Larvicidal nanoemulsion based on jatropha oil.

Description

1 / 9 “NANOEMULSION LARVICIDE BASED ON JATROPHA OIL” FIELD OF THE INVENTION

[001] . This invention patent consists of a nanoemulsion based on physic nut (Jatropha curcas L) vegetable oil for the control of Aedes aegypti larvae. This invention is applicable in the technical sector of public health, specifically in the area of ​​surveillance and control of zoonoses. FUNDAMENTALS OF THE INVENTION

[002] . There is a significant problem of insect resistance to synthetic chemical insecticides commonly used in vector control.

[003] . The Aedes aegypti mosquito is the vector of several serious diseases, such as dengue, yellow fever, Chikungunya and Zika.

[004] . Plant products with pesticidal properties are desirable, especially because such products have a lower environmental impact than synthetic chemical insecticides, which can persist in the environment and cause damage to ecosystems and non-target organisms. The complexity of natural components is also advantageous because it makes mosquito selection more difficult, unlike synthetic insecticides, which are often associated with the selection of resistant populations.

[005] . Vegetable oils, including physic nut oil (Jatropha curcas L.), have limitations in chemical stability and water solubility, making their use in bodies of water difficult. This makes it challenging to develop formulations that achieve uniform oil dispersion in water, improving the effectiveness of larvicidal control in aquatic environments where larvae develop.

[006] . Biocides composed of natural products can be integrated with other Petition 870240111086, dated 12 / 30 / 2024, pp. 44 / 56 2 / 9 Vector control practices, such as environmental sanitation and biological control, provide a more comprehensive and sustainable approach to vector management.

[007] . Patent document US2020390110A1 discloses compositions consisting of oil-in-water emulsions with added polymer. The formulations may also include a wetting agent (with HLB between 7 and 10) for the purpose of reducing surface tension in the final product. Unlike the technology disclosed in the present description, such formulations involve the use of vegetable oils such as corn or canola oils, alkalized and combined with one or more emulsifiers, followed by the addition of a polymer, such as carrageenan, agar or chitin, and optionally, pesticidal essential oils. The resulting compositions create an oil / polymer film on the plant surface for the purpose of controlling arthropod pests.

[008] . Document CN1351834A discloses a plant-based insecticidal composition consisting of an oil / water emulsion containing croton oil. The emulsion is applied for insect control in vegetables and fruit trees, such as pomegranate. The insecticidal composition of that document, unlike that disclosed in the present description, contains 20% croton oil or a combination thereof with Luffa cylindrica oil in a 1:1 ratio, and only Tween-80.

[009] . Document CN102388929A discloses an oil / water microemulsion composed of an alcoholic extract of Jatropha curcas oil flour. The pesticidal properties are attributed to jatrophine, a protein present in the extract, without, therefore, revealing larvicidal activity achieved by a formulation containing Jatropha curcas oil.

[0010] . Oliveira and colleagues (2016, doi.org / 10.1371 / journal.pone.0145835) reveal a nanoemulsion produced with sucupira oil (Pterodon emarginatus Vogel) which, in turn, is recognized for its larvicidal activity against Aedes aegypti larvae. The document suggests that the mechanism of action Petition 870240111086, dated 12 / 30 / 2024, pp. 45 / 56 3 / 9 may involve the reversible inhibition of acetylcholinesterase, attributed to the presence of diterpenes and the compound methyl 6a,7e-dihydroxyvouacapan-17-e-oate, described as a larvicide.

[0011] . Therefore, in the state of the art, biopesticides for arthropods are found that are based on emulsions containing vegetable oils as main ingredients, but a composition in the form of an oil-in-water nanoemulsion comprising jatropha curcas L. oil in its composition is not disclosed. Nor is a composition in the form of an oil-in-water nanoemulsion comprising vaccenic acid disclosed.

[0012] . The nanoemulsion, the subject of this patent, has a larvicidal effect against arthropods, specifically against insects. Since the composition of the nanoemulsion allows the active compounds of a lipophilic nature to be available in aqueous media, this nanoemulsion has application as a pesticide for the elimination of insect larvae in aquatic environments, such as, for example, mosquito larvae. The biodegradable nature of its components allows application in a greater number of locations where Aedes aegypti larvae develop, and provides a practical and efficient solution for vector control. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] . Figure 1 is a chromatogram showing the fatty acid composition of jatropha curcas L. oil after transesterification reaction. 1: Palmitoleic acid; 2: Palmitic acid; 3: Linoleic acid methyl ester; 4: Oleic acid methyl ester; 5: Cisvaccenic acid methyl ester; and 6: Stearic acid methyl ester.

[0014] . Figure 2 is a graph showing the cumulative survival of Aedes aegypti larvae exposed to three nanoemulsions containing Jatropha curcas L. oil at 0.5% (v / v) for up to 72 hours after exposure. Petition 870240111086, dated 12 / 30 / 2024, pages 46 / 56 4 / 9

[0015] . Figure 3 consists of 3 graphs for particle size distribution and Polydispersity Index (PdI), where: A - is a graph showing the particle size distribution and Polydispersity Index (PdI), represented by the mean and standard deviation of the Zeta potential (Z-ave, bars) and PdI (line) of 14 formulations on the day of preparation; B - is a graph showing the particle size distribution and Polydispersity Index (PdI) of an optimized formulation of Jatropha curcas L. crude oil over time; C - is a graph showing the particle size distribution and Polydispersity Index (PdI) of an optimized formulation of Jatropha curcas L. degummed oil over time. DESCRIPTION OF THE INVENTION

[0016] . This patent application relates to a nanoemulsion for controlling Aedes aegypti larvae, whose composition contains as its main ingredient the vegetable oil of physic nut (Jatropha curcas L.).

[0017] . The larvicidal nanoemulsion, the subject of this patent, consists of the following composition: 1.0 g of physic nut oil (Jatropha curcas L.); 0.30 g of Span 80; 0.30 g of Tween 80; 50 mL of ultrapure water; and 25 mL of acetone. The physic nut oil (Jatropha curcas L.) acts as a source of vaccenic acid, the main larvicidal substance. The surfactants used can be replaced by other non-ionic surfactants. Acetone acts as a solvent in the preparation of the oil phase.

[0018] . The preparation of the nanoemulsion, the subject of this patent, comprises the following steps: (i) preparation of the oil phase, by adding crude or degummed oil of Jatropha curcas L., or a fatty acid derived from such oil, the surfactant is Span 80 and acetone; Petition 870240111086, dated 12 / 30 / 2024, pp. 47 / 56 5 / 9 (ii) preparation of the aqueous phase, adding water and the surfactant is Tween 80; and (iii) heating the oil and aqueous phases separately, under stirring, at 37 °C, until they form homogeneous phases; (v) slowly pour the oily aqueous phase over the aqueous phase, keeping the system under constant agitation for 5 minutes; (vi) remove the acetone by evaporation, using a rotary evaporator between 45 °C and 60 °C until a stable emulsion is obtained. Characterization of nanoemulsions:

[0019] . For the purpose of testing the larvicidal nanoemulsion, compositions with a final volume of 10 mL were prepared, comprising: a) Oil phase, consisting of 0.3-0.6 g of Span 80 surfactant; 1.0-2.0 g of Jatropha curcas L. oil; 25-28 mL of acetone; b) Aqueous phase, consisting of: 0.25-0.6 g of Tween 80 surfactant; 50-57 mL of ultrapure water.

[0020] . A stability test against particle size was performed with different formulations, which showed stability between zero and 28 days. The analyses were performed macroscopically and considered color, visual appearance, phase separation, presence of creaming and sedimentation, and Zeta potential: Table 1: Stability against particle size analyzed over 28 days. Formulation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Stability (days) 7 7 7 14 0 14 7 7 0 0 7 28 7 7

[0021] . The fatty acid composition of jatropha curcas L. oil was determined by gas chromatography (GC) coupled with mass spectrometry (MS) analysis. Petition 870240111086, dated 12 / 30 / 2024, pages 48 / 56 6 / 9

[0022] . As shown in Figure 1, the oil from jatropha curcas L. presented 6 peaks identified as palmitoleic acid; palmitic acid; linoleic acid methyl ester; oleic acid methyl ester; cis-vaccenic acid methyl ester, and stearic acid methyl ester. The most prominent peak is formed by the compounds oleic acid methyl ester and linoleic acid methyl ester. Cis-vaccenic acid methyl ester has low polarity and similarity to oleic acid methyl ester, making its separation difficult. The percentage of each fatty acid can be seen in the table below: Table 2: List of chemical constituents of jatropha curcas L. oil S. No. Retention Time Chemical Constituent Peak Area % 1 46.838 Palmitoleic acid 0.718% 2 48.112 Palmitic acid 13.476% 3 55.864 Linoleic acid methyl ester 35.517% 4 56.247 Oleic acid methyl ester 42.944% 5 56.334 Cisvaccenic acid methyl ester 1.841% 6 57.259 Stearic acid methyl ester 5.504%

[0023] . To test the insecticidal activity of nanoemulsions containing physic nut oil (Jatropha curcas L.), tests were performed in triplicate, using formulations with different concentrations of physic nut oil, in addition to surfactants, as shown in Table 3. Table 3: Quantities in grams of the constituents of the formulations. Formulation: Tween 80 (g) Span 80 (g) Jatropha curcas L. oil (g) Z- ave (nm) 12 0.3064 0.3025 1.0005 318.2 Petition 870240111086, dated 12 / 30 / 2024, pp. 49 / 56 7 / 9 15 0.2423 0.2443 0.8050 248.7 17 0.2413 0.2440 0.8090 247.9

[0024] . The formulations whose tests are disclosed in this description contain between 8% and 10% oil (w / v), 2.4% to 3% (w / v) Span 80 and 2.4% to 3% (w / v) Tween 80.

[0025] . Each test group consisted of 10 L3 stage larvae of Aedes aegypti. As shown in Figure 2, the compositions were able to reduce larval survival by 95% in the first 36 hours of use, reaching 1% survival after 60 hours. Table 4: Paired log-rank comparisons of survival rates and mean survival time (S50) of 3rd instar Aedes aegypti larvae exposed to nanoemulsions and water. S50 Treatment nanoemulsions H2O F12 F15 F17 S50 H2O X2=2.000 P =0.1573 χ2= 0.2089 P= 0.6476 P=0.0128 ND F15 - - χ2=1.103 P=0.2936 ND F17 - - - ND Statistical significance (P-value) among Aedes aegypti exposed to nanoemulsions 12, 15, and 17 in water. ND (Not determined) and χ2 (chi-square value).

[0026] . The prediction of the biological activity of vaccenic acid was performed using online PASS servers, and different targets were identified. Petition 870240111086, dated 12 / 30 / 2024, pages 50 / 56 8 / 9 potential molecular factors related to insect physiology and lipid interactions, described in Table 5. Table 5. Summary of selected cis-vaccenic acid molecular targets from analysis on the PASS online platform: Cis-vaccenic acid activity Probability of inhibition Probability Activity / molecular target Lipid alteration induction 0.928 0.002 Lipoprotein lipase inhibitor 0.908 0.004 Lipid metabolism regulator 0.856 0.003 Fatty-acyl-CoA synthase inhibitor 0.606 0.003 Phospholipase C inhibitor 0.621 0.022 Membrane integrity antagonist 0.579 0.004 Membrane permeability enhancer 0.415 0.004 Phospholipase inhibitor 0.399 0.010 Cholesterol synthesis inhibitor Cytochrome P450 (CYP) 0.974 0.001 CYP2J substrate 0.808 0.005 NADPH-cytochrome-c2 reductase inhibitor 0.733 0.002 Cytochrome-c3 hydrogenase inhibitor 0.692 0.015 Nitrate reductase (cytochrome) inhibitor 0.503 0.010 dismutase inhibitor 0.754 0.003 Nitrite reductase (NO-forming) inhibitor 0.749 0.003 Catalase inhibitor 0.724 0,005 TNF expression inhibitor 0.688 0.020 Glutathione thiolesterase inhibitor, Petition 870240111086, dated 12 / 30 / 2024, pages 51 / 56 9 / 9 0.540 0.501 0.031 0.053 Immunostimulant JAK2 expression inhibitor 0.321 0.021 Transcription factor NF kappa A inhibitor 0.306 0.042 NOS2 expression inhibitor 0.897 0.002 Glutarate-semialdehyde dehydrogenase inhibitor Nervous system 0.861 0.004 Acetylesterase inhibitor 0.820 0.003 GABA aminotransferase inhibitor 0.578 0.003 Glutamate-1 -semialdehyde 2,1-aminomutase inhibitor 0.547 0.025 Neurotransmitter antagonist 0.346 0.149 Octopamine antagonist 0.366 0.187 Antineurotic Chitin synthesis q g5g 0.008 Chitinase inhibitor 0.417 0.002 Chitin deacetylase inhibitor 0.802 0.003 Ecdysone 20-monooxygenase inhibitor Insecticide 0.318 0.014 Insecticide Digestion 0.934 0.002 Carboxypeptidase Taq inhibitor 0.909 0.003 Glucan endo-1,3-beta-D-glucosidase inhibitor 0.875 0.004 Fucosterol-epoxide lyase inhibitor CONCLUSION

[0027] . The best way to carry out the invention, considering the stability of the composition and effectiveness as a larvicide, consists of the following composition: 1.0g of jatropha curcas L oil; 0.30g of Span 80; 0.30g of Tween 80; 50 mL of ultrapure water; and 25 mL of acetone.

[0028] . However, other embodiments of the invention are possible by varying the quantities of the ingredients in the composition between: 1.0-2.0g of jatropha curcas L. oil; 0.3-0.6g of Span 80 surfactant; 25-28mL of acetone; 0.25-0.6g of Tween 80 surfactant; and 50-57 mL of ultrapure water. Petition 870240111086, dated 12 / 30 / 2024, pp. 52 / 56

Claims

1 / 1 CLAIM 1. Larvicidal nanoemulsion characterized by consisting of the following composition: 1.0g of jatropha curcas L. oil; 0.30g of Span 80; 0.30g of Tween 80; 50 mL of ultrapure water; and 25 mL of acetone.

2. Larvicidal nanoemulsion, according to claim 1, characterized in that jatropha oil acts as a source of vaccenic acid.

3. Larvicidal nanoemulsion, according to claims 1 and 2, characterized in that vaccenic acid has the larvicidal effect in the composition.

4. Larvicidal nanoemulsion, according to claim 1, characterized by the fact that it uses two combined non-ionic surfactants, Span 80 and Tween 80.

5. Larvicidal nanoemulsion, according to claim 1, characterized by using a polar and aprotic organic solvent in the preparation of the oil phase. Petition 870240111086, dated 12 / 30 / 2024, pp. 53 / 56

Images