ratio of 2:1. The mixture was stirred at a constant speed in a magnetic stirrer at 25°C until homogeneous, yielding a mixed emulsifier. This mixture was then combined with d-limonene at a mass ratio of 5:1 and stirred at a constant speed in a magnetic stirrer at 25°C until homogeneous. Distilled water was added dropwise while stirring, and the formation of a clear, transparent nanoemulsion was observed.

[0030] Selection of co-emulsifiers

[0031] The selected emulsifier and co-emulsifier combinations were mixed with d-limonene at mass ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, respectively. The mixtures were stirred at a constant speed in a magnetic stirrer at 25°C, and distilled water was slowly added dropwise, recording the amount of distilled water used when the solution changed from turbid to clear. The most suitable co-emulsifier was screened using a pseudo-ternary phase diagram.

[0032] Results and Analysis:

[0033] The emulsifying performance of the combination of emulsifier and co-emulsifier was evaluated by observing whether a clear and transparent solution could be formed. Table 1 shows that Ethylan NS-500LQ can form a clear and transparent solution when combined with n-butanol, ethylene glycol, or glycerol. TERMUL 2507 can also form a clear and transparent solution when combined with ethylene glycol, but this solution is not stable and becomes cloudy upon standing. Furthermore, when Ethylan NS-500LQ is used as the emulsifier, it can form nanoemulsion formulations with a wider variety of co-emulsifiers; therefore, Ethylan NS-500LQ was chosen as the emulsifier for nanoemulsions.

[0034] Depend on Figure 1-3 The comparison shows that the area of ​​the nanoemulsion region formed when n-butanol is used as a co-emulsifier is larger than that formed when ethylene glycol or glycerol is used as a co-emulsifier. Therefore, n-butanol was chosen as the co-emulsifier.

[0035] Table 1: Emulsifying properties of d-limonene nanoemulsions

[0036]

[0037] Example 2: Optimization of d-limonene nanoemulsion formulation

[0038] Experimental method: Selected emulsifiers and co-emulsifiers were mixed in mass ratios of 1:1, 3:1, 6:1, and 9:1. Then, the emulsifiers and essential oils were mixed in the following mass ratios and stirred at room temperature. At the same time, d-limonene was added dropwise, and the stability was observed.

[0039] Conclusions and Analysis:

[0040] As shown in Table 2, when the mass ratio of Ethylan NS-500LQ to n-butanol is 1:1-6:1, the nanoemulsion formulation is clear and transparent; when the mass ratio of Ethylan NS-500LQ to n-butanol is 9:1, the nanoemulsion formulation is unstable and becomes cloudy when left to stand.

[0041] Table 2: Screening of Ethylan NS-500LQ and n-butanol ratios

[0042]

[0043]

[0044] As shown in Table 3, clear and transparent nanoemulsions can be formed when the mass ratio of the mixed emulsifier to d-limonene is between 1:2 and 1:9. However, the nanoemulsions formed when the mass ratio of the mixed emulsifier to d-limonene is 4:4 are unstable.

[0045] Table 3: Screening of mixed emulsifiers and d-limonene mass ratio

[0046]

[0047] Unstable: Turns cloudy when left to stand

[0048] Example 3: Nanoemulsion Formulation

[0049] 5% d-limonene nanoemulsion contains the following ingredients in weight percentage:

[0050] d-Limonene 5%, EthylanNS-500LQ 12%, n-Butanol 6%, distilled water to make up.

[0051] 8% d-limonene nanoemulsion contains the following ingredients in weight percentage:

[0052] d-Limonene 8%, EthylanNS-500LQ 13%, n-Butanol 5%, distilled water to make up.

[0053] 2% d-limonene nanoemulsion contains the following ingredients in weight percentage:

[0054] d-Limonene 2%, EthylanNS-500LQ 10%, n-Butanol 2%, distilled water to make up.

[0055] Example 4: Performance Testing of d-Limonene Nanoemulsion Formulation

[0056] Test metrics:

[0057] Formulation type: The diffusion rate of Sudan Red dye and Methylene Blue dye in nanoemulsion was used for determination.

[0058] Particle size test: Take 1 mL of nanoemulsion and dilute it 1000 times. Measure the average particle size of the nanoemulsion using a laser particle size analyzer at room temperature.

[0059] Dilution stability: The nanoemulsion was diluted 500 times and 1000 times with deionized water, and phenomena such as layering were observed.

[0060] Temperature stability: Take an appropriate amount of nanoemulsion preparation and place it at -20 and 40℃ for 48 hours respectively, then restore it to room temperature and observe its appearance and layering changes.

[0061] Table 4: Performance Testing of d-Limonene Nanoemulsion Formulations

[0062]

[0063] Example 5: Field efficacy test of d-limonene nanoemulsion formulation against tomato gray mold

[0064] Crop diseases: Tomato gray mold

[0065] Test reagent: Example 3 nanoemulsion formulation;

[0066] 5% d-limonene soluble concentrate (Puriwan), manufactured by Oro Agra International Ltd.

[0067] Experimental methods: A treatment group and a control group were set up, with four replicates in a randomized block design. The plot area was 45 m². Foliar spraying was used, applied three times at 7-day intervals, with a spray volume of 40 kg / mu. Leaf disease severity was assessed before the first spray and again 21 days after the third application. Disease severity grading criteria were as follows:

[0068] Grade 1: No lesions

[0069] Grade 3: Three lesions on a single leaf

[0070] Grade 5: 4-6 lesions on a single leaf

[0071] Grade 7: 7-10 lesions on a single leaf

[0072] Level 9: Dysplasia is densely distributed on a single leaf, covering more than a quarter of the leaf area.

[0073] Formula for calculating drug efficacy:

[0074]

[0075]

[0076] In the formula: CK0—disease index of the blank control area before drug administration;

[0077] CK1 – Disease index after drug administration in the blank control area;

[0078] PT0—Disease index in the treatment area before application of pesticides;

[0079] PT1 – Disease index after drug treatment in the treatment area.

[0080] Experimental results:

[0081] The test results in Table 4 show that, compared with the known 5% d-limonene soluble concentrate, the d-limonene nanoemulsion preparation prepared in this invention can significantly improve the control effect of the agent on tomato gray mold, and field observations show that it is safe for crops.

[0082] Table 5: Field efficacy test of d-limonene nanoemulsion formulation against tomato gray mold

[0083]

[0084]

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. A nanoemulsion formulation comprising d-limonene, characterized in that, It contains the following ingredients by weight percentage: d-limonene 1-15%, emulsifier 2-30%, co-emulsifier 1-15%, and distilled water to make up to 100%; The emulsifier mentioned is Ethylan NS-500LQ; The co-emulsifier is n-butanol; The mass ratio of the emulsifier to the co-emulsifier is 1:1 to 6:1; The mass ratio of d-limonene to the total of emulsifiers and co-emulsifiers is 1:2 to 1:

9.

2. The use of the nanoemulsion formulation according to claim 1 for the prevention and control of crop diseases and pests.

3. The use according to claim 2, characterized in that, Used for the prevention and control of crop diseases.

4. The use according to claim 3, characterized in that, Used for the prevention and control of gray mold in tomatoes.

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